Methods of eliciting immune tolerance using soluble immune checkpoint proteins

Abstract

The present disclosure provides, in part, methods of inducing immune tolerance using truncated membrane binding and soluble immune checkpoints. Constructs comprising an immune checkpoint protein or a portion thereof capable of binding to and activating an immune checkpoint protein receptor are provided. A nucleic acid encoding the immune checkpoint protein or part thereof is operably linked to the promoter, and optionally also linked to a secretion signal to allow secretion of the immune checkpoint protein or part thereof. The constructs may comprise viral vectors that may be used to deliver or introduce the constructs into a transplantable article or organ.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 411,298, filed on September 29, 2022, the content of which is incorporated herein by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The content of the electronic sequence listing (155554.00711.xml; size: 18,509 bytes; and creation date: September 29, 2023) is incorporated herein by reference in its entirety.

[0005] Background

[0006] The number of patients expected to have heart failure is projected to increase by nearly 46% by 2030, reaching approximately 8 million. Nearly 50% of these patients are expected to die within 5 years of diagnosis. For those patients with end - stage heart failure, heart transplantation is the gold - standard treatment. Long - term survival after transplantation is limited by graft dysfunction, rejection, vasculopathy, and long - term systemic immunosuppression. There is an urgent need to apply significant advances in the field of gene therapy to the transplantation field to reduce transplant rejection, reduce the use of immunosuppressive agents, and expand the donor pool. Given the above, it is desirable to provide new targets and methods to minimize organ rejection.

[0007] Summary

[0008] The present disclosure provides constructs encoding immune checkpoint proteins (including PDL1) and methods of using the constructs to transduce organs and reduce transplant rejection.

[0009] 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 the 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 functionally linked to the nucleic acid encoding the immune checkpoint protein or a portion thereof. In some embodiments, the immune checkpoint protein includes PDL1 or PD1. In some embodiments, the construct includes a viral vector, such as an AAV vector. In some embodiments, the composition includes a pharmaceutical composition comprising the construct described herein.

[0010] The 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 the construct or pharmaceutical composition described herein into the transplantable article. In some embodiments, the method comprises perfusing the construct or pharmaceutical composition ex vivo or in vitro into the article. 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 tissue.

[0011] Another aspect of the present disclosure provides a method for preventing or reducing rejection of a transplanted article in a subject. In some embodiments, the method comprises introducing the construct or pharmaceutical composition described herein into the article prior to transplanting the transplantable article into the subject. In some embodiments, the method further comprises transplanting the article into the subject.

[0012] Another aspect of the present disclosure provides a method for introducing a construct into a transplantable article, the method comprising perfusing the construct ex vivo into the article prior to transplantation.

[0013] Another aspect of the present disclosure provides a method for preventing or reducing rejection of a transplanted article in a subject. In some embodiments, the method comprises introducing the construct or pharmaceutical composition described herein into the transplantable article prior to transplantation into the subject. In some embodiments, the method further comprises transplanting the article into the subject. Brief Description of the Drawings

[0015] The present technology can be better understood by reference to the accompanying drawings. The drawings are merely exemplary and illustrate certain features that can be used alone or in combination with other features, and the present technology should not be limited to the embodiments shown.

[0016] Figure 1 . Plot of PD-L1 variant-mediated T cell inactivation. The PD-L1 variant constructs include full-length PD-L1, ICD-truncated PD-L1, and soluble PD-L1.

[0017] Figure 2 . In vitro expression of PD-L1 (full-length, truncated, and secreted isoforms) in triple-negative breast cancer cells.

[0018] Figure 3 . Elevated PD-L1 (pg / mL) in the sera of tumor-bearing mice with the PD-L1 secreted isoform.

[0019] Figure 4 . Transplantation of PD-L1 isoform-expressing immunogenic cell lines in mice.

[0020] Figure 5 . Schematic of construct targeting the ROSA26 locus.

[0021] Figure 6 . In vivo PD-L1 expression was confirmed by A) histology and B) ELISA to be evident in transgenic mice with truncated and secreted variants.

[0022] Figure 7 . Transgenic mice containing the truncated PD-L1 construct showed increased tolerance to rejection and prolonged graft survival.

[0023] Figure 8 . Introduction of a viral vector with the PDL1 secreted construct into an allograft prior to transplantation resulted in graft survival being prolonged for over 80 days.

[0024] DETAILED DESCRIPTION

[0025] The present disclosure is in part based on the inventors' discovery of using gene therapy methods to ex vivo or in vitro transduce transplanted organs with immunosuppressive genes. Constructs encoding immune checkpoint proteins, including PDL1, and methods of using the constructs to transduce organs and reduce transplant rejection are disclosed herein.

[0026] Constructs:

[0027] In a first aspect, the 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 appropriately activating the receptor to allow immune checkpoint signaling to occur. A construct or expression vector, also referred to as an expression construct, is generally a plasmid or virus designed for gene expression in cells. Constructs are used to introduce a specific gene into target cells and can commandeer the cell's protein synthesis machinery to produce the protein encoded by that gene.

[0028] The term "construct" or "polynucleotide construct" is a polynucleotide that permits the replicated and / or expressed encoding sequences in a target cell. The construct may contain an exogenous promoter operably linked to any of the polynucleotides described herein. As used herein, a polynucleotide is "operably linked" or "operably linked to" when 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, which may be found 5' or 3' to the polynucleotide described herein, or within the coding region of the polynucleotide. Generally, 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 is bounded at its 3' end by a transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. The transcription start site (conveniently defined by nuclease S1 mapping), as well as the protein-binding domain (consensus sequence) responsible for RNA polymerase binding, are within the promoter sequence.

[0029] In some embodiments, the construct is an expression construct, vector, or viral vector. A vector is any particle that serves as a vehicle to artificially carry a foreign nucleic acid sequence (usually DNA) into another cell, in which the foreign nucleic acid sequence can replicate and / or be expressed. A construct containing foreign DNA is called recombinant DNA. The four main types of constructs and vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. An expression construct contains a heterologous promoter and a nucleic acid sequence encoding a protein of interest (e.g., an immune checkpoint protein), which is capable of being expressed in the cell into which it is introduced. Expression constructs include constructs capable of directing the expression of an exogenous gene operably linked thereto. Such constructs are referred to herein as "recombinant constructs", "expression constructs", "recombinant expression vectors" (or simply "expression vectors" or "vectors") and can be used interchangeably. Suitable constructs are known in the art and contain the elements necessary to express the gene encoded within the construct as a protein in a host cell. The terms "vector" and "construct" are used interchangeably herein and refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment (such as a foreign DNA segment encoding a mutant α-gal protein) can be ligated. Another type of vector is a viral vector, into which an additional DNA segment can be ligated into the viral genome. The viral vector is incorporated into a viral particle, which is then used to transport the viral polynucleotide encoding the protein of interest into a target cell. Certain constructs are capable of autonomous replication in the host cell into which they are introduced. Other constructs can integrate into the genome of the host cell after being introduced into the host cell and thus replicate with the host genome (e.g., lentiviral vectors). In addition, certain vectors are capable of directing the expression of an exogenous gene operably linked thereto. Generally, constructs useful in recombinant DNA technology are often in the form of plasmids. In this specification, "vector" includes expression vectors such as viral vectors (e.g., replication-defective retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses (AAV)), which perform equivalent functions.

[0030] A construct is a heterologous foreign construct containing sequences from two or more different sources. Suitable constructs or vectors include, but are not limited to, plasmids, retroviruses, adenoviruses, oncoretroviruses, lentiviruses, foamy viruses, adeno-associated viruses, herpes simplex viruses, etc., and include constructs capable of expressing a protein of interest. A preferred vector is an adeno-associated vector (AAV). Suitable methods for preparing viral particles capable of transforming cells to express a protein of interest as described herein are known in the art.

[0031] Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive promoters, inducible promoters, temporally regulated promoters, developmentally regulated promoters, chemically regulated promoters, tissue-preferred promoters, tissue-specific promoters, and cell type-specific promoters. The heterologous promoter can be an animal promoter, a bacterial promoter, a fungal promoter, a viral promoter, or a synthetic promoter. Suitable promoters are known and described in the art. In mammalian cells, typical promoters include, but are not limited to, the Rous sarcoma virus (RSV) promoter, the human immunodeficiency virus (HIV-1) promoter, the cytomegalovirus (CMV) promoter, the SV40 virus promoter, the chicken β-actin promoter, the 3-phosphoglycerate kinase promoter, and the translation elongation factor EF-1α promoter or ubiquitin promoter.

[0032] In some embodiments, the constructs described herein can include a secretion signal. A secretion signal, sometimes referred to as a signal peptide, signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide, is or encodes a short peptide (usually 16-30 amino acids in length) present at the N-terminus, C-terminus, or internally in most newly synthesized proteins that enter the secretory pathway. Generally, the function of the secretion signal is to cause the cell to translocate the protein to the cell membrane and / or the extracellular space. The signal peptide follows a general three-domain structure, containing a basic N-domain, a hydrophobic H-domain, and a slightly polar C-domain. As a representative example, the signal sequence from murine follicle-stimulating hormone B (5'-atgatgaagttgatccagctttgcatcttattctggtgctggagagcaatctgctgc-3' SEQ ID NO:13) was used in murine constructs and experiments.

[0033] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound that includes a nucleobase and an acidic moiety, such as a nucleoside, nucleotide, or nucleotide polymer. Nucleic acids generally refer to polymers that include nucleotides or nucleotide analogs linked together by a backbone linkage (such as, but not limited to, a phosphodiester bond). Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as messenger RNA (mRNA), transfer RNA (tRNA), and the like. Generally, polymeric nucleic acids (such as nucleic acid molecules that include 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 (such as nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain that includes three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to a nucleotide polymer (such as, a string of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids can be naturally occurring, such as in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or include non-naturally occurring nucleotides or nucleosides. In addition, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, that is, analogs having a backbone other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, synthesized chemically, and the like. In suitable cases, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars and backbone modifications. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' direction.In some embodiments, the nucleic acid is or comprises 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-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 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); intercalating bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

[0034] In some embodiments, the constructs described herein encode an immune checkpoint protein or an extracellular portion of an immune checkpoint protein. Immune checkpoints are inhibitory regulators of the immune system that are essential for maintaining self-tolerance, preventing autoimmunity, and controlling the duration and extent of immune responses to minimize collateral tissue damage. When checkpoint proteins and ligand partner proteins (which are most often expressed on T cells) bind together, they send a "shut off" signal to the T cells, thus reducing the immune response. Immune checkpoint proteins are often overexpressed on tumor cells or non-transformed cells within the tumor microenvironment and impair the ability of the immune system to mount an effective anti-tumor response, thus allowing tumor cells to proliferate. Thus, immune checkpoint inhibitors are the standard of care for treating some cancers. As shown in the examples, overexpression or induced expression of immune checkpoint proteins on non-HLA-matched tumors allows tumor cells to proliferate in a mouse model in which the growth of non-HLA-typed tumors would otherwise be inhibited by the animal's immune response. See Figure 3 and Figure 4Immune checkpoint proteins lacking transmembrane domains or prepared in a secreted form are significantly better at allowing tumors to form in a mouse model, indicating that these forms may be more suitable for transfer into transplantable products or organs to reduce the risk of immune-based rejection of the transplantable product 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, HLA-E. In some embodiments, the immune checkpoint protein is CTLA4.

[0035] In some embodiments, the immune checkpoint proteins described herein or the extracellular portion of an immune checkpoint protein is capable of binding its receptor. 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 are capable of interacting with more than one receptor or more than one ligand. For example, CTLA4 is a receptor for CD80 as well as CD86. The truncated immune checkpoint proteins (portions of an immune checkpoint protein) provided herein may remain capable of eliciting the same or significantly the same changes in immune function as the full-length protein.

[0036] 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 (also known as PD-1 and CD279). Engagement of PD-L1 with its receptor PD-1 on T cells delivers signals that inhibit TCR-mediated IL-2 production and activation of T cell proliferation, which downregulates the immune system and promotes self-tolerance by inhibiting T cell inflammatory activity. PD-L1 is a type I transmembrane glycoprotein encoded by the CD274 gene on human chromosome 9. Transcription of this gene can generate 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 can comprise full-length PD-L1, including 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, including 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, including 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).

[0037] In some embodiments of the present disclosure, the pharmaceutical composition comprises the 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. An aqueous liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffers, chelating agents, complexing agents, solubilizing agents, wetting agents, solvents, suspending agents, and / or thickening agents, tonicity agents, wetting agents, or other biocompatible materials. A list of ingredients in the above categories can be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990).

[0038] Some examples of materials that can serve as pharmaceutically acceptable carriers are: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethanol, and phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. Depending on the expectations of the formulator, wetting agents, emulsifying agents, and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, and fragrances, preservatives, and antioxidants may also be present in the composition.

[0039] Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0040] In another embodiment, the formulations of the present application may further comprise other suitable agents such as stabilizing delivery vehicles, carriers, supports, or complex-forming substances. The coordinated administration methods and combination formulations of the present invention may optionally incorporate effective carriers, processing agents, or delivery vehicles to provide improved formulations for delivering the constructs or vectors described herein.

[0041] The pharmaceutical formulation may additionally contain a biologically acceptable buffer to maintain a near-neutral pH (7.0 - 7.3). Such buffers that are preferably used are generally 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 about 0.0001 - 5% (w / v), more preferably about 0.001 - 1% (w / v) of the vaccine formulation. If desired, other excipients may be included as part of the final pharmaceutical formulation.

[0042] Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0043] Method:

[0044] In a second aspect, the present invention provides a method for introducing one or more of the constructs described herein into a transplantable article. In some embodiments, the method includes introducing any of the constructs described herein or the pharmaceutical compositions described herein into the transplantable article, optionally wherein the introduction includes ex vivo or in vitro perfusion of the construct or pharmaceutical composition into the article.

[0045] For the purpose of expressing the immune checkpoint protein of the construct, the constructs described herein can be introduced into the article. The constructs can be introduced into cells by any means known in the art. These means can include transfection or transduction. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process of introducing foreign DNA into another cell by a viral vector. These are common tools for introducing foreign genes into host cells. Among others, additional means include transformation and conjugation. Additionally, methods employing targeted endonucleases for knock-in of constructs such as CRISPR / Cas gene editing can also be used.

[0046] In some embodiments, the construct can be introduced into the article by ex vivo perfusion. Ex vivo perfusion is also known as normothermic perfusion and includes machines that maintain an organ at body temperature by continuously pumping or perfusing blood, or a bloodless solution of nutrients, proteins and oxygen through the organ. Ex vivo perfusion can reduce the ischemic injury time and allow graft evaluation. In some embodiments, the constructs described herein can be included in the solution perfused through the article.

[0047] In some embodiments, the method further includes analyzing the article or the cells of the article to determine the presence or expression of the construct or the immune checkpoint protein encoded by the construct in the article. Evaluation of the construct or the immune checkpoint protein can be performed by any means known in the art. The results of the evaluation can be used to inform the dose, function or effect of the construct, or if the article is used as a transplanted organ, can be used to determine the level or requirement of additional immunosuppressive therapy.

[0048] In some embodiments, the article may consist of organs, cell populations, and tissues. Articles for transplantation may include organs. Organs may include, but are not limited to, the heart, heart valves, lungs, kidneys, liver, pancreas, skin, spleen, middle ear, connective tissue, intestine, colon, eyes, stomach, ovaries, testes, bladder, uterus, and adrenal glands. Cell populations may include stem cells, bone marrow, and immune cells. Tissues may include bone, tendons, ligaments, skin, heart valves, blood vessels, islets of Langerhans, nerves, veins, and limbs.

[0049] In some embodiments, the method further includes transplanting the article into a subject. Organ transplantation is a medical procedure in which an organ is removed from one body and placed into the body of a recipient 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 site to another location. Allograft transplantation is the transplantation of an organ or tissue between two genetically non-identical members of the same species. Due to the genetic differences between the organ and the recipient, the recipient's immune system may recognize the organ as foreign and attempt to destroy it, causing transplant rejection. Additionally, in the case of stem cells, bone marrow, or other hematopoietic grafts, the immune cells of the graft attack the host cells. This is known as graft-versus-host disease (GvHD). A "graft" refers to the transplanted or donated tissue, and a "host" refers to the recipient's tissue. Transplant recipients often receive prophylactic treatment to suppress the immune system after transplantation. These treatments continue after transplantation. Immunosuppressive therapies include, but are not limited to, ruxolitinib, belumosudil, ibrutinib, corticosteroids, and photopheresis. Human leukocyte antigen (HLA) typing or HLA matching is used to match the recipient and donor of the graft. HLA is a protein found on most cells of your body and is used by the immune system to recognize foreign cells. The HLA genes of the donor and recipient must be identical or as closely matched as possible for the transplantation to be successful and to reduce the chance of developing GvHD or transplant rejection. Two main classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) allow each cell to be recognized as "self," while HLA class II antigens (DR, DP, and DQ in humans) stimulate the immune system. The methods provided herein may allow for an increase in the level of HLA type mismatching between the donor and recipient to allow for a wider use of donated organs, or may reduce the need or level of immunosuppressive therapy required to avoid transplant rejection or GvHD.

[0050] Another aspect of the present invention provides an ex vivo method for preventing or reducing rejection of a transplant product in a subject. In some embodiments, the method comprises introducing a construct into a transplantable product 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 is capable of binding to its receptor and can activate the receptor similarly to a native immune checkpoint protein.

[0051] As used herein, "a subject in need" may refer to a subject in need of treatment for a disease or disorder associated with organ transplantation. A subject in need may include a subject having any condition in which transplantation of an organ, cell, or tissue would be beneficial. The subject may be experiencing failure or injury of the heart, lung, kidney, liver, pancreas, spleen, intestine, colon, eye, stomach, ovary, testis, 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.

[0052] In some embodiments, the compositions and methods provided herein can be used in xenotransplantation. Xenotransplantation or heterologous transplantation is the transplantation, implantation, or infusion of living cells, tissues, or organs from one species into another species, such as from a non-human animal source into a human recipient. Such cells, tissues, or organs are referred to as xenografts or xenotransplants. Non-human organs, cells, or tissues can be genetically modified by ex vivo viral or non-viral transduction with vectors that allow expression of the PD-l1 genes and variants described herein. Such methods can inhibit the immune mechanism response responsible for organ rejection and allow for more successful xenotransplantation.

[0053] Additional definitions

[0054] The present disclosure is not limited to the specific details of the constructions, component arrangements, or method steps described herein. The compositions and methods disclosed herein can be made, practiced, used, implemented, 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 language and terminology used herein are for descriptive purposes only and should not be regarded as limiting the scope of the claims. Ordinal indicators such as first, second, and third, as used in the specification and claims, refer to various structural or method steps and are not intended to be construed as indicating any particular structure or step, or any particular order or configuration of such structures or steps. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise required, any and all examples or exemplary language (e.g., "such as") provided herein are merely intended to facilitate the disclosure and do not imply any limitation on the scope of the disclosure. No language in this specification and no structure shown in the drawings should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The terms "comprising," "including," or "having" and variations thereof as used herein are intended to cover the recited elements and their equivalents as well as additional elements. Embodiments recited as "comprising," "including," or "having" certain elements are also contemplated as "consisting essentially of the recited certain elements" and "consisting of the recited certain elements."

[0055] Unless the context otherwise indicates or implies, the terms "a / an" and "the" mean "one or more." For example, "a molecule" should be construed to mean "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 it is not clear to those of ordinary skill in the art how the terms are being used given the context, "about" and "approximately" will mean plus or minus ≤10% of the particular term, and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0056] Unless otherwise indicated herein, a range of values recited herein is merely intended to be a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were recited individually herein. For example, if a concentration range is described as 1% to 50%, values such as 2% to 40%, 10% to 30%, or 1% to 3% are intended to be expressly recited in the specification. These are merely examples of what is specifically intended, and all possible combinations of values between the recited minimum and maximum values (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 values very close to the recited amount are included within the amount, such as values that might be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. Unless otherwise indicated, all percentages of amounts are by weight.

[0057] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it should be understood that unless otherwise stated, the citation of any document in this text does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or any other country. Any discussion of a reference states the claims of its author, and the applicant reserves the right to challenge the accuracy and relevance of any document cited herein. Unless otherwise expressly indicated, all references cited herein are incorporated by reference in their entirety. If there is any inconsistency between any definition and / or description found in the cited references, the present disclosure shall govern.

[0058] The following examples are illustrative only and are not intended to limit the scope of the invention or the appended claims. Examples

[0059] Example 1:

[0060] There is an urgent need to apply the significant progress in the field of gene therapy to the transplantation field to reduce transplant rejection, reduce the use of immunosuppressive agents, and expand the donor pool. The development of successful gene therapy strategies depends on four components: 1) delivery methods, 2) vectors (usually virus-based), 3) well-defined disease or injury states and disease models, and 4) molecular targets that are therapeutically appropriate for the disease or indication. Previously, the lack of an effective delivery method for targeting transgenes to the donor heart was an obstacle to gene therapy for heart transplantation. Recently, an ex vivo warm blood perfusion system (TransMedics, Organ Care System (OCS)) has been approved by the FDA for extending donor heart transport time and reducing ischemic injury. This system can also be used timely for viral vector delivery to genetically modify the transplanted organ prior to implantation. The present inventors have successfully demonstrated the ability to uniformly deliver a reporter gene to the entire myocardium of allografts in a porcine transplantation model via a viral vector. 2 Ex vivo warm blood perfusion-mediated delivery of viral vectors to the heart has been well established in the laboratory of the present inventors.

[0061] In the following examples, the present inventors describe therapeutic targets for gene therapy to minimize organ rejection. The role of T cells in the context of transplant rejection has been well established, and similarly well established are the multiple co-stimulatory pathways that mediate T cell activation. Organ rejection is mainly driven by T cells that recognize foreign allogeneic antigens on the donor organ. This recognition leads to subsequent effector responses and ultimately to organ injury and rejection. However, there are many inhibitory pathways to limit T cell activity. Among them, the PD-1 / PD-L1 axis is a central pathway for CD8+ T cell inhibition. Therapies targeting the PD-1 / PD-L1 pathway through a series of blocking antibodies have revolutionized the clinical practice for cancer treatment. Interestingly, it has been reported that in cancer patients with heart transplantation, the use of PD-1 / PD-L1 blocking antibodies is associated with both heart and kidney allograft rejection. 3,4 In a recent study of biopsies from human heart allografts from acute and chronic rejection, the present inventors observed dysregulation of the protein expression of PD-1 and PD-L1, supporting the relevance of this pathway in heart rejection. 5 Bracamonte-Baran and colleagues confirmed that in 23 endomyocardial biopsies, allograft endothelial PD-L1 expression was negatively correlated with CD8+ T cell infiltration. 6 In addition, PD-L1 transgene expression in a transgenic pig model led to a reduced ability to stimulate CD4+ T cell proliferation. 7 Preclinical experiments using donor PD-L1 knockout and recipient PD-1 knockout mice by others have determined that heart allograft rejection is significantly promoted by the lack of PD-L1. 8-10Conversely, overexpression of PD-1 in murine T cells prior to heart transplantation confirmed both graft survival benefits and reduced rejection. 11 This survival benefit was dependent on PD-L1 expression on the donor allograft - grafts from PD-L1 knockout donor mice and T cells overexpressing PD-1 in recipient mice did not show a survival benefit. However, in a clinical setting, overexpression of organ-specific PD-L1 rather than systemic T cells would be more useful to patients and would minimize potential systemic adverse effects. In a transgenic islet transplantation murine model, prevention of immune allograft destruction has been demonstrated for over 50 weeks even in the absence of immunosuppression. 12 Whether cardiac PD-L1 overexpression can reverse cardiac allograft rejection remains to be explored.

[0062] Results:

[0063] Molecular targets for transplant rejection. In a recent study at our institution of cardiac samples from 18 rejecting and non-rejecting human heart grafts, we found dysregulation of the PD-1 / PD-L1 pathway, supporting its relevance in cardiac rejection. 5 Acute rejection was associated with near absence of PD-L1 in myocytes. PD-L1 was also significantly lower in lymphocyte populations compared to PD-1. A trend towards decreased PD-L1 in cardiomyocytes was observed compared to PD-1. However, these studies were performed in rejecting allografts that had been exposed to immunosuppression.

[0064] Cell-based models to assess the ability of PD-L1 variants to resist cellular rejection. The PD-L1 protein has three main components: an intracellular domain, an extracellular domain, and a transmembrane domain. The intracellular domain enables reverse signaling (after binding to PD-1) that can alter various cellular behaviors. However, these have not been fully characterized. 13 These could potentially include negative feedback loops that may limit the effectiveness of gene therapies aimed at overexpressing PD-L1. Additionally, a secreted isoform of PD-L1 has been described in human studies; this soluble form of PD-L1 has been reported to be elevated during pregnancy 14 , and is associated with poor outcomes in different cancers. 15 Plasmid constructs contained three PD-L1 variants (full-length, intracellular domain truncated, and secreted isoform)( Figure 1 ). Lentiviral vectors encoding these constructs under the control of the CMV promoter were generated and used to transduce different tumor models, and it has been confirmed that they allow in vitro ( Figure 2 ) and in vivo ( Figure 3) for robust expression. To evaluate the ability of these genes to resist immune-mediated T cell attack, they were engineered into breast cancer (E0771) or colorectal cancer (MC38) cells, which also express an immunogenic form of ovalbumin (membrane-associated) that can trigger rejection. These cells were then implanted to determine which genes could resist immune attack by syngeneic C57B16 / J cells, as demonstrated by the ability of tumors to form in mice( Figure 4 ). We found that control cells infected with empty vector were rejected in 80% of cases, which was significantly comparable to cells containing the full-length form of PDL1 in both colorectal and breast cells. Significantly, tumor cells containing truncated PDL1 and those containing the secreted form resisted rejection in most mice, indicating that the lack of reverse signaling is crucial for establishing local immunosuppression, which may be mediated by the absence of specific feedback signaling circuits.

[0065] References:

[0066] 1. Benjamin, E.J. et al. Heart Disease and Stroke Statistics - 2017 Update: A Report From the American Heart Association. Circulation 135, e146 - e603.

[0067] 2. Bishawi et al. A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. Sci Rep. 2019 May 29;9(1):8029. doi:10.1038 / s41598 - 019 - 43737 - y.

[0068] 3. Owonikoko, et al. Cardiac allograft rejection as a complication of PD - 1 checkpoint blockade for cancer immunotherapy: a case report. Cancer Immunol Immunother. 2017 Jan;66(1):45 - 50. doi:10.1007 / s00262 - 016 - 1918 - 2. Epub 2016 Oct 22.

[0069] 4. Lipson et al. Tumor Regression and Allograft Rejection after Administration of Anti-PD-1. N Engl J Med. 2016 Mar 3; 374(9): 896-8. doi: 10.1056 / NEJMc1509268.

[0070] 5. Bishawi et al. PD-1 and PD-L1 expression in cardiac transplantation. Cardiovasc Pathol. Sep-Oct 2021; 54: 107331. doi: 10.1016 / j.carpath.2021.107331. Epub 2021 Mar 16.

[0071] 6. Bracamonte-Baran, W, et al. Endothelial Stromal PD-L1 (Programmed Death Ligand 1) Modulates CD8+ T-Cell Infiltration After Heart Transplantation. Circ Heart Fail. 2021 Oct; 14(10): e007982. doi: 10.1161 / CIRCHEARTFAILURE 120.007982.

[0072] 7. Buermann A, et al. Pigs expressing the human inhibitory ligand PD-L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties. Xenotransplantation. 2018 Sep; 25(5): e12387. doi: 10.1111 / xen.12387.

[0073] 8. Ozkaynak et al. Programmed death-1 targeting can promote allograft survival. J Immunol. 2002 Dec 1; 169(11): 6546-53. doi: 10.4049 / jimmunol.169.11.6546.

[0074] 9.Yang et al.Critical role of donor tissue expression of programmeddeath ligand-1in regulating cardiac allograft rejection and vasculopathy.Circulation.2008Feb

[0075] 5;117(5):660-9.doi:10.1161 / CIRCULATIONAHA.107.741025.Epub 2008Jan 22.

[0076] 10.Wang et al.Protective role of programmed death 1ligand 1(PD-L1)innonobese diabetic mice:the paradox in transgenic models.Diabetes.2008Jul;57(7):1861-9.doi:10.2337 / db07-1260.Epub 2008Apr 16.

[0077] 11.Borges,TJ,et al.Overexpression of PD-1on T cells promotestolerance in cardiac transplantation via ICOS-dependent mcchanisms.JCIInsight.2021 Dcc 22;6(24):e142909.doi:10.1172 / jci.insight.142909.

[0078] 12.Paul PK,et al.Islet allografts expressing a PD-L1and IDO fusionprotein evade immune rejection and reverse preexisting diabetes inimmunocompetent mice without systemic immunosuppression.Am JTransplant.2022Jul 27.doi:10.1111 / ajt.17162.

[0079] 13. Lucas et al. PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity. Cell Rep. 2020 Oct 13; 33(2): 108258. doi: 10.1016 / j.celrep.2020.108258.

[0080] 14. Okuyama et al. Elevated Soluble PD-L1 in Pregnant Women′s Serum Suppresses the Immune Reaction. Front Immunol. 2019 Feb 18; 10: 86. doi: 10.3389 / fimmu.2019.00086.eCollection 2019.

[0081] 15. Han et al. The clinical implication of soluble PD-L1 (sPD-L1) in patients with breast cancer and its biological function in regulating the function of T lymphocyte. Cancer Immunol Immunother. 2021 Oct; 70(10): 2893 - 2909. doi: 10.1007 / s00262-021-02898-4. Epub 2021 Mar 10.

[0082] Example 2:

[0083] Targeted allograft-specific PD-L1 overexpression increases the time to rejection after heterotopic heart transplantation in a transgenic mouse model

[0084] After demonstrating the utility of the PD-L1 variants in inhibiting cellular immune responses in a manner superior to full-length PD-L1, we sought to test their potential in a murine animal model by generating transgenic mice and by vector-based transduction studies. To generate the mice, we cloned PD-L1 (full-length), PD-L1-TM (ICD-truncated), and PD-L1-SS (soluble form) into a ROSA targeting vector equipped with a CAG promoter that is activated after CRE recombination ( Figure 5)。These plasmids were generated and verified by sequencing, then electroporated into embryonic stem cells and selected using G418. After selection, positive clones were screened by multiplex PCR and the targeted intact clones were verified. These ES cells were used to generate chimeric mice, and these mice were bred to establish germline transmission of these transgenes. After transgenesis, the mice were mated with Myh6CRE mice. After generating multiple litters, it was determined that PD-L1 x Myh6-cre resulted in embryonic lethality, with no surviving double-positive mice. In contrast, a Mendelian ratio was observed for the cross of PDL1-FL mice with non-transgenic C57BL6 / J mice. In contrast, we did observe that PD-L1-TM and PD-L1-SS mice could be generated after crossing with Myh6-CRE, indicating a non-lethal and potentially more beneficial cardiac phenotype. To determine whether this was related to CRE expression in the developing heart (or other tissues), we additionally crossed the mice with Myh6-CRE / ER mice that allowed temporal control of CRE after tamoxifen administration. In this case, we were able to generate transgenic mice at a Mendelian ratio, indicating that lack of expression resulted in embryonic lethality of PD-L1-FL. To examine the temporal control of PD-L1 expression, tamoxifen was administered for five days (75 mg / kg), and cardiac tissue was evaluated 1 week after the last tamoxifen injection (12 days after the first injection). These studies confirmed that, compared to PDL1-FL and PDL1-SS, PD-L1-TM had robust PDL1 expression on cardiomyocytes (determined by IHC)( Figure 6 )。Using a PD-L1 ELISA, we were also able to detect significant amounts of PD-L1 in the sera of mice with PD-L1-SS, some increase from PD-L1-TM, but no detectable expression from PD-L1-FL or control non-transgenic mice. We therefore conclude that a secondary mechanism that inhibits PD-L1 expression by signaling through its intracellular domain (ICD) may be suppressing expression, as has been found to affect several different signaling pathways in cancer models (Jalali et al., Blood Cancer J 2019; Tamburini, Cell Reports 2020). Thus, confirmation of the lack of tissue-specific PD-L1 expression indicates a potential obstacle to the success of this strategy mediated by protein expression controlled by the ICD, which is unexpected but critical for implementing this approach.

[0085] To determine whether transgenic hearts could inhibit immune rejection, we then initiated studies to perform major mismatched transplantation between transgenic and non-transgenic hearts.

[0086] Experimental design:

[0087] The donor Myh6-Cre / PD-L1 transgenic mice have a C57BL6 background, and the recipient mice are wild-type BALB / c. There are three experimental groups - one for each transgenic PD-L1 variant donor, and an HLA-mismatched wild-type positive control (C57BL6 donor heart into BALB / c recipient). Heterotopic heart transplantation will be performed as follows: The donor mice are anesthetized and prepared. After sternotomy, the heart is removed aseptically. The heart is arrested by delivering cold buffer into the aortic root. The heart is then implanted into the HLA-mismatched recipient. The ascending aorta of the donor will be attached end-to-side to the abdominal aorta of the recipient, and the donor pulmonary artery will be attached end-to-side to the inferior vena cava of the recipient. The graft is degassed. The abdomen is closed and the recipient mice are allowed to recover.

[0088] Assessment of rejection: The assessment of heart rejection is mainly carried out by the following three methods: 1) Daily physical examination. Graft function can be easily evaluated by simple palpation of the animal's abdomen and graded according to the 4-point standard International Society for Heart and Lung Transplantation scale. 16 ; 2) Echocardiography is performed weekly; 3) Euthanasia is performed on subgroups of animals (n = 5 at each time point) weekly to obtain blood and tissues. Peripheral blood mononuclear cells (PBMCs) are isolated and stored for immunophenotyping. Immunophenotyping is performed by flow cytometry to characterize the cellular composition of the immune response during rejection when PD-L1 is overexpressed compared to control animals. The heart is harvested and processed for sectioning and H&E staining. The mice are continuously examined for signs of intimal hyperplasia or other cardiac allograft obliterative vasculopathy / rejection.

[0089] In our first set of experiments, we preferentially used PD-L1-TM transgenic hearts because of their elevated PD-L1 surface expression levels. In these experiments, hearts from C57BL6 were transplanted into BALB / c recipients, and these recipients were also treated with a single dose of Abatacept (250 μg / mouse on the day of implantation). In this case, the transgenic hearts expressing PD-L1-TM were not rejected, whereas, by 21 days post-transplantation, 50% of the control hearts were rejected. Figure 7)。To test the potential of vector-mediated delivery of this method, an AAV vector expressing PD-L1-SS was generated and used to transduce the mouse heart in vivo (2×10E12 vg AAV was intravenously injected into each mouse 14 days before transplantation). The hearts of these mice were then removed and used for major mismatched experiments, and a single dose of belatacept (a less potent immunosuppressant in mice) was used to induce immunosuppression (250 μg / mouse on the day of implantation). These experiments revealed that the PD-L1→SS-transduced hearts survived significantly longer than the control hearts, and all of the control hearts were rejected 14 days after implantation ( Figure 8 ). Some of these hearts survived for more than 80 days and were then sacrificed to evaluate the heart tissue. These experiments confirmed the potential of PD-L1→TM and PD-L1→SS to prevent solid organ rejection through genetically engineered transgenic organs and through the use of viral vectors that can express these genes after ex vivo tissue transduction. Although these experiments were conducted in the heart tissue of mice, we believe that this method can be used for different organs in humans and potentially enable xenotransplantation by using organs from different species.

[0090] References:

[0091] 1. Benjamin, E.J. et al. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation 135, e146-e603, (2017)

[0092] 2. Bishawi et al. A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. Sci Rep. 2019 May 29;9(1):8029. doi: 10.1038 / s41598-019-43737-y.

[0093] 3.Owonikoko,et al.Cardiac allograft rejection as a complication ofPD-1 checkpoint blockade for cancer immunotherapy:a case report.CancerImmunol Immunother.2017Jan;66(1):45-50.doi:10.1007 / s00262-016-1918-2.Epub2016Oct 22.

[0094] 4.Lipson et al.Tumor Regression and Allograft Rejection afterAdministration of Anti-PD-1.NEngl J Med.2016Mar 3;374(9):896-8.doi:10.1056 / NEJMc1509268.

[0095] 5.Bishawi et al.PD-1 and PD-L1 expression in cardiactransplantation.Cardiovasc Pathol.Sep-Oct 2021;54:107331.doi:10.1016 / j.carpath.2021.107331.Epub 2021 Mar 16.

[0096] 6.Bracamonte-Baran,W,et al.Endothelial Stromal PD-L1(Programmed DeathLigand 1)Modulates CD8+T-Cell Infiltration After Heart Transplantation.CircHeart Fail.2021 Oct;14(10):e007982.doi:10.1161 / CIRCHEARTFAILURE.120.007982.

[0097] 7. Buermann A, et al. Pigs expressing the human inhibitory ligand PD-L1(CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties. Xenotransplantation. 2018 Sep;25(5):e12387. doi:10.1111 / xen.12387.

[0098] 8. Ozkaynak et al. Programmed death-1 targeting can promote allograft survival. J Immunol. 2002 Dec 1;169(11):6546-53. doi:10.4049 / jimmunol.169.11.6546.

[0099] 9. Yang et al. Critical role of donor tissue expression of programmed death ligand-1 in regulating cardiac allograft rejection and vasculopathy. Circulation. 2008 Feb 5;117(5):660-9. doi:10.1161 / CIRCULATIONAHA.107.741025. Epub 2008 Jan 22.

[0100] 10. Wang et al. Protective role of programmed death 1 ligand 1(PD-L1) in nonobese diabetic mice: the paradox in transgenic models. Diabetes. 2008 Jul;57(7):1861-9. doi:10.2337 / db07-1260. Epub 2008 Apr 16.

[0101] 11. Borges, TJ, et al. Overexpression of PD-1 on T cells promotes tolerance in cardiac transplantation via ICOS-dependent mechanisms. JCI Insight. 2021 Dec 22; 6(24): e142909. doi: 10.1172 / jci.insight.142909.

[0102] 12. Paul PK, et al. Islet allografts expressing a PD-L1 and IDO fusion protein evade immune rejection and reverse preexisting diabetes in immunocompetent mice without systemic immunosuppression. Am J Transplant. 2022 Jul 27. doi: 10.1111 / ajt.17162.

[0103] 13. Lucas et al. PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity. Cell Rep. 2020 Oct 13; 33(2): 108258. doi: 10.1016 / j.celrep.2020.108258.

[0104] 14. Okuyama et al. Elevated Soluble PD-L1 in Pregnant Women's Serum Suppresses the Immune Reaction. Front Immunol. 2019 Feb 18; 10: 86. doi: 10.3389 / fimmu.2019.00086. eCollection 2019.

[0105] 15. Han et al. The clinical implication of soluble PD-L1 (sPD-L1) in patients with breast cancer and its biological function in regulating the function of T lymphocyte. Cancer Immunol lmmunother. 2021 Oct;70(10):2893-2909. doi:10.1007 / s00262-021-02898-4. Epub 2021 Mar 10.

[0106] 16. Stewart S, et al. Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant 2005;24(11):1710-20.

[0107] Example 3:

[0108] The present disclosure provides constructs and methods that can be used to ex vivo transduce organs with immunosuppressive genes. To illustrate such gene therapy methods, articles for transplantation can be preserved by any method known in the art, including but not limited to preservation methods that maintain the article at room temperature and aerobic metabolism. The preserved article can be ex vivo exposed to one or more of the constructs described herein. These constructs can comprise a promoter operably linked to an immune checkpoint protein (such as PDL1, CTLA4, Galectin-3, etc.) or an extracellular portion of an immune checkpoint protein. These constructs can further comprise a secretion signal operably linked to the promoter and functionally linked to a nucleic acid encoding an immune checkpoint protein or an extracellular portion of an immune checkpoint protein such that the immune checkpoint protein is secreted. For example, the heart can undergo ex vivo normothermic perfusion, wherein the perfusion can comprise an AAV vector. The AAV vector can encode a cell type promoter, such as a cardiac promoter operably linked to a secretion signal and a nucleic acid encoding secreted PDL1. Expression of the vector or immunosuppressive gene can be evaluated after perfusion into the organ or article. The organ perfused with the vector can then be transplanted into a recipient. Organs that have been ex vivo perfused with a construct encoding an immunosuppressive gene are expected to improve transplantation outcomes, including reducing rejection of the transplanted organ, reducing the immune response to the graft, and reducing ischemic injury. Ex vivo perfusion with a construct encoding an immunosuppressive gene can also reduce the amount, duration, or frequency of administration of immunosuppressive therapy after transplantation. The methods and constructs described herein can also allow transplantation of organs with a greater degree of HLA mismatch. Without wishing to be bound by theory, organs ex vivo perfused with a construct encoding an immunosuppressive gene (such as PDL1 or a variant thereof) can have a lower incidence of rejection and a higher incidence of HLA mismatch compared to organs not perfused with the construct. The method can also be useful in suppressing the immune mechanisms responsible for organ rejection of non-human organs, thus allowing more successful xenotransplantation by genetically modifying animal organs or by ex vivo viral (or non-viral) transduction using vectors that allow 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. The construct according to claim 1, further comprising a secretion signal operably linked to the promoter and functionally linked to the nucleic acid encoding the immune checkpoint protein or the extracellular portion of the immune checkpoint protein.

3. The construct according to claim 2, wherein the secretion signal encodes a signal peptide and allows the extracellular portion of the immune checkpoint protein to be secreted.

4. The construct according to any one of claims 1-3, 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 according to claim 1 or 2, wherein the immune checkpoint protein comprises PDL1.

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

7. The construct according to 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 and activating PD1.

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

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

10. The construct according to any one of the preceding claims, wherein the construct comprises a viral vector.

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

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

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

14. An ex vivo or in vitro method for expressing an immune checkpoint protein or a portion thereof in a transplantable article, the method comprising introducing the construct according to any one of claims 1-12 or the pharmaceutical composition according to claim 13 into the transplantable article, optionally wherein introducing comprises perfusing the construct or the pharmaceutical composition ex vivo or in vitro into the article.

15. The method according to claim 14, further comprising analyzing the article or cells of the article to determine the presence or expression of the construct or the immune checkpoint protein encoded by the construct in the article.

16. The method according to claim 15, further comprising transplanting the article into a subject.

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

18. The method according to claim 17, wherein the one or more additional therapeutic agents are administered before, during, or after transplanting the article into the subject.

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

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

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

22. The method according to claim 21, further comprising analyzing the article or cells of the article to determine the presence or expression of the construct or the immune checkpoint protein encoded by the construct in the article.

23. The method according to claim 22, further comprising transplanting the article into a subject.

24. The method according to claim 23, wherein one or more additional therapeutic agents are administered before, during, or after transplanting the article into the subject.

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

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

27. An ex vivo method for introducing a construct into a transplantable article, the method comprising perfusing the construct ex vivo into the article prior to transplantation, 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, wherein the immune checkpoint protein or the extracellular portion of the immune checkpoint protein is capable of binding to its receptor.

28. The method of claim 27, further comprising transplanting the article into a subject.

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

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, testis, bladder, uterus, adrenal gland, and combinations thereof.

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

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. 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 capable of binding and activating PD1.

34. 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, foamy virus, adeno-associated virus, herpes simplex virus, and combinations thereof.

35. An ex vivo method for preventing or reducing rejection of a transplanted article in a subject, the method comprising introducing a construct into the transplantable article before 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. The method according to claim 35, further comprising transplanting the article into the subject.

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

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

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

40. The method according to any one of claims 35-39, 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. The method according to claim 40, 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 and activating PD1.

42. The method according to claim 41, wherein the construct is a viral vector, and the viral vector is selected from the group consisting of: retroviruses, adenoviruses, oncoretroviruses, lentiviruses, spumaviruses, adeno-associated viruses, herpes simplex viruses, and combinations thereof.

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