Compositions and methods for cardiomyocyte transplantation

Abstract

The present disclosure provides treatments for cardiac diseases and disorders using in vitro differentiated cardiomyocytes. Such methods can utilize both autologous and allogeneic pluripotent stem cells. TIFF2024507865000002.tif180165

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 152,737, filed February 23, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant No. R01 HL128362 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] background The field of regenerative medicine for the treatment of cardiac diseases and disorders is evolving, and the use of in vitro differentiated cardiomyocytes is being explored in clinical settings. Such methods can utilize both autologous and allogeneic pluripotent stem cells. To minimize cardiac graft rejection after transplantation, there is a need to identify therapies that can be used to prevent short-term rejection and promote long-term maintenance of cardiac grafts. Summary of the Invention

[0004] overview The methods and compositions described herein are based, in part, on the discovery of a combination of a calcineurin inhibitor and a CD80 / CD86 signaling inhibitor that can prevent or reduce graft rejection of in vitro differentiated cardiomyocytes in subjects being treated for a cardiac disease or disorder.

[0005] Thus, in one aspect, provided herein is a method of cardiomyocyte transplantation comprising: (i) administering in vitro differentiated cardiomyocytes or cardiomyocyte progenitor cells to a graft site in cardiac tissue of a recipient in need thereof; and (ii) administering a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling to the recipient.

[0006] In one embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are allogeneic to the recipient.

[0007] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are autologous to the recipient.

[0008] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor is selected from the group consisting of tacrolimus, cyclosporine, voclosporin, and pimecrolimus. Immunosuppressant derivatives of tacrolimus, cyclosporine, voclosporin, and pimecrolimus are also contemplated herein.

[0009] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 comprises an extracellular domain fragment of CTLA4.

[0010] In another embodiment of this aspect and all other aspects provided herein, the extracellular domain fragment of CTLA4 is fused to a heterologous polypeptide.

[0011] In another embodiment of this aspect and all other aspects provided herein, the heterologous polypeptide increases the serum half-life of the inhibitor of CD80 and / or CD86.

[0012] In another embodiment of this aspect and all other aspects provided herein, the heterologous polypeptide comprises an IgG Fc domain polypeptide.

[0013] In another embodiment of this aspect and all other aspects provided herein, the extracellular domain fragment of CTLA4 is conjugated to polyethylene glycol (PEG).

[0014] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 signaling comprises abatacept or belatacept.

[0015] In another embodiment of this aspect and all other aspects provided herein, abatacept is administered via intravenous or subcutaneous administration at a dose of 5 mg / kg to 20 mg / kg. In another embodiment of this aspect and all other aspects provided herein, abatacept is administered via intravenous or subcutaneous administration at a dose of 10 mg / kg to 15 mg / kg (e.g., at a dose of 12.5 mg / kg).

[0016] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 signaling is administered (i) every 2 or 3 weeks, (ii) every 12-16 days, (iii) every 10-18 days, and / or (iv) by subcutaneous, oral, or intravenous administration, i.e., the inhibitor of CD80 and / or CD86 signaling can be administered every 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days, or longer.

[0017] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor comprises tacrolimus or an immunosuppressant derivative thereof.

[0018] In another embodiment of this and all other aspects provided herein, tacrolimus is administered to achieve a serum or plasma concentration of 5-20 ng / mL. In other embodiments, tacrolimus is administered intravenously or subcutaneously to achieve a blood, plasma or serum concentration of 10 mg / kg to 15 mg / kg.

[0019] In another embodiment of this aspect and all other aspects provided herein, tacrolimus is administered by continuous infusion every 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours (i.e., daily).

[0020] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling are each administered subcutaneously or intravenously. Alternatively, the calcineurin inhibitor can be administered subcutaneously while the inhibitor of CD80 and / or CD86 is administered intravenously, or vice versa.

[0021] In another embodiment of this aspect and all other aspects provided herein, immune rejection of the administered cardiomyocytes is reduced (e.g., by at least 10%) compared to rejection of cardiomyocytes administered without the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling.

[0022] In another embodiment of this aspect and all other aspects provided herein, the need for steroid immunosuppression to suppress immune rejection of the transplanted cardiomyocytes is reduced compared to the need for steroid immunosuppression when the cardiomyocytes are transplanted without administration of a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling.

[0023] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced compared to infiltration that occurs in the absence of administration of the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling.

[0024] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced 8 weeks after administration of the cardiomyocytes.

[0025] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site remains reduced 16 weeks after administration of the cardiomyocytes.

[0026] Another aspect provided herein relates to a method of reducing immune rejection of transplanted, in vitro differentiated cardiomyocytes in a recipient comprising administering to the recipient a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling.

[0027] In one embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are allogeneic to the recipient.

[0028] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are autologous to the recipient.

[0029] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor is selected from the group consisting of tacrolimus, cyclosporine, voclosporin, and pimecrolimus.

[0030] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 comprises an extracellular domain fragment of CTLA4.

[0031] In another embodiment of this aspect and all other aspects provided herein, the extracellular domain fragment of CTLA4 is fused to a heterologous polypeptide.

[0032] In another embodiment of this aspect and all other aspects provided herein, the heterologous polypeptide increases the serum half-life of the inhibitor of CD80 and / or CD86.

[0033] In another embodiment of this aspect and all other aspects provided herein, the heterologous polypeptide comprises an IgG Fc domain polypeptide.

[0034] In another embodiment of this aspect and all other aspects provided herein, the extracellular domain fragment of CTLA4 is conjugated to polyethylene glycol (PEG).

[0035] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 signaling comprises abatacept or belatacept.

[0036] In another embodiment of this aspect and all other aspects provided herein, abatacept is administered by intravenous or subcutaneous administration at a dose of 1.25 mg / kg to 125 mg / kg.

[0037] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 signaling is administered (i) every 2 or 3 weeks, (ii) every 12-16 days, (iii) every 10-18 days, and / or (iv) by subcutaneous, oral, or intravenous administration, i.e., the inhibitor of CD80 and / or CD86 signaling can be administered every 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days, or longer.

[0038] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor comprises tacrolimus or an immunosuppressant derivative thereof.

[0039] In another embodiment of this aspect and all other aspects provided herein, tacrolimus is administered to achieve a serum or plasma concentration of 5-20 ng / mL.

[0040] In another embodiment of this aspect and all other aspects provided herein, tacrolimus is administered by continuous infusion every 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or daily.

[0041] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling are each administered subcutaneously or intravenously or a combination thereof.

[0042] In another embodiment of this aspect and all other aspects provided herein, immune rejection of the administered cardiomyocytes is reduced compared to rejection of cardiomyocytes administered without the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling.

[0043] In another embodiment of this aspect and all other aspects provided herein, the need for steroid immunosuppression to suppress immune rejection of the transplanted cardiomyocytes is reduced compared to the need for steroid immunosuppression when the cardiomyocytes are transplanted without administration of a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling.

[0044] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced compared to infiltration that occurs in the absence of administration of the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling.

[0045] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced 8 weeks after administration of the cardiomyocytes.

[0046] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site remains reduced 16 weeks after administration of the cardiomyocytes.

[0047] Another aspect provided herein is a method of treating a cardiovascular disease or disorder, comprising: (i) administering in vitro differentiated cardiomyocytes to a graft site in cardiac tissue of a subject in need thereof; and (ii) administering to the subject a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling.

[0048] In one embodiment of this aspect and all other aspects provided herein, the cardiovascular disease or disorder comprises myocardial infarction, ischemia / reperfusion injury, tachyarrhythmia, bradyarrhythmia, cardiomyopathies, congenital defects, and muscular dystrophy-associated cardiomyopathies.

[0049] In another embodiment of this aspect and all other aspects provided herein, the congenital defect comprises a septal defect or a hypoplastic syndrome.

[0050] In another embodiment of this aspect and all other aspects provided herein, the muscular dystrophy associated cardiomyopathy comprises cardiomyopathy associated with Duchenne muscular dystrophy (DMD).

[0051] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are allogeneic to the recipient.

[0052] In another embodiment of this aspect and all other aspects provided herein, the in vitro differentiated cardiomyocytes are autologous to the recipient.

[0053] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor is selected from the group consisting of tacrolimus, cyclosporine, voclosporin, and pimecrolimus.

[0054] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 comprises an extracellular domain fragment of CTLA4.

[0055] In another embodiment of this aspect and all other aspects provided herein, the extracellular domain fragment of CTLA4 is fused to a heterologous polypeptide.

[0056] In another embodiment of this aspect and all other aspects provided herein, the heterologous polypeptide increases the serum half-life of the inhibitor of CD80 and / or CD86.

[0057] In another embodiment of this aspect and all other aspects provided herein, the heterologous polypeptide comprises an IgG Fc domain polypeptide.

[0058] In another embodiment of this aspect and all other aspects provided herein, the extracellular domain fragment of CTLA4 is conjugated to polyethylene glycol (PEG).

[0059] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 signaling comprises abatacept or belatacept.

[0060] In another embodiment of this aspect and all other aspects provided herein, abatacept is administered by intravenous or subcutaneous administration at a dose of 5 mg / kg to 20 mg / kg.

[0061] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of CD80 and / or CD86 signaling is (i) administered every 2 weeks or every 3 weeks, (ii) administered every 12-16 days, (iii) administered every 10-18 days, and / or (iv) administered by subcutaneous, oral, or intravenous administration.

[0062] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor comprises tacrolimus or an immunosuppressant derivative thereof.

[0063] In another embodiment of this aspect and all other aspects provided herein, tacrolimus is administered to achieve a serum or plasma concentration of 5-20 ng / ml.

[0064] In another embodiment of this aspect and all other aspects provided herein, tacrolimus is administered by continuous infusion every 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or daily.

[0065] In another embodiment of this aspect and all other aspects provided herein, the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling are each administered subcutaneously or intravenously or a combination thereof.

[0066] In another embodiment of this aspect and all other aspects provided herein, immune rejection of the administered cardiomyocytes is reduced compared to rejection of cardiomyocytes administered without the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling.

[0067] In another embodiment of this aspect and all other aspects provided herein, the need for steroid immunosuppression to suppress immune rejection of the transplanted cardiomyocytes is reduced compared to the need for steroid immunosuppression when the cardiomyocytes are transplanted without administration of a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling.

[0068] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced compared to infiltration that occurs in the absence of administration of the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling.

[0069] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced 8 weeks after administration of the cardiomyocytes.

[0070] In another embodiment of this aspect and all other aspects provided herein, infiltration of CD3+ T cells or CD20+ B cells into the graft site remains reduced 16 weeks after administration of the cardiomyocytes.

[0071] Another aspect provided herein relates to the use of a calcineurin inhibitor in combination with an inhibitor of CD80 and / or CD86 signaling as described herein for the treatment or prevention of immune-mediated rejection of a cardiac graft.

[0072] Another aspect provided herein relates to the use of in vitro differentiated cardiomyocytes or cardiomyocyte progenitor cells for cardiomyocyte transplantation, comprising: (i) in vitro differentiated cardiomyocytes or cardiomyocyte progenitor cells administered to a graft site in the cardiac tissue of a recipient in need thereof; and (ii) a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling administered to the recipient.

[0073] In another aspect, also provided herein is a use of in vitro differentiated cardiomyocytes for treating a cardiovascular disease or disorder, the use comprising (i) administering to a graft site in cardiac tissue of a subject in need thereof, and (ii) administering to the subject a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling. [Brief description of the drawings]

[0074] [Figure 1] Histological analysis showing that in the absence of immunosuppression, cardiomyocyte grafts survived for 2 weeks without rejection, but by 8 weeks the grafts had undergone severe cellular rejection with extensive inflammatory infiltrates. The steroid-sparing combination of tacrolimus + abatacept revealed mild to moderate immune rejection of the grafts with patchy infiltrates of CD20+ (B cell) and CD3+ (T cell) populations at 16 weeks.

[0075] [Diagram 2] FIG. 1 is a schematic showing an exemplary method for in vitro differentiation of cardiomyocytes from human embryonic stem cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] Detailed Description Provided herein are methods and compositions including combination treatment with a calcineurin inhibitor (e.g., tacrolimus) and an inhibitor of CD80 and / or CD86 signaling (e.g., abatacept) that improve cardiac repair methods using pluripotent stem cell-derived cardiomyocytes (PSC-CMs) by reducing or preventing immune-mediated graft rejection. A combination immunosuppressive regimen of a calcineurin inhibitor (e.g., tacrolimus (TAC)) and an inhibitor of CD80 and / or CD86 signaling (e.g., abatacept (ABT)) can be used to support long-term PSC-CM transplantation.

[0077] definition As used herein, the term "recipient in need thereof" refers to a subject with cardiovascular disease or disorder that can be treated with the cardiac graft that comprises in vitro differentiated cardiomyocytes.For example, the recipient in need thereof can include cardiovascular disease or disorder, including injury such as myocardial infarction, arrhythmia (e.g., tachyarrhythmia or bradyarrhythmia), ischemia / reperfusion injury, cardiomyopathy, muscular dystrophy-related cardiomyopathy (e.g., Duchenne muscular dystrophy (DMD)), or congenital defect (e.g., hypoplastic syndrome, septal defect), etc.

[0078] The terms "differentiate" or "differentiated" are relative terms indicating that a "differentiated cell" is a cell that has progressed further down the developmental pathway than its progenitor cell. Thus, in some aspects, stem cells, as that term is defined herein, can differentiate into lineage-restricted progenitor cells (e.g., human cardiac progenitor cells or central primitive streak cardiac mesoderm progenitor cells), which in turn can differentiate further down the pathway into other types of progenitor cells (e.g., tissue-specific precursors such as cardiomyocyte progenitors) and then into terminally differentiated cells (e.g., cardiomyocytes), which may or may not retain the ability to play a characteristic role in a particular tissue type and to further proliferate.

[0079] The term "pluripotent" or "pluripotent stem cell (PSC)" as used herein refers to cells that have the ability to differentiate under different conditions into cell types characteristic of all three germ layers (endoderm, mesoderm and ectoderm). Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. Although the preferred test for pluripotency is the demonstration of differentiation potential into cells of each of the three germ layers, pluripotency is also demonstrated by the expression of embryonic stem (ES) cell markers.

[0080] As used herein, the terms "induced pluripotent stem cells," "iPSCs," "hPSCs," and "human pluripotent stem cells" are used interchangeably herein and refer to pluripotent cells that are artificially derived (e.g., by reprogramming using one or more methods known in the art) from differentiated somatic cells. iPSCs are capable of self-renewal and differentiation into various types of mature cells, as well as cell fate-committed stem cells, including cells of the cardiac lineage.

[0081] As used herein, "in vitro differentiated cardiomyocytes" refers to cardiomyocytes that are generated in culture, but not necessarily undergo stepwise differentiation from progenitor cells, such as human embryonic stem cells, induced pluripotent stem cells, early mesoderm cells, lateral plate mesoderm cells, or cardiac precursor cells. Thus, while in vivo cardiomyocytes are ultimately derived from stem cells, i.e., during tissue or organ development, the stem cell-derived cardiomyocytes described herein are generated by in vitro differentiation from stem cells. As used herein, a cell differentiated in vitro from a stem cell, e.g., an induced pluripotent stem (iPS) cell or an embryonic stem cell ("ES cell" or "ESC"), is a "stem cell-derived cardiomyocyte" or "in vitro differentiated cardiomyocyte" if it has expression of cardiac troponin T (cTnT). Methods for differentiating stem cells in vitro into cardiomyocytes are known in the art and described elsewhere herein. In one embodiment, the cardiomyocytes are differentiated from pluripotent stem cells (e.g., PSC-CM). An exemplary protocol for in vitro differentiation of cardiomyocytes is illustrated in FIG.

[0082] As used herein, the term "cardiomyocyte" refers to a cardiac muscle cell. Cardiomyocytes generally comprise phenotypic and / or structural characteristics associated with cardiac muscle (e.g., electrical phenotype, expression of sarcomeres, actin, myosin and cardiac troponin T, etc.). Typically, cardiomyocytes are terminally differentiated.

[0083] As used herein, the term "cardiomyoprogenitor cells" refers to cells that are committed to the cardiac lineage but are not fully differentiated cardiomyocytes. Cardiomyocyte progenitor cells can differentiate into cardiomyocytes in vivo within a cardiac graft. In some embodiments, "cardiomyoprogenitor cells" are cells that are partially differentiated along the cardiac lineage in vitro but not fully differentiated. In one embodiment, cardiomyocyte progenitor cells for use in the methods and compositions described herein refer to partially differentiated cardiomyocytes in vitro that have ceased the differentiation process after inhibition of the Wnt pathway (see, for example, FIG. 2). In another embodiment, cardiomyocyte progenitor cells include cells after day 5 of an in vitro differentiation protocol to generate cardiomyocytes (but before reaching terminal differentiation of cardiomyocytes).

[0084] The term "isolated cell" as used herein refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, for example in the presence of other cells. Optionally, the cell is later introduced into a second organism, or reintroduced into the organism from which it (or its ancestor) was isolated.

[0085] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the total cell population. That is, the terms "substantially pure" or "essentially purified" with respect to a cardiomyocyte population refers to a cell population that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, and most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1%, respectively, of cells that are not cardiomyocytes.

[0086] The term "marker" as used herein is used to describe the characteristics and / or phenotype of a cell. Markers can be used, for example, for the selection of cells containing a characteristic of interest and can vary with the particular cell. A marker is a morphological, structural, functional, or biochemical (enzymatic) characteristic of a cell of a particular cell type, or a molecule expressed by a cell type. In one aspect, such a marker is a protein. Such a protein can have an epitope for an antibody or other binding molecule available in the art. However, a marker can consist of any molecule found in or on a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to take up or exclude a particular dye, the ability to migrate under particular conditions, and the ability to differentiate along a particular lineage. Markers can be detected by any method available to one of skill in the art. A marker can also be the absence of a morphological feature or the absence of a protein, lipid, etc. A marker can be a combination of a panel of unique features, the presence and / or absence of polypeptides and other morphological or structural features. In one embodiment, the marker is a cell surface marker.

[0087] The term "derived from" when used in reference to stem cells means that the stem cell was generated by reprogramming a differentiated cell to a stem cell phenotype. The term "derived from" when used in reference to a differentiated cell means that the cell is the result of differentiation of a stem cell, e.g., differentiation in vitro. As used herein, "iPSC-CM" or "induced pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from induced pluripotent stem cells. Similarly, "PSC-CM" or "pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from pluripotent stem cells. In some embodiments, the term "hPSC-CM" or "human pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from human pluripotent stem cells.

[0088] As used herein, the term "mature" or "mature phenotype" or "mature cardiomyocyte" when applied to cardiomyocytes refers to a phenotype of a cell that includes a phenotype similar to an adult cardiomyocyte, but does not include at least one characteristic of a fetal cardiomyocyte. In some embodiments, markers indicative of increased maturity of in vitro differentiated cells include, but are not limited to, electrical maturity, metabolic maturity, genetic marker maturity, and contractile maturity. The methods and compositions can include one or more methods for inducing cardiomyocyte maturation, for example, as described in US2020 / 008588 or WO2020 / 190939, each of which is incorporated herein by reference in its entirety).

[0089] As used herein, the terms "transplanting," "administering," or "engraftment" are used in the context of placing the cells described herein, e.g., stem cell-derived cardiomyocytes or cardiac progenitor cells, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, so that a desired effect is produced. The cells, e.g., cardiac stem or progenitor cells or cardiomyocytes, can be directly implanted into the heart, or alternatively, administered by any suitable route that results in delivery to a desired location in a subject, where at least a portion of the implanted cells or cell components remain viable. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to several days or years, i.e., long-term engraftment. As one skilled in the art will recognize, long-term engraftment of cardiomyocytes is desirable because cardiomyocytes do not proliferate to the extent that the heart can heal from acute injury, including cell death. Thus, grafts can be used to replace lost cells that occur during injury. In other aspects, the cells can be administered via an indirect systemic route of administration, such as intraperitoneal or intravenous routes. For example, methods for improving engraftment or preventing engraftment arrhythmia, such as those described in US2020-0085880, WO2020 / 190739, or WO2021 / 163037, the contents of each of which are incorporated by reference in their entireties, can be combined with the methods and compositions described herein.

[0090] As used herein, the term "contacting" when used in reference to a cell includes introducing an agent, surface, scaffold, etc., into a cell in a manner that allows physical contact of the cell with the agent, surface, scaffold, etc.

[0091] As used herein, the term "cardiac disease" refers to the disease that affects the cardiac tissue of a subject.Non-limiting examples of cardiac disease include cardiomyopathy, cardiac arrhythmia, myocardial infarction, heart failure, cardiac hypertrophy, long QT syndrome, arrhythmogenic right ventricular dysplasia (ARVD), catecholamine-induced polymorphic ventricular tachycardia (CPVT), Barth syndrome, congenital defects, and Duchenne muscular dystrophy.

[0092] As used herein, the term "treatment" refers herein to a reduction in immune rejection of a cardiac graft in the presence of a combination treatment of a calcineurin inhibitor (e.g., tacrolimus) and an inhibitor of CD80 and / or CD86 signaling (e.g., abatacept) of at least 50% compared to a substantially similar amount of immune rejection of a cardiac graft in a subject not treated with a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling. In other embodiments, the reduction in immune rejection in a subject treated as described herein is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or even 100% reduction (i.e., no detectable immune rejection).

[0093] As used herein, the term "immune rejection" refers to immune-mediated death of cells transplanted into the cardiac graft site in a recipient or subject in need thereof. Immune rejection can be assessed by measuring increased inflammation at the graft site, for example, by an increase in circulating inflammatory markers (e.g., C-reactive protein, cytokines, etc.), leukocyte infiltration at the graft site, or the need for treatment with immunosuppressants, or an increase in the dose or frequency of administration of immunosuppressants. Alternatively or additionally, immune rejection can be assessed by monitoring cardiac function or its loss during immune rejection, for example, by the appearance or increase of arrhythmia or another impaired cardiac function measurement.

[0094] As used herein, the term "infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced" refers to at least a 20% reduction in the number of CD3+ or CD20+ B cells into the graft site in a subject treated with a calcineurin inhibitor and a CD80 / CD86 signaling inhibitor, compared to the number of CD3+ or CD20+ B cells in the graft site in a subject not treated with such a combination. In other embodiments, the number of CD3+ or CD20+ B cells is reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% (i.e., no CD3+ or CD20+ B cells) when a subject is treated with such a combination, compared to the number of CD3+ or CD20+ B cells in a subject not treated with a calcineurin inhibitor and a CD80 / CD86 signaling inhibitor.

[0095] The terms "patient", "subject" and "individual" are used interchangeably herein and refer to animals, particularly humans, to which treatment, including preventative treatment, is provided. The term "subject" as used herein refers to humans and non-human animals. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include all vertebrates, such as mammals, including non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals, such as chickens, amphibians, reptiles, and others. In one embodiment of any aspect, the subject is a human. In another embodiment of any aspect, the subject is an experimental animal or surrogate animal as a disease model. In another embodiment of any aspect, the subject is a livestock animal, including companion animals (e.g., dogs, cats, rats, guinea pigs, hamsters, and others). The subject may have previously been treated for a disease or has never previously been treated for a disease. The subject may have been previously diagnosed with the disease or may not have been previously diagnosed with the disease. The subject can be of any age, including, for example, a fetus, a newborn, a toddler, a child, an adolescent, an adult, a geriatric subject, etc.

[0096] The terms "reduce", "reduce", "reduction" or "inhibit" are all used herein to mean a statistically significant amount of reduction or decrease in a property, level, or other parameter. In some embodiments, "reduce", "reduce" or "reduce" or "inhibit" typically means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduce" or "inhibit" does not encompass a complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease can preferably be down to a level that is accepted as within the normal range for individuals without a given disorder.

[0097] The terms "enhanced," "enhancement," "enhancement," or "enhancement" or "activation" are all used herein generally to mean an increase in a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms "enhanced," "enhancement," or "enhancement" or "activation" mean an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% increase, or an increase up to 100%, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, at least about 20-fold increase, at least about 50-fold increase, at least about 100-fold increase, or greater increase compared to a reference level.

[0098] As used herein, the terms "comprising" or "comprising" are used in reference to compositions, methods, and their respective components that are essential to the invention, but do not exclude the inclusion of unspecified elements, whether or not essential.

[0099] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic, novel or functional characteristics of that embodiment of the invention.

[0100] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0101] Example devices, methods, and systems are described herein. The words "example," "exemplary," and "illustrative" should be understood as used herein to mean "serving as an example, example, or illustration." Any aspect or feature described herein as being "example," "exemplary," or "illustrative" should not necessarily be construed as preferred or advantageous over other aspects or features. The example aspects described herein are not meant to be limiting. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0102] Additionally, the particular arrangements shown in the drawings should not be considered limiting. It should be understood that other embodiments may include more or less of each element shown in a given drawing. Additionally, some of the illustrated elements may be combined or omitted. Still further, example embodiments may include elements not shown in the drawings. As used herein with respect to measurements, "about" means ±5%.

[0103] The details shown in this specification are by way of example and for the sole purpose of illustrative description of preferred embodiments of the invention, and are presented to provide what is believed to be the most useful and readily understood explanation of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description provided by way of the figures and / or examples will make clear to those skilled in the art how some forms of the invention may be embodied in practice.

[0104] As used herein, and unless otherwise indicated, the terms "a" and "an" are employed to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms as used herein shall include pluralities and plural terms shall include the singular.

[0105] Unless the context requires otherwise, throughout this specification and the claims, the words "comprises," "including," and other terms are to be construed in an inclusive sense, i.e., "including but not limited to," as opposed to an exclusive or exhaustive sense. Words using the singular or plural also include the plural and singular, respectively. Additionally, when used in this application, the words "herein," "on," and "below," and words of similar meaning, shall refer to this application as a whole and not to any particular portions of this application.

[0106] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize.

[0107] All references cited herein are incorporated by reference. Aspects of the present disclosure can be modified, as necessary, to employ the systems, functions, and concepts of the above references and applications to provide still further aspects of the present disclosure. These and other changes can be made to the present disclosure in light of the detailed description.

[0108] Particular elements of any of the above-mentioned embodiments can be combined with or substituted for elements of other embodiments. Moreover, the inclusion of particular elements in at least some of these embodiments can be optional, whereby further embodiments can include one or more embodiments that specifically exclude one or more of these particular elements. Moreover, although advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0109] Although specific embodiments of the invention have been described herein for purposes of illustration, it will be appreciated that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

[0110] cardiovascular disease The methods and compositions described herein relate to methods and compositions for extending cardiac graft viability or preventing / reducing cardiac graft rejection in subjects treated with a combination of a calcineurin inhibitor (e.g., tacrolimus) and an inhibitor of CD80 and / or CD86 signaling (e.g., abatacept). The methods and compositions provided herein include administering cardiomyocytes derived in vitro from pluripotent stem cells. The methods described herein can be used to treat, ameliorate, prevent, or slow the progression of several diseases or their symptoms, such as those that result in pathological damage to the structure and / or function of the heart.

[0111] Cardiovascular disease is the disease that affects the heart and / or circulatory system of a subject.Such heart disease or heart-related disease includes, but is not limited to, myocardial infarction, cardiac arrhythmia, heart failure, coronary artery sclerosis, cardiomyopathy, congenital heart defect (such as myocardial compaction disorder, septal defect, hypoplastic left heart), hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac hypertrophy, myocarditis, arrhythmogenic right ventricular dysplasia (ARVD), long QT syndrome, catecholamine-induced polymorphic ventricular tachycardia (CPVT), Barth syndrome, valve stenosis, regurgitation, ischemia, fibrillation, polymorphic ventricular tachycardia, and muscular dystrophy or related heart disease such as Duchenne type, and cardiac hypertrophy.

[0112] Symptoms of cardiovascular disease can include, but are not limited to, fainting, fatigue, shortness of breath, chest pain, and palpitations.Cardiovascular disease is generally diagnosed by physical examination, blood test, and / or electrocardiogram (EKG).Abnormal EKG is an indication that a subject has abnormal heart rhythm or cardiac arrhythmia.Methods for diagnosing arrhythmia are known in the art.

[0113] In some embodiments of any of the aspects, the subject has or is at risk of having cardiovascular disease or a cardiac event.

[0114] In some embodiments of any of the aspects, the subject having cardiovascular disease is in need of, is receiving, or has received a cardiac cell graft.

[0115] Pluripotent stem cell source The methods and compositions described herein can use cardiomyocytes differentiated in vitro from, for example, embryonic stem cells, pluripotent stem cells, such as induced pluripotent stem cells, or other stem cells that allow such differentiation. Various stem cells that can be used to prepare cardiomyocytes are described below.

[0116] Stem cells are cells that retain the ability to self-renew via mitotic cell division and can differentiate into more specialized cell types. Three broad types of mammalian stem cells include: embryonic stem (ES) cells found in blastocysts, induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells, and adult stem cells found in adult tissues. Other sources of pluripotent stem cells can include amniotic or placenta-derived stem cells. In the developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In the adult organism, stem and progenitor cells replenish specialized cells to act as a repair system for the body, but also maintain normal turnover of regenerative organs such as blood, skin or intestinal tissues. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0117] Cardiomyocytes useful for the methods and compositions described herein can be differentiated from both embryonic stem cells and induced pluripotent stem cells, among others.In one aspect, the compositions and methods provided herein use human cardiomyocytes differentiated from embryonic stem cells.Alternatively, in some aspects, the compositions and methods provided herein do not include the generation or use of human cardiogenic cells made from cells taken from living human embryos.

[0118] Embryonic stem cells and their recovery methods are well known in the art and will not be described in detail herein.If a cell possesses one or more of the unique characteristics of embryonic stem cells, thereby allowing the cell to be distinguished from other cells, the cell has the phenotype of embryonic stem cell.Exemplary characteristic embryonic stem cell characteristics include but are not limited to morphology, gene expression or marker profile, proliferation ability, differentiation ability, karyotype, response to specific culture conditions, etc.

[0119] Cells derived from embryonic sources can include embryonic stem cells or stem cell lines obtained from stem cell banks or other recognized depositories. Other means of producing stem cell lines include methods involving the use of blastomere cells from early embryos (approximately the 8-cell stage) prior to blastocyst formation. Such techniques correspond to preimplantation genetic diagnosis techniques routinely practiced in assisted reproduction clinics. Single blastomere cells are co-cultured with established ES cell lines and then separated from them to form fully competent ES cell lines.

[0120] Embryonic stem cells are considered undifferentiated when they are not committed to a specific differentiation lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated embryonic stem (ES) cells are easily recognized by those skilled in the art and typically appear as colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli in two dimensions under a microscope. In some aspects, the human cardiomyocytes described herein are not derived from embryonic stem cells or any other cells of embryonic origin.

[0121] Adult stem cells are stem cells derived from tissues of postnatal or postneonatal organisms or from adult organisms. Adult stem cells differ structurally from embryonic stem cells by the presence of epigenetic differences, such as differences in DNA methylation patterns, as well as markers that they do or do not express compared to embryonic stem cells.

[0122] In some embodiments, the methods and compositions described herein utilize cardiomyocytes differentiated in vitro from induced pluripotent stem cells. The advantage of using iPSCs to generate cardiomyocytes for the compositions described herein is that the desired human cardiomyocytes are derived from the very subject to which they are administered. That is, somatic cells can be obtained from the subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into human cardiomyocytes that are administered to the subject (e.g., autologous cells). Since the cardiomyocytes (or their differentiated progeny) are essentially derived from an autologous source, the risk of engraftment rejection or allergic response is reduced compared to the use of cells from another subject or group of subjects. However, even cardiac grafts from autologous sources pose some risk of graft rejection, especially due to the presence of neoepitopes (e.g., mitochondrial gene mutations that can recognize cells as foreign).

[0123] In some aspects, cardiomyocytes useful in the compositions described herein are derived from a non-autologous or allogeneic source.

[0124] In some embodiments, iPSCs are cells that have been reprogrammed, which is a process that changes or reverses the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming is a process that drives the differentiation of cells backwards to more undifferentiated or more primitive types of cells. Reprogramming of somatic cells to induced pluripotent stem cells is known in the art and will not be described in detail herein. iPS cells can be generated or derived from terminally differentiated somatic cells as well as adult stem cells or somatic stem cells. That is, non-pluripotent progenitor cells can be made pluripotent or multipotent by reprogramming.

[0125] To confirm the induction of pluripotent stem cells for use by the methods described herein, isolated clones can be tested for the expression of stem cell markers. Such expression in cells derived from somatic cells identifies cells as induced pluripotent stem cells. Stem cell markers can be selected from a non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR, and immunological methods to detect the presence of encoded polypeptides, such as Western blot or flow cytometry analysis. In some embodiments, detection does not only involve RT-PCR, but also includes detection of protein markers. Although intracellular markers may best be identified via RT-PCR, cell surface markers are readily identified by, for example, immunocytochemistry.

[0126] The reprogrammed somatic cells disclosed herein express or express any of the following proteins: alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; beta-III-tubulin; alpha-smooth muscle actin (alpha-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Da x1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ESC-associated transcript 1 (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14; blastocytic cell transcription factor (Utf1); Rex1; p53; G3PDH; T Any number of pluripotent cell markers can be expressed, including telomerase including ERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3 / Stella; DPPA4; other general markers for pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3; Grb2; β-catenin, and Bmi1. Such cells can also be characterized by downregulation of markers characteristic of the somatic cell from which the induced pluripotent stem cell was derived.

[0127] In vitro differentiation of cardiomyocytes The methods and compositions described herein can use grafts that contain in vitro differentiated cardiomyocytes. Methods for differentiation of cardiomyocytes from ESCs or IPSCs are known in the art. See, for example, LaFlamme et al., Nature Biotech 25:1015-1024 (2007), which describes the differentiation of cardiomyocytes. These approaches use various factors and conditions to activate and guide the differentiation programs that lead to their respective cell types. The pathways and certain factors involved in them are described below.

[0128] In certain embodiments, the stepwise differentiation of ESCs or iPSCs into cardiomyocytes proceeds in the following order: ESCs or iPSCs>cardiac mesoderm>cardiomyocyte precursor cells>cardiomyocytes (see, e.g., US 2017024086, the entire contents of which are incorporated herein by reference).

[0129] As will be appreciated by those skilled in the art, in vitro differentiation of cardiomyocytes produces a final result of cells having phenotypic and morphological characteristics of cardiomyocytes, but the differentiation steps of in vitro differentiation need not be the same as those that occur naturally in an embryo. That is, during differentiation into cardiomyocytes, it is specifically contemplated herein that the stepwise differentiation approach utilized to produce such cells does not need to proceed through every precursor cell type specified during embryogenesis, but can essentially "skip" certain developmental periods that occur during embryogenesis.

[0130] Exemplary methods for generating cardiomyocytes or cardiomyocyte progenitor cells from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) are described, for example, in US2020-0085880, the contents of which are incorporated by reference in their entirety herein.

[0131] Calcineurin inhibitors Calcineurin is a calcium and calmodulin-dependent serine / threonine protein phosphatase (also known as protein phosphatase 3 and calcium-dependent serine-threonine phosphatase) that is ubiquitously expressed throughout the body.Calcineurin serves to activate the T cells of the immune system, and there are some agents that can inhibit this effect of calcineurin.Exemplary calcineurin inhibitors include small molecules such as tacrolimus or pimecrolimus, or peptides.

[0132] In one embodiment, the calcineurin inhibitor is a small molecule. Non-limiting small molecule inhibitors of calcineurin include cyclosporine A (CsA) and CsA derivatives (e.g., [(R)α-methylsarcosine 3 ]CsA, [Dimethylaminoethylthiosarcosine 3]CsA, [MeBm2t]1-CsA, ISA247 (voclosporin); FK506 (tacrolimus) and derivatives (e.g., FK520 (ascomycin), pimecrolimus (SDZ AM 981, 33-epi-chloro-33-desoxy-ascomycin), L-732,531 (32-O-(1-hydroxyethylindol-5-yl)-ascomycin), L-685,818 (FK506BD) and V-10,367; FMPP (4-(fluoromethyl)phenylphosphate), tyrphostin, norcantharidin, okadaic acid, endosal, kaempferol, barbiturates, 1,5-dibenzoyloxymethyl-norcantharidin, gossypol, Lie120, PD144795, diberufin, dipyridamole, NCI3, INCA compound, BTP or 3,5-bis(trifluoromethyl)pyrazole, BTP1, BTP2 (YM-58483), BTP3, BTP4, BTP5, BTP6, BTP7, BTP8, BTP9, BTP10, BTP11, BTP12, BTP13, BTP14, BTP15, BTP16, BTP17, BTP18, BTP19, BTP21, BTP22, BTP23, BTP34, BTP45, BTP46, BTP47, BTP50, BTP51, BTP52, BTP53, BTP54, BTP55, BTP56, BTP57, BTP58, BTP59, BTP61, BTP62, BTP63, BTP64, BTP65, BTP66, BTP67, BTP68, BTP69, BTP69, BTP61, BTP62, BTP63, BTP64, BTP65, BTP66, BTP67, BTP68, BTP69, BTP70, BTP71, BTP72, BTP73, BTP74, BTP75, These include, but are not limited to, A-285222, ST1959, AM404, UR-1505, triflusal, rocoglamide derivatives, WIN 53071, trifluoroperazine, KRM-III, caffeic acid phenylethyl ester (CAPE), YM-53792, quinazolinediones, pyrrolopyrimidinediones, NFAT-68, NFAT-133, punicalagin, imperatorine and quinolone alkaloids.

[0133] In one embodiment, the calcineurin inhibitor is CN585 (6-(3,4-dichlorophenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine).

[0134] In another embodiment, the calcineurin inhibitor is a peptide. Exemplary peptides include AID fragments (derived from the autoinhibitory domain of calcineurin), e.g., AID 424-521 , A.I.D. 457-482 , A.I.D. 420-511 , A.I.D. 328-511 , 11R-AID 457-482PxIxIT peptide (derived from the conserved calcineurin docking motif PxIxIT found in NFATc), VIVIT 16-nt oligopeptide, NFATc2 106-121 -SPRIEIT peptide, AKAP79 330-357 Peptide, RCANI peptide, RCANI-4 141-197 - Exon 7, RCAN1-4 143-163 -CIC peptide, LxVPcI peptide, RCANI-4 95-118 -SP repeat peptide, VacA peptide, A238L and A238L 200-213 Another example of a peptide is the pS3 peptide (derived from the cofilin phosphorylation domain).

[0135] In certain embodiments, the calcineurin inhibitor is FK506 (tacrolimus) or an FK506 derivative that contains calcineurin inhibitor activity, such as FK520 (ascomycin), FK523, pimecrolimus (SDZ AM 981, 33-epi-chloro-33 desoxy-ascomycin), L-732,531 (32-O-(1-hydroxyethylindol-5-yl)-ascomycin), L-685,818 (FK506BD), L-732-731, 15-0-DeMe-FK-520, meridamycin, 31-O-demethyl-FK506, V-10,367, L-683,590, L-685,818, C 18-OH-ascomycin; 9-deoxo-31-O-demethyl-FK506; L-688,617; A-1 19435 and AP1903.

[0136] In one embodiment, the calcineurin inhibitor comprises tacrolimus. Without wishing to be bound by theory, the mechanism of action of tacrolimus may involve, in part, inactivation of T lymphocytes and immunosuppression.

[0137] In some embodiments, calcineurin inhibitors include tacrolimus derivatives, such as those described in US Pat. No. 9,505,779, the entire contents of which are incorporated herein by reference.

[0138] Inhibitors of CD80 and / or CD86 signaling CD80 is a transmembrane protein that provides costimulatory signals for T cell activation. It works in tandem with CD86 to prime T cells. Regulatory T cells secrete soluble CTLA-4 (sCTLA-4), which binds to CD80 and blocks costimulatory activation of T cells.

[0139] The methods and compositions described herein use inhibitors of CD80 and / or CD86 signaling to reduce or prevent cardiac graft rejection. The CD80 and / or CD86 signaling inhibitors can include (i) a soluble extracellular domain of CTLA4; (ii) a mutant soluble extracellular domain of CTLA4 in which (a) the alanine at position 29 is substituted with an amino acid selected from the group consisting of tyrosine, leucine, tryptophan, and threonine, and (b) the leucine at position 104 is substituted with glutamic acid; or (iii) a soluble extracellular domain of (i) or (ii) and a moiety that alters the solubility of the extracellular domain of CTLA4 and / or its affinity for CD80 and / or CD86. In some embodiments, the moiety that alters the solubility of the extracellular domain of CTLA4 and / or its affinity for CD80 and / or CD86 can be a non-proteinaceous moiety, such as polyethylene glycol (PEG), or it can be a portion of an immunoglobulin molecule, such as the Fc, constant region of IgG.

[0140] In one embodiment, the CD80 and / or CD86 signaling inhibitor comprises abatacept, which is sold under the trade name ORENCIA® in both the United States and Europe and is a genetically engineered fusion protein consisting of a modified Fc region of immunoglobulin IgG1 fused to the extracellular domain of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4).

[0141] In another embodiment, the CD80 and / or CD86 signaling inhibitor comprises belatacept. Belatacept is a derivative of abatacept that contains two amino acid changes in the CD80 / 86 binding portion of the abatacept compound. Belatacept contains a 10-fold increase in the ability to inhibit T cell activation in vitro compared to abatacept.

[0142] Dosage and Administration A pharmaceutical or therapeutic composition comprising a calcineurin inhibitor and / or a CD80 / CD86 signaling inhibitor for reducing or preventing cardiac graft rejection can contain a physiologically tolerable carrier, in which the therapeutic agent is dissolved or dispersed as an active ingredient. In a preferred embodiment, the pharmaceutical composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes. As used herein, the terms "pharmaceutical acceptable", "physiologically tolerable" and grammatical variations thereof are used interchangeably when they refer to compositions, carriers, diluents and reagents and indicate that these substances can be administered to or on a mammal without producing undesired physiological effects such as nausea, dizziness, stomach upset, etc. A pharmaceutical acceptable carrier does not promote the production of an immune response against the agent with which it is incorporated unless so desired. The preparation of pharmacological or pharmaceutical compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on the formulation. Typically, such compositions are prepared for injection as either liquid solutions or suspensions, although solid forms suitable for solution or suspension in liquid prior to use can also be prepared. Preparations can also be emulsified or presented as liposomal compositions. The active ingredient can be mixed with an excipient that is pharma- ceutically acceptable and compatible with the active ingredient, in an amount suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. Therapeutic compositions containing therapeutic agents for reducing or preventing cardiac graft rejection can include pharma- ceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids, such as hydrochloric or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, and the like.Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0143] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no substances other than the active ingredient and water, or both, such as a buffer such as sodium phosphate, saline, or phosphate buffered saline at physiological pH values. Still further, aqueous carriers can contain salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes, as well as multiple buffer salts. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein that is effective in treating or preventing cardiac graft rejection depends on several factors and can be determined by standard clinical techniques.

[0144] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.The preparations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with optional addition of preservatives.The compositions can be suspensions, solutions, or emulsions in oily or aqueous media, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0145] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions.

[0146] Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0147] Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, aqueous solution, before use.

[0148] In some embodiments, the therapeutic agent can be delivered in an immediate release form. In other embodiments, the therapeutic agent can be delivered in a controlled or sustained release system. Controlled or sustained release pharmaceutical compositions can have a common goal of improving drug therapy over the results achieved by their non-controlled or non-sustained release counterparts. The advantages of controlled or sustained release compositions include extended activity of the therapeutic agent, reduced dosing frequency, and increased compliance. In addition, controlled or sustained release compositions can favorably affect the time of onset of action or other characteristics, such as blood levels of the therapeutic agent, and therefore reduce the occurrence of adverse side effects. Controlled or sustained release of active ingredients can be stimulated by various conditions, including, but not limited to, changes in pH, temperature, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0149] In one embodiment, a pump can be used for administration (Langer, Science 249:1527-1533 (1990); Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N. Engl. J. Med 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); and Howard et al., J. Neurosurg. 71:105 (1989)).

[0150] In the case of tablet or pill dosage forms, the pharmaceutical compositions described herein can be coated (e.g., enteric coated) to delay disintegration and absorption in the digestive tract, thereby providing a sustained action over an extended period of time. A selectively permeable membrane surrounding an osmotically active driving compound is also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is absorbed by the driving compound, which swells and displaces the agent or agent composition through an opening. In contrast to the spiked profile of immediate release formulations, these delivery platforms can provide an essentially zero-order delivery profile. Time-delay materials such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one aspect, the excipients are pharmaceutical grade.

[0151] The pharmaceutical compositions described herein can also be formulated as rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0152] The appropriate dosage range for a given therapeutic agent depends on potency and includes an amount large enough to produce the desired effect, for example, reduction of at least one symptom of cardiac graft rejection. The dosage of a therapeutic agent should not be so large as to cause unacceptable or life-threatening side effects, or should be used under close supervision by a medical professional. In general, dosage varies with the type of agent, as well as the age, condition, and sex of the patient. Dosage can be determined by those skilled in the art and can be adjusted by individual physicians in the event of any complications.

[0153] The administration of the dose as described above or as adopted by a skilled clinician can be repeated for a limited, predetermined period of time. In some embodiments, the dose is given once a day, or multiple times a day, for example, but not limited to, three times a day. Typically, the administration regimen is informed by the half-life of the agent, as well as the local minimum therapeutic concentration of the agent in blood, serum, or in a given biological tissue. In a preferred embodiment, the dose is administered daily for several weeks or months. The duration of treatment depends on the subject's clinical progression and continued response to therapy. After an initial higher therapeutic dose, a continuous, relatively low maintenance dose is contemplated.

[0154] A therapeutically effective amount is that amount of an agent sufficient to produce a statistically significant and measurable change in a given symptom of cardiac graft rejection (see "Measuring Efficacy" below). Such effective amounts can be measured in animal studies as well as in clinical trials for a given agent. For example, reduction or prevention of graft rejection can indicate adequate therapeutic efficacy of the agent.

[0155] Agents useful in the methods and compositions described herein can be administered intravenously (by bolus or continuous infusion), orally, intraperitoneally, intramuscularly, subcutaneously, into a body cavity, if desired, can be delivered by peristaltic means, or by other means known to those of skill in the art. Agents can be administered systemically if so desired.

[0156] The therapeutic composition containing at least one therapeutic agent can be conventionally administered in unit doses.The term "unit dose" when used in relation to therapeutic compositions refers to a physically separate unit suitable as a unitary administration for a subject, each unit containing a predetermined amount of therapeutic agent calculated to produce a desired therapeutic effect in association with a required physiologically acceptable diluent, i.e., carrier, or vehicle.

[0157] The composition is administered in a therapeutically effective amount, so as to be compatible with the dosage formulation.The amount and timing of administration depend on the subject to be treated, the ability of the subject's system to utilize active ingredient, and the degree of desired therapeutic effect.The active substance can be targeted by targeting moiety such as antibody or targeting liposome technology.

[0158] The exact amount of active ingredient that needs to be administered depends on the judgment of the practitioner and is specific to each individual.However, the dosage range suitable for systemic application is disclosed herein and depends on the route of administration.The mode suitable for administration also varies, but a typical feature is an initial administration followed by one or more repeated administrations at intervals by subsequent injections or other administrations.Alternatively, continuous intravenous infusion sufficient to bring blood concentration to the range specified for in vivo therapy is contemplated.

[0159] In certain embodiments, the calcineurin inhibitor is tacrolimus, which can be administered by subcutaneous, intravenous or oral administration. Exemplary therapeutic doses of tacrolimus for use in the methods and compositions described herein are administered to achieve a blood, plasma or serum concentration of 1-50 ng / mL, e.g., 1-45 ng / mL, 1-40 ng / mL, 1-35 ng / mL, 1-30 ng / mL, 1-25 ng / mL, 1-20 ng / mL, 1-15 ng / mL, 1-10 ng / mL, 1-9 ng / mL, 1-8 ng / mL, 1-7 ng / mL, 1-6 ng / mL, 1-5 ng / mL, 1-4 ng / mL, 1-3 ng / mL, 1-2 ng / mL, 1-4 ng / mL, 1-5 ng / mL, 1-4 ng / mL, 1-3 ng / mL, 1-2 ng / mL, 1-5 ng / mL, 1-6 ...5 ng / mL, 1-6 ng / mL, 1-5 ng / mL, 1-5 ng / mL, 1-6 ng / mL, 1-7 ng / mL, 1-8 ng / mL, 1-8 ng / mL, 1-9 ng / mL, 1-8 ng / mL, 1-9 ng / mL, 1-9 ng / mL, 1-9 ng / mL / mL, 45-50ng / mL, 40-50ng / mL, 35-50ng / mL, 30-50ng / mL, 25-50ng / mL, 20-50ng / mL, 15-50ng / mL, 10-50ng / mL, 5-50ng / mL, 5-25ng / mL, 5-20ng / mL, 5-30ng / mL, 1-25ng / mL, 10-20ng / mL, 10-30ng / mL, 10-15ng / mL, 7.5-20ng / mL, or any integer therebetween, or equivalent doses administered using alternating treatment regimens. Dosages for tacrolimus derivatives such as pimecrolimus can be similar or can be adjusted up or down by one of skill in the art to account for differences in pharmacokinetics or other parameters.

[0160] In certain embodiments, the calcineurin inhibitor comprises cyclosporine, which can be administered orally or intravenously. In embodiments in which cyclosporine is administered orally, the dose of cyclosporine is 0.1-50 mg / kg / day, 0.1-40 mg / kg / day, 0.1-30 mg / kg / day, 0.1-25 mg / kg / day, 0.1-20 mg / kg / day, 0.1-15 mg / kg / day, 0.1-10 mg / kg / day, 0.1-5 mg / kg / day, 0.1-2 mg / kg / day, 40-50 mg / kg / day, 30-50 mg / kg / day, 20-50 mg / kg / day, 10-50 mg / kg / day, 5 mg / kg / day, 0.5-20 mg / kg / day, 0.5-15 mg / kg / day, 1-15 mg / kg / day, 2-15 mg / kg / day, / day, 3-15 mg / kg / day, 4-15 mg / kg / day, 5-15 mg / kg / day, 6-15 mg / kg / day, 7-15 mg / kg / day, 8-15 mg / kg / day, 9-15 mg / kg / day, 10-15 mg / kg / day, 11-15 mg / kg / day, 12-15 mg / kg / day, 13-15 mg / kg / day, 14-15 mg / kg / day, 15-20 mg / kg / day, 16-20 mg / kg / day, 17-20 mg / kg / day, 18-20 mg / kg / day, 19-20 mg / kg / day, or any integer therebetween, or an equivalent dose administered using an alternating treatment regimen. In one embodiment, the dose of cyclosporine administered orally comprises 1-15 mg / kg / day.

[0161] In embodiments in which cyclosporine is administered intravenously, the dose of cyclosporine is 0.1 to 15 mg / kg / day, 0.1 to 14 mg / kg / day, 0.1 to 13 mg / kg / day, 0.1 to 12 mg / kg / day, 0.1 to 10 mg / kg / day, 0.1 to 8 mg / kg / day, 0.1 to 5 mg / kg / day, 0.1 to 4 mg / kg / day, 0.1 to 3 mg / kg / day, 0.1 to 2 mg / kg / day, 0.1~1mg / kg / day, 1~15mg / kg / day, 1~14mg / kg / day, 1~13mg / kg / day, 1~12mg / kg / day, 1~11mg / kg / day, 1~10mg / kg / day, 1~9mg / kg / day, 1~8mg / kg / day, 1~7mg / kg / day, 1~6 mg / kg / day, 1~5mg / kg / day, 1~4mg / kg / day, 1~3mg / kg / day, 1~2mg / kg / day, 2~15mg / kg / day, 2~14mg / kg / day, 2~13mg / kg / day, 2~12mg / kg / day, 2~11mg / kg / day, 2~10mg / k g / day, 2-9mg / kg / day, 2-8mg / kg / day, 2-7mg / kg / day, 2-6mg / kg / day, 2-5mg / kg / day, 2-4mg / kg / day, 2-3mg / kg / day, 3-15mg / kg / day, 3-14mg / kg / day, 3-13mg / kg / day, 3-12mg / kg / day, 3-11mg / kg / day, 3-10mg / kg / day, 3-9mg / kg / day, 3-8mg / kg / day, 3-7mg / kg / day, 3-6mg / kg / day, 3-5mg / kg / day, 3-4mg / kg / day, 4-15mg / kg / day, 4-14mg / kg / day g / day, 4-13mg / kg / day, 4-12mg / kg / day, 4-11mg / kg / day, 4-10mg / kg / day, 4-9mg / kg / day, 4-8mg / kg / day, 4-7mg / kg / day, 4-6mg / kg / day, 4-5mg / kg / day, 5-15mg / kg / day, 5-14mg / kg / day, 5-13mg / kg / day, 5-12mg / kg / day, 5-11mg / kg / day, 5-10mg / kg / day, 5-9mg / kg / day, 5-8mg / kg / day, 5-7mg / kg / day, 5-6mg / kg / day, 6-15mg / kg / day, 6-14 In one embodiment, the dose of cyclosporine administered intravenously is within the range of 1-6 mg / kg / day, 6-13 mg / kg / day, 6-12 mg / kg / day, 6-11 mg / kg / day, 6-10 mg / kg / day, 6-9 mg / kg / day, 6-8 mg / kg / day, 6-7 mg / kg / day, 7-15 mg / kg / day, 8-15 mg / kg / day, 9-15 mg / kg / day, 10-15 mg / kg / day, 11-15 mg / kg / day, 12-15 mg / kg / day, 13-15 mg / kg / day, 14-15 mg / kg / day, or any integer therebetween, or an equivalent dose administered using an alternating treatment regimen. In one embodiment, the dose of cyclosporine administered intravenously is comprised between 1 and 6 mg / kg / day.

[0162] In certain embodiments, the calcineurin inhibitor is administered at a dose of 0.01-5 mg / kg, 0.01-4 mg / kg, 0.01-3 mg / kg, 0.01-2 mg / kg, 0.01-1 mg / kg, 0.1-5 mg / kg, 0.1-4 mg / kg, 0.1-3 mg / kg, 0.1-2 mg / kg / day, 0.1-1 mg / kg, 0.1-0.5 mg / kg, 0.5-5 mg / kg, 0.5-4 mg / kg, 0.5-3 mg / kg, 0. The present invention includes voclosporin that can be administered orally twice daily at doses in the ranges of 5-2 mg / kg, 0.5-1 mg / kg, 1-5 mg / kg, 1-4 mg / kg, 1-3 mg / kg, 1-2 mg / kg, 2-5 mg / kg, 2-4 mg / kg, 2-3 mg / kg, 3-5 mg / kg, 3-4 mg / kg, 4-5 mg / kg, or any integer therebetween, or an equivalent dose administered using a different treatment regimen. In one embodiment, the dose of voclosporin administered twice daily via oral administration comprises 0.1-1 mg / kg.

[0163] In certain embodiments, the inhibitor of CD80 and / or CD86 signaling comprises abatacept or belatacept, which can be administered either intravenously or subcutaneously. In some embodiments, the dose of abatacept or belatacept is 1.25-125 mg / kg administered intravenously or subcutaneously at a given interval (e.g., every 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 days, every 3 weeks, every 4 weeks, or longer). In certain embodiments, the intravenous or subcutaneous dose of abatacept or belatacept is 1.25-100 mg / kg, 1.25-90 mg / kg, 1.25-80 mg / kg, 1.25-75 mg / kg, 1.25-70 mg / kg, 1.25-60 mg / kg, 1.25-50 mg / kg, 1.25-40 mg / kg, 1.25-30 mg / kg, 1.25-20 mg / kg, 1.25-10 mg / kg, 1.25-5 mg / kg, 2-125 mg / kg, 5-125 mg / kg, 10-125 mg / kg, 10-125 mg / kg, 20-125 mg / kg, 30-325 mg / kg, 35-40 mg / kg, 30-40 mg / kg, 35-50 mg / kg, 35-40 mg / kg, 30-50 mg / kg, 35 ... 0-125mg / kg, 40-125mg / kg, 50-125mg / kg, 60-125mg / kg, 70-125mg / kg, 75-125mg / kg, 80-125mg / kg, 90-125mg / kg, 100-125mg / kg, 110-125mg / kg, 115-125mg / kg, 5-20mg / kg, 5-15mg / kg, 5-25mg / kg, 10-20mg / kg, 10-25mg / kg, 20-50mg / kg, 25-75mg / kg, or any integer therebetween, or an equivalent dose administered using an alternating treatment regimen.

[0164] In certain embodiments, the dose of abatacept or belatacept is 5-25 mg / kg and the therapeutic dose of tacrolimus administered to achieve a blood, plasma or serum concentration is 1-50 ng / mL, e.g., 1-45 ng / mL, 1-40 ng / mL, 1-35 ng / mL, 1-30 ng / mL, 1-25 ng / mL, 1-20 ng / mL, 1-15 ng / mL, 1-10 ng / mL, 1-9 ng / mL, 1-8 ng / mL, 1-7 ng / mL, 1-6 ng / mL, 1-5 ng / mL, 1-4 ng / mL, 1-3 ng / mL, 1-4 ng / mL, 1-5 ng / mL, 1-6 ng / mL, 1-7 ng / mL, 1-8 ng / mL, 1-8 ng / mL, 1-9 ... 2ng / mL, 45-50ng / mL, 40-50ng / mL, 35-50ng / mL, 30-50ng / mL, 25-50ng / mL, 20-50ng / mL, 15-50ng / mL, 10-50ng / mL, 5-50ng / mL, 5-25ng / mL, 5-20ng / mL, 5-30ng / mL, 1-25ng / mL, 10-20ng / mL, 10-30ng / mL, 10-15ng / mL, 7.5-20ng / mL, or any integer therebetween, or an equivalent dose administered using an alternating treatment regimen.

[0165] In certain embodiments, the dose of tacrolimus is 1.25-100 mg / kg, 1.25-90 mg / kg, 1.25-80 mg / kg, 1.25-75 mg / kg, 1.25-70 mg / kg, 1.25-60 mg / kg, 1.25-50 mg.kg, 1.25-40 mg / kg, 1.25-30 mg / kg, 1.25-20 mg / kg, 1.25-10 mg / kg, 1.25-5 mg / kg, 2-125 mg / kg, 5-125 mg / kg, 10-125 mg / kg, 10-125 mg / kg, 20-125 mg / kg, 30-125 mg / kg, 40-125 mg / kg, 50-125 mg / kg, 60-125 mg / kg, 70-125 mg / kg, 75-125 mg / kg, 80-125 mg / kg, 90-125 mg / kg, 100-125 mg / kg, 110-125 mg / kg, 115-125 mg / kg, 5-20 mg / kg, 5-15 mg / kg, 5-25 mg / kg, 10-20 mg / kg, 10-25 mg / kg, 20-50 mg / kg, 25-75 mg / kg, or any integer therebetween, or an equivalent dose administered using an alternating treatment regimen, to achieve a blood, plasma or serum concentration of 5-30 ng / mL in combination with a dose of abatacept or belatacept selected from the group consisting of

[0166] In certain embodiments, the dose of tacrolimus is administered to achieve a blood, plasma or serum concentration of 5-25 ng / mL and the dose of abatacept or belatacept is 5-20 mg / kg.

[0167] In some embodiments of the combination treatment, the calcineurin inhibitor is selected from the group consisting of tacrolimus, cyclosporine, voclosporine, and pimecrolimus. In some embodiments of the combination treatment, the CD80 and / or CD86 inhibitor comprises an extracellular domain fragment of CTLA4. In some embodiments of the combination treatment, the CD80 and / or CD86 signaling inhibitor comprises abatacept or belatacept. In some embodiments, the combination treatment comprises the calcineurin inhibitor tacrolimus and the CD80 and / or CD86 signaling inhibitor abatacept. In some embodiments, the combination treatment comprises the calcineurin inhibitor cyclosporine and the CD80 and / or CD86 signaling inhibitor abatacept. In some embodiments, the combination treatment comprises the calcineurin inhibitor voclosporine and the CD80 and / or CD86 signaling inhibitor abatacept. In some embodiments, the combination treatment includes the calcineurin inhibitor pimecrolimus and the inhibitor of CD80 and / or CD86 signaling, abatacept. In some embodiments, the combination treatment includes the calcineurin inhibitor tacrolimus and the inhibitor of CD80 and / or CD86 signaling, belatacept. In some embodiments, the combination treatment includes the calcineurin inhibitor cyclosporine and the inhibitor of CD80 and / or CD86 signaling, belatacept. In some embodiments, the combination treatment includes the calcineurin inhibitor voclosporine and the inhibitor of CD80 and / or CD86 signaling, belatacept. In some embodiments, the combination treatment includes the calcineurin inhibitor pimecrolimus and the inhibitor of CD80 and / or CD86 signaling, belatacept. In some embodiments, the combination treatment includes the calcineurin inhibitor tacrolimus and the inhibitor of CD80 and / or CD86 signaling includes an extracellular domain fragment of CTLA4. In some embodiments, the combination treatment comprises the calcineurin inhibitor cyclosporine and the inhibitor of CD80 and / or CD86 signaling comprises an extracellular domain fragment of CTLA4.In some embodiments, the combination treatment comprises the calcineurin inhibitor voclosporin and the inhibitor of CD80 and / or CD86 signaling comprises an extracellular domain fragment of CTLA4. In some embodiments, the combination treatment comprises the calcineurin inhibitor pimecrolimus and the inhibitor of CD80 and / or CD86 signaling comprises an extracellular domain fragment of CTLA4.

[0168] In some embodiments, it is contemplated that the combination therapy will last at least the lifespan of the graft, up to the remaining lifespan of the recipient in need thereof. Long-term treatment as described herein is contemplated because the immune system is dynamic and may flare in response to viral or bacterial infection, which may then initiate autoimmune rejection of the graft.

[0169] Combination therapy Treatment of a subject with both a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling is considered a "combination therapy." In other embodiments, the therapeutically effective agents described herein (i.e., a calcineurin inhibitor and a CD80 / CD86 signaling inhibitor) are administered to a subject simultaneously with an additional combination therapy, such as an immunosuppressant. Exemplary immunosuppressants can include, but are not limited to, methylprednisolone, betamethasone, cortisone, dexamethasone, hydrocortisone, prednisolone, prednisone, or triamcinolone. An exemplary treatment regimen for cardiac graft rejection includes methylprednisolone 500 mg administered IV until separation from cardiopulmonary bypass, followed by methylprednisolone 125 mg administered IV every 8 hours for the first 24 hours, then the subject is switched to oral prednisone at a dose of 0.1-10 mg / kg PO daily.

[0170] As used herein, the term "simultaneously" is not limited to administration of two or more agents at exactly the same time, but means that they are administered to a subject in sequence, within a time interval such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered alone). For example, a combination of therapeutic agents can be administered sequentially at the same time or at different times in any order. However, if not administered at the same time, they are administered sufficiently close in time to provide the desired therapeutic effect, preferably synergistically. The agents can be administered separately in any suitable form and by any suitable route. It is understood that if each of the combined therapeutic agents is not administered as the same pharmaceutical composition, they can be administered to a subject in need thereof in any order. For example, the first therapeutic agent can be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the second therapeutic agent (or vice versa).In other embodiments, the delivery of either therapeutic agent ends before the delivery of the other agent / treatment begins.In some embodiments in either case, the treatment is more effective due to the combined administration. For example, therapeutic agents used in combination are more effective than seen with either agent alone. In some embodiments, delivery is such that the reduction in symptoms or other parameters related to the disorder is greater than observed with either therapeutic agent alone. The effect of such combinations can be partially additive, fully additive, or greater than additive. The agents and / or other therapeutic agents, procedures, or modalities can be administered during periods of active disease or during periods of persistent or less active disease.

[0171] When administered in combination, one or more of the therapeutic agents can be administered in an amount or dosage that is higher, lower, or the same as the amount or dosage of a given agent used individually, for example, as monotherapy.In certain embodiments, the administered amount or dosage of a first therapeutic agent when administered in combination with a second therapeutic agent is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the first agent when used individually.In other embodiments, the amount or dosage of a first therapeutic agent when administered in combination with a second therapeutic agent that results in a desired effect (e.g., improved cognitive function) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the first (or second) agent required to achieve the same therapeutic effect when administered alone.

[0172] Effectiveness The effectiveness of a given treatment to reduce or prevent cardiac graft rejection can be determined by a skilled clinician. However, if any one or all of the signs or symptoms of cardiac graft rejection are favorably altered, or other clinically acceptable symptoms or markers of the disease are improved, or, for example, at least 10% restored after treatment with a therapeutic agent for cardiac graft rejection, the treatment is considered to be an "effective treatment" as this term is used herein. Efficacy can also be measured by the individual not worsening as assessed by stabilization of the disease, or the need for medical intervention (i.e., the progression of the disease is stopped or at least slowed). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals) including (1) inhibiting the disease, e.g., arresting or slowing the progression of graft rejection, or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of onset or preventing a second disease / disorder associated with graft rejection (e.g., cardiomyopathy, etc.).

[0173] An effective amount for the treatment of a disease means an amount sufficient to result in effective treatment of the disease, as that term is defined herein, when administered to a mammal in need thereof. The effectiveness of an agent can be determined by assessing physical indicators of the disease, such as reduction or cessation of arrhythmias, reduction of inflammatory markers (e.g., C-reactive protein (CRP)), etc.

[0174] In some embodiments, the efficacy of a given combination treatment (e.g., tacrolimus and abatacept) can be determined by the reduction in the severity of cellular rejection in endomyocardial biopsies according to the International Society for Heart and Lung Transplantation (ISHLT) Acute Cellular Rejection Grading Scheme. The grading scheme includes four different rejection levels: (i) 0R, no rejection as evidenced by histopathological findings; (ii) 1R, mild, interstitial and / or perivascular infiltration with up to one focus of myocyte damage; (iii) 2R, moderate, two or more focal infiltration with myocardial damage; and (iv) 3R, severe, diffuse infiltration with multifocal myocyte damage ± edema ± hemorrhage ± vasculitis. Thus, a given combination treatment is effective if the subject moves down at least one level on the ISHLT acute cellular rejection grading scheme (e.g., from severe to moderate, moderate to mild, mild to asymptomatic, severe to mild, mild to asymptomatic, moderate to asymptomatic, etc.). Ideally, treatment is optimized to achieve a grading of 2R, 1R, or 0R.

[0175] In some embodiments, efficacy can be measured by a reduction in the need for co-immunosuppressive therapy (e.g., glucocorticoids) without an increase in graft rejection. A reduction in the need for one or more immunosuppressive therapies can include, for example, reducing the dose of a given agent, removing a co-immunosuppressive therapy from the treatment regimen, reducing dosing frequency, reducing the duration of treatment, or replacing a given immunosuppressive therapy with a similar agent that is less potent.

[0176] In other embodiments, efficacy can be assessed by measuring the length of graft viability in subjects treated with a calcineurin inhibitor and a CD80 / CD86 signaling inhibitor compared to the length of graft viability in subjects not treated with such agents. For example, cardiac graft viability is extended by at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, at least 10 years, at least 20 years or more when a subject is treated with a combination of a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling than the viability of a substantially similar cardiac graft when the subject is not treated with such a combination.

[0177] In other embodiments, efficacy can also be expressed as a reduction or elimination of cell loss in cardiac grafts when subjects are treated with a combination of calcineurin inhibitors and inhibitors of CD80 and / or CD86 signaling, compared to the loss that occurs in untreated subjects with similar cardiac grafts.For example, the reduction of cell loss in cardiac grafts can be less than 0.5% when subjects are treated with calcineurin inhibitors and inhibitors of CD80 and / or CD86 signaling, compared to the graft at initial engraftment (e.g., graft size at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks); in other embodiments, treatment can be less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, or less than 60% of the cell loss in cardiac grafts when subjects are treated with calcineurin inhibitors and inhibitors of CD80 and / or CD86 signaling. EXAMPLES

[0178] Example 1: Steroid-sparing chronic immunosuppression for transplantation of allogeneic stem cell-derived cardiomyocytes Transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is an emerging strategy to treat recent myocardial infarction (MI). Most studies to date have used xenotransplantation of human PSC-CMs, which does not inform how to achieve successful allogeneic transplantation in patients. To identify a minimal, steroid-sparing immunosuppressive regimen for first-in-human allogeneic transplantation, a non-human primate (NHP) model was developed in which rhesus macaque iPSC-CMs were transplanted into normal or infarcted hearts of MHC-mismatched recipients. Allogeneic iPSCs derived from rhesus macaques (Macaca mulatta) were differentiated into a highly enriched population of cardiomyocytes (riPSC-CMs). Approximately 200 × 10 6riPSC-CMs were transplanted into 11 MHC-mismatched rhesus macaque recipients. The primary endpoint of the study was the degree of histopathological immune rejection. In the absence of immunosuppression, cardiomyocyte grafts survived for 2 weeks without rejection, but by 8 weeks they underwent severe cellular rejection with extensive inflammatory infiltration by CD3+ T cells and CD20+ B cells resulting in graft destruction. Subsequent studies focused on 8-week engraftment, with promising regimens further studied up to 16 weeks. Low-dose subcutaneous tacrolimus (TAC) alone (trough 5–10 ng / mL) or subcutaneous abatacept (ABT, CTLA4-Ig) alone (12.5 mg / kg, biweekly) was inadequate as grafts showed moderate to severe rejection 8 weeks after transplantation. In contrast, the combination of mid-dose TAC (trough 10–15 ng / mL) and ABT (12.5 mg / kg, biweekly) resulted in graft preservation without significant myocardial rejection at 8 weeks post-transplantation. However, evaluation of TAC+ABT at 16 weeks post-transplantation revealed low-to-moderate immune rejection of the graft, with one animal having patchy infiltration of T and B cells and a second NHP having no significant rejection. The presence or absence of MI did not affect the immunoreactive outcomes. In summary, allogeneic iPSC-CMs are less immunogenic than whole cardiac allografts, and data to date suggest that a steroid-sparing immunosuppressive regimen may have the potential to support long-term PSC-CM transplantation. Thus, a combined immunosuppressive regimen of TAC and ABT can be used to support long-term PSC-CM transplantation. This study also suggests that long-term observation up to 16 weeks is required to detect late rejection.

[0179] All heading and section names are used for clarity and reference purposes only and should not be construed as limiting in any manner For example, those skilled in the art will recognize the utility of combining various aspects from different headings and sections as appropriate and consistent with the spirit and scope of the technology described herein.

[0180] All references cited in this specification are hereby incorporated by reference in their entirety into this specification to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety into this specification.

[0181] As will be apparent to those skilled in the art, many modifications and variations of this application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the scope of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A pharmaceutical for use in a method of cardiomyocyte transplantation comprising in vitro differentiated cardiomyocytes or cardiomyocyte progenitor cells, the method comprising: administering the in vitro differentiated cardiomyocytes or cardiac progenitor cells to a graft site in cardiac tissue of a recipient in need thereof; administering to said recipient a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling; A pharmaceutical comprising:

2. The pharmaceutical composition of claim 1, wherein the in vitro differentiated cardiomyocytes are allogeneic to the recipient.

3. The pharmaceutical composition of claim 1, wherein the in vitro differentiated cardiomyocytes are autologous to the recipient.

4. 4. The pharmaceutical composition of claim 1, 2, or 3, wherein the calcineurin inhibitor is selected from the group consisting of tacrolimus, cyclosporine, voclosporin, and pimecrolimus.

5. The pharmaceutical agent according to any one of claims 1 to 4, wherein the inhibitor of CD80 and / or CD86 signaling comprises an extracellular domain fragment of CTLA4.

6. The pharmaceutical composition of claim 5, wherein the extracellular domain fragment of CTLA4 is fused to a heterologous polypeptide and / or conjugated to polyethylene glycol (PEG).

7. The pharmaceutical agent of claim 6, wherein the heterologous polypeptide increases the serum half-life of the inhibitor of CD80 and / or CD86 signaling, or the heterologous polypeptide comprises an IgG Fc domain polypeptide.

8. The pharmaceutical agent according to any one of claims 1 to 7, wherein the inhibitor of CD80 and / or CD86 signaling comprises abatacept or belatacept.

9. The method of claim 8, wherein abatacept is administered intravenously or subcutaneously at a dose of 5 mg / kg to 20 mg / kg.

10. Inhibitors of CD80 and / or CD86 signaling (i) administered every 2 weeks or every 3 weeks; (ii) administered every 12 to 16 days; (iii) administered every 10 to 18 days; or (iv) administered subcutaneously, orally or intravenously; The pharmaceutical composition according to any one of claims 1 to 9.

11. The pharmaceutical agent according to any one of claims 1 to 10, wherein the calcineurin inhibitor comprises tacrolimus or an immunosuppressant derivative thereof.

12. The pharmaceutical composition of claim 11, wherein the administration of tacrolimus is selected from the following: administered to achieve a serum or plasma concentration of 5-20 ng / mL; or It may be given by continuous infusion every 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 hours, or daily.

13. The pharmaceutical agent according to any one of claims 1 to 12, wherein the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling are each administered subcutaneously or intravenously, or a combination thereof.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein administering a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling results in one or more of the following: reducing immune rejection of the administered cardiomyocytes compared to rejection of cardiomyocytes administered without the calcineurin inhibitor and the inhibitor of CD80 and / or CD86 signaling; reducing the need for steroid immunosuppression to prevent immune rejection of the transplanted cardiomyocytes compared to the need for steroid immunosuppression when the cardiomyocytes are transplanted without administration of a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling; reducing infiltration of CD3+ T cells or CD20+ B cells into the graft site compared to infiltration that occurs in the absence of the administration of a calcineurin inhibitor and an inhibitor of CD80 and / or CD86 signaling; reduce infiltration of CD3+ T cells or CD20+ B cells into the graft site 8 weeks after administration of the cardiomyocytes; or Infiltration of CD3+ T cells or CD20+ B cells into the graft site is reduced 16 weeks after administration of cardiomyocytes.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the recipient has a cardiovascular disease or disorder.

16. The pharmaceutical of claim 15, wherein the cardiovascular disease or disorder comprises tachyarrhythmia, bradyarrhythmia, cardiomyopathies, congenital defects, and muscular dystrophy-associated cardiomyopathies.

17. 17. The method of claim 16, wherein the congenital defect comprises a septal defect or a hypoplastic syndrome.

18. 17. The method of claim 16, wherein the muscular dystrophy associated cardiomyopathy comprises cardiomyopathy associated with Duchenne muscular dystrophy (DMD).

19. A pharmaceutical comprising a calcineurin inhibitor used in combination with an inhibitor of CD80 and / or CD86 signaling for the treatment or prevention of immune-mediated rejection of a cardiac graft or for the treatment or prevention of a cardiovascular disease or disorder.

20. A medicament according to claim 19, comprising any of the features according to claims 1 to 18.

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