WO2025043075A1 - Methods of treating myocardial infarction, ischemia, and ischemia reperfusion injury - Google Patents

Abstract

Compositions and methods are provided for treating ischemic conditions such as myocardial infarction, myocardial ischemia, or ischemia reperfusion injury in a subject in need thereof. In particular, the methods comprise administering a therapeutically effective amount of programmed death ligand-1 (PD-L1) to a subject.

Description

METHODS OF TREATING MYOCARDIAL INFARCTION, ISCHEMIA, AND ISCHEMIA

REPERFUSION INJURY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63/578,431 , filed August 24, 2023, which application is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF A SEQUENCE LISTING

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML file, “STAN-2134WO”, created on August 12, 2024, and having a size of 6,717 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

[0003] Myocardial infarction (Ml) represents a major global cause of morbidity and mortality. In patients who experience Ml, immediate ischemia and cell death occurs which result in decreased cardiac function and therefore high morbidity and mortality.

[0004] Recently, Zhang et al. showed that patients with acute Ml had increases in the levels of granulocytic and monocytic populations of myeloid-derived suppressor cells in the peripheral blood and upregulation of expression of death-ligand 1 (PD-L1 ) on the surface of both granulocytic and monocytic myeloid-derived suppressor cells (Zhang et al. (2023) Biomed. Rep. 19(2):55). Upregulation of PD-L1 expression may induce expansion of regulatory T-cells by mediating binding to PD-1 on the surface of regulatory T-cells, thus playing a crucial role in acute Ml.

[0005] Currently, there are no minimally invasively deliverable therapeutics that can be administered to patients to treat Ml, to stop or even reverse the adverse effect of myocardial ischemia and the associated immune response that result in further deterioration of cardiac function and reverse remodeling of a heart ventricle.

SUMMARY OF THE I VENTION

[0006] Compositions and methods are provided for treating ischemic conditions such as myocardial infarction, myocardial ischemia, and ischemia reperfusion injury in a subject in need thereof. In particular, the methods comprise administering a therapeutically effective amount of programmed death ligand-1 (PD-L1 ) to a subject.

[0007] In certain embodiments, the PD-L1 is administered intramyocardially, intraperitoneally, or intravenously. [0008] In certain embodiments, the PD-L1 is administered locally to a site of an ischemic injury.

[0009] In certain embodiments, the PD-L1 is administered systemically.

[0010] In certain embodiments, the treatment increases the left ventricular ejection fraction compared to the left ventricular ejection fraction before treatment.

[0011] In certain embodiments, the treatment decreases infarct size compared to the infarct size without treatment.

[0012] In certain embodiments, the treatment causes a change in immune cell composition.

[0013] In certain embodiments, multiple cycles of treatment are administered to the subject. In some embodiments, the PD-L1 is administered according to a daily dosing regimen or intermittently.

[0014] In certain embodiments, the method further comprises administering a statin, an angiotensin- converting-enzyme inhibitor, a beta blocker, an angiotensin II receptor antagonist, an antiplatelet agent, an anticoagulant, a calcineurin inhibitor, or a combination thereof to the subject.

[0015] In certain embodiments, the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or a variant comprising a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, or a biologically active fragment thereof, wherein the variant or fragment retains the ability to increase left ventricular ejection fraction, decrease infarct size, and/or decrease, delay, or prevent remodeling of a heart ventricle.

[0016] In another aspect, a composition comprising PD-L1 for use in a method of treating an ischemic condition is provided. In certain embodiments, the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

[0017] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0018] In certain embodiments, the composition further comprises a statin, an angiotensin- converting-enzyme inhibitor, a beta blocker, an angiotensin II receptor antagonist, an antiplatelet agent, an anticoagulant, a calcineurin inhibitor, or a combination thereof.

[0019] In certain embodiments, the composition is formulated for administration intramyocardially, intravenously, or intraperitoneally.

[0020] In certain embodiments, the composition is formulated for administration locally to a site of an ischemic injury.

[0021] In certain embodiments, the PD-L1 is formulated for systemic administration.

[0022] In another aspect, the use of programmed death ligand-1 (PD-L1 ) in the manufacture of a medicament or pharmaceutical composition for treating an ischemic condition is provided. In some embodiments, the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

[0023] In another aspect, a method of preventing, delaying, or decreasing remodeling of a heart ventricle in a subject is provided, the method comprising administering a therapeutically effective amount of PD-L1 to the subject. In some embodiments, the subject has a myocardial infarction, myocardial ischemia, or an ischemia reperfusion injury.

[0024] In certain embodiments, the PD-L1 is administered intramyocardially, intravenously, or intraperitoneally.

[0025] In certain embodiments, the PD-L1 is administered systemically.

[0026] In certain embodiments, the PD-L1 is administered prophylactically.

[0027] In certain embodiments, the PD-L1 is administered multiple times to the subject.

[0028] In certain embodiments, the PD-L1 is administered according to a daily dosing regimen or intermittently.

[0029] In another aspect, a composition comprising PD-L1 for use in a method of treating heart ventricle remodeling is provided.

[0030] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0031] In certain embodiments, the composition is formulated for administration intramyocardially, intravenously, or intraperitoneally.

[0032] In certain embodiments, the composition is formulated for administration systemically or locally to the heart ventricle.

[0033] In another aspect, the use of PD-L1 in the manufacture of a medicament or pharmaceutical composition for treating heart ventricle remodeling is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIGS. 1A-1D. Treatment of Ml with programmed death ligand-1 (PD-L1 ). When delivered immediately intramyocardially (IM) following Ml via left anterior descending artery (LAD) ligation in a rodent model, PD-L1 , significantly improved the left ventricular ejection fraction compared to control animals that did not receive PD-L1 treatment. (FIG. 1A) We have found that by delivering 30 ig of PD-L1 intramyocardially in mice, the left ventricular ejection fraction (LVEF) increased from 28% without treatment to 42% with PD-L1 treatment (1 .5-fold increase) 14 days after the event. (FIG. 1 B) Increasing the PD-L1 dose to 50 pg intramyocardial delivery was found to have similar outcomes with LVEF increasing from 21% without treatment to 41% with PD-L1 treatment (2-fold increase) 14 days after the event. (FIG. 1 C) We also studied the effect of 50 pg PD-L1 for intraperitoneal (IP) delivery as systemic delivery and found that LVEF similarly increased from 38% without treatment to 51 % with PD-L1 treatment (1 .3-fold increase) 14 days after the event. (FIG. 1D) We also found that PD-L1 treatment, regardless of the dose and delivery route, reduced LV infarct size compared to controls without treatment.

[0035] FIGS. 2A-2E. Representative images of mice hearts 14 days after Ml with LAD ligation treated with (FIG. 2A) programmed death-ligand 1 (PD-L1 ) injected intramyocardially, (FIG. 2B) PD- L1 injected intraperitoneally, and (FIG. 2C) PBS. Smaller scar area in general was noted after the PD-L1 treatment compared with PBS treatment. Scale bar = 1 mm. (FIG. 2D) Composite average LVEF measured from baseline and 14 days after Ml. Mice that received 50 p.g PD-L1 delivered intramyocardially or intraperitoneally demonstrated significantly improved ejection fractions compared with those that received PBS. Error bars represent standard errors. (FIG. 2E) Flow cytometry data demonstrating peripheral blood and myocardial PD-1 + monocyte percentage changes after Ml. Note an increase of the PD-1 + monocyte population was detected in the myocardium and in peripheral blood 3 days after Ml. Error bars represent standard errors.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Compositions and methods are provided for treating ischemic conditions such as myocardial infarction, myocardial ischemia, or ischemia reperfusion injury in a subject in need thereof. In particular, the methods comprise administering a therapeutically effective amount of programmed death ligand-1 (PD-L1 ) to a subject.

[0037] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions or methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0038] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0040] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0041] As used herein the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins and reference to "the drug" includes reference to one or more drugs and equivalents thereof (e.g., therapeutics, medicines, medicaments), known to those skilled in the art, and so forth.

[0042] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definitions

[0043] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0044] The term “ischemic condition” is used herein to refer to any disease or condition causing reduced blood flow through a tissue or organ. Ischemic conditions may result in inadequate oxygenation and supply of nutrients to a tissue or organ and cell death. Exemplary ischemic conditions include, without limitation, myocardial infarction, myocardial ischemia, and ischemia reperfusion injury, which may lead to deterioration of cardiac function, including decreased left ventricular ejection fraction, remodeling of heart ventricles, increased levels of granulocytic and monocytic populations of myeloid-derived suppressor cells in the peripheral blood, and increased morbidity and mortality. [0045] The term “ventricle remodeling” is used herein to refer to adverse structural changes in a heart ventricle associated with an ischemic condition such as myocardial infarction, myocardial ischemia, or ischemia reperfusion injury. Ventricle remodeling may include, without limitation, pathological changes in myocardial structure, ventricle shape, and end-diastolic and end-systolic volumes. In particular, the left ventricle may undergo the most pronounced morphological changes, including a transition from the normal ellipsoid shape to a more spherical shape with increased end- diastolic and end-systolic volumes. Ventricle remodeling may be accompanied by myocyte hypertrophy, loss of cardiomyocytes and contractile function, death of myocytes leading to an immune response including an influx of inflammatory cells, destruction of the extracellular collagen matrix, ventricular dilation, vascular stiffness, wall thinning, microvascular obstruction, intramyocardial hemorrhage, myocardial strain, and myocardial fibrosis. Ventricle remodeling can be diagnosed by detecting changes in ventricular cavity diameter, mass, and geometry. See, e.g., Calvier et al. (2023) J. Clin, Med. 12(1):334; Burchfield et al. (2013) Circulation 128(4) :388-400, Leanca et al. (2022) Life (Basel) 12(8):11 11 , Cohn et al. (2000) J. Am. Coll. Cardiol. 35(3):569-582; herein incorporated by reference.

[0046] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. , arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0047] A "therapeutically effective amount" is intended for an amount of an active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with a disease, or which improves resistance to a disorder. [0048] The terms "pharmacologically effective amount" or "therapeutically effective amount" of PD- L1 refers to an amount of the PD-L1 sufficient to provide the desired response, such as increasing the left ventricular ejection fraction, decreasing infarct size, and/or decreasing, delaying, or preventing remodeling of a heart ventricle. Additionally, a therapeutically effective dose or amount may increase survival or reduce morbidity of a subject. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0049] The term "survival" as used herein means the time from the start of treatment to the time of death.

[0050] "Substantially" or "essentially" means nearly totally or completely, for instance, 95% or greater of some given quantity.

[0051] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0052] By "isolated" is meant, when referring to a polypeptide or protein, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

[0053] As used herein, the terms “increase”, “increasing”, “enhance”, and “enhancing” (and grammatical variations thereof) describes, unless the context indicates otherwise, a detectable elevation compared to a reference value. An increase can comprise an elevation of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more such as compared to another measurable property or quantity (e.g., a control value). [0054] The terms "protein," "peptide" and "polypeptide" refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and/or imino molecules. No particular size is implied by use of the terms "protein," "peptide" or "polypeptide" and these terms are used interchangeably. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, linearly linked peptides), cyclized, branched molecules and the like, are included within the definition. The terms also include molecules comprising one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831 ,005; 5,877,278; and 5,977,301 ; Nguyen et al. (2000) Chem Biol. 7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20) :9367-9371 for descriptions of peptoids).

[0055] Thus, references to proteins, polypeptides, or peptides also include derivatives of the amino acid sequences including one or more non-naturally occurring amino acids. A first polypeptide or peptide is "derived from" a second polypeptide or peptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide encoding the second polypeptide or peptide, or (ii) displays sequence identity to the second polypeptide or peptide as described herein. Sequence (or percent) identity can be determined as described below. Preferably, derivatives exhibit at least about 50% percent identity, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value therebetween) to the sequence from which they were derived. Such derivatives can include post-expression modifications of the protein, polypeptide, or peptide, for example, glycosylation, acetylation, phosphorylation, hydroxylation, lipidation, PEGylation, and the like.

[0056] Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein (or fragment thereof) maintains the desired activity (e.g., PD-L1 biological activity, ability to increase left ventricular ejection fraction, decrease infarct size, decrease, delay, or prevent remodeling of a heart ventricle). These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. Furthermore, modifications may be made that have one or more of the following effects: increasing ability to increase left ventricular ejection fraction, decrease infarct size, decrease, delay, or prevent remodeling of a heart ventricle, or facilitating purification, delivery, or cell processing. Proteins or biologically active fragments thereof can be made recombinantly, synthetically, or in tissue culture.

[0057] The term “programmed death ligand- 1 ” or “PD-L1” as used herein encompasses all forms of PD-L1 , including isoforms a, b, and c, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (e.g., ability to increase left ventricular ejection fraction, decrease infarct size, decrease, delay, or prevent remodeling of a heart ventricle).

[0058] A PD-L1 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism but may be synthetically or recombinantly produced. A number of PD-L1 nucleic acid and protein sequences are known. Representative sequences of a human PD-L1 protein (SEQ ID NO:1) and a human PD-L1 mRNA (SEQ ID NO:2) are presented in the Sequence Listing. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NP_001254635, NP_001300958, NP 054862, XP 054218785, NP_068693, NP_001278901 , XP_038509460, NP.001178883, NP_001156884, XP_059745144, XP_059745143, NP_001020392, NM_001267706, NR_052005, NM 001314029, NM_014143, XM_054362810, NM_021893, NM_021396, NM_001291972, XM 038653532, NM_001163412, XM_059889161 , XM_059889160, NM_001191954, and NM 001025221 ; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the variant PD-L1 protein retains PD-L1 biological activity (i.e. , the ability to increase left ventricular ejection fraction, decrease infarct size, decrease, delay, or prevent remodeling of a heart ventricle), can be used to produce a PD-L1 protein or recombinant polynucleotide comprising a coding sequence encoding a PD-L1 protein for use in the methods described herein.

[0059] The terms “variant,” “analog” and “mutein” refer to biologically active derivatives of the reference molecule that retain desired activity, such as PD-L1 biological activity (e.g., the ability to increase left ventricular ejection fraction, decrease infarct size, decrease, delay, or prevent remodeling of a heart ventricle), as described herein. In general, the terms “variant” and “analog” refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are “substantially homologous” to the reference molecule as defined below. In general, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned. Often, the analogs will include the same number of amino acids but will include substitutions, as explained herein. The term “mutein” further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and/or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like. The term also includes molecules comprising one or more N-substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831 ,005; 5,877,278; and 5,977,301 ; Nguyen et al., Chem Biol. (2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions of peptoids). Preferably, the analog or mutein has at least the same biological activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.

[0060] As explained above, analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1 ) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5- 25, so long as the desired function of the molecule remains intact. One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.

[0061] By “derivative” is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0062] By "fragment" is intended a molecule consisting of only a part of the intact full-length sequence and structure. The fragment can include a C-terminal deletion an N-terminal deletion, and/or an internal deletion of the polypeptide. Active fragments of a particular protein or polypeptide will generally include at least about 5-14 contiguous amino acid residues of the full length molecule, but may include at least about 15-25 contiguous amino acid residues of the full length molecule, and can include at least about 20-50 or more contiguous amino acid residues of the full length molecule, or any integer between 5 amino acids and the full length sequence, provided that the fragment in question retains biological activity (e.g., PD-L1 biological activity, ability to increase left ventricular ejection fraction, decrease infarct size, decrease, delay, or prevent remodeling of a heart ventricle).

[0063] As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length protein or protein fragment. A reference sequence can comprise, for example, a sequence identifiable in a database such as GenBank and UniProt and others identifiable to those skilled in the art.

[0064] Algorithms and programs for comparing primary biological sequence information between any two sequences are identifiable by a skilled person. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (Myers and Miller 1988), the local homology algorithm of Smith et al. (Smith and Waterman 1981 ); the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch 1970)); the search-for-similarity-method of Pearson and Lipman (Pearson and Lipman 1988).); the algorithm of Karlin and Altschul (Karlin and Altschul 1990)., modified as in Karlin and Altschul (Karlin and Altschul 1993)). Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA (Pearson and Lipman 1988).);, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters and allowing a user to identify database sequences that resemble the reference sequence (query sequence) above a certain threshold of confidence.

[0065] Algorithms and programs for comparing primary biological sequence information between any two sequences typically provide an output comprising percent identity between the sequence retrieved and the reference sequence. [0066] A person skilled in the art would understand that identity between sequences is typically measured by a process that comprises the steps of aligning the two polypeptide or polynucleotide sequences to form aligned sequences, then detecting the number of matched characters, i.e. characters similar or identical between the two aligned sequences, and calculating the total number of matched characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity result is expressed as a percentage of identity.

[0067] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0068] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human (or non-human animal in the case of veterinary use). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0069] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0070] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0071] By "subject" is meant any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.

Treating an Ischemic Condition with PD-L1

[0072] The present disclosure provides compositions and methods for treating an ischemic condition with PD-L1 . Ischemic conditions include any disease or condition causing reduced blood flow through a tissue or organ. In particular, ischemic conditions that affect the heart include, without limitation, myocardial infarction, myocardial ischemia, and ischemia reperfusion injury, which may lead to deterioration of cardiac function, including decreased left ventricular ejection fraction, remodeling of heart ventricles, increased levels of granulocytic and monocytic populations of myeloid-derived suppressor cells in the peripheral blood, and increased morbidity and mortality.

[0073] In some embodiments, PD-L1 is used to prevent, delay, or decrease remodeling of a heart ventricle caused by an ischemic condition. By "ventricle remodeling” is meant any adverse structural changes in a heart ventricle associated with an ischemic condition such as myocardial infarction, myocardial ischemia, or ischemia reperfusion injury. Ventricle remodeling may include, without limitation, pathological changes in myocardial structure, ventricle shape, and end-diastolic and end- systolic volumes. In particular, the left ventricle may undergo the most pronounced morphological changes, including a transition from the normal ellipsoid shape to a more spherical shape with increased end-diastolic and end-systolic volumes. Ventricle remodeling may be accompanied by myocyte hypertrophy, loss of cardiomyocytes and contractile function, death of myocytes leading to an immune response including an influx of inflammatory cells, destruction of the extracellular collagen matrix, ventricular dilation, vascular stiffness, wall thinning, microvascular obstruction, intramyocardial hemorrhage, myocardial strain, and myocardial fibrosis. Ventricle remodeling can be diagnosed by detecting changes in ventricular cavity diameter, mass, and geometry. See, e.g., Calvier et al. (2023) J. Clin, Med. 12(1):334; Burchfield et al. (2013) Circulation 128(4) :388-400, Leanca et al. (2022) Life (Basel) 12(8) :11 11 , Cohn et al. (2000) J. Am. Coll. Cardiol. 35(3):569-582; herein incorporated by reference.

[0074] A PD-L1 protein or biologically active variants or fragments thereof can be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g., native, fusions, labeled, lipidated, amidated, acetylated, PEGylated, etc.). The PD-L1 protein may include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing proteins are well understood in the art. Proteins are preferably prepared in substantially pure form (i.e. substantially free from other host cell or non-host cell proteins).

[0075] PD-L1 nucleic acid and protein sequences may be derived from any source. A number of PD-L1 nucleic acid and protein sequences are known. Representative sequences of a human PD- L1 protein (SEQ ID NO:1) and a human PD-L1 mRNA (SEQ ID NO:2) are presented in the Sequence Listing. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NP 001254635, NP_001300958, NP_054862, XP_054218785, NP_068693, NP_001278901 , XP_038509460, NP_001178883, NP_001156884, XP_059745144, XP_059745143, NP_001020392,

NM 001267706, NR_052005, NM_001314029, NM_014143, XM_054362810, NM_021893, NM 021396, NM 001291972, XM_038653532, NM_001163412, XM_059889161 , XM_059889160, and NM 001191954, and NM 001025221 ; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the variant PD-L1 protein retains PD-L1 biological activity (i.e., the ability to increase left ventricular ejection fraction, decrease infarct size, decrease, delay, or prevent remodeling of a heart ventricle), can be used to produce a PD-L1 protein or recombinant polynucleotide comprising a coding sequence encoding a PD-L1 protein for use in the methods described herein.

[0076] In certain embodiments, the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or a variant comprising a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, or a biologically active fragment thereof, wherein the variant or fragment retains the ability to increase left ventricular ejection fraction, decrease infarct size, and/or decrease, delay, or prevent remodeling of a heart ventricle.

[0077] A biologically active fragment of PD-L1 can include a C-terminal deletion an N-terminal deletion, and/or an internal deletion of the polypeptide. In some embodiments, a biologically active fragment of PD-L1 comprises at least about 15-25 contiguous amino acid residues of the full length molecule, at least about 25-50 contiguous amino acid residues of the full length molecule, at least about 50-100 contiguous amino acid residues of the full length molecule, or at least about 100-200 or more contiguous amino acid residues of the full length molecule, or any integer between 15 amino acids and the full length sequence, provided that the fragment in question retains biological activity (e.g., PD-L1 biological activity, ability to increase left ventricular ejection fraction, decrease infarct size, and/or decrease, delay, or prevent remodeling of a heart ventricle).

[0078] In one embodiment, a PD-L1 protein is generated using recombinant techniques. One of skill in the art can readily determine nucleotide sequences that encode PD-L1 using standard methodology and the teachings herein. Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual (3^rd edition, Cold Spring Harbor Laboratories, New York) for a description of techniques used to obtain and isolate DNA.

[0079] The sequences encoding PD-L1 can also be produced synthetically, for example, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) A/a/we 292:756: Nambair et al. (1984) Science 223:1299: Jay et al. (1984) J. Biol. Chem. 259:6311 ; Stemmer et al. (1995) Gene 164:49-53.

[0080] Recombinant techniques are readily used to clone sequences encoding PD-L1 that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes. Alternatively, the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0081 ] Once coding sequences have been isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression. (See, also, Examples). As will be apparent from the teachings herein, a wide variety of vectors encoding modified polypeptides can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding polypeptides having deletions or mutations therein.

[0082] Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage A (E coll), pBR322 (E coll , pACYC177 (E coll), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRI (gram-negative bacteria), pME290 (non-E coll gram-negative bacteria), pHV14 (E co// and Bacillus subtilis), pBD9 (Bacillus), plJ61 (Streptomyces), pUC6 (Streptomyces), Ylp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, generally, DNA Cloning-. Vols. I & II, supra-, Sambrook et al., supra-, Perbal et al. A Practical Guide to Molecular Cloning (Wiley-Liss; 2^nd edition, 1988).

[0083] Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit).

[0084] Plant expression systems can also be used to produce PD-L1 . Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems, see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221 ; and Hackland et al., Arch. Virol. (1994) 139:1 -22.

[0085] Viral systems, such as a vaccinia-based infection/transfection system, as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-1113, will also find use with the present invention. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).

[0086] The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. With the present invention, both the naturally occurring signal peptides or heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431 ,739; 4,425,437; 4,338,397. Such sequences include, but are not limited to, the TPA leader, as well as the honeybee mellitin signal sequence.

[0087] Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0088] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

[0089] In some cases, it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

[0090] The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, human aortic smooth muscle cells (HASMC), human aortic endothelial cells (HAEC), Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, as well as others. Similarly, bacterial hosts such as E coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0091] Depending on the expression system and host selected, the PD-L1 protein is produced by growing host cells transformed by an expression vector described above under conditions whereby the protein is expressed. The selection of the appropriate growth conditions is within the skill of the art.

[0092] In one embodiment, the transformed cells secrete the PD-L1 protein product into the surrounding media. Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TPA) leader sequence, an interferon (yor a) signal sequence or other signal peptide sequences from known secretory proteins. The secreted PD-L1 protein product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0093] Alternatively, the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant peptides or polypeptides substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001 ).

[0094] For example, methods of disrupting cells for use with the present invention include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freezethaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. The particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pre-treatment used.

[0095] Following disruption of the cells, cellular debris is removed, generally by centrifugation, and the intracellularly produced peptides or polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, sizeexclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.

[0096] For example, one method for obtaining the intracellular polypeptides involves affinity purification, such as by immunoaffinity chromatography using antibodies (e.g., previously generated antibodies), or by lectin affinity chromatography. Particularly preferred lectin resins are those that recognize mannose moieties such as but not limited to resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LOA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (AUA). The choice of a suitable affinity resin is within the skill in the art. After affinity purification, the peptides or polypeptides can be further purified using conventional techniques well known in the art, such as by any of the techniques described above.

[0097] The PD-L1 protein can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. See, e.g., Fmoc Solid Phase Peptide Synthesis: A Practical Approach (\N. C. Chan and Peter D. White eds., Oxford University Press, 1^st edition, 2000) ; N. Leo Benoiton, Chemistry of Peptide Synthesis (CRC Press; 1 ^st edition, 2005); Peptide Synthesis and Applications (Methods in Molecular Biology, John Howl ed., Humana Press, 1^st ed., 2005); and Pharmaceutical Formulation Development of Peptides and Proteins (The Taylor & Francis Series in Pharmaceutical Sciences, Lars Hovgaard, Sven Frokjaer, and Marco van de Weert eds., CRC Press; 1 ^st edition, 1999); herein incorporated by reference.

[0098] In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) are removed sequentially or concurrently, to render the final peptide or polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, IL 1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1 , for classical solution synthesis. These methods are typically used for relatively small polypeptides, i.e., up to about 50- 100 amino acids in length, but are also applicable to larger polypeptides.

[0099] Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o- bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl and the like.

[00100] Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene- benzhydrylaminopolystyrene copolymers.

[00101 ] The PD-L1 protein can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131 -5135; U.S. Patent No. 4,631 ,211 .

Pharmaceutical Compositions

[00102] A PD-L1 protein can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[00103] A composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[00104] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the PD-L1 , or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[00105] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[00106] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[00107] The amount of PD-L1 (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint. [00108] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1 % to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[00109] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional preferred compositions include those for oral, ocular, or localized delivery.

[00110] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use. Preferably, the compositions comprising PD-L1 are in unit dosage form, meaning an amount of a conjugate or composition appropriate for a single dose, in a premeasured or pre-packaged form.

[00111 ] The compositions herein may optionally include one or more additional agents, such as other drugs for treating an ischemic condition, or other medications used to treat a subject for a condition or disease. Compounded preparations may include PD-L1 and one or more drugs for treating myocardial infarction, myocardial ischemia, or ischemia reperfusion injury including, but not limited to, statins such as atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin; angiotensin-converting-enzyme (ACE)-inhibitors such as captopril, enalapril, lisinopril, benazepril, fosinopril, quinapril, ramipril, perindopril, moexipril, and trandolapril; beta blockers such as metoprolol, carvedilol, propranolol, bucindolol, carteolol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, nebivolol, esmolol, butaxamine, ICI-118,551 , and SR 59230A; angiotensin II receptor antagonists such as losartan, irbesartan, olmesartan, candesartan, valsartan, and fimasartan; aldosterone antagonists such as spironolactone, eplerenone, canrenone, finerenone, and mexrenone; an antiplatelet agent such as aspirin, clopidogrel, and ticagrelor; a calcineurin inhibitor such as cyclosporin, tacrolimus, sirolimus, everolimus, and zotarolimus; an anticoagulant such as heparin, dabigatran, rivaroxaban, apixaban, betrixaban, edoxaban, coumarin, warfarin, acenocoumarol, phenprocoumon, atromentin, and phenindione; or other medications. Alternatively, such agents can be contained in a separate composition from the composition comprising PD-L1 and co-administered concurrently, before, or after the composition comprising the PD-L1 .

Administration

[00112] At least one therapeutically effective cycle of treatment with PD-L1 will be administered to a subject for treatment of an ischemic condition or heart ventricle remodeling. Ischemic conditions include, but are not limited to, myocardial infarction, myocardial ischemia, and ischemia reperfusion injury. By “therapeutically effective dose or amount” of PD-L1 , is intended an amount that, when administered, as described herein, brings about a positive therapeutic response, such as improved recovery from an ischemic condition. Improved recovery may include increasing the left ventricular ejection fraction, decreasing infarct size, and/or decreasing, delaying, or preventing remodeling of a heart ventricle. Additionally, a therapeutically effective dose or amount may increase survival or reduce morbidity of a subject.

[00113] In certain embodiments, multiple therapeutically effective doses of compositions comprising PD-L1 and/or one or more other therapeutic agents, such as other drugs for treating an ischemic condition, or other medications will be administered. The compositions of the present invention are typically, although not necessarily, administered orally, via injection (subcutaneously, intravenously, intramuscularly, intramyocardially, intraperitoneally, or intra-arterially), by infusion, or locally. Additional modes of administration are also contemplated, such as, by intracardiac injection or central venous catheter, or rectal, pulmonary, or transdermal administration, and so forth. In some embodiments, the compositions are administered systemically.

[00114] The preparations are also suitable for local treatment. In a particular embodiment, a composition is used for localized delivery of PD-L1 for the treatment of an ischemic condition. For example, compositions may be administered locally in the vicinity of an ischemic injury. The particular preparation and appropriate method of administration are chosen to target the PD-L1 to the site where an ischemic condition is in need of treatment. [00115] The pharmaceutical preparation can be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, or the like. The pharmaceutical compositions comprising PD-L1 and/or other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.

[00116] In another embodiment, the pharmaceutical compositions comprising PD-L1 and/or other agents are administered prophylactically, e.g., to prevent or reduce ischemic injury and/or ventricle remodeling. Such prophylactic uses will be of particular value for subjects at risk of myocardial infarction, myocardial ischemia, ischemia reperfusion injury, or ventricle remodeling. In another embodiment, the pharmaceutical compositions comprising PD-L1 and/or other agents are administered therapeutically to subjects with myocardial infarction, myocardial ischemia, ischemia reperfusion injury, or ventricle remodeling.

[00117] In another embodiment, the pharmaceutical compositions comprising PD-L1 and/or other agents are in a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.

[00118] The disclosure also provides a method for administering a conjugate comprising PD-L1 to a patient suffering from an ischemic condition that is responsive to treatment with PD-L1 contained in the conjugate or composition. The method comprises administering, via any of the herein described modes, a therapeutically effective amount of the conjugate or drug delivery system, preferably provided as part of a pharmaceutical composition. The method of administering may be used to treat any condition that is responsive to treatment with PD-L1 .

[00119] Those of ordinary skill in the art will appreciate which conditions a specific PD-L1 protein can effectively treat. The actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the particular disorder associated with protein misfolding being treated, the severity of the condition being treated, the judgment of the health care professional, and the particular PD-L1 protein or conjugate being administered. Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.

[00120] In certain embodiments, multiple therapeutically effective doses of PD-L1 will be administered according to a daily dosing regimen or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, every other week, and so forth. For example, in some embodiments, a composition comprising PD-L1 will be administered once-weekly, twice-weekly or thrice-weekly for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present disclosure, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods described herein, a subject can receive intermittent therapy (i.e., once-weekly, twice-weekly or thrice- weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below. The amount administered will depend on the potency of the specific PD-L1 protein, the particular ischemic condition that is treated, the magnitude of the effect desired, and the route of administration.

[00121] A purified PD-L1 protein (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents such as, but not limited to, statins such as atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin; angiotensin-converting-enzyme (ACE)- inhibitors such as captopril, enalapril, lisinopril, benazepril, fosinopril, quinapril, ramipril, perindopril, moexipril, and trandolapril; beta blockers such as metoprolol, carvedilol, propranolol, bucindolol, carteolol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, nebivolol, esmolol, butaxamine, ICI-118,551 , and SR 59230A; angiotensin II receptor antagonists such as losartan, irbesartan, olmesartan, candesartan, valsartan, and fimasartan; aldosterone antagonists such as spironolactone, eplerenone, canrenone, finerenone, and mexrenone; an antiplatelet agent such as aspirin, clopidogrel, and ticagrelor; a calcineurin inhibitor such as cyclosporin, tacrolimus, sirolimus, everolimus, and zotarolimus; an anticoagulant such as heparin, dabigatran, rivaroxaban, apixaban, betrixaban, edoxaban, coumarin, warfarin, acenocoumarol, phenprocoumon, atromentin, and phenindione; or other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring dosing no more than once a day.

[00122] PD-L1 can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the PD-L1 can be provided in the same or in a different composition. Thus, PD-L1 and/or other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising PD-L1 , and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating an ischemic condition such as myocardial infarction, myocardial ischemia, or ischemia reperfusion injury, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, PD-L1 and one or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[00123] The methods described herein can be used for treating a human subject for an ischemic condition or ventricle remodeling. The methods described herein will also find use in veterinary applications for treatment of ischemic conditions or ventricle remodeling, for example, in domestic animals including, without limitation, pets, such as dogs and cats, and farm animals, such as sheep, goats, pigs, horses, and cattle.

Kits

[00124] Also provided are kits for treating a patient for an ischemic with PD-L1 or ventricle remodeling, as described herein. The PD-L1 and optionally other therapeutic agents may be contained in separate compositions or in the same composition.

[00125] In certain embodiments, the kit comprises a PD-L1 protein comprising or consisting of the amino acid sequence of SEQ ID NO:1 , or a variant comprising a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto, or a biologically active fragment thereof, wherein the variant or fragment retains the ability to increase left ventricular ejection fraction, decrease infarct size, and/or decrease, delay, or prevent remodeling of a heart ventricle.

[00126] A subject kit may include at least one container comprising a solution comprising a unit dose of the PD-L1 , and a pharmaceutically acceptable excipient; and instructions to administer a unit dose according to a desired regimen or exemplary regimen dependent upon the particular ischemic condition being treated, age, weight, and the like.

[00127] Kits may include unit doses of the formulations comprising PD-L1 suitable for use in the treatment methods described herein, e.g., in tablets or injectable dose(s). In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use of PD-L1 and attendant benefits of the treatment for an ischemic condition or ventricle remodeling. The kit can include, for example, a dosing regimen for the PD-L1 .

[00128] Formulations suitable for intravenous, intramyocardial, or intraperitoneal administration are of particular interest, and in such embodiments the kit may further include a syringe or other device to accomplish such administration, which syringe or device may be pre-filled with the PD-L1. The instructions can be printed on a label affixed to the container or can be a package insert that accompanies the container.

[00129] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

Utility

[00130] The compositions and methods of the present disclosure find use in a variety of different applications, including the treatment of ischemic conditions, including any disease or condition causing reduced blood flow through a tissue or organ, which may result in inadequate oxygenation and/or supply of nutrients to the tissue or organ and cell damage or cell death. Ischemic conditions include, but are not limited to, myocardial infarction, myocardial ischemia, and ischemia reperfusion injury. T reatment with PD-L1 may increase the left ventricular ejection fraction, decrease infarct size, or decrease, delay, or prevent remodeling of a heart ventricle. Additionally, a therapeutically effective dose or amount may increase survival or reduce morbidity.

Examples of Non-Limiting Aspects of the Disclosure

[00131] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1 -37 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

1 . A method of treating an ischemic condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of programmed death ligand-1 (PD-L1) to the subject.

2. The method of aspect 1 , wherein the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

3. The method of aspect 1 or 2, wherein the PD-L1 is administered intramyocardially, intravenously, or intraperitoneally.

4. The method of aspect 1 or 2, wherein the PD-L1 is administered locally to a site of an ischemic injury.

5. The method of aspect 1 or 2, wherein the PD-L1 is administered systemically.

6. The method of any one of aspects 1 -5, wherein treatment increases a left ventricular ejection fraction compared to the left ventricular ejection fraction before treatment.

7. The method of any one of aspects 1 -6, wherein treatment decreases infarct size compared to the infarct size without treatment. 8. The method of any one of aspects 1 -7, wherein multiple cycles of treatment are administered to the subject.

9. The method of aspect 8, wherein the PD-L1 is administered according to a daily dosing regimen or intermittently.

10. The method of any one of aspects 1 -9, further comprising administering a statin, an angiotensin-converting-enzyme inhibitor, a beta blocker, an angiotensin II receptor antagonist, an antiplatelet agent, an anticoagulant, a calcineurin inhibitor, or a combination thereof to the subject.

11 . The method of any one of aspects 1 -10, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

12. A composition comprising programmed death ligand-1 (PD-L1) for use in a method of treating an ischemic condition.

13. The composition of aspect 12, wherein the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

14. The composition of aspect 12 or 13, further comprising a pharmaceutically acceptable excipient.

15. The composition of any one of aspects 12-14, further comprising a statin, an angiotensin-converting-enzyme inhibitor, a beta blocker, an angiotensin II receptor antagonist, an antiplatelet agent, an anticoagulant, a calcineurin inhibitor, or a combination thereof.

16. The composition of any one of aspects 12-15, wherein the composition is formulated for administration intramyocardially, intravenously, or intraperitoneally.

17. The composition of any one of aspects 12-15, wherein the composition is formulated for administration locally to a site of an ischemic injury. 18. The composition of any one of aspects 12-15, wherein the PD-L1 is formulated for systemic administration.

19. The composition of any one of aspects 12-18, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

20. Use of programmed death ligand-1 (PD-L1 ) in the manufacture of a medicament or pharmaceutical composition for treating an ischemic condition.

21 . The use of aspect 20, wherein the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

22. The use of aspect 20 or 21 , wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NOU , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NOU .

23. A method of preventing, delaying, or decreasing remodeling of a heart ventricle in a subject, the method comprising administering a therapeutically effective amount of programmed death ligand-1 (PD-L1 ) to the subject.

24. The method of aspect 23, wherein the PD-L1 is administered intramyocardially, intravenously, or intraperitoneally.

25. The method of aspect 23, wherein the PD-L1 is administered systemically.

26. The method of any one of aspects 23-25, wherein the PD-L1 is administered prophylactically.

27. The method of any one of aspects 23-26, wherein the subject has a myocardial infarction, myocardial ischemia, or an ischemia reperfusion injury.

28. The method of any one of aspects 23-27, wherein the PD-L1 is administered multiple times to the subject. 29. The method of aspect 28, wherein the PD-L1 is administered according to a daily dosing regimen or intermittently.

30. The method of any one of aspects 23-29, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

31 . A composition comprising programmed death ligand-1 (PD-L1) for use in a method of treating heart ventricle remodeling.

32. The composition of aspect 31 , further comprising a pharmaceutically acceptable excipient.

33. The composition of aspect 31 or 32, wherein the composition is formulated for administration intramyocardially, intravenously, or intraperitoneally.

34. The composition of aspect 31 or 32, wherein the composition is formulated for administration systemically or locally to the heart ventricle.

35. The composition of any one of aspects 31-34, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

36. Use of programmed death ligand-1 (PD-L1 ) in the manufacture of a medicament or pharmaceutical composition for treating heart ventricle remodeling.

37. The use of aspect 36, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NOU , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NOU .

[00132] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. EXPERIMENTAL

[00133] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[00134] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[00135] The present invention has been described in terms of particular embodiments found or proposed by the present inventors to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.

Example 1

Programmed Death Ligand-1 was Effective in Preserving Heart Function in a Myocardial Ischemia Model

[00136] In patients who experience myocardial infarct, immediate ischemia and cell death occurs which result in decrease in cardiac function and therefore high morbidity and mortality. Currently, there is no minimally invasively deliverable therapeutics that could be administered to these patients to stop or even reverse the adverse effect of myocardial ischemia and the associated immune response that result in further deterioration of cardiac function and reverse remodeling of a heart ventricle.

[00137] Programmed death-1 (PD-1 ) is an inhibitory immune checkpoint protein expressed on activated immune cells. PD-1 binding its ligands, programmed death ligand-1 (PD-L1 ) or programmed death ligand-2 (PD-L2), limits collateral damage in the setting of chronic infection and protects against autoimmunity. [00138] We discovered that programmed death ligand-1 (PD-L1 ), when delivered immediately intramyocardially following Ml via LAD ligation in a rodent model, left ventricular ejection fraction was significantly improved compared to control animals who did not receive PD-L1 treatment. PD-L1 when delivered systemically was found to have similar beneficial impact on heart function. We believe that when translating this into clinical application, PD-L1 could be given intravenously as soon as a Ml occurs to preserve as much cardiac function as possible to significantly enhance patient outcomes. We have found that by delivering 30 |ig of PD-L1 intramyocardially in mice, left ventricular ejection fraction (LVEF) increased from 28% without treatment to 42% with PD-L1 treatment (1.5- fold increase) 14 days after the event. Increasing the PD-L1 dose to 50 |ig intramyocardial delivery was found to have similar outcomes with LVEF increasing from 21% without treatment to 41% with PD-L1 treatment (2-fold increase) 14 days after the event. We also studied the effect of 50 j g PD- L1 for intraperitoneal delivery as systemic delivery and found that LVEF similarly increased from 38% without treatment to 51% with PD-L1 treatment (1.3 fold increase) 14 days after the event. We also found that PD-L1 treatment, regardless of the dose and delivery route, significantly reduced LV infarct size compared to controls without treatment.

[00139] Current methodologies for salvaging the consequences of Ml focus primarily on revascularization through PCI or CABG, for example. There is, so far, no effective therapeutics that can be minimally invasively delivered to preserve cardiac function following Ml. Our discovery regarding the PD-L’s effect on Ml models is significant, as this therapeutic can be easily delivered to patients intravenously, it does not preclude patients from receiving the traditional revascularization treatment. The compounded effect of blood flow restoration and preventing secondary immunologic injuries can potentially further improve patient outcomes.

Example 2

Programmed Death-Ligand 1 Attenuates Myocardial Injury following Myocardial Infarction

Introduction

[00140] Ml represents a major global cause of morbidity and mortality. Immune therapy to mitigate the secondary injury to myocardial tissues following Ml is lacking. Programmed death-1 (PD-1) is an inhibitory protein expressed by immune cells during activation. Programmed death-ligand 1 (PDL1 ) binds to PD-1 and limits collateral damage in diseases with chronic inflammatory response. The objective of this study was to evaluate the impact of PDL1 on myocardial injury following Ml. Methods

[00141] 56 C57BL/6 male and female mice underwent LAD ligation. Mice were randomized to receive

50 pg of PDL1 (14 intramyocardial, 14 intraperitoneal) or PBS (12 intramyocardial, 16 intraperitoneal) immediately after LAD ligation. Echocardiography was obtained at baseline and 2 weeks after Ml. Mice hearts were then explanted and sectioned for Masson’s trichrome staining. An additional 25 mice after Ml without treatment were terminated on postoperative day 1 , 3, 5, 7, and 14. Peripheral blood and myocardium were stained for CD45, CD11 b, Ly6c, and PD-1 for flow cytometry.

Results

[00142] Myocardial infarct size reduction was observed after PDL1 treatment (FIGS. 2A-2C). Compared with controls, PDL1 delivered intramyocardially significantly improved ejection fraction from 29.8 ± 12.4 % to 41 .4 ± 14.0 % (p = 0.04), and when delivered intraperitoneally, from 32.5 ± 9.4 % to 47.7 ± 8.0 % (p < 0.0001 , FIG. 2D). In the myocardium, the percentage of monocytes that were PD-1 + surged from 0.2 ± 0.2 % on day 1 to 83.8 ± 17.7 % on day 3 and remained elevated 14 days after Ml at 31 .5 ± 23.6 % (FIG. 2E).

Conclusions

[00143] PDL1 can attenuate myocardial injury and partially preserve cardiac function following Ml in a rodent model. PD-1 + monocytes may be one of the key mediators of secondary myocardial injury after Ml.

Claims

WHAT IS CLAIMED IS:

1 . A method of treating an ischemic condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of programmed death ligand-1 (PD-L1) to the subject.

2. The method of claim 1 , wherein the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

3. The method of claim 1 or 2, wherein the PD-L1 is administered intramyocardially, intravenously, or intraperitoneally.

4. The method of claim 1 or 2, wherein the PD-L1 is administered locally to a site of an ischemic injury.

5. The method of claim 1 or 2, wherein the PD-L1 is administered systemically.

6. The method of any one of claims 1-5, wherein treatment increases a left ventricular ejection fraction compared to the left ventricular ejection fraction before treatment.

7. The method of any one of claims 1-6, wherein treatment decreases infarct size compared to the infarct size without treatment.

8. The method of any one of claims 1-7, wherein multiple cycles of treatment are administered to the subject.

9. The method of claim 8, wherein the PD-L1 is administered according to a daily dosing regimen or intermittently.

10. The method of any one of claims 1-9, further comprising administering a statin, an angiotensin-converting-enzyme inhibitor, a beta blocker, an angiotensin II receptor antagonist, an antiplatelet agent, an anticoagulant, a calcineurin inhibitor, or a combination thereof to the subject.

11 . The method of any one of claims 1-10, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

12. A composition comprising programmed death ligand-1 (PD-L1) for use in a method of treating an ischemic condition.

13. The composition of claim 12, wherein the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

14. The composition of claim 12 or 13, further comprising a pharmaceutically acceptable excipient.

15. The composition of any one of claims 12-14, further comprising a statin, an angiotensin-converting-enzyme inhibitor, a beta blocker, an angiotensin II receptor antagonist, an antiplatelet agent, an anticoagulant, a calcineurin inhibitor, or a combination thereof.

16. The composition of any one of claims 12-15, wherein the composition is formulated for administration intramyocardially, intravenously, or intraperitoneally.

17. The composition of any one of claims 12-15, wherein the composition is formulated for administration locally to a site of an ischemic injury.

18. The composition of any one of claims 12-15, wherein the PD-L1 is formulated for systemic administration.

19. The composition of any one of claims 12-18, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

20. Use of programmed death ligand-1 (PD-L1 ) in the manufacture of a medicament or pharmaceutical composition for treating an ischemic condition.

21 . The use of claim 20, wherein the ischemic condition is myocardial infarction, myocardial ischemia, or ischemia reperfusion injury.

22. The use of claim 20 or 21 , wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

23. A method of preventing, delaying, or decreasing remodeling of a heart ventricle in a subject, the method comprising administering a therapeutically effective amount of programmed death ligand-1 (PD-L1 ) to the subject.

24. The method of claim 23, wherein the PD-L1 is administered intramyocardially, intravenously, or intraperitoneally.

25. The method of claim 23, wherein the PD-L1 is administered systemically.

26. The method of any one of claims 23-25, wherein the PD-L1 is administered prophylactically.

27. The method of any one of claims 23-26, wherein the subject has a myocardial infarction, myocardial ischemia, or an ischemia reperfusion injury.

28. The method of any one of claims 23-27, wherein the PD-L1 is administered multiple times to the subject.

29. The method of claim 28, wherein the PD-L1 is administered according to a daily dosing regimen or intermittently.

30. The method of any one of claims 23-29, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

31 . A composition comprising programmed death ligand-1 (PD-L1) for use in a method of treating heart ventricle remodeling.

32. The composition of claim 31 , further comprising a pharmaceutically acceptable excipient.

33. The composition of claim 31 or 32, wherein the composition is formulated for administration intramyocardially, intravenously, or intraperitoneally.

34. The composition of claim 31 or 32, wherein the composition is formulated for administration systemically or locally to the heart ventricle.

35. The composition of any one of claims 31-34, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NO:1 , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 .

36. Use of programmed death ligand-1 (PD-L1 ) in the manufacture of a medicament or pharmaceutical composition for treating heart ventricle remodeling.

37. The use of claim 36, wherein the PD-L1 comprises or consists of the amino acid sequence of SEQ ID NOU , or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NOU .