Immunocytokines for heart failure prevention

Abstract

In various aspects and embodiments, the disclosure provides a chimeric IL-4 polypeptide comprising interleukin-4 (IL-4), a myocardial infarct targeting agent, and a peptide linker between IL-4 and the targeting agent. In a further embodiment, the chimeric IL-4 polypeptide further comprises an IL-4 mask and a cardiovascular inflammatory protease-sensitive linker between IL-4 and the IL-4 mask, wherein the IL-4 activity is unmasked following cleavage of the cardiovascular inflammatory protease-sensitive linker and release of the IL-4 mask. Also described are methods for treating myocardial infarction, heart failure, ischemia / reperfusion injury with the chimeric IL-4 polypeptide or cells expressing the chimeric IL-4 polypeptide.

Description

[0001] Attorney Docket No. 046483-7492WO 1(04069)

[0002] IMMUNOCYTOKINES FOR HEART FAILURE PREVENTION CROSS-REFERENCE TO RELATED APPLICATION

[0003] The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S.

[0004] Provisional Patent Application No. 63 / 719,486 filed on November 12, 2024, which is herein incorporated by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] The XML file named “046483_7492WOl_SequenceListing.xml” created on November 11, 2025, comprising 67,983 bytes, is hereby incorporated by reference in its entirety.

[0007] BACKGROUND

[0008] Inflammation after an acute myocardial infarction (MI) strongly predicts heart failure (HF) severity. A cascade of events after an MI contributes to HF via several mechanisms. The immediate consequence of an MI is massive cardiomyocyte death from the sudden lack of blood supply, which acutely impairs cardiac output. The drop in cardiac output reflexively activates the sympathetic nervous system and renin-angiotensin-aldosterone system. These pathways seek to preserve blood perfusion to other organs, resulting in compensatory enhancement of heart rate and contractility. However, chronic activation of these pathways worsens cardiac contractility by altering proper excitation-contraction coupling and increasing ventricular pressure, wall stress, and dilation. These adaptations progressively strain the remaining viable myocardium, eventually culminating in its failure.

[0009] Medications like beta-blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and mineralocorticoid receptor antagonists are aimed at stalling these recognized mechanisms of HF after an MI. Despite these highly effective therapies, the risk remains. HF still occurs in 10-40% of patients and leads to 695,000 deaths in the U.S. per year, in part because residual myocardial inflammation cannot yet be managed.

[0010] Although many mechanisms clearly catalyze HF, it remains less appreciated is how immune cells, in particular macrophages, influence these “classic concepts” of HF pathophysiology. In response to an MI, macrophages infiltrate the injured heart to facilitate Attorney Docket No. 046483-7492WO 1(04069)

[0011] tissue repair. Emerging data have identified a lingering pro-inflammatory macrophage subset that promotes defective myocardial remodeling, thereby contributing to HF.

[0012] Cytokines that regulate macrophage phenotype have the potential to stave off HF by dampening inflammation and promoting myocardial repair. Interleukin-4 (IL-4), for example, is a potent endogenous cytokine that converts macrophages from an inflammatory to a reparative phenotype. Unfortunately, the clinical potential of IL-4 therapy is hampered by its systemic side effects on other immune cells.

[0013] Therefore, there is a pressing need for developing chimeric IL-4 compositions engineered with selective activity in a myocardial infarct to both safely and effectively treat MI and prevent HF.

[0014] SUMMARY

[0015] In one aspect, a chimeric IL-4 polypeptide comprises interleukin-4 (IL-4), a myocardial infarct targeting agent, and a peptide linker between IL-4 and the targeting agent.

[0016] In an embodiment, the myocardial infarct targeting agent is linked to the N-terminus of IL-4. In another embodiment, the myocardial infarct targeting agent is linked to the C-terminus of IL-4. In an embodiment, the myocardial infarct targeting agent specifically binds an extracellular matrix (ECM) protein expressed in a myocardial infarct. In an embodiment, the ECM protein is fibronectin or a collagen, or periostin, or a hyaluronic acid, or an elastin. In an embodiment, the myocardial infarct targeting agent specifically binds Fn-EIIIB, Fn-EIIIA, or Type I collagen. In an embodiment, the myocardial infarct targeting agent specifically binds Fn-EIIIB. In another embodiment, the myocardial infarct targeting agent specifically binds Fn-EIIIA. In another embodiment, the myocardial infarct targeting agent specifically binds Type I collagen.

[0017] In some embodiments, the myocardial infarct targeting agent is an antibody or antigen binding fragment thereof. In some embodiments, the myocardial infarct targeting agent is a nanobody. In one embodiment, the nanobody comprises the amino acid sequence of SEQ ID NO: 1.

[0018] In an embodiment, the myocardial infarct targeting agent is a protein that specifically binds an ECM protein. In one embodiment, the ECM protein is a collagen. In certain embodiments, the protein is lumican. In one embodiment, the lumican comprises the amino acid sequence of SEQ ID NO: 61. Attorney Docket No. 046483-7492WO 1(04069)

[0019] In an embodiment, the chimeric IL-4 polypeptide further comprises human serum albumin (HSA) linked to the myocardial infarct targeting agent. In one embodiment, the chimeric IL-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 65.

[0020] In some embodiments, the chimeric IL-4 polypeptide further comprises an IL-4 masking moiety and a cardiovascular inflammatory protease-sensitive linker between IL-4 and the IL-4 masking moiety, wherein the activity of IL-4 is unmasked following cleavage of the cardiovascular inflammatory protease-sensitive linker and release of the IL-4 masking moiety. In an embodiment, the IL-4 masking moiety comprises an IL-4 receptor alpha- 1 (IL-4Ral) extracellular domain (ECD). In one embodiment, the IL-4Ral ECD comprises the amino acid sequence of SEQ ID NO: 6.

[0021] In an embodiment, the IL-4Ral masking moiety is a mutant IL-4Ral ECD with reduced binding to IL-4. In some embodiments, the mutant IL-4Ral ECD comprises one or more alanine substitutions relative to the wild-type IL-4Ral ECD. In some embodiments, the mutant IL-4Ral ECD comprises one or more mutations of the mature wild-type IL-4Ral polypeptide at any one of amino acid residues 13-15, 39, 41-43, 66-70, 72-74, 91-94, 125-129, 183, or a combination thereof. In certain embodiments, the mutant IL-4Rul ECD comprises one or more mutations selected from the group consisting of L39A, F41A, L42A, L43A, D72A, and Y74A. In certain embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL -4 is between about 25 nm to about 250 nm. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is between about 50 nm to about 150 nm.

[0022] In an embodiment, the cardiovascular inflammatory protease-sensitive linker is an MMP-2 or MMP-9 cleavable linker. In certain embodiments, the MMP-2 or MMP-9 cleavable peptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10-47.

[0023] In an embodiment, the peptide linker between IL-4 and the targeting agent comprises an amino acid sequence set forth in any one of SEQ ID NOs: 48-58.

[0024] In another aspect, the present invention provides an immune cell expressing or treated with a chimeric IL-4 polypeptide described herein. In an embodiment, the immune cell is a bone marrow derived macrophage (BMDM).

[0025] In another aspect, the present invention provides a pharmaceutical composition comprising a chimeric IL-4 polypeptide or immune cell expressing or treated with a chimeric IL-4 polypeptide as described herein in combination with a pharmaceutically acceptable carrier. Attorney Docket No. 046483-7492WO 1(04069)

[0026] In another aspect, the present invention provides a nucleic acid encoding a chimeric IL-4 polypeptide as described herein. In another embodiment, the present invention provides a vector comprising a nucleic acid encoding a chimeric IL-4 polypeptide as described herein. In another embodiment, the present invention provides an expression vector comprising a nucleic acid encoding a chimeric IL-4 polypeptide as described herein. In another embodiment, the present invention provides an mRNA encoding a chimeric IL-4 polypeptide as described herein. In another embodiment, the present invention provides an LNP composition comprising an mRNA encoding a chimeric IL-4 polypeptide as described herein.

[0027] In another aspect, the present invention provides a pharmaceutical composition, comprising: a chimeric IL-4 polypeptide described herein, an immune cell expressing the chimeric IL-4 polypeptide described herein, or an LNP composition comprising an mRNA encoding a chimeric IL-4 polypeptide described herein; and a pharmaceutically acceptable carrier.

[0028] In another aspect, a method for treating a myocardial infarction comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered via intracoronary or intramyocardial delivery. In some embodiments, the pharmaceutical composition is administered between 2-4 hours after a myocardial infarction. In some embodiments, the pharmaceutical composition is further administered on at least 5-7 consecutive days following the myocardial infarction.

[0029] In another aspect, a method for treating or reducing the severity of heart failure comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the subject has undergone a myocardial infarction. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered via intracoronary or intramyocardial delivery. In some embodiments, the pharmaceutical composition is administered between 2-4 hours after the myocardial infarction. In some embodiments, the pharmaceutical composition is further administered on at least 5-7 consecutive days following the myocardial infarction. Alternatively, or in addition, the pharmaceutical composition is administered at least once a week following the myocardial infarction. In some embodiments, the pharmaceutical Attorney Docket No. 046483-7492WO 1(04069)

[0030] composition is administered at least once every two weeks following the myocardial infarction. In some embodiments, the pharmaceutical composition is administered at least once a month following the myocardial infarction.

[0031] In another aspect, a method for treating or reducing the severity of heart failure comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an immune cell as described herein. In some embodiments, the immune cell is a bone marrow derived macrophage (BMDM).

[0032] In another aspect, a method for treating ischemia / reperfusion injury, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the ischemia / reperfusion injury is a cardiac ischemia / reperfusion injury selected from the group consisting of acute cardiac ischemia, chronic cardiac ischemia, chronic coronary disease, and acute coronary syndrome. In some embodiments, the ischemia / reperfusion injury is caused by cardiac surgery. In some embodiments, the ischemia / reperfusion injury is caused by stroke, surgery, or organ transplantation. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered via intracoronary or intramyocardial delivery. In some embodiments, the pharmaceutical composition is administered between 2-4 hours after a myocardial infarction. In some embodiments, the pharmaceutical composition is further administered on at least five consecutive days following the myocardial infarction.

[0033] In another aspect, a method of performing cardiovascular surgery in a subject in need thereof comprises performing a surgical procedure and administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the surgical procedure is an angioplasty or stent placement. In some embodiments, the pharmaceutical composition is administered between 2-6 hours after the myocardial infarction.

[0034] In another aspect, a method for making a chimeric IL -4 polypeptide described herein comprises culturing a host cell comprising a nucleic acid encoding a chimeric IL-4 polypeptide described herein in a culture medium under conditions such that the chimeric IL-4 polypeptide is expressed. In a further embodiment, the chimeric IL-4 polypeptide is recovered and / or purified from the culture medium or the host cell. Attorney Docket No. 046483-7492WO 1(04069)

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0037] FIG. 1 : Engineering infarct selective delivery of IL-4. Predicted complex of murine IL-4 and IL-4Ral extracellular domain (ECD) (left) as an MMP-cleavable conditional mask fused to infarct binding nanobody LMJ2.5I (right).

[0038] FIGs. 2A-2D: EIIIB -targeted nanobody accumulates in a myocardial infarct. (A) Depiction of engineered nanobodies. (B) Schematic of study timeline. Mice underwent left anterior descending coronary artery ligation (LAD) surgery to stimulate myocardial infarction on day 0. Mice received a retro-orbital injections on day 7 of AF750 conjugated nanobodies after MI. Hearts were excised 24 hours later. (C) Quantification of fluorescent imaging of heart tissues in (D). (D) Fluorescent imaging of hearts tissue sliced 1 mm from apex to base. Far-red imaging of AF750 indicates presence of nanobody (top). Autofluorescence in the red channel was used to demarcate the infarct area (bottom).

[0039] FIGs. 3A-3H: EIIIB -targeted IL-4 improves cardiac repair. (A) Depiction of engineered nanobodies or collagen binding lumican fused to IL-4. (B). Schematic of study timeline. Mice underwent left anterior descending coronary artery ligation (LAD) surgery to stimulate myocardial infarction on day 0. Mice received retro-orbital injections (morning and night) on day 3, 5, 7 after MI of indicated treatment. (C) Representative parasternal long-axis echocardiography images of the left ventricle. Outline depicts left ventricular end systolic volume (left) and left ventricular end diastolic volume (right). (D-H) Quantification of cardiac ejection fraction (D), longitudinal strain (E), radial strain (F), end diastolic volume (G) and end systolic volume (H) of each treatment group as measured by echocardiography. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0040] FIGs. 4A-4B: Cardiac functional improvement as measured by echocardiography. (A) Quantification of left ventricular mass, heart rate, cardiac output, end diastolic volume, and end systolic volume of mice in FIG. 3 as measured by echocardiography. (B) Additional day 28 Attorney Docket No. 046483-7492WO 1(04069)

[0041] echocardiology measurements. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0042] FIGs. 5A-5C: EIIIB -targeted IL-4 drive improvement do not lead to persistent immunological changes in peripheral blood. (A) Schematic of study timeline. Mice underwent left anterior descending coronary artery ligation (LAD) surgery to stimulate myocardial infarction on day 0. Mice received retro-orbital injections (morning and night) on day 3, 5, 7 after MI of indicated treatment. (B-C) Peripheral blood counts of immune subsets on day 10 (B) and day 28 (C). Only statistically significant comparisons are displayed.

[0043] FIG. 6 shows the flow cytometry gating strategy for obtaining the peripheral blood counts of the immune subsets depicted in FIGs. 5B-5C.

[0044] FIGs. 7A-7F: IL-4 immunocytokines alter macrophage phenotype. (A) Schematic of study design. Bone marrow derived macrophages (BMDM) from C57BL / 6 were established with M-CSF incubation for 5 days. Once established, BMDM were treated with IL-4 immunocytokines at varying IL-4. LPS and saline were only evaluated as single dose conditions. (B-C) Expression of surface CD86, CX3CR1, CD301b, CD206, and Argl in terms of median fluorescence intensity (MFI, (B)) and percentage of expressing cells (C). (D) Fn-EIIIB binding as determined by ELISA for LMJ2.5I-IL4 and NJT6-IL4. (D-F) Commassie Blue stained SDS-Page gel with LMJ2.5I-IL4, NJT6-IL4 (E) and Lumi can-MS A-IL4 (F).

[0045] FIGs. 8A-8I. (A) Schematic showing Fn-EIIIB targeted nanobody (LMJ2.5I) and size matched, untargeted control (NJT6). (B) Schematic of timeline of study. Mice underwent LAD surgery at day 0, 7 days later were injected with AF750 labeled nanobodies and on day 8 organs were collected to determine biodistribution. (C) Quantification of infarct to remote ratio of fluorescent signal as measured on flatbed scanner. (D) Representative images of infarcted heart tissue slices, AF750 signal (top) and autofluorescent infarcted tissue (bottom). (E) Representative images of clearance organ systems. (F) Quantified mean AF750 signal in clearance organs. (G) Adjacent murine cross axis slices of mouse infarct showing cardiac troponin staining (left) and Fn-EIIIB staining (right) in 5x magnification. 25X magnification images showing infarct and remote expression of Fn-EIIIB. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0046] FIGs. 9A-9F. (A) Publicly available spatial RNA-sequencing data of mice 1, 3, 7 days after LAD surgery showing Tnnt2 (gene encoding cardiac troponin) and Collal (gene encoding Attorney Docket No. 046483-7492WO 1(04069)

[0047] collagen type I alpha 1). (B) Quantified mean expression of Col lai in infarct and remote. (C) Publicly available spatial RNA-sequencing data of mice 1, 3, 7 days after LAD surgery showing Tnnt2 (gene encoding cardiac troponin) and Col3al (gene encoding collagen type III alpha 1). (D) Quantified mean expression of Col3al in infarct and remote. (E) Publicly available spatial RNA-sequencing data of mice 1, 3, 7 days after LAD surgery showing Tmit2 (gene encoding cardiac troponin) and Col4al (gene encoding collagen type IV alpha 1). (F) Quantified mean expression of Col4al in infarct and remote. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0048] FIGs. 10A-10J: Fn-EIIIB targeted IL-4 immunocytokine improves cardiac outcomes after MI. (A) Schematic of IL -4 conjugated nanobodies. LMJ2.5LIL4 is targeted to Fn-EIIIB and NJT6-IL4 is control non-targeted. (B) Fn-EIIIB binding of each immunocytokine (arbitrary units) (n = 2). (C) Mean fluorescence intensity of Arginase-1 expression in murine F4 / 80+ bone marrow derived macrophages (n = 3) 36 hours after treatment with IL-4 or immunocytokines. Curve fits were generated by EC50 fit (least-squares fit) with X as concentration. (D) Mean fluorescence intensity of CD206 in CD68+ peripheral blood monocyte derived macrophages (n = 2) 36 hours after treatment with IL-4 or LMJ2.5I-huIL4. Curve fits were generated by EC50 fit (least-squares fit) with X as concentration. (E) Experimental outline. C57BL / 6 mice were randomized to LAD surgery or sham and then randomized into treatment groups. Mice were treated with saline, NJT6-IL4, or LMJ2.5I-IL4 on days 3, 5, 7 after surgery with endpoint echo performed on day 28. (F) Long axis echocardiogram images of murine hearts at day 28 in systole (contraction) or diastole (relaxation). Left ventricular space is outlined in yellow. (G) Day 28 ejection fraction measured by echocardiogram (mean with SD, n = 9-18). Data were analyzed by one-way ANOVA and Tukey’s post hoc test. (H) Short axis section of murine hearts after LAD stained with picrosirius red. (I) left ventricular wall thickness (mm) measured from picrosirius red stained hearts using unbiased matlab code (n = 3 biological replicates with n = 6 technical per heart). Data were analyzed by one-way ANOVA and Tukey’s post hoc test. (J) Percent fibrosis of entire cross sectional murine cardiac slice analyzed from picrosirius red stained slices (w = 3 biological replicates with n = 6 technical per heart). Data were analyzed by one-way ANOVA and Tukey’s post hoc test.

[0049] FIGs. 11A-1 IB depict Coomassie blue stained gels showing purified IL-4

[0050] immunocytokines. Attorney Docket No. 046483-7492WO 1(04069)

[0051] FIGs. 12A-12I: (A) Experimental design explained in FIG 10E. (B) Percent weight change of mice after MI, with no statistical difference, evaluated by one-way ANOVA on each time point and Tukey’s post hoc test. (C-H) Echocardiogram measurements taken on day 28 of the study. All data were analyzed by one-way ANOVA and Tukey’s post hoc test.

[0052] FIGs. 13A-13I: (A) Day 10 flow plot depicting CD31+ cells per treatment group. CD31+ cells were gated as cells, singlets, singlets, live, CD31+CD45- (B) CD31+ cells per mg of tissue for each group, quantifying representative flow plots in (A). Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (C) Representative images of PEC AM- 1 (red) and DAPI (blue) stained short axis tissue sections to determine vascularization. (D) Quantification of vascularization. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (E) Representative images of interstitial fibrosis. (F) Quantification of interstitial fibrosis. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (G) Representative flow plots of myofibroblasts per group. Myofibroblasts were identified as cells, singlets, singlets, live, CD31-CD45-, mEF-SK4+. (H) Myofibroblasts per mg of tissue for each group, quantifying representative flow plots in (G). Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (I) Percent of Ly6Chlmonocytes (of total monocytes) in peripheral blood over time course of the study. Data were analyzed as one-way ANOVA per each timepoint with Tukey’s post hoc test.

[0053] FIGs. 14A-14M: Fn-EIIIB targeted IL-4 supports remodeling of infarcted myocardium. (A) Short axis slices of mice day 8 after surgery stained for cardiac troponin (cTNT), scale bar is 1000 pm. (B) Percent of total heart that is positive for cTNT as defined by area that is DAPI positive (mean with SD, n = 3 biological replicates with n = 3 technical per heart). (C) Short axis slices of mice day 8 after surgery stained with TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) (green) and DAPI (blue), scale bar is 50 pm. (D) Percent of total short axis heart slice that is positive for TUNEL as defined by area that is DAPI positive (mean with SD, n = 3 biological replicates with n = 3 technical per heart). (E) Representative flow plots of infarct (top) and remote (bottom) regions of murine hearts on day 10 after surgery. This is the terminal flow plot where cells have been gated to be live, CD45+, CD1 lb+, and CD64+. (F) Mean fluorescence intensity of Arginase- 1 in CCR2+ and CCR2- macrophage populations in the infarct (top) and remote (bottom). Macrophages are defined as live, CD45+, CD1 lb+ and CD64+ and then split into CCR2+ / - (mean with SD, n = 6-7). Data were analyzed by one-way Attorney Docket No. 046483-7492WO 1(04069)

[0054] ANOVA and Tukey’s post hoc test per macrophage subset. (G) Experimental outline. Bone marrow derived macrophages (BMDM) were established and then treated with PBS, LPS, LMJ2.5I-IL4, or both LPS and LMJ2.5LIL4 for 36 hours. After 36 hours, BMDMs were phenotype and co-cultured with pHrodo labeled apoptotic cells. (H) Trem2 mean fluorescence intensity in live, F4 / 80+ BMDMs (mean with SD, n = 4). Data were analyzed by one-way ANOVA and Tukey’s post hoc test per macrophage subset. (I) MerTK mean fluorescence intensity in live, F4 / 80+ BMDMs (mean with SD, n = 4). Data were analyzed by one-way ANOVA and Tukey’s post hoc test per macrophage subset. (J) Percent BMDM that are pHrodo positive after 2 hours of co-culture (mean with SD, n = 3). Data were analyzed by one-way ANOVA and Tukey’s post hoc test per macrophage subset. (K) Experimental outline. Peripheral blood monocyte derived macrophages (PBMDM) were established and treated with PBS, LPS, LMJ2.5I-huIL4, or both LPS and LMJ2.5I-huIL4 for 36 hours. After 36 hours, PBMDMs were phenotyped and co-cultured with apoptotic cells. (L) Trem2 mean fluorescence intensity in live, CD68+ PBMDMs (mean with SD, n = 4). Data were analyzed by one-way ANOVA and Tukey’s post hoc test per macrophage subset. (M) MerTK mean fluorescence intensity in live, CD68+ PBMDMs (mean with SD, n = 4). Data were analyzed by one-way ANOVA and Tukey’s post hoc test per macrophage subset.

[0055] FIGs. 15A-15B: (A) Day 10 flow cytometry results of infarcted tissue. Flow cytometry analysis where the following cell types were identified: CD31+, myofibroblasts, CD45+, monocytes, neutrophils, macrophages, and then the phenotypic markers for macrophages were looked at: Argl, MHCII, CCR2. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (B) Day 10 flow cytometry results of remote tissue. Flow cytometry analysis where the following cell types were identified: CD31+, myofibroblasts, CD45+, monocytes, neutrophils, macrophages, and then the phenotypic markers for macrophages were looked at: Argl, MHCII, CCR2. Data were analyzed using one-way ANOVA and Tukey’s post hoc test.

[0056] FIGs. 16A-16F: Model predicts immunocytokine availability in infarct. (A) Model design schematic. After intravenous injection, immunocytokine can diffuse into the infarct or be cleared by blood. Once in the tissue, immunocytokine can bind or unbind to matrix, and at each stage can be degraded by proteases. (B) NJT6-IL4 (purple) and LMJ2.5I-IL4 (pink) predicted exposure after 72 hours by modeling an intravenous injection (right bar graph). Measured mean fluorescence intensity of Arginase- 1 in Live, CD45+CD1 lb+CD64+cells on day 10, 72 hours Attorney Docket No. 046483-7492WO 1(04069)

[0057] after final injection (left bar graph). (C) Fractional activity of immunocytokines with varying infarct target concentration. (D) Fractional activity of immunocytokines with varying sizes, each corresponding to typical biologic size conjugated to IL-4 (nanobody-IL4, scFv-IL4, antibody-IL4). (E) Predicted infarct exposure of different size immunocytokines with varying infarct target concentration. (F) Spatial RNA-seq from publicly available dataset (X) and reanalyzed for extracellular matrix gene expression level in infarct and remote myocardium 1, 3, 7 days after MI.

[0058] FIGs. 17A-17E: (A) Table of values used in building model. (B) Model output of concentration of each immunocytokine in infarct over 48 hours. (C) Model output of each immunocytokine in blood over 48 hours. (D) Fractional activation over 48 hours in infarct as predicted by model. (E) Fractional activation of each immunocytokine in blood over 48 hours. For (B-E) Pink is LMJ2.5I-IL4 and purple is NJT6-IL4.

[0059] FIGs. 18A-18M: Collagen targeted IL-4 improves cardiac function after MI but induces systemic immune side effects. (A) Schematic of collagen targeted immunocytokine, with lumican conjugated to mouse serum albumin and IL-4. (B) Experimental outline. C57BL / 6 mice were randomized to LAD surgery or sham and then randomized into treatment groups. Mice were treated with saline or Lumican-MSA-IL4 on days 3, 5, 7 after surgery with endpoint echo performed on day 28. (C) Long axis echocardiogram images of murine hearts at day 28 in systole (contraction) or diastole (relaxation). Left ventricular space is outlined in yellow. (D) Day 28 ejection fraction measured by echocardiogram (mean with SD, n = 9-18). Data were analyzed by one-way ANOVA and Tukey’s post hoc test. (E) Short axis section of murine hearts after LAD stained with picrosirius red. (F) Left ventricular wall thickness (mm) measured from picrosirius red stained hearts using unbiased matlab code n = 3 biological replicates with n = 6 technical per heart). Data were analyzed by one-way ANOVA and Tukey’s post hoc test, j, Percent fibrosis of entire cross sectional murine cardiac slice analyzed from picrosirius red stained slices (n = 3 biological replicates with n = 6 technical per heart). Data were analyzed by one-way ANOVA and Tukey’s post hoc test.

[0060] FIGs. 19A-19B: (A) Commassie Blue stained SDS-Page gel with Lumican-MSA-IL4. (B) Mean fluorescence intensity of Arginase- 1 expression in murine F4 / 80+ bone marrow derived macrophages (n = 3) 36 hours after treatment with IL-4 or immunocytokines. Curve fits were generated by EC50 fit (least-squares fit) with X as concentration. Attorney Docket No. 046483-7492WO 1(04069)

[0061] FIGs. 20A-20I: (A) Experimental design explained in FIG 10E. (B) Percent weight change of mice after MI, with no statistical difference, evaluated by one-way ANOVA on each time point and Tukey’s post hoc test. (C-H) Echocardiogram measurements taken on day 28 of the study. All data were analyzed by one-way ANOVA and Tukey’s post hoc test. Pink dashed line indicates value for LMJ2.51-114 treated mice.

[0062] FIGs. 21A-21H: (A) Representative images of interstitial fibrosis. (B) Quantification of interstitial fibrosis. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (C) Representative images of PEC AM- 1 (red) and DAPI (blue) stained short axis tissue sections to determine vascularization. (D) Quantification of vascularization. Data were analyzed using oneway ANOVA and Tukey’s post hoc test. (E) Representative images of cardiac troponin (cTNT) staining. (B) Representative images of TUNEL stained hearts. (C) Quantification of cTNT staining from whole cross-sectional hearts. (F) Quantification of cTNT staining. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (G) Quantification of TUNEL staining from whole cross-sectional hearts. (H) Quantification of TUNEL staining. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. Pink line indicates corresponding LMJ2.5I-IL4 results.

[0063] FIGs. 22A-22E: (A) Day 28 heart measurements normalized to both tibia length and bodyweight (BW). (B) Day 28 infarct weight normalized to the whole heart weight. (C) Day 28 spleen weights normalized by tibia and bodyweight. (D) % Body weight change after surgery per group. (E) Kaplan-Meier curve describing survival. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s. For % weight change, on each day a two-way ANOVA with Dunnett’s test was performed to determine significance. For survival a Kaplan-Meier with survival analysis was performed.

[0064] FIG. 23: Complete serum analysis describing serum levels of liver enzymes, proteins, carbs, molecules, and gas. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s (all non-significant with p<0.05 as threshold).

[0065] FIGs. 24A-24G: (A) Representative images of picrosirius stained day 28 hearts per treatment group. (B) Quantified amount of fibrosis per picrosirius staining. (C) Left ventricular wall thickness calculated from picrosirius stained images. (D) PECAM-1 and DAPI stained day 28 heart slices. (E) Representative images of interstitial fibrosis taken from whole heart picrosirius stained hearts. (F) Quantification of PECAM-1 staining. (G) Quantification of Attorney Docket No. 046483-7492WO 1(04069)

[0066] interstitial fibrosis. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0067] FIGs. 25A-25K: (A) Representative images of cardiac troponin (cTNT) staining. (B) Representative images of TUNEL stained hearts. (C) Quantification of cTNT staining from whole cross-sectional hearts. (D) Quantification of TUNEL staining from whole cross-sectional hearts. (E) Schematic of efferocytosis assay and phagocytosis receptor analysis. (F) Mean fluorescence intensity of MerTK expression in F4 / 80+ cells. (G) Mean fluorescence intensity of Trem2 in F4 / 80+ cells. (H) Percent of BMDM that were positively stained for pHrodo after treatment with apoptotic cells. (I) Schematic of human efferocytosis assay and phagocytosis receptor analysis of peripheral blood monocyte derived macrophages. (J) Mean fluorescence intensity of MerTK in CD68+ cells. (K) Mean fluorescence intensity of Trem2 in CD68+ cells. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0068] FIGs. 26A-26I: (A) Schematic of the therapeutic timeline, mice underwent LAD permanent occlusion on day 0, were injected on days 3, 5, 7 and then had day 10 echo. (B) Day 10 flowplots of infarcted heart tissue with gating for Argl+ macrophages. (C) Day 10 flowplots of remote heart tissue with gating for Argl + macrophages. (D) Macrophage cell counts in the infarct. E) Number of Argl+ macrophages in infarct. (E) Mean fluorescence intensity of Argl staining in macrophages in infarct tissue at day 10. (F / G) Argl mean fluorescence intensity in CCR2+ and CCR2- cells in the infarct. (H / I) Argl mean fluorescence intensity in CCR2+ and CCR2- cells in the remote tissue of the heart. Statistical significance was calculated with oneway ANOVA with post-hoc Tukey’s.

[0069] FIGs. 27A-27B: All cell types quantified by flow cytometry at day 10 in both infarct and remote regions of the heart. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0070] FIG. 28: Argl MFI in infarct and remote regions of the heart within macrophage subsets at day 10. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0071] FIG. 29. Echocardiography results at day 10 after MI per treatment group. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0072] FIG. 30. Day 10 heart and spleen measurements normalized to both tibia length and bodyweight (BW). Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s. Attorney Docket No. 046483-7492WO 1(04069)

[0073] FIG. 31. Day 10 serum and cardiac interstitial fluid cytokine measurements as measured by legendplex. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0074] FIG. 32. All cell types quantified by flow cytometry at day 28 in both infarct and remote regions of the heart. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0075] FIG. 33. CD206 MFI in infarct and remote regions of the heart within macrophage subsets at day 28. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0076] FIGs. 34A-34B: (A) Schematic of the therapeutic timeline, mice underwent LAD permanent occlusion on day 0, were injected on days 3, 5, 7 and then blood was collected at day 10 to be analyzed by flow cytometry. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s. (B) All cell types that were quantified as cells / mL of blood. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0077] FIGs. 35A-35C: (A) Schematic of the therapeutic timeline, mice underwent LAD permanent occlusion on day 0, were injected on days 3, 5, 7 and then blood was collected at day 28 to be analyzed by flow cytometry. (B) All cell types that were quantified as cells / mL of blood. (C) Ly6Chi and Ly6Clo monocytes as a percent of total monocytes in the blood across the study. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0078] FIGs. 36A-36B: (A) Schematic of the therapeutic timeline, mice underwent LAD permanent occlusion on day 0, were injected on days 3, 5, 7 and then organs were harvested on day 8. (B) Cell counts per mg of tissue in the spleen measured on day 8. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s.

[0079] FIGs. 37A-37B: (A) Schematic of the therapeutic timeline, mice underwent LAD permanent occlusion on day 0, were injected on days 3, 5, 7 and then organs were harvested on day 8. (B) Cell counts per mg of tissue in the liver measured on day 8.

[0080] FIGs. 38A-38C: (A) Schematic of therapeutic timeline, mice underwent LAD permanent occlusion on day 0, were injected on days 3, 5, 7 and then organs were harvested on day 8. (B) Cell counts per mg of infarcted heart tissue measured on day 8. (C) Cell counts per mg of remote heart tissue measured on day 8. Statistical significance was calculated with one-way ANOVA with post-hoc Tukey’s. Attorney Docket No. 046483-7492WO 1(04069)

[0081] FIG. 39. Exemplary flow plots showing gating to determine blood cell counts

[0082] FIG. 40: Exemplary flow plots showing gating to determine heart cell counts

[0083] FIGs. 41A-41K: (A) Schematic of compartments being modeled. (B, left) Predicted exposure as determined by fractional activation output after modeling 72 hours after initial injection (left). (B, right) Argl mean fluorescence intensity of macrophages in infarct on day 10 treated mice (72 hours after last treatment). (C) Model output for fractional activation of immunocytokines that have different concentrations of infarct extracellular matrix target. (D) Model output for fractional activation of immunocytokines of different sizes. (E) Heatmap depicting infarct exposure of different size immunocytokines with different concentrations of extracellular matrix target. (F) Mean expression of extracellular matrix proteins in infarct and remote 1, 3, 7 days after MI (from analyzed spatial- RNA sequencing data). (G) Table of constants input into the model. (H) Model output of concentration of LMJ2.5I-IL4 and NJT6-IL4 in infarct for 48 hours after injection. (I) Model output of blood concentration of LMJ2.5I-IL4 and NJT6-IL4 in infarct for 48 hours after injection. (J) Model output of infarct fractional activity of LMJ2.5I-IL4 and NJT6-IL4 in infarct for 48 hours after injection. (K) Model output of infarct fractional activity of LMJ2.5I-IL4 and NJT6-IL4 in infarct for 48 hours after injection.

[0084] FIGs. 42A-42D: (A) Schematic of study where human peripheral blood monocyte derived macrophages were differentiated over 5 days and then treated. (B) Quantification of macrophage phenotypic markers as determined by flow cytometry in LMJ2.5I-huIL4 treated PBMDMs. (C) Quantification of macrophage phenotypic markers as determined by flow cytometry in LMJ2.5I-porIL4 treated PBMDMs. (D) Commassie Blue stained SDS-Page gel with LMJ2.5I-IL4, NJT6-IL4 and Lumi can-MS A-IL4.

[0085] FIGs. 43A-43E: (A) Experimental design where mice only received one dose of immunocytokine after LAD surgery. (B-D) Echocardiogram measurements at day 28. End diastolic volume and end systolic volume were directly measured and lead to resultant ejection fraction. Data were analyzed using one-way ANOVA and Tukey’s post hoc test. (E) Short axis heart sections stained with picrosirius red to visualize fibrosis.

[0086] FIGs. 44A-44B: (A) Schematic of stroke biodistribution study where radiolabeled proteins were injected into different stroke models. (B) Organ and brain biodistribution in each surgical and protein case. Proteins have the same nanobodies (LMJ2.5I and NJT6) with size matched deactivated cytokine (deIL2). Attorney Docket No. 046483-7492WO 1(04069)

[0087] DETAILED DESCRIPTION

[0088] Definitions

[0089] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0090] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0091] That the disclosure may be more readily understood, select terms are defined below. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0092] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Attorney Docket No. 046483-7492WO 1(04069)

[0093] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0094] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0095] As used herein, the term “cardiomyopathy” refers to a disease or condition affecting the heart, wherein a heart muscle (e.g., cell of the heart muscle) is damaged or the function of a heart muscle (e.g., cell of the heart muscle) is impaired (e g., relative to a healthy fully functioning heart, heart muscle, of heart muscle cell). An exemplary cardiomyopathy that may be treated with a compound or method provided herein include heart muscle damage, alcoholic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, noncompaction cardiomyopathy, heart failure, (congestive) heart failure, hypertensive cardiomyopathy, cardiomyopathy associated with cardiac surgery, cardiomyopathy associated with coronary intervention or myocardial infarction, cardiomyopathy caused by genetic changes in cardiac proteins, cardiomyopathy associated with genetic mutations in one or more cardiac proteins, and cardiomyopathy associated with aberrant expression or function of one or more cardiac proteins. In some embodiments, treating a cardiomyopathy includes treating a condition or symptom caused by a cardiomyopathy. In some embodiments, cardiomyopathy is caused by another disease (e.g., a cardiovascular disease) and treatment of cardiomyopathy includes Attorney Docket No. 046483-7492WO 1(04069)

[0096] treating the causative disease (e.g. cardiovascular disease) of the cardiomyopathy. In some embodiments, the cardiomyopathy is dilated cardiomyopathy. In some embodiments, the cardiomyopathy is hypertrophic cardiomyopathy. In some embodiments, the cardiomyopathy is hypertrophic, restrictive, or dilated.

[0097] The term “cardiovascular inflammatory protease” refers to an inflammatory protease upregulated following inflammation or tissue damage at a cardiovascular tissue site of inflammation (e.g. cardiac tissue, cardiovascular arteries, etc.), proteases that are specifically directed to the cardiovascular tissue site of inflammation through biological processes, or proteases that perform specific biological functions related to inflammation at the cardiovascular tissue site of inflammation. Cardiovascular inflammatory proteases may be expressed at and / or targeted to the site of cardiovascular inflammation. In embodiments, the cardiovascular inflammatory protease is found at the site of cardiovascular inflammation (e.g., inflammation at cardiac tissue resulting from stent placement, angioplasty, or surgical procedure). Non-limiting examples of a cardiovascular inflammatory protease include membrane bound MMP (e.g., MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP), serine protease (e g., plasmin, cathepsin G), lysosomal cysteine protease (cathepsin B), tryptase, chymase, collagenase (e.g., MMP-1, MMP-8, MMP-13), gelatinase (MMP-2, MMP-9), stromelysin (e.g., MMP-3), or membrane type (e.g., MMP-14). In embodiments, the cardiovascular inflammatory protease is MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, or MMP-14.

[0098] The term "chimeric” is used with reference to a polypeptide having amino acid sequences derived from multiple different proteins, including mutant proteins thereof. As used herein, the term “chimeric IL-4 polypeptide” refers to a polypeptide comprising amino acid sequences from IL-4, IL-4Ra ECD, and a cardiovascular inflammatory protease-responsive targeting moiety (e.g., anti-Fn EIIIB nanobody).

[0099] A “disease” or “condition”, as used herein, refers to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. In some embodiments, the disease is a disease related to (e.g. caused by) heart muscle damage (e.g. myocardial infarction, cardiomyopathy, heart failure). In some instances, “disease” or “condition” refers to myocardial infarction, cardiomyopathy, or heart failure. In some embodiments, the disease is heart muscle damage. In some embodiments, the disease is Attorney Docket No. 046483-7492WO 1(04069)

[0100] myocardial infarction. In some embodiments, the disease is heart failure. In some embodiments, the disease is cardiomyopathy.

[0101] The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes.

[0102] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of the chimeric IL-4 polypeptide effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit.

[0103] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0104] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0105] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0106] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system. Attorney Docket No. 046483-7492WO 1(04069)

[0107] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0108] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0109] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen). The term, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0110] The terms “fused” and “fusion” are used in reference to the joining of the two polypeptide sequences through a backbone peptide bond. Two polypeptides may be fused directly or through a peptide linker that is one or more amino acids long. A fusion polypeptide may be made by recombinant technology from a coding sequence containing the respective coding sequences for the two fusion partners, with or without a coding sequence for a peptide linker in between. In some embodiments, fusion encompasses chemical conjugation.

[0111] The terms “heart failure” or “congestive heart failure” are used according to their ordinary meaning and refer to a condition affecting the heart, wherein a heart muscle (e.g., cell of the heart muscle) is damaged or the function of a heart muscle (e.g., cell of the heart muscle) is impaired (e.g., relative to a healthy fully functioning heart, heart muscle, of heart muscle cell). In embodiments, heart failure is a condition affecting the heart, wherein the heart exhibits reduced blood flow. Attorney Docket No. 046483-7492WO 1(04069)

[0112] The term “immune cell” refers to a cell which is capable of affecting or inducing an immune response upon recognition of an antigen. In some embodiments, the immune cell is a macrophage, myeloid cell, T-cell, a natural killer (NK) cell, a or a dendritic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0113] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0114] The term “ischemia-reperfusion injury (IR injury)” refers to tissue damage caused by ischemia, reperfusion, or reperfusion after ischemia. Therefore, the term "IR damage" may include ischemia-induced injury, reperfusion injury, or injury by reperfusion after ischemia. As used in the present specification, "an event that causes an IR injury" may include ischemia, reperfusion, thromboembolism, vasoconstriction, cardiac standstill, myocardial infarction, stroke, or surgeries (e.g., organ transplantation, heart surgery, etc.) in which blood flow to tissues is blocked or reduced, and the like. Myocardial infarction (Ml) is a type of cardiac ischemia that can cause an IR injury.

[0115] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0116] The terms “mask”, “masking moiety”, and “masking” are used herein with reference to an IL-4 moiety masking IL-4 activity by virtue of being bound to IL-4. Upon release of the IL-4 masking moiety (e.g., IL-4Ra extracellular domain (ECD)) from the bound complex, IL-4 activity is “unmasked” (or no longer blocked) such that IL-4 can carry out its functional activities

[0117] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0118] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the Attorney Docket No. 046483-7492WO 1(04069)

[0119] subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0120] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0121] The term “myocardial infarction” is used according to its ordinary meaning and refers to heart tissue damage and or myocardial cell death resulting from a decrease in blood flow. In embodiments, myocardial infarction is caused by prolonged ischemia. The myocardial infarction may encompass ST-segment elevation myocardial infarction (STEMI) or non-ST-segment elevation myocardial infarction (NSTEMI). A patient having symptoms of myocardial infarction can be diagnosed by clinical features, such as electrocardiographic (ECG) findings, elevated values of biochemical markers (e.g., proteins) of myocardial necrosis (e.g., cardiac troponins), and / or by imaging. Symptoms associated with myocardial infarction include myocardial ischemia, tachycardia arrhythmia, bradycardia arrhythmia, arrhythmia, heart failure, heart tissue damage, or renal failure.

[0122] The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T ”

[0123] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0124] “Parenteral” administration of an immunogenic composition includes, e. ., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0125] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s Attorney Docket No. 046483-7492WO 1(04069)

[0126] sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0127] The term “polynucleotide” as used herein is defined as a chain of nucleotides.

[0128] Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0129] As used herein, the term “proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells. In particular aspects, “proliferation” refers to the symmetric or asymmetric division of T cells. “Increased proliferation” occurs when there is an increase in the number of cells in a treated sample compared to cells in a non-treated sample.

[0130] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen.

[0131] However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical Attorney Docket No. 046483-7492WO 1(04069)

[0132] species, to mean that the interaction is dependent upon the presence of a particular structure (e. ., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0133] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g, mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0134] The term "surgical procedure" is used with reference to an invasive therapy involving an incision performed in a patient. Non-limiting examples of surgical procedures include coronary ligation, coronary artery bypass grafting, transmyocardial laser revascularization, pacemaker placement, heart transplant, left ventricular assist device placement, aneurysm repair, mitrial valve surgery, cardiothoracic surgery, angioplasty, and stent placement.

[0135] The term “targeting moiety” is used with reference to a moiety that can be covalently or noncovalently attached to the IL-4 component of the chimeric IL-4 polypeptide which serves as a recognition segment for an inflammation-associated product upregulated in a cardiovascular condition, such as cardiomyopathy, including myocardial infarction, and thereby helps in localization of the chimeric IL-4 polypeptide provided herein to a target of interest. In embodiments, the targeting moiety is covalently attached. The targeting moiety may be an amino acid sequence, peptide or protein. The targeting moiety may be an antibody, nanobody, or antigen binding fragment thereof.

[0136] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0137] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. Attorney Docket No. 046483-7492WO 1(04069)

[0138] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0139] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0140] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0141] Myocardial Infarction

[0142] The present invention provides polypeptide compositions for treating a myocardial infarction (MI) and preventing or reducing the severity of heart failure (HF). In response to an MI, macrophages expand in the heart. These macrophages primarily arise from blood monocytes recruited to the injury as well as increased local proliferation. Different subsets of macrophages pursue divergent but complementary functions to clean and to repair the injured myocardium. They are generally mobilized in two sequential phases: the inflammatory and reparative phases. The initial inflammatory phase, beginning upon ischemia onset, is dominated by inflammatory macrophages, which release cytokines like IL-ip, IL-6, TNF-a, and chemokines to stimulate the recruitment of leukocytes, secrete proteases and phagocytose necrotic cells to prepare infarcted Attorney Docket No. 046483-7492WO 1(04069)

[0143] tissue for repair. This inflammatory phase peaks around three days after an Ml and then gives way to reparative macrophages, which release anti-inflammatory cytokines like IL- 10, TGF-0, and VEGF to initiate wound healing through angiogenesis and extracellular matrix reconstruction. By 2-3 weeks post-MI, most macrophages have been resolved. Finally, the infarcted tissue matures into a scar with cross-linked collagen. A coordinated bimodal response by macrophages is necessary for proper healing after an MI, but, if dysregulated, it may become a mechanism of HF.

[0144] In patients after an MI, the number of circulating inflammatory monocytes, precursors to inflammatory macrophages, rises, and the magnitude of this increase correlates with worse patient outcomes and death. MI risk factors such as obesity have been linked to a chronic state of inflammation and predispose a subset of patients towards a pro-inflammatory skewed response. Unfortunately, excessive inflammation is particularly deleterious to a struggling heart. The cytokines IL- 1(3, IL-6, TNF-a secreted by inflammatory macrophages can directly injure cardiomyocytes and limit the necessary conversion and recruitment of reparative macrophages. This imbalance provokes further cardiomyocyte death and delayed angiogenesis, which impair the contractile function of the remaining viable myocardium. Unfortunately, depleting macrophages after an MI only worsens tissue remodeling, deteriorates cardiac function, and even causes emergent arrhythmias. Also, due to their indispensable role in protecting against pathogens across the body, the broad suppression of macrophages also poses great risk. All macrophage subsets are clearly necessary and benefit recovery after an MI, but there are clearly harmful consequences if inflammatory macrophage subsets become overly abundant.

[0145] Interleukin-4 (IL-4) is a cytokine that signals through two different heterodimeric receptor complexes. The type II complex, composed of IL-4Ra and IL-13Ral chains, is expressed on nonhematopoietic cells, whereas the type I complex, composed of IL-4Ra and the common y chain, is expressed on leukocytes with its highest expression on monocytes and macrophages. IL-4 polarizes inflammatory macrophages toward the reparative phenotype.

[0146] Encouragingly, preclinical studies by us and others have shown IL-4 can reduce infarct size and improve left ventricular ejection fraction (LVEF) and wall thickness post-MI. However, like the treatments evaluated in clinical trials, these IL -4 treatments were not targeted to the heart. As a result, they also perturbed the immune system in healthy tissues. In fact, IL-4 has been explored as a potential cancer treatment, but toxicities related to its systemic immune effects have limited Attorney Docket No. 046483-7492WO 1(04069)

[0147] its use in the clinic. The crux of the issue with all immunotherapies is that on-target (correct receptor) off-tissue (wrong tissue localization) activity can result in severe dose-limiting toxicities and diminished therapeutic effects. The present invention addresses these shortcomings by appending tissue-targeting moieties in combination with conditional cytokine masking.

[0148] Chimeric IL-4 Compositions

[0149] In one aspect, a chimeric IL-4 polypeptide comprises interleukin-4 (IL-4), a myocardial infarct targeting agent, and a peptide linker between IL-4 and the targeting agent. In some embodiments, the chimeric IL-4 polypeptide further comprises an IL-4 masking moiety and a cardiovascular inflammatory protease-sensitive linker between IL-4 and the IL-4 masking moiety, wherein the activity of IL-4 is unmasked following cleavage of the cardiovascular inflammatory protease-sensitive linker and release of the IL-4 masking moiety as further described below. In some embodiments, the IL-4 masking moiety is a mini-protein, a singleheavy chain nanobody, a peptide, or an antibody fragment e.g., scFv). In certain embodiments, the IL-4 masking moiety comprises an IL-4 receptor alpha-1 (IL-4Ral) extracellular domain (ECD).

[0150] In some embodiments, the myocardial infarct targeting agent is operatively linked or fused to the N-terminus of IL-4. In some embodiments, the myocardial infarct targeting agent is operatively linked or fused to the C-terminus of IL-4.

[0151] In some embodiments, the IL-4 in the chimeric IL-4 polypeptide is of human origin. In one embodiment, the IL-4 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the IL-4 is of murine origin. In one embodiment, the IL-4 comprises the amino acid sequence of SEQ ID NO: 3.

[0152] In some embodiments, the IL-4Ral ECD in the chimeric IL-4 polypeptide is of human origin. In one embodiment, the IL-4Ral ECD comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IL-4Ral ECD is of murine origin. In one embodiment, the IL-4Ral ECD comprises the amino acid sequence of SEQ ID NO: 5.

[0153] In some embodiments, the IL-4Ral ECD is a mutant IL-4Ral ECD with reduced binding to IL-4. In some embodiments, the mutant IL-4Ral ECD comprises one or more alanine substitutions relative to a corresponding wild-type IL-4Ral ECD. In some embodiments, the mutant IL-4Ral ECD comprises one or more mutations (e.g., alanine substitutions) of the mature Attorney Docket No. 046483-7492WO 1(04069)

[0154] wild-type IL-4Ral polypeptide at any one of amino acid residues 13-15, 39, 41-43, 66-70, 72-74, 91-94, 125-129, 183, or a combination thereof. In certain embodiments, the mutant IL-4Ral ECD comprises one or more mutations selected from the group consisting of L39A, F41A, L42A, L43A, D72A, and Y74A.

[0155] In certain embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is between about 15 nm to about 500 nm. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is between about 25 nm to about 250 nm. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is between about 50 nm to about 150 nm. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is between about 75 nm to about 125 nm.

[0156] In some embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is reduced at least 5-fold relative to the binding affinity of the corresponding wild-type IL-4Ral ECD. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is reduced at least 10-fold relative to the binding affinity of the corresponding wild-type IL-4Ral ECD. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is reduced at least 20-fold relative to the binding affinity of the corresponding wild-type IL-4Ral ECD. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is reduced at least 40-fold relative to the binding affinity of the corresponding wild-type IL-4Ral ECD. In other embodiments, the binding affinity of the mutant IL-4Ral ECD to the IL-4 is reduced at least 100-fold relative to the binding affinity of the corresponding wild-type IL-4Ral ECD.

[0157] Exemplary IL-4Ral ECD mutants are described in US 2021 / 0206862, the disclosure of which is expressly incorporated by reference herein.

[0158] In an embodiment, the myocardial infarct targeting agent specifically binds an inflammation marker upregulated during the inflammation process accompanying myocardial infarction. The inflammation process accompanying myocardial infarction promotes alternative splicing of the matrix protein fibronectin (Fn), resulting in the inclusion of alternative exons extra domain A (Fn-EIIIA) and extra domain B (Fn-EIIIB). In certain embodiments, the myocardial infarct targeting agent specifically binds Fn-EIIIB. In other embodiments, the myocardial infarct targeting agent specifically binds Fn-EIIIA. Attorney Docket No. 046483-7492WO 1(04069)

[0159] In an embodiment, the myocardial infarct targeting agent is an antibody or antigen binding fragment thereof. The myocardial infarct targeting agent can include any domain that binds to an inflammation marker upregulated during the inflammation process accompanying myocardial infarction, such as Fn-IIIB or Fn-IIIA, and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, or any antigen binding fragment thereof, which is capable of specifically binding the inflammation marker (e.g., Fn-IIIB or Fn-IIIA).

[0160] In some embodiments, the myocardial infarct targeting agent is a nanobody. In certain embodiments, the nanobody is humanized. In certain embodiments, the myocardial infarct targeting agent is a nanobody that specifically binds Fn-EIIIB. In one embodiment, the nanobody that specifically binds Fn-EIIIB comprises the amino acid sequence of SEQ ID NO: 1. In other embodiments, the myocardial infarct targeting agent is a nanobody that specifically binds Fn-EIIIA.

[0161] In some embodiments, the Fn-EIIIB-specific or Fn-IIIA binding domain is of human origin. In some embodiments, the Fn-EIIIB-specific or Fn-IIIA binding domain is of murine origin. In some embodiments, the Fn-EIIIB-specific or Fn-IIIA binding domain is a humanized Fn-EIIIB-specific or Fn-IIIA binding domain. Fn-EIIIB-specific nanobodies are described in US 2019 / 0225693 Al and US 2023 / 0295284A1, the disclosures of which are expressly incorporated by reference in their entirety herein.

[0162] In some embodiments, the myocardial infarct targeting agent is a protein that specifically binds an ECM protein upregulated in myocardial infarcts. In some embodiments, the ECM protein is a collagen or a fibrillar collagen, such as Type I collagen. In some embodiments, the collagen-binding protein is lumican. Lumican is a proteoglycan Class II member of the small leucine-rich proteoglycan (SLRP) family that includes decorin, biglycan, fibromodulin, keratocan, epiphycan, and osteoglycin. In some embodiments, the lumican is of human origin. In one embodiment, lumican of human origin comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the lumican is of murine origin. In one embodiment, lumican of murine origin comprises the amino acid sequence of SEQ ID NO: 62. Other collagen-binding proteins for use as myocardial infarct targeting agents are described in US 2020 / 0102370, which is incorporated by reference in its entirety herein. Attorney Docket No. 046483-7492WO 1(04069)

[0163] In some embodiments, the linker between the IL-4 and the collagen-binding protein (e.g., lumican) provides a steric separation such that the immunomodulatory domain retains its activity (e.g., promote receptor / ligand engagement). In some embodiments, the linker between IL-4 and the collagen-binding protein is of sufficient length or mass to reduce adsorption of the immunomodulatory domain onto collagen fibrils. Methods for measuring adsorption are known to those of skill in the art. For example, adsorption can be measured by ellipsometry (ELM), surface plasmon resonance (SPR), optical waveguide lightmode spectroscopy (OWLS), attenuated total internal reflectance-infrared spectroscopy (ATR-IR), circular dichroism spectroscopy (CD), total internal reflectance-infrared spectroscopy (TIRF), and other high resolution microscopy techniques. In some embodiments, these methods show the spatial arrangement between the domains of the chimeric IL-4 polypeptide.

[0164] In some embodiments, the linker between IL-4 and the collagen-binding protein (e.g., lumican) provides sufficient molecular weight to slow or reduce diffusion from the tissue.

[0165] Methods for measuring diffusion from the tissue are known to those of skill in the art. For example, diffusion can be measured by in vivo imagining, or via microscopy of tissue sections over time. Exemplary methods are described in at least Schmidt & Wittrup, Mol Cane Ther. 2009 and Wittrup et al., Methods in Enzymol 2012, each of which is herein incorporated by reference in their entirety. In some embodiments, the linker is a hydrophilic polypeptide comprising “N” amino acids in length, where N=l-1000, 50-800, 100-600, or 200-500. In some embodiments, the linker is an albumin. Suitable albumins for use in the IL-4 chimeric polypeptides proteins can be from human, primate, rodent, bovine, equine, donkey, rabbit, goat, sheep, dog, chicken, or pig. In some embodiments, the albumin is a serum albumin, for example, a human serum albumin (HSA) comprising e.g., the amino acid sequence of SEQ ID NO: 63, mouse serum albumin (MSA) comprising e.g., the amino acid sequence of SEQ ID NO: 62.

[0166] In some embodiments, the chimeric IL-4 polypeptide comprises, from N-terminus to C-terminus, lumican, albumin (e.g, HSA or MSA), and IL-4. In another embodiment, the chimeric IL-4 polypeptide comprises, from N-terminus to C-terminus, IL-4, albumin, and lumican. In one embodiment, the chimeric IL-4 polypeptide comprises, from N-terminus to C-terminus, human lumican, HSA, and human IL-4, optionally comprising the amino acid sequence of SEQ ID NO: 67. In another embodiment, the chimeric IL-4 polypeptide comprises, from N-terminus to C- Attorney Docket No. 046483-7492WO 1(04069)

[0167] terminus, mouse lumican, MSA, and mouse IL-4, optionally comprising the amino acid sequence of SEQ IDNO: 66.

[0168] In another aspect, the present invention provides an immune cell expressing or treated with a chimeric IL-4 polypeptide described herein. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells, NK cells, NKT cells, and monocytes or granulocytes, including myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the immune cell is a bone marrow derived macrophage (BMDM). Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).

[0169] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. The source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject’s bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is a human.

[0170] In another aspect, the present invention provides a nucleic acid encoding a chimeric IL-4 polypeptide. In some embodiments, the nucleic acid is a vector or expression vector. In some embodiments, the nucleic acid encoding the chimeric IL-4 polypeptide is encapsulated in a lipid nanoparticle (LNP) as further described below.

[0171] In another aspect, the present invention provides a pharmaceutical composition comprising a chimeric IL-4 polypeptide, an immune cell expressing or treated with a chimeric IL-4 polypeptide, or a nucleic acid encoding a chimeric IL-4 polypeptide as described herein in combination with a pharmaceutically acceptable carrier.

[0172] Peptide Linkers

[0173] In certain embodiments, the chimeric IL-4 polypeptide comprises a cardiovascular inflammatory protease-sensitive linker between IL-4 and IL-4Ral ECD in the chimeric IL-4 polypeptide, which is sensitive to cleavage by a protease upregulated during the inflammatory process accompanying a myocardial infarction. In one embodiment, the cardiovascular inflammatory protease-sensitive linker is sensitive to cleavage by a serine protease. In another Attorney Docket No. 046483-7492WO 1(04069)

[0174] embodiment, the cardiovascular inflammatory protease-sensitive linker is sensitive to cleavage by a matrix metalloproteinase (MMP).

[0175] Non-limiting examples of a cardiovascular inflammatory protease include membrane bound MMP (e.g., MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP), serine protease (e.g., plasmin, cathepsin G), lysosomal cysteine protease (cathepsin B), tryptase, chymase, collagenase (e.g., MMP-1, MMP-8, MMP-13), gelatinase (MMP-2, MMP-9), stromelysin (e.g., MMP-3), and membrane type (e.g., MMP-14). In certain embodiments, the cardiovascular inflammatory protease is MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, or MMP-14.

[0176] In certain embodiments, the cardiovascular inflammatory protease-sensitive linker between IL-4 and IL-4Ral ECD in the chimeric IL-4 polypeptide is an MMP-2 or MMP-9 cleavable linker. MMP-2 and MMP-9 cleavable linker sequences are known in the art and include, without limitation, PLGLAG (SEQ ID NO: 10), PLGLWA (SEQ ID NO: 11), PLGVRGK (SEQ ID NO: 12), AIPVSLR (SEQ ID NO: 13), PQGIAMG (SEQ ID NO: 14), HPVGLLAR (SEQ ID NO: 15), KGPLGVRG (SEQ ID NO: 16), HPVGLLAR (SEQ ID NO: 17), GPLGVRGK (SEQ ID NO: 18), GPLGLWAGG (SEQ ID NO: 19), GPQGIAGQR (SEQ ID NO: 20), KPVSLSYR (SEQ ID NO: 21), PLGMTS (SEQ ID NO: 22), PRALM (SEQ ID NO: 23), PRGMAS (SEQ ID NO: 24), AAALGNVAP (SEQ ID NO: 25), PQGLAG (SEQ ID NO: 26), GPQGARGQ (SEQ ID NO: 27), AVRWLLTA (SEQ ID NO: 28), GPLGLWAQ (SEQ ID NO: 29), VPLSLYSG (SEQ ID NO: 30), CGLDD (SEQ ID NO: 31), PVGLIG (SEQ ID NO: 32), PLGVRG (SEQ ID NO: 33), PLGLYL (SEQ ID NO: 34), GPLGIAGQ (SEQ ID NO: 35), GPLGMLSQ (SEQ ID NO: 36), GPQGIWGQ (SEQ ID NO: 37), PLGL (SEQ ID NO: 38), PLGLYAL (SEQ ID NO: 39), GPLGLAGGWGERDGS (SEQ ID NO: 40), LAGGWGERDGS (SEQ ID NO: 41), GPLGLAGGERDG (SEQ ID NO: 42), and PR(S / T)(L / I)(S / T) (SEQ ID NO: 43), and combinations thereof.

[0177] In some embodiments, the cardiovascular inflammatory protease-sensitive linker comprises 2, 3, 4, or 5 copies of a protease-sensitive sequence. In some embodiments, the cardiovascular inflammatory protease-sensitive linkers include and / or are flanked by glycine serine (GS) linkers. For example, in certain embodiments, the MMP-2 or MMP-9-sensitive linker sequence is GSGGPGPAGMHGLPGGS (SEQ ID NO: 44), SGGPGPAGLKGAPGS Attorney Docket No. 046483-7492WO 1(04069)

[0178] (SEQ ID NO: 45), (GGGS)2(HPVGLLAR)3(GGGS)2(SEQ ID NO: 46), or (GGGS)2(VPLSLYSG)3(GGGS)2(SEQ ID NO: 47).

[0179] In an embodiment, the peptide linker between IL-4 and the targeting agent in the chimeric IL-4 polypeptide comprises an amino acid sequence including, but not limited to, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO: 48), (GGGS)n (SEQ ID NO: 49), and (GGGGS)n(SEQ ID NO: 50), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 51), GGSGG (SEQ ID NO: 52), GSGSG (SEQ ID NO: 53), GSGGG (SEQ ID NO: 54), GGGSG (SEQ ID NO: 55), GSSSG (SEQ ID NO: 56), GGGGS (SEQ ID NO: 57), (GGGGS (SEQ ID NO: 58), (GGGGS)4(SEQ ID NO: 59), and the like.

[0180] Amino sequences of exemplary functional domains for use in the present invention can be found in Table 1. It should be understood that where an amino acid sequence is identified by a SEQ ID NO: as shown in Table 1, the disclosure should be further construed as including its corresponding nucleic acid, such as described in Table 1 below.

[0181] Table 1: Amino Acid Sequences

[0182]

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[0184]

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[0186]

[0187] Attorney Docket No. 046483-7492WO 1(04069)

[0188]

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[0190]

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[0194]

[0195] Nucleic Acids

[0196] In another aspect, the present invention provides a nucleic acid which may be used to express the chimeric IL-4 polypeptide as described herein. Suitable vectors include, e.g., plasmids, expression vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in any way as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR40, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0197] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Th c. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., murine leukemia Attorney Docket No. 046483-7492WO 1(04069)

[0198] virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0199] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0200] In certain embodiments, the vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g, WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0201] In some embodiments, the expression vector comprises additional elements that will aid in the functional expression of the chimeric IL-4 polypeptide described therein. In some embodiments, an expression vector comprises a mammalian promoter. In one embodiment, the vector comprises an elongation-factor-1 -alpha (EF-la) promoter.

[0202] In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., a lentiviral vector) of the present invention.

[0203]

[0204] In some embodiments, the nucleic acids are encapsulated in a lipid nanoparticle (LNP). As used herein, the term “lipid nanoparticle (LNP)” refers to lipid-based particles in the submicron range which include one or more lipid components as described herein. LNPs can have structural characteristics of liposomes and / or have alternative non-bilayer types of Attorney Docket No. 046483-7492WO 1(04069)

[0205] structures, which can be used to systemically deliver nucleic acid based drugs, including, for example, the antisense and RNAi molecules of the present disclosure.

[0206] In some embodiments, the LNP formulation comprises one or more cationic lipids.

[0207] Cationic lipids are lipids that carry a net positive charge at any physiological pH. The positive charge is useful for association with negatively charged therapeutic agents, including the antisense and RNAi molecules of the present disclosure.

[0208] In some embodiments, the sizes and charge ratios and other physical properties (e.g., membrane fluidity) of LNPs are optimized for increased cell transfection and delivery.

[0209] Lipid particles can comprise, for example, cationic lipids, neutral lipids, amino acid- or peptide-based lipids, polyethylene glycol (PEG)-lipids, e.g., lipids with PEG chains such as hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHE), 1, 2-distearoyl-glycero-3-phosphoethanolamine-N-[methoxy (PEG)-2000] (DSPE-PEG2000), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (PEG)-2000] modified with a maleimidic group in the distal end of the chain 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (PEG)-2000], DSPE-PEG2000-MAL, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (DMPE-PEG550), 1, 2-dioleoyl- 1-3 -trimethylammonium propane (DOTAP), and those with a glycerol backbone e.g., DMG-PEG, DSG-PEG (DMG-PEG2000) etc. As used herein, a liposome is a structure comprising lipid-containing membranes enclosing an aqueous interior. For example, lipid-based formulations can be used to deliver nucleic acid agents of the present invention, e.g., siRNAs, miRNAs, oligonucleotides, modified mRNAs and other types of nucleic acid molecules.

[0210] Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including l,2-diacyl-glycero-3 -phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingogly colipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; l,2-diacyl-sn-glycero-3 -phosphoethanolamine, including, but not limited to, 1,2-di oleylphosphoethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine (DHPE), 1,2-di stearoylphosphatidylcholine (DSPC), 1,2- Attorney Docket No. 046483-7492WO 1(04069)

[0211] dipalmitoyl phosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and / or synthetic (e.g., saturated and unsaturated l,2-diacyl-sn-glycero-3-phosphocholines, l-acyl-2-acyl-sn-glycero-3 -phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids.

[0212] In some embodiments, the LNP is conjugated to a liver-binding moiety to target the loaded LNP to the liver. Thus, in certain embodiments, the LNP is conjugated to GalNAc, a high affinity ligand for the asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalizing and subsequent clearing circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to the LNP of the instant disclosure can be used to target loaded LNPs (i.e., LNP encapsulating a polynucleotide) to the ASGPR expressed on cells. In some embodiments, an LNP of the instant disclosure is conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target a loaded LNP to an ASGPR expressed on human liver cells (e.g., human hepatocytes).

[0213] In some embodiments, an LNP of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the LNP is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3 or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an LNP is conjugated to one or more bivalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties.

[0214] Compositions and methods for preparing LNP formulations are well known to those skilled in the art, and are further described in e.g., U.S. Patent Application Nos.

[0215] US2021 / 0317461 Al, US2015 / 0118288 Al, and U.S. Patent No. 9,579,338B2, the disclosures of which are expressly incorporated by reference herein.

[0216] Method

[0217]

[0218] the Chimeric IL-4

[0219]

[0220] In another aspect, a method for producing the chimeric IL-4 polypeptide comprises culturing a host cell containing a nucleic acid encoding a chimeric IL-4 polypeptide described herein in a culture medium under conditions such that the chimeric IL-4 polypeptide is Attorney Docket No. 046483-7492WO 1(04069)

[0221] expressed. The chimeric IL-4 polypeptide may be recovered and / or purified from the culture medium (when secreted) or the host cells in which the polypeptide is produced.

[0222] The chimeric IL-4 polypeptide of the present invention may be produced using virtually any method known to the skilled artisan. Typically, recombinant polypeptides are produced by transforming a suitable host cell with a polypeptide-encoding nucleic acid molecule in a suitable expression vehicle and culturing the transformed cells in an appropriate culture medium.

[0223] Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. A polypeptide of the invention may be produced in a prokaryotic host (e.g., E. co / / ) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp.

[0224] 1987).

[0225] A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, retroviruses, lentiviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowlpox viruses, herpesviruses, and vectors derived from combinations thereof.

[0226] In some embodiments, the method comprises culturing a cell transiently or stably expressing the chimeric IL-4 polypeptide from the cultured cells. Any cell capable of producing the chimeric IL-4 polypeptide may be used. In certain embodiments, the chimeric IL-4 polypeptide-expressing cell is of eukaryotic or mammalian origin. Cells from various tissue cell types and cell lines may be used to express the chimeric IL-4 polypeptide. In some embodiments, the cell is a yeast cell, an insect cell or a bacterial cell. Attorney Docket No. 046483-7492WO 1(04069)

[0227] An expression vector encoding the chimeric IL-4 polypeptide can be introduced into a cell by any conventional method, such as by naked DNA technique, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated infection, physical or chemical agents or treatments, electroporation, etc. In addition, cells may be transfected with an expression vectors containing a selectable marker for facilitating selection of stably transformed clones expressing the chimeric IL-4 polypeptide. The chimeric IL-4 polypeptides produced by such cells may be collected and / or purified according to techniques known in the art, such as by centrifugation, chromatography, etc.

[0228] Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are CHO DHFR' cells and mouse LTK' cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in

[0229] non-supplemented media.

[0230] The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, or hygromycin. The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puromycin. Attorney Docket No. 046483-7492WO 1(04069)

[0231] Exemplary cells for expressing the chimeric TL-4 polypeptide include human Jurkat, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, and mouse WEHI fibrosarcoma cells. In addition, stably transformed, chimeric IL-4 polypeptide producing cell lines may be produced using primary cells immortalized with c-myc or other immortalizing agents.

[0232] In some embodiments, the cell line expresses at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg of the chimeric IL-4 polypeptide / liter of culture.

[0233] The chimeric IL-4 polypeptides may be isolated from cells following culture and maintenance in any appropriate culture medium, such as RPMI, DMEM, and AIM V® . The chimeric IL-4 polypeptides can be purified using conventional protein purification methodologies (e.g., affinity purification, chromatography, etc.), including the use ofProtein-A or Protein-G immunoaffinity purification. In some embodiments, the chimeric IL-4 polypeptides are engineered for secretion into culture supernatants for isolation therefrom. Once the chimeric IL-4 polypeptide is expressed, it may be isolated, for example, using affinity chromatography. In some embodiments, to facilitate purification of the recombinant polypeptide, the polypeptide comprises an epitope tag fused thereto. The polypeptide is then isolated using an antibody against the epitope tag. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra).

[0234] Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, which binds to a nickel column. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

[0235] Methods of Treatment

[0236] In another aspect, a method for treating a cardiovascular condition characterized by excessive inflammation. In some embodiments, the disease is a disease related to (e.g. caused by) heart muscle damage (e.g. myocardial infarction, cardiomyopathy, heart failure). In some instances, “disease” or “condition” refers to myocardial infarction, cardiomyopathy, or heart failure. In some embodiments, the disease is heart muscle damage. In some embodiments, the Attorney Docket No. 046483-7492WO 1(04069)

[0237] disease is myocardial infarction. In some embodiments, the disease is heart failure. In embodiments, the method improves cardiac function following administration.

[0238] In some embodiments, a method for treating myocardial infarction comprises administering to a subject in need thereof a therapeutically effective amount of a chimeric IL-4 polypeptide as described herein.

[0239] In another aspect, a method for treating ischemia / reperfusion injury comprises administering to a subject in need thereof a therapeutically effective amount of a chimeric IL-4 polypeptide as described herein. In some embodiments, the ischemia / reperfusion injury is a cardiac ischemia / reperfusion injury selected from the group consisting of acute cardiac ischemia, chronic cardiac ischemia, chronic coronary disease and acute coronary syndrome. In some embodiments, the ischemia / reperfusion injury is caused by stroke, organ transplantation (e.g., kidney and liver transplantation), ovarian torsion, small bowel obstruction.

[0240] In another aspect, a method for preventing or reducing the severity of heart failure in a subject having experienced a myocardial infarction comprises administering to a subject in need thereof a therapeutically effective amount of a chimeric IL-4 polypeptide as described herein.

[0241] In some embodiments, the chimeric IL-4 polypeptide is administered intravenously. In some embodiments, the chimeric IL-4 polypeptide is administered via intracoronary delivery. In some embodiments, the chimeric IL-4 polypeptide is administered via intramyocardial delivery. In some embodiments, the chimeric IL-4 polypeptide or the pharmaceutical composition is administered via intracoronary infusion, retrograde injection, or injection into adventitial space. In some embodiments, the chimeric IL-4 polypeptide or the pharmaceutical composition is administered following angioplasty or following stent placement.

[0242] In another aspect, a method for treating or reducing the severity of heart failure in a subject having experienced a myocardial infarction, comprises administering to a subject in need thereof a therapeutically effective amount of a cell expressing the chimeric IL-4 polypeptide. In certain embodiments, the cell is a bone marrow derived macrophage (BMDM).

[0243] In some embodiments, the chimeric IL-4 expressing cells are administered intravenously. In some embodiments, the chimeric IL-4 expressing cells are administered via intracoronary or intramyocardial delivery. In some embodiments, the chimeric IL-4-expressing cells are administered between 2-4 hours after a myocardial infarction. In some embodiments, the chimeric IL-4-expressing cells are administered on at least seven consecutive days following the Attorney Docket No. 046483-7492WO 1(04069)

[0244] myocardial infarction. In some embodiments, the chimeric IL-4-expressing cells are administered via intramyocardial delivery. In some embodiments, the chimeric IL-4-expressing cells are administered via intracoronary infusion, retrograde injection, or injection into adventitial space. In some embodiments, the IL-4-expressing cells are administered following angioplasty or following stent placement.

[0245] In another aspect, a method of performing cardiovascular surgery in a subject in need thereof comprises performing a surgical procedure and administering to the subject a therapeutically effective amount of a chimeric IL-4 as described herein. In some embodiments, the surgical procedure is an angioplasty or stent placement. In some embodiments, the chimeric IL-4 polypeptide is administered during the surgical procedure between 2-6 hours after the myocardial infarction.

[0246] For the prevention or treatment of disease, the appropriate dosage may depend on the severity and course of the disease, whether the chimeric IL-4 polypeptide is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the chimeric IL-4 polypeptide, and the discretion of the attending physician.

[0247] A dose of the chimeric IL-4 polypeptide or chimeric IL-4-expressing cells may be administered to the subject in a single dose or multiple doses. In some embodiments, the chimeric IL -4 polypeptide or chimeric IL-4-expressing cells are administered between 2-4, 2-6, or 3-4 hours after the myocardial infarction. In some embodiments, the chimeric IL-4 polypeptide or chimeric IL-4-expressing cells are administered at least once a week following the myocardial infarction. In some embodiments, the chimeric IL-4 polypeptide or chimeric IL-4-expressing cells are administered at least once every two weeks, every 3 weeks, every 4 weeks following the myocardial infarction. In some embodiments, the chimeric IL-4 polypeptide or chimeric IL-4-expressing cells are administered at least once a month following the myocardial infarction. In some embodiments, a dose of the chimeric IL-4 polypeptide or chimeric IL-4-expressing cells are administered by rapid intravenous infusion.

[0248] In embodiments, the method improves cardiac function (e.g., promotes myocardial salvage), increases cardiomyocyte survival, increases neovascularization, modulates (e.g., reduces relative to the absence of the block copolymer or pharmaceutical composition) the inflammatory response, modulates (e.g., reduces relative to the absence of the block copolymer or pharmaceutical composition) cardiomyocyte metabolism, reduces infarct size, reduces fibrosis Attorney Docket No. 046483-7492WO 1(04069)

[0249] (eg., cardiac fibrosis), reduces or inhibits negative LV remodeling, reduces LV volume (e.g. diastolic and / or systolic), increases infarct wall thickness, inhibits MMP activity, or prevents extracellular matrix degradation following administration.

[0250] In embodiments, the method improves cardiac function (e.g., promotes myocardial salvage). In embodiments, the method increases cardiomyocyte survival. In embodiments, the method increases neovascularization. In embodiments, the method modulates (e.g., reduces relative to the absence of the block copolymer or pharmaceutical composition) the inflammatory response. In embodiments, the method modulates (e.g., reduces relative to the absence of the block copolymer or pharmaceutical composition) cardiomyocyte metabolism. In embodiments, the method reduces infarct size. In embodiments, the method reduces fibrosis (e.g., cardiac fibrosis). In embodiments, the method reduces or inhibits negative LV remodeling. In embodiments, the method reduces LV volume (e.g. diastolic and / or systolic). In embodiments, the method increases infarct wall thickness.

[0251] Pharmaceutical Compositions and Formulations

[0252] Also provided are compositions including the masked IL-4 composition of the present invention for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent. The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In Attorney Docket No. 046483-7492WO 1(04069)

[0253] some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0254] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0255] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active cardiovascular agents or drugs. The pharmaceutical composition contains the chimeric IL- Attorney Docket No. 046483-7492WO 1(04069)

[0256] 4 polypeptide in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the polypeptide.

[0257] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0258] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g, methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0259] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, Attorney Docket No. 046483-7492WO 1(04069)

[0260] for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0261] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e. ., by fdtration through sterile filtration membranes.

[0262] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0263] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0264] EXPERIMENTAL EXAMPLES

[0265] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein. Attorney Docket No. 046483-7492WO 1(04069)

[0266] masked IL-4

[0267]

[0268] In a pilot study, mice treated with intravenous (iv) injections of a collagen-binding IL-4 immunocytokine after permanent coronary artery ligation exhibited a trend towards improved left ventricular ejection fraction compared to the vehicle-treated mice after one month. Although promising, this approach is not infarct-targeted due to collagen’s ubiquitous expression.

[0269] Furthermore, a worrisome increase in CD8+ T cells was observed in treated mice, indicating uptake by IL-4R+ cells in the blood. On-target, off-tissue uptake is an issue for many immunocytokines that limits their clinical application. While these data highlight the therapeutic potential of IL-4 immunocytokines for HF, they also underscore a need to engineer infarct selectivity.

[0270] To engineer IL -4 with infarct selectively, conditional masking is combined with infarct targeting. Conditional masking describes obscuring a cytokine’s active domain until it has reached an intended tissue. One method to achieve conditional masking relies on fusing a cytokine to its own receptor binding domain via a matrix metalloproteinase (MMP) cleavable linker as depicted in FIG. 1. Since MMPs are proteases that are locally upregulated in inflamed tissues, this strategy constrains the activity of an immunocytokine in an infarct.

[0271] To ensure mask engagement when the linker is intact and disengagement upon linker cleavage, the affinity between the cytokine and its mask is modified by identifying residues critical for a cytokine / receptor interaction. When inflamed, the tissue’s ECM undergoes a unique remodeling. These changes, although mostly studied in cancer, are also detectable as early as 72 hours after an MI. For example, inflammation promotes alternative splicing of the ECM protein fibronectin (Fn), resulting in the inclusion of alternative exons extra domain A (Fn-EIIIA) and extra domain B (Fn-EIIIB). Fn-EIIIB is an effective target to diagnose patients with excessive inflammation and to improve the infarct exposure of a conditionally masked IL-4, since it is scarcely expressed in healthy tissues.

[0272] To mask IL-4, a flexible, MMP-cleavable linker is used to attach LMJ2.5I-IL-4 to the IL-4Ral extracellular domain (ECD). Because of the strong affinity between IL-4 and IL-4Ral ECD (4 nM), to enable mask disengagement upon linker cleavage, an IL-4Ral ECD mutant with a lower binding affinity (e.g., 20 nm-100 nM) is selected for use in a conditional IL-4 mask. Attorney Docket No. 046483-7492WO 1(04069)

[0273] Computational analysis has revealed three key binding epitopes between IL-4 and IL-4Ral ECD vital to their interaction. After mutating each epitopes’ residues to alanine to produce lower affinity IL-4Rod ECD mutants, MMP-cleavable linkers are fused between LMJ2.5LIL4 and the IL-4Ral ECD mutants.

[0274] To identify an appropriate IL-4Ral ECD mutant for use in the immunocytokine, mouse macrophage RAW264.7 cells and mouse bone marrow derived macrophages are exposed to varying immunocytokine doses before and after incubation with MMPs. The magnitude of IL-4-stimulated STAT6 phosphorylation, CD206, CD301b, CX3CR1, CD86, and Argl expression is quantified by flow cytometry. After incubation with MMPs, an LMJ2.5I-IL4-Mask is produced, which similarly polarizes macrophages toward a reparative phenotype as both unmasked constructs, LMJ2.5I-IL4 and NJT6-IL4. Unmasked IL-4 (LMJ2.5I-IL4) and permanently masked IL-4 (LMJ2.5IIL4- permMask, with a non-cleavable linker) serve as additional positive and negative controls.

[0275] Example 2: Therapeutic effects of LMJ2.5I-IL4-Mask on post-MI HF

[0276] To evaluate the therapeutic efficacy of the LMJ2.5I-IL4-Mask selected in Example 1, the gold-standard mouse model of HF after MI may be utilized. To induce the MI, the left anterior descending coronary artery (LAD) is permanently ligated surgically. Mice are randomized to treatment cohorts, receiving either the vehicle saline or a single dose of LMJ2.5I-IL4-Mask on either 1, 3 or 7 days after ML All mice are evaluated on day 28. Left ventricular ejection fraction and wall thickness are used as primary endpoints for assessing efficacy. Secondary endpoints of efficacy include flow cytometry, histology, RT-qPCR, and ELISA to detail myocardial immune cell frequencies and phenotypes, tissue remodeling and vascularization, infarct size, inflammatory cytokines, and chemokines. Blood leukocyte frequency and serum concentration of inflammatory mediators measured by flow cytometry and ELISA provide a primary assessment of treatment toxicity.

[0277] To parse the contribution of targeting and conditional masking, the performance of LMJ2.5I-IL4-Mask is compared to the following additional controls: untargeted, unmasked IL-4 (NJT6-IL4); Fn-EIIIB -targeted, unmasked IL-4 (LMJ2.5IIL4); and Fn-EIIIB-targeted, permanently masked IL-4 (LMJ2.5I-IL4-permMask, with a non-cleavable linker). A day 3 intervention is beneficial for attenuating infarct inflammation, since day 3 post-MI is the natural Attorney Docket No. 046483-7492WO 1(04069)

[0278] turning point for an inflammatory to reparative phenotype and coincides with increased Fn expression in an infarct. As compared to the aforementioned control immunocytokines, the LMJ2.5I-IL4-Mask improves cardiac function with minimal peripheral immune effects.

[0279] Example 3: EIIIB-targeted nanobody accumulates in a myocardial infarct

[0280] In order to determine accumulation of targeted nanobodies in the infarct compared to remote regions of the heart, fluorescent distribution of AF750 conjugated nanobodies was determined. FIG. 2A depicts the engineered nanobodies used. FIG.2B depicts a schematic of the study timeline. Mice underwent left anterior descending coronary artery ligation (LAD) surgery to stimulate myocardial infarction on day 0. Mice received a retro-orbital injections on day 7 of AF750 conjugated nanobodies after MI. Hearts were excised 24 hours later. FIG. 2C shows a quantification of fluorescence of AF750 conjugated nanobodies as seen in FIG. 2D. Fluorescence was quantified using ImageJ by diving fluorescence of infarct area by remote area to generate infarct-to-remote AAF750 ratio. FIG.2D shows fluorescent imaging of hearts tissue sliced 1 mm from apex to base. Far-red imaging of AF750 indicates presence of nanobody (top). Autofluorescence in the red channel was used to demarcate the infarct area (bottom). Infarct-to-remote ratio and accompanying fluorescence images of the heart portray the specificity of EIIIB targeted nanobody to the infarct.

[0281] Example 4: EIIIB-targeted IL-4 improves cardiac repair

[0282] The effect of EIIB-targeted IL-4 constructs in the relevant mouse model for MI, LAD permanent ligation was examined. FIG.3A depicts Fn-EIIIB targeted IL-4, non-Fn-EIIIB targeted IL-4 (i.e., anti-huTNC), and collagen-targeted (Lumican-MSA) IL-4 used in this study.

[0283] FIG. 3B depicts a schematic of the study timeline. Mice underwent left anterior descending coronary artery ligation (LAD) surgery to stimulate myocardial infarction on day 0. Mice received retro-orbital injections (morning and night) on day 3, 5, 7 after MI of indicated treatment. FIG. 3C shows representative parasternal long-axis echocardiography images of the left ventricle. Outline depicts left ventricular end systolic volume (left) and left ventricular end diastolic volume (right). (FIGs. 3D-3F) Quantification of cardiac ejection fraction (FIG. 3D), longitudinal strain (FIG. 3E), radial strain (FIG. 3F), end diastolic volume (FIG. 3G), and end systolic ) (FIG. 3H) as measured by echocardiography. Attorney Docket No. 046483-7492WO 1(04069)

[0284] The results show improved ejection fraction and other relevant heart measures, such as longitudinal and radial strain, following administration of EIIIB -targeted IL-4 improve ejection fraction and other relevant heart measures, such as longitudinal and radial strain, consistent with improved cardiac healing following post-MI treatment. These improvements were also observed following administration of collagen-targeted IL-4 immunocytokines (data not shown).

[0285] Example 5: Cardiac functional improvement as measured by echocardiography Cardiac functional improvement was measured by echocardiography as shown in FIG. 4. FIGs. 4A-4B show quantitated analyses of left ventricular mass, heart rate, cardiac output, end diastolic volume, end systolic volume, radial strain, and longitudinal strain obtained from the mice in FIG. 3 as measured by echocardiography. The results further supplement FIG. 3 to show that mice treated with EIIIB targeted IL-4 and collagen targeted IL-4 exhibit improved cardiac function.

[0286] Example 6: EIIIB-targeted IL-4 drive improvement do not lead to persistent immunological changes in peripheral blood

[0287] IL-4 is a cytokine naturally existing in the body and both IL-4 receptor subsets are on many immune cells. Thus, it is important to determine the effects of an intravenous injection of IL-4 on all immune cell types in the blood after treatment. Blood was collected retro-orbitally on days 10 and 28 after MI to evaluate immunological changes in peripheral blood. The improvements using Fn-EIIIB targeted IL-4 were not accompanied by persistent immunological changes in peripheral blood. FIG. 5A depicts a schematic of the study timeline. Mice underwent left anterior descending coronary artery ligation (LAD) surgery to stimulate myocardial infarction on day 0. Mice received retro-orbital injections (morning and night) on day 3, 5, 7 after MI of indicated treatment. Peripheral blood counts of immune subsets on day 10 and day 28 are shown in FIGs. 5B-5C, respectively. Only statistically significant comparisons are displayed. FIG. 6 shows the flow cytometry gating strategy for obtaining the peripheral blood counts of the immune subsets depicted in FIGs. 5B-5C.

[0288] The results show that Fn-EIIIB -targeted IL-4 improvement in cardiac function do not lead to persistent immunological changes in peripheral blood, since most immune cell subsets were shown to be unaffected by treatment. Only collagen targeted IL-4 (Lumican) and EIIIB Attorney Docket No. 046483-7492WO 1(04069)

[0289] targeted IL-4 (LMJ2.5I) were shown to significantly impact CD8+ T cells in the blood.

[0290] Nevertheless, these changes do not remain for EIIIB -targeted IL-4 as evidenced by the fact that the immune cell subsets were not significantly different 28 days after MI as compared to saline treated mice in the EIIIB -targeted IL-4 group.

[0291] Example 7; IL-4 Immunocytokines alter macrophage phenotype

[0292] It was of interest to demonstrate that all IL-4 immunocytokines have the same potency as wild type IL-4, even when conjugated to the targeted nanobodies. FIGs. 7A-7C shows that IL-4 immunocytokines alter macrophage phenotype. FIG. 7A depicts a schematic of the study design. Bone marrow derived macrophages (BMDM) from C57BL / 6 were established with M-CSF incubation for 5 days. Once established, BMDM were treated with IL-4 immunocytokines at varying IL-4. LPS and saline were only evaluated as single dose conditions. FIGs. 7B-7C, show expression of surface CD86, CX3CR1, CD301b, CD206, and Argl in terms of median fluorescence intensity ((MFI, FIG. 7B) and percentage of expressing cells ( FIG. 7C). FIG. 7D shows Fn-EIIIB binding as determined by ELISA for LMJ2.5I-IL4 and NJT6-IL4. FIGs. 7E-7F show Commassie Blue stained SDS-Page gels with LMJ2.5I-IL4 and NJT6-IL4 (FIG. 7E) and Lumican-MSA-IL4 (FIG. 7F).

[0293] As demonstrated by both MFI and percentage of expressing cells, all engineered IL-4 immunocytokines have the same potency as wild-type IL-4 and display an appropriate dose response. Additionally, the expression of IL-4 treated cells is different than LPS and saline controls.

[0294] Example 8; Specifically targeting infarcted myocardium by binding nascent Fn-EIIIB After myocardial infarction (MI), the cardiac extracellular matrix undergoes dynamic changes in abundance and composition, positioning these nascent matrix proteins as promising targets for infarct therapeutic delivery. To gain insights into the onset of this remodeling, previously deposited spatial RNA-sequencing was analyzed to examine the temporal changes in gene expression of extracellular matrix components after MI in mice. We observed that fibronectin (Fnl') (FIGs. 8A-8B) and several collagen types (Collal, Col3ctl, Col4al) (FIGs.

[0295] 9A-9F) are significantly upregulated in infarcts as early as 3 days post-MI (FIG. 8B) confirming Attorney Docket No. 046483-7492WO 1(04069)

[0296] the nascent matrix can serve as a tissue target for the delivery of therapeutics during the reparative phase of cardiac remodeling.

[0297] Specific delivery to an infarct is important, particularly for pleiotropic cytokines which may have effects on many cell types in other tissues. During matrix remodeling, most matrix genes generate transcripts that undergo extensive alternative splicing, producing multiple protein variants from one gene. Although these variants are not discernable from most transcriptomics datasets, alternatively spliced variants of the nascent matrix could serve as infarct-specific targets for therapy. Fibronectin extra domain B (Fn-EIIIB), which has been shown to be uniquely enriched in tissues undergoing extensive remodeling such as cancer and arthritis and is absent from healthy tissues, was a focus of this study. To determine whether Fn-EIIIB protein is expressed after myocardial infarction, a mouse model of MI was employed in which the left anterior descending (LAD) coronary artery is permanently ligated. Infarcts showed deposition of Fn-EIIIB in the infarct, and not the remote region (FIG. 8G). Fn-EIIIB is sequence conserved across several species. Consistent with the findings in the mice, human infarcted myocardium also expressed Fn-EIIIB (FIG. 8D placeholder). Importantly, Fn-EIIIB was not observed in the myocardium of healthy individuals (FIG. 8D placeholder).

[0298] To assess whether infarct Fn-EIIIB is a tractable target for therapeutic delivery, a small 13 kDa nanobody called LMJ2.5I was leveraged which had previously engineered to bind Fn-EIIIB with a 25 picomolar affinity (FIG. 8E). As a non-targeted, size-matched control in the mouse, a nanobody against human tenascin C (referred to as NJT6) was used (FIG. 8A). Using dye-labeled constructs, we found that intravenously injected LMJ2.5I could selectively accumulate in an infarct, unlike NJT6 (FIGs. 8C-8D). Negligible accumulation of LMJ2.5I was observed relative to NJT6 in the lung, spleen, kidney, and liver (FIGs. 8E-8F). These observations indicate to that Fn-EIIIB binding LMJ2.5I could indeed be employed to target therapeutic payloads specifically to the infarcts, even when not reperfused, in the days following an MI.

[0299]

[0300] infarct-taraeted interleukin-4 (IL-4) imm

[0301]

[0302] Interleukin-4 (IL -4) is widely recognized for its ability to polarize macrophages toward a more tissue reparative phenotype, making it a compelling therapeutic candidate for infarct targeting post-MI. To test this, we generated immunocytokine fusion proteins with IL-4. Murine Attorney Docket No. 046483-7492WO 1(04069)

[0303] IL-4 was fused to the C-terminus of the Fn-EIIIB nanobody LMJ2.5I separated by a glycineserine linker (hereafter LMJ2.5I-IL4). We also synthesized a non-targeted, size-matched immunocytokine using the control nanobody NJT6 (hereafter NJT6-IL4) (FIG. 10A). LMJ2.5L IL4 bound to Fn-EIIIB and triggered expression of arginase- 1 (Argl), a canonical marker associated with a reparative phenotype, in murine bone marrow derived macrophages (BMDM) (wild-type IL-4 ECso = 0.25 nM; LMJ2.5LIL4; EC50 = 0.27 nM; NJT6-IL4 EC50 = 0.61 nM) (FIGs. 10B-10C). To determine whether similar bioactivity could be observed with human macrophages, we designed a human-equivalent immunocytokine using the same LMJ2.5I nanobody, since it is cross-reactive, fused to human IL-4 instead. Because Argl is not a reliable marker of IL-4 polarization in human macrophages, we instead assessed expression of the mannose receptor CD206. Human peripheral blood monocyte-derived macrophages (PBMDM) treated with LMJ2.5I-IL4 exhibited an increase in CD206 expression (FIG. 10D). In comparison to native human IL-4 though, we observed a slight decrease in maximal signaling and potency (huIL-4 EC50 = 0.065 nM, LMJ2.5I-huIL4 ECso= 0.42 nM), likely due to modest steric hindrance of this construct on human cells (FIG. 10D).

[0304] Example 10: Infarct-targeted IL-4 immunocvtokine promotes cardiac after MI Localized interleukin-4 has been shown to accelerate repair in other injured tissues such as the skin after topical administration and skeletal muscle after intramuscular injection (Lauterbach, A.L. et al., NPJ Regen Med. 2023 Sep 11: 8(1):49; Raimondo, T.M. et al., Proc. Natl. Acad. Sci. USA 2018 Oct. 16; 115(42): 10648-10653), but has not yet been targeted to an infarcted heart. It was hypothesized that localization of IL-4 to an infarcted myocardium using LMJ2.5I-mediated Fn-EIIIB targeting might exhibit new biological effects relative to the untargeted form of this cytokine. The impact of murine IL-4 immunocytokines, LMJ2.5I-IL4 and NJT6-IL4, were evaluated for cardiac repair in a mouse model of MI. After a permanent LAD ligation to induce MI, mice were treated with intravenous IL-4 immunocytokine or saline 3, 5 and 7 days later (FIG. 10E), coinciding with nascent Fn-EIIIB infarct expression (FIG. 10E). The additional rationale for this sequencing was to allow the IL-4 immunocytokine to reinforce the reparative phase of myocardial remodeling, rather than intervene in the initial recruitment of inflammatory leukocytes which occurs in the first hours to days immediately after MI which has already been shown to improve cardiac repair. Strikingly, mice treated with our Fn-EIIIB infarct Attorney Docket No. 046483-7492WO 1(04069)

[0305] targeted immunocytokine, LMJ2.5LIL4, exhibit robust improvements in cardiac function 28 days post-MI compared to saline-treatments as quantified by left ventricular end-diastolic volume (EDV), end-systolic volume (ESV), and ejection fraction (LVEF: saline = 29%;

[0306] LMJ2.5I-IL4 = 45%) (FIGs. 10F-10G, FIGs. 12C-12D). By contrast, these parameters of cardiac function were not significantly improved with untargeted immunocytokine NJT6-IL4 (LVEF: NJT6-IL4 = 39%). (FIGs. 10F-10G, FIGs. 12C-12D). Notably, these data show that the therapeutic effect elicited by specifically targeting IL-4 to the infarct is superior in improving cardiac function compared to systemic exposure of the same IL-4 dose. Moreover, treated mice did not exhibit signs of cytokine toxicity, as assessed by weight loss and changes in abundance of peripheral blood immune cells.

[0307] Example 11: IL-4, irrespective of tissue targeting, reduced fibrosis

[0308] To understand the mechanisms underlying the observed therapeutic effect, we inspected the myocardial tissue after immunocytokine therapy, 28 days post-MI (FIG. 10H). The left ventricular walls of mice treated with LMJ2.5-IL4 were remarkably thicker compared to those treated with NJT6-IL4 or saline, indicative of improved tissue remodeling which is consistent with the observed improvement in cardiac function (FIGs. 10H-10I). LMJ2.5-IL4 also induced a reduction in myocardial fibrosis and matrix-producing myofibroblasts (FIG. 10J, FIGs. 13E-13G). Excessive fibrosis certainly impairs cardiac function. However, a comparable decrease in fibrosis and myofibroblasts was elicited by NJTJ-IL4 treatment, which failed to induce comparable therapeutic outcomes in terms of cardiac function and left ventricular tissue remodeling (FIG. 10J, FIGs. 13E-13G)

[0309] Example 12: Infarct-targeted IL-4 immunocvtokine stalls infarct expansion

[0310] Other tissue-level changes were investigated within the infarcts shortly after treatment to understand the differential impact of infarct-targeted IL-4 immunocytokine on myocardial repair. Since vascularization is critical for proper tissue repair and IL-4 has been implicated in angiogenesis after ischemic injury, the extent of vascularization was assessed three days after the last treatment. However, no differences in vessel density or abundance of CD31 cells in the infarct were observed across all treatment groups (FIGs. 13A-13B). Attorney Docket No. 046483-7492WO 1(04069)

[0311] Next, the extent of myocardial injury was quantified by immunostaining for cardiac troponin T (cTnT) on day 8 post-MI, one day after the final treatment (FIG. 14A). Intriguingly, despite identical permanent LAD ligation across all mice, LMJ2.5I-IL4 treatment resulted in smaller infarcts compared to NJT6-IL4 and saline (FIGs. 14A-14B). Since cardiomyocyte death can continue for days after Ml-a phenomenon called infarct expansion- we examined if infarct-targeted IL-4 reduced cardiomyocyte death ensuing later. Hearts excised on day 8 from mice treated with LMJ2.5I-IL4 had fewer terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells compared to NJT6-IL4 and saline controls (FIGs. 14C-14D). Altogether, these data suggest that infarct-targeting uniquely endows IL-4 with the ability to limit cardiomyocyte death, halt infarct expansion and thereby preserve cardiac function after Mi-effects not achieved with untargeted IL-4 delivery.

[0312] Example 13; Infarct-targeted IL-4 immunocytokine robustly reprograms infarct resident macrophages

[0313] To understand the cellular mechanisms underlying infarct-targeted IL-4’s protection, the immune response after MI was examined. Although influx of immune cells predominantly occurs in the initial three days post-MI, and thus prior to treatment, leukocyte abundance was nonetheless profiled to look for changes in treated animals, which may influence infarct expansion. By flow cytometry on infarct and remote myocardium, no differences were found in recruited neutrophils, monocytes, and macrophages between LMJ2.5-IL4, NJT6-IL4 and saline-treated mice (FIGs. 15A-15B). Changes in immune cell phenotype in macrophages, which comprise the vast majority (>70%) of the infarct leukocyte population during the time of treatment and is associated with tissue repair were further investigated (FIG. 15A). The extent of IL-4 polarization of macrophages was quantified by their Argl expression (FIGs. 14E-14F). Probing macrophage polarization 24 hours after the last dose of immunocytokine revealed that 67% of infarct macrophages in LMJ2.5I-IL4-treated mice expressed Argl, substantially greater than in NIT6-IL4- (50%) or saline-treated (21%) hearts (FIGs. 14E, FIGs. 15A-15B). When these infarct macrophages were stratified by their CCR2 expression, it was found that both NJT6-IL4 and LMJ2.5LIL4 increased Argl expression in CCR2+ macrophages (FIG. 14F). However, in the CCR2- macrophage subset, only LMJ2.5LIL4 robustly induced Argl (FIG. 14F). This effect is consistent with CCR2+ macrophages being monocyte-derived cells recently Attorney Docket No. 046483-7492WO 1(04069)

[0314] infiltrated from circulation, where both targeted and untargeted immunocytokines transit, whereas CCR2- macrophages represent tissue-resident cells within the infarct microenvironment, where LMJ2.5I-IL4 is preferentially retained. Importantly, Argl induction was restricted to the infarct, as Argl expression in remote myocardium remained at baseline across all treatment groups, demonstrating the infarct-specific activity of LMJ2.5LIL4 therapy.

[0315] Example 14: IL-4 polarization of inflammatory macrophages enhances efferocytosis To interrogate the molecular and functional consequences of robust IL-4 macrophage polarization, murine BMDM cells were treated with LMJ2.5I-IL4 alone or in combination with lipopolysaccharide to mimic the inflammatory milieu of the infarct in vitro (FIG. 14G). Since the days after an MI, macrophages are primarily responsible for engulfing dead and dying cells in a process called efferocytosis, the receptors that mediate this essential process were evaluated. MerTK and TREM2 expression was highest on BMDMs treated with both inflammatory stimuli and IL-4 (FIGs. 14H-14I). To assess their efferocytic capacity, pHRodo-labeled apoptotic cells were introduced to these polarized BMDMs. BMDMs treated with IL-4, with or without LPS, exhibited equivalent uptake of pHRodo-labeled apoptotic cells compared to unpolarized macrophages (FIG. 14J). Human PBMDM polarized with LPS exhibited a decrease in MerTK and TREM2 compared to baseline, which was partially recovered by the addition of LMJ2.5-hIL4 (FIGs. 14K-14M). Collectively, the in vivo and in vitro studies suggest that robust reprogramming with infarct-targeted IL-4 endows resident macrophages with enhanced efferocytic capacity.

[0316] Example 15: Modeling predicts immunocvtokine availability in infarct

[0317] Given the importance of infarct retention in mediating the reparative effects of IL-4 post-MI, a better understanding of how the features of these immunocytokines impact its infarct exposure was sought. To this end, a simple ordinary differential equation system was built that modeled our immunocytokines' disposition between two compartments — the blood and infarct — after an intravenous injection. In this model, the diffusive transport of the immunocytokine between the blood and infarct, kinetic binding of the immunocytokine to its matrix target Fn-EIIIB, and two modes of immunocytokine removal — clearance from the blood or through proteolytic degradation within the infarct are simulated (FIG. 16A). By parameterizing this Attorney Docket No. 046483-7492WO 1(04069)

[0318] model using empirically- measured kinetic rates and transport relationships, the concentration of immunocytokines in blood or infarct over time was determined (FIGs. 17B-17C). To connect these concentrations to the bioactivity of IL-4, fractional activities of the immunocytokines in the blood and infarct were evaluated (FIGs. 17D-17E) based on the ECso of IL-4 (4 nM). When immunocytokine concentration far exceeds the ECso, fractional activity approaches 100%, whereas at the ECso — eliciting half-maximal signaling — fractional activity equals 50% (FIGs.

[0319] 6D-6E) The area under the curve (AUC) of fractional activity over time yields immunocytokine exposure, or the duration of immunocytokine activity within the infarct or the blood.

[0320] To validate this model, the predicted exposure of LMJ2.5I-IL4 and NJT6-IL4 to experimentally measured Argl expression was compared in infarct macrophages after these treatments. The model predicted a 5.3-fold increase in infarct exposure for LMJ2.5I-IL4 relative to NJT6-IL4, closely matching the observed 5.0-fold increase in Argl MFI observed three days after the last treatment (FIG. 16B).

[0321] Equipped with this predictive model, it was of interest to investigate how the choice of immunocytokine’s matrix target or format could alter IL-4’s infarct exposure. Since matrix target deposition in an infarct is heterogeneous and challenging to exactly quantify, a broad range of concentrations of infarct matrix targets was considered to account for both rare (IE- 10 M) and abundant matrix targets (IE-6 M). According to the model, increased infarct matrix target expression improves a nanobody -based immunocytokine’s exposure, with a plateau in exposure occurring when all immunocytokine that has diffused into the infarct is bound to a target, no matter if there is excess available matrix target binding sites (FIG. 16C). Separately, three common formats for immunocytokines were investigated: nanobody- (shown in this work), a single chain variable fragment (scFv)-, and a full-length antibody fused to IL-4. The model predicted that a larger immunocytokine, such as that formatted with an antibody, would increase infarct exposure (FIG. 16D). To understand how both matrix-targeting and size interplay, a heat map relating immunocytokines varied in their matrix-target’s concentration and format to their duration of activity, i.e. exposure, in the infarct was generated. The results of this analysis found that large immunocytokines exhibit higher infarct exposure across a wide spectrum of infarct target concentrations. In contrast, small immunocytokines, such as the LMJ2.5I-IL4 nanobody, only exhibit a high infarct exposure when a target is highly expressed (FIG. 16E). Attorney Docket No. 046483-7492WO 1(04069)

[0322] 16: Collagen-binding IL-4 exhibits im cardiac remodeling, at the

[0323]

[0324] of immune-related

[0325]

[0326] Based on the model predictions of infarct exposure across a spectrum of immunocytokine permutations, it was hypothesized that a larger IL-4 immunocytokine targeting a more abundant matrix protein could further enhance therapeutic efficacy. Thus, collagen as a new target, as it is the most abundant component of the nascent extracellular matrix post-MI. Although collagen is not infarct-specific, it is infarct-associated and upregulated beginning at day 3 post-MI (FIG. 16F). Moreover, since the Fn-EIIIB -targeted nanobody immunocytokines exhibited no toxicity after repeated intravenous dosing, it was reasoned that the therapeutic window for IL-4 remained sufficiently broad to accommodate higher systemic exposure from a larger immunocytokine format.

[0327] To test the hypothesis that exposure-driven improvements in IL-4 therapy are attainable, Lumican-MSA-IL4 (FIG. 18A), an immunocytokine format was generated in which lumican, a native collagen I and IV binding protein is fused to large mouse serum albumin (MSA) and IL-4. First, it was confirmed that Lumican-MSA-IL4 (117.3 kDa) was able to polarize BMDMs in vitro (FIG. 19B). Following an identical treatment regimen post-MI as performed previously, the Lumican-MSA-IL4 immunocytokine was found to improve cardiac function as measured by LVEF relative to saline-treatment (Lumi can-MS A-IL4 = 47%) (FIGs. 18C-18D, FIGs. 20C-20D), but this was identical to the improvement achieved with LMJ2.5LIL4.

[0328] To identify whether Lumican-MSA-IL4 differentially affected tissue remodeling, ventricular wall thickness and fibrosis was examined. Left ventricular wall thickness was increased in Lumican-MSA-IL4-treated mice, similar to LMJ2.5I-IL4. However, unlike LMJ2.5I-IL4 and NJT6-IL4 which decreased fibrosis, Lumican-MSA-IL4 only exhibited a nonsignificant trend towards reduced fibrosis compared to saline controls (FIGs. 18E-18G, FIGs.

[0329] 21A-21B). At the cellular-level, analogous to LMJ2.5I-IL4, Lumican-MSA-IL4 treatment reduced cardiomyocyte apoptosis (FIGs. 21E-21H).

[0330] In light of many equivalent effects between LMJ2.5I-IL4 and Lumican-MSA-IL4, it was confirmed that increased infarct exposure with Lumican-MSA-IL4 was achieved as predicted by the model. Consistent with enhanced IL-4 delivery, Lumican-MSA-IL4 induced a robust Argl expression in all macrophage subsets within the infarct, including both CCR2+ and CCR2-populations. The magnitude of Argl induction was 2.87-fold higher than LMJ2.5I-IL4, slightly Attorney Docket No. 046483-7492WO 1(04069)

[0331] higher than the model's prediction of 1.25-fold increased infarct exposure with larger formats targeting abundant matrix proteins. Notably, Argl+ polarization also occurred in CCR2-macrophages in the remote myocardium (FIG. 181), indicating that Lumican-MSA-IL4 binds collagen beyond the infarct and exhibits broader cardiac distribution than the infarct-specific LMJ2.5I-IL4.

[0332] Next, it was of interest to examine whether the broader tissue distribution of Lumican-MSA-IL4 resulted in effects in other tissues. In profiling peripheral blood immune populations over the course of treatment, Lumican-MSA-IL4 treated mice exhibited a significant increase in Ly6Chi inflammatory monocytes compared to LMJ2.5I-IL4, NJT6-IL4, and saline controls (FIG. 18J). This effect was accompanied by persistent elevated CD8+ T cells in the blood and splenomegaly. (FIGs. 18K-18M). In contrast, LMJ2.5I-IL4 did not induce these systemic immunological changes despite achieving comparable cardiac functional improvement. Taken together, while infarct-associated Lumican-MSA-IL4 increased overall IL-4 delivery to the infarct relative to infarct-specific LMJ2.5I-IL4, both achieved comparable improvements in myocardial remodeling and cardiac function.

[0333] Example 17: Materials and methods

[0334] Mice

[0335] C57BL / 6 (stock 000664) mice were purchased from The Jackson Laboratory. All experiments were performed with 6-to-8-week-old male animals with age-matched control groups. Where appropriate, mice were randomly assigned to groups. Housing facilities followed a 12-hour dark / light cycle, room temperature was 18-22 °C and maintained humidity was between 40% and 60%. Mice were fed with respective diets ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania in accordance with federal, state and local guidelines.

[0336] Cell lines

[0337] Expi293 cells were obtained from Thermo Fisher Scientific and maintained shaking in suspension at 125 rpm in Expi293 Expression medium. H9C2(2-1) (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine, 4500 mg / L glucose, 1 mM sodium pyruvate, 1500 mg / L sodium bicarbonate and supplemented with 10% fetal bovine serum (FBS). All cells and cell assays were maintained at 37°C and 5% CO2. Attorney Docket No. 046483-7492WO 1(04069)

[0338] In vivo myocardial infarction studies and treatment

[0339] At day 0, mice underwent left anterior descending coronary artery permanent ligation. Mice received 0.1 mg / 10 g Ket / Xy / Ace as anesthetic agent via intraperitoneal injection. Mice also received 2 mg / kg of bupivacaine and 6 mg / kg of Mel oxi cam prior to surgery. To stimulate myocardial infarction, mice underwent a left thoracotomy, revealing the left ventricle and left main coronary artery system. The left anterior descending brand (LAD) of the left coronary artery was ligated by a 9-0 nylon suture at a height of 14 distance from the apex to induce a small distal MI. Mice received 2 mg / kg of buprenorphine on day 2 after surgery. On day 3, 5, 7 mice received a morning and evening retro-orbital injection of 0.373 nanomoles in 100 pL dose of each immunocytokine or saline. Mice were randomized into treatment groups immediately after surgery.

[0340] Echocardiography

[0341] End stage cardiac function was assessed via echocardiography via 2D VevoStrain echocardiography. Echocardiography was performed using a 2D VevoStrain (FUJIFILM VisualSonics Inc., Toronto, Canada). Prior to echocardiography, mice were anesthetized with an intraperitoneal injection (0.05 mg / g) of 2% avertin in order to maintain heart rate close to 600 beats per minute or above for full evaluation of left ventricular function. Complete 2D and Doppler ultrasound examinations were performed by utilizing multiple views. Heart rate (HR), End diastolic volume (EDV), end systolic volume (ESV), ejection fraction (EF), average peak radial strain % (S-rad), average peak longitudinal strain (S-long), and left ventricular mass (LVM) were all assessed by echocardiography.

[0342] In vivo stroke studies and biodistribution

[0343] Surgeon was blinded to protein. Proteins were labeled with 1251, resulting in 98% binding as measured by thin layer chromatography. Both transient and permanent middle cerebral artery occlusion were performed to model stroke. All groups were injected with radiolabeled protein 24 hours after injury. 30 minutes after injection, mice were perfused and organs collected to determine biodistribution.

[0344] In vivo nanobody biodistribution

[0345] LMJ2.5I and NJT6 were labeled with NHS-AF750. After MI LAD surgery, mice were injected with fluorescently labeled nanobodies. 24 hours after injection, mice were sacked and organs perfused and harvested. Whole organs were scanned on a flatbed scanner for AF750 as Attorney Docket No. 046483-7492WO 1(04069)

[0346] well as autofluorescence. Hearts were sliced and laid out on the scanner from apex to base.

[0347] AF750 and autofluorescence were quantified using FIJI.

[0348] Cloning

[0349] Codon optimized DNA encoding murine IL-4 was purchased as a GeneArt string fragment (Thermo Fisher). This fragment was cloned into the gWIZ vector (Gelantis) via InFusion (Clonetech) using custom primers (Thermo Fisher). This cloning generated two His-tagged proteins: NJT6-IL4 and LMJ-IL4. The sequences for LMJ2.5I and NJT6 were derived from prior published work [Lutz et al. 2022. PNAS Nexus] . Sequences for all fusion proteins are in Table 1. Plasmid DNA that encoded each fusion protein was transformed and amplified in Stellar Competent Cells (Takara Bio Inc) and then further purified using NucleoBond Xtra Midi E endotoxin-free midi-prep kit (Takara Bio Inc).

[0350] Protein purification

[0351] Suspension Expi293 cells were transfected with sterile-filtered plasmid DNA using ExpiFectamine 293 Transfection Kit (Thermo Fisher). All proteins were His-tagged and thus isolated from Expi293 media using TALON Metal Affinity Resin (Takara Bio Inc). Purified proteins were run with Novex Prestained Sharp Protein Ladder on a 4-12% NuPAGE Bis-Tris protein gel (Life Technologies) with 1% MES running to confirm molecular weight. Proteins were buffer exchanged into sterile PBS (Gibco), confirmed to contain less than 0.1 EU per injection by chromogenic LAL assay (Lonza) and 0.2 pm sterile-filtered. All proteins were stored at -80°C and were thawed on ice before use.

[0352] Cell collection

[0353] Peripheral blood (70 pL) was collected by retro-orbital bleeding with heparinized capillary tubes (BD) on days 10 and 28. Red blood cells were lysed with lx ACK lysis buffer (Gibco) prior to flow cytometry staining, acquisition, and analysis.

[0354] Bone marrow macrophage isolation

[0355] Femurs from C57BL / 6 were flushed with PBS and passed through a 100 pm nylon mesh, to isolate bone marrow cells. Cells were counted using a cell counting chamber and plated at a density between 4-5 x 106cells T-75 flask, and cultured in Rosewell Park Memorial Institute (RMPI) media (Gibco) enriched with 10% heat-inactivated FBS (Gibco) and 1% Pen strep (Gibco) supplemented with 25 ng / mL of M-CSF. Media was replenished 2 days after plating. After 5 total days of culture, bone-marrow derived macrophages (BMDM) were established. Attorney Docket No. 046483-7492WO 1(04069)

[0356] Cells were gently removed from T-75 flasks using TrypLE (Gibco), counted, and then replated into 12 well plates at a density of 2.5 x 105cells per well. All cells and cell assays were maintained at 37°C and 5% CO2.

[0357] In vitro bone marrow -derived macrophage assessment of IL-4 function

[0358] Six hours after BMDM were established at initial seeding density of 2.5 x 105cells per well in 12 well plates, cells were treated with different concentrations of IL-4 immunocytokines in the following groups: IL-4, NJT6-IL4, LMJ2.5I-IL4, or PBS. The following doses were used: 9.89 x IO’12moles per mL of media, 2.93 x 10'12moles per mL of media, 2.93 x 10'13moles per mL of media, 2.93 x 10’14moles per mL of media, and 2.93 x 10‘15moles per mL of media. 36 hours later, cells were collected, and stained for viability with LIVE / DEAD Fixable Violet (Invitrogen) at 4°C in lx PBS for 30 minutes. Cells were washed and resuspended in FACS buffer (PBS containing 0.5% w / v of bovine serum albumin and 2 mM of EDTA). Cell suspensions were blocked with CD16 / CD32 antibody (clone 93, 1:100, Biolegend 101302) and then stained with CD45-BV711 (clone 30-F11, 1:600, Biolegend 103147), CD86-PE-Dazzle594 (clone GL-1, 1:450, Biolegend 105042), CD301b-PE (clone URA-1, 1:300, Biolegend 146804), F4 / 80-APC-Fire750 (clone BM8, 1:300, Biolegend 123152), CX3CRl-PE-Fire700 (clone SA0011F11, 1:300, Biolegend 149052) at 4°C for 15 minutes. Cells were then fixed and permeabilized using the Foxp3 Transcription Factor Staining Kit (eBiosciences) for 30 minutes at room temperature. Cells were then stained with Argl-APC (clone AlexF5, 1:50, Thermo Fisher 17-3697-82) and CD206-AF488 (clone C068C2, 1:50, Biolegend 141710) for 30 minutes at room temperature. Cells were resuspended with 300 pL of FACS buffer prior to flow analysis.

[0359] Human macrophage polarization

[0360] Human monocytes were isolated by the Penn Human Immunology Core from PBMCs from a donor. Monocytes were plated in non-TC treated T-75s and differentiated to macrophages using M-CSF for 5 days. Cells were seeded at 1 x IO3cells per well in 24 well plates and then treated with a range of concentrations (0.0001 nM-100 nM is the entire range) of each of the following groups: human IL-4, LMJ2.5I-huIL4, LMJ2.5I-porIL4. 36 hours later, cells were collected, and stained for viability with LIVE / DEAD Fixable Violet (Invitrogen) at 4°C in IX PBS for 30 minutes. Cells were washed and resuspended in FACS buffer (PBS containing 0.5% w / v of bovine serum albumin and 2 mM of EDTA). Cell suspensions were blocked with CD16 / CD32 antibody (clone S1701 IE, 1:600, Biolegend 422302) and then stained with CD86- Attorney Docket No. 046483-7492WO 1(04069)

[0361] BV711 (clone IT2.2, 1 :450, Biolegend 305440) at 4°C for 15 minutes. Cells were then fixed and permeabilized using the Foxp3 Transcription Factor Staining Kit (eBiosciences) for 30 minutes at room temperature. Cells were then stained with CD68-PE (clone Y1 / 82A, 1:100, Biolegend 333808) and CD206-APC (clone 15-2, 1:100, Biolegend 321110). In other instances, the cells were stained with Argl-APC (clone AlexF5, 1:50, Thermo Fisher 17-3697-82) and CD206-AF488 (clone C068C2, 1:50, Biolegend 141710) for 30 minutes at room temperature. Cells were resuspended with 300 pL of FACS buffer prior to flow analysis.

[0362] Phagocytic receptor analysis and efferocytosis assay

[0363] Murine BMDM were treated with PBS, 1 ng LPS, 10 nM LMJ2.5I-IL4 , or LPS and LMJ2.5I-IL4. Trem2 and Mertk expression were confirmed 36 hours after treatment via flow cytometry. Cell suspensions were blocked with CD16 / CD32 antibody (clone 93, 1:100, Biolegend 101302) and then stained with F4 / 80-PE-Cy7 (clone BM8, 1:300, Biolegend 123114), Trem2-PE (clone 237920, R&D Systems FAB17291P, 1:50), and MerTK-FITC (clone 2B10C42, Biolegend 151504, 1:200).

[0364] Cell apoptosis was induced by incubating Expi293 cells with 2.5 pM staurosporine for 2 hours at 37 C. Cells were then labeled with the pHrodo™ Deep Red Mammalian and Bacterial Cell Labeling Kit (Thermo Fisher). Labeled apoptotic cells were co-cultured with BMDMs for 2 hours at a ratio of 1 :6 (BMDM: apoptotic cells). All cells were then removed from the plate and washed with FACS buffer. Cell suspensions were blocked with CD16 / CD32 antibody (clone 93, 1:100, Biolegend 101302) and then stained with CD45-BV711 (clone 30-F11, 1:300, Biolegend 103147). Data was acquired using an BD LSRFortessa Flow Cytometer with BD FACSDiva Software v8 and subsequently analyzed using FLowJo vlO.10.0.

[0365] Human monocytes were isolated by the Penn Human Immunology Core from PBMCs from a donor. Monocytes were plated in non-TC treated T-75s and differentiated to macrophages using M-CSF for 5 days. Human PBMDM Murine BMDM were treated with PBS, 1 ng LPS, 100 nM LMJ2.5I-IL4 , or both LPS and LMJ2.5I-huIL4. Trem2 and Mertk expression were confirmed 36 hours after treatment via flow cytometry. Cells were collected, and stained for viability with LIVE / DEAD Fixable Violet (Invitrogen) at 4°C in IX PBS for 30 minutes. Cells were washed and resuspended in FACS buffer (PBS containing 0.5% w / v of bovine serum albumin and 2 mM of EDTA). Cell suspensions were blocked with CD16 / CD32 antibody (clone S1701 IE, 1 :600, Biolegend 422302) and then stained for MerTK-PE-Cy7 (clone 590H11G1E3, Attorney Docket No. 046483-7492WO 1(04069)

[0366] Biolegend 367610, 1 :200), and Trem2-PE (clone 237920, R&D Systems FAB17291P, 1 :50). Cells were then fixed and permeabilized using the Foxp3 Transcription Factor Staining Kit (eBiosciences) for 30 minutes at room temperature. Cells were then stained with CD68-FITC (clone Y 1 / 82A, 1 : 100, Biolegend 333808). Data was acquired using an BD LSRFortessa Flow Cytometer with BD FACSDiva Software v8 and subsequently analyzed using FLowJo vlO.10.0.

[0367] Fn-EIIIB Binding ELISA

[0368] Prior to performing the ELISA, purified Fn-EIIIB was biotinylated using SulfoChromalink Biotin (Vector Labs). Biotinylation was performed at pH=8 and 5X molar excess of sulfo-chromalink biotin was used. Biotinylated Fn-EIIIB was purified using a zeba column. A Nunc MaxiSorp flat-bottom 96-well plate (Thermo Fisher) was coated with 10 ug / mL of purified LMJ-IL4 or NJT6-IL4 overnight. The plate was then washed with ELISA wash buffer (IX PBS, pH=7.4, containing 0.05% v / v Tween-20) 2 times, and then IX PBS 2 times. The plate was then blocked with blocking buffer (IX PBS pH=7.4, 0.05 v / v Tween-20, and 1% w / v BSA) for 1 hour, and then washed twice with wash buffer and IX PBS. The plate was then incubated with biotinylated Fn-EIIIB for 2 hours, and then washed twice with wash buffer and IX PBS. To detect Fn-EIIIB binding, streptavadin horseradish peroxidase (Vector Laboratories) was added to the plate at 0.5 ug / mL for 45 mins, and then washed twice with wash buffer and IX PBS. 100 uL of 1-Step Ultra TMB ELISA (Thermo Fisher) was added to each well and then quenched with 100 uL of 2 M sulfuric acid. The plate was read at 450 nm to determine absorbance, with absorbance at 570 nm subtracted for background.

[0369] Flow cytometry staining of in vivo tissues, acquisition and analysis

[0370] To detect cellular subset in the process blood samples, cell suspensions were stained at 4°C in FACS buffer (PBS containing 0.5% w / v of bovine serum albumin and 2 mM of EDTA) on ice for 30 minutes. Blood single cell suspensions were first blocked with CD16 / CD32 antibody (clone 93, 1:100, Biolegend 101302). Cells were then stained with CD115-BV605 (clone AFS98, 1:300, BioLegend 135517), CD45-BV711 (clone 30-F11, 1:300, Biolegend 103147), B220-BV786 (clone RA3-6B2, 1:150, Biolegend 103246), Ly6G-FITC (clone 1A8, 1:300, Biolegend 127606), Ly6C-PerCP-Cy5.5 (clone HK1.4, 1:300, Biolegend 128012), CD3-PE (clone 17A2, 1:100, Biolegend 100206), CD4-PE-Cy7 (clone GK1.5, 1:150, Biolegend 100421), CD8a-AF700 (clone 53-6.7, 1:300, Biolegend 100729), and CD1 lb-APC-Cy7 (clone MI / 70, 1 :300, Biolegend 101226). Viability was determined by Live / Dead FxViolet DAPI Attorney Docket No. 046483-7492WO 1(04069)

[0371] (Thermo Scientific) was used at a 1.300 dilution (1 pL per sample). Data was acquired using an BD LSRII Flow Cytometer with BD FACSDiva Software v6 and subsequently analyzed using FLowJo vlO.10.0.

[0372] To detect cellular subsets in the heart tissue, infarct and remote tissue were split under microscope. Tissues were digested with Collagenase XI (Sigma), Hyaluronidase (Sigma), Collagenase I (Sigma), DNase (Sigma) for 60 minutes at 37°C at 700 rpm. Samples were then strained through a 0.22 micron cell strainer and stained. Viability was assessed with LIVE / DEAD Fixable Violet (Invitrogen) at 4°C in IX PBS for 30 minutes. Cells were washed and resuspended in FACS buffer (PBS containing 0.5% w / v of bovine serum albumin and 2 mM of EDTA). Cells suspensions were blocked with CD16 / CD32 antibody (clone 93, 1:100, Biolegend 101302) for 10 minutes, and then stained with CD31-BV421 (clone 390, 1:360, Biolegend 102424), CD1 Ib-Pacific Blue (clone MI / 70, 1:360, Biolegend 101224), CCR2-BV605 (clone SA203G11, 1:240, Biolegend 150613), CD45-BV711 (clone 30-F11, 1:600, Biolegend 103147), Ly6G-FITC (clone 1A8, 1:600, Biolegend 127606), Ly6C-PerCP-Cy5.5 (clone HK1.4, 1:600, Biolegend 128012), mEF-SK4-PE (clone mEF-SK4, 1:120, Miltenyi Biotech Inc), CD64-PE-Cy7 (clone X54-5 / 7.1, 1:240, Biolegend 139314), MHCII-AF700 (clone M5 / 114.15.2, 1:600, Biolegend 107622) at 4°C for 20 minutes. Cells were then fixed and permeabilized using the Foxp3 Transcription Factor Staining Kit (eBiosciences) for 30 minutes at room temperature. Cells were then stained with Argl-APC (clone AlexF5, 1:50, Thermo Fisher 17-3697-82). Cells were resuspended in 400 uL of FACS buffer prior to analysis. Data was acquired using a BD Symphony A5 Flow Cytometer with BD FACSDiva Software v8 and subsequently analyzed using FLowJo vlO.10.0.

[0373] To detect cellular subsets in liver and spleen tissue, tissues were digested with Collagenase XI (Sigma), Hyaluronidase (Sigma), Collagenase I (Sigma), DNase (Sigma) for 30 minutes at 37°C at 700 rpm. Samples were then strained through a 0.22 micron cell strainer and stained. Viability was assessed with LIVE / DEAD Fixable Violet (Invitrogen) at 4°C in IX PBS for 30 minutes. Cells were washed and resuspended in FACS buffer (PBS containing 0.5% w / v of bovine serum albumin and 2 mM of EDTA). Cells suspensions were blocked with CD16 / CD32 antibody (clone 93, 1:100, Biolegend 101302) for 10 minutes, and then stained with CD31-BV421 (clone 390, 1:360, Biolegend 102424), CD1 Ib-Pacific Blue (clone MI / 70, 1:360, Biolegend 101224), CCR2-BV605 (clone SA203G11, 1:240, Biolegend 150613), CD45-BV711 Attorney Docket No. 046483-7492WO 1(04069)

[0374] (clone 30-F11, 1 :600, Biolegend 103147), B220-BV786 (clone RA3-6B2, 1:100, Biolegend 103246), Ly6G-FITC (clone 1A8, 1:600, Biolegend 127606), Ly6C-PerCP-Cy5.5 (clone HK1.4, 1:600, Biolegend 128012), mEF-SK4-PE (clone mEF-SK4, 1:120, Miltenyi Biotech Inc), F4 / 80-PE-Cy7 (clone BM8, 1:100, Biolegend 123114), CD3-APC-Cy7 (clone 17A2, 1:100, Biolegend 100222). Cells were then fixed and permeabilized using the Foxp3 Transcription Factor Staining Kit (eBiosciences) for 30 minutes at room temperature. Data was acquired using a BD Symphony A5 Flow Cytometer with BD FACSDiva Software v8 and subsequently analyzed using FLowJo vlO.10.0.

[0375] Flow cytometry gating

[0376] Leukocytes were identified as CD45+. B cells were identified as CD45+CD3 B220+. CD4+T cells were identified as CD45+B220 CD3+CD4+. CD8+ T cells were identified as CD45+B220 CD3+CD8+. Classical monocytes were identified as CD45+CD3 B220‘

[0377] CD1 lb+CDl 15hiLy6Chi. Nonclassical monocytes were identified CD45+CD3’B220‘

[0378] CD1 Ib CDl 15hiLy6Clo. Neutrophils were identified as CD45 'CD3’B220’

[0379] CD1 lb+CDl 151oiLy6Ghi. Macrophages were identified as CD45+F4 / 80+and then further phenotype based on CD86, CX3CR1, CD206, CD301b, and Argl expression.

[0380] Histology

[0381] On day 29, after echocardiography assessment, organs were harvested for subsequent histology following picrosirius red staining. Organs were fixed in 10% neutral buffered formalin overnight, and then gradually dehydrated in ethanol over 48 hours, and finally stored in 100% ethanol at -20°C. Hearts were embedded in paraffin and then 8 um slices were mounted onto slides for further analysis. Fibrosis was determined by picrosirius red staining (ab 150681) according to the manufacturer's protocol. CD31 expression was determined by staining for PECAM-1 (Histo Biotech DIA-310 and Impress Red (Vector Labs). TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was performed using the In Situ Cell Death Detection Kit (Fluorescein, Roche). Cardiac Troponin was stained with anti-cardiac troponin T (c-TNT) antibodies. Fn-EIIIB was determined by staining with Fn-EIIIB primary (Thermo Fisher MA5-48025), and Impress Red (Vector Labs). Percent vascularization, TUNEL%, cTNT% were determined by quantifying area of DAPI + and area of

[0382] CD31+ / TUNEL+ in each field of view in FIJI. Attorney Docket No. 046483-7492WO 1(04069)

[0383] For each slice, whole heart fibrosis and interstitial fibrosis were quantified using a custom, unbiased Python script that used color thresholds to identify fibrosed regions (total tissue threshold = 50, healthy tissue threshold = 210). An unbiased Matlab script was used to quantify wall thickness from picrosirius red stained day 28 heart slices.

[0384] Serum Analysis

[0385] Serum markers were analyzed by Children’s hospital of Pennsylvania (CHOP) Translational Core. Complete serum analysis was completed.

[0386] Computational ODE Model

[0387] Computational ordinary differential model (ODE) to stimulate drug transport from blood to infarct built in python. ODEs were solved computationally using scipy. integrate. solve_ivp(). Fractional activation was calculated and overall exposure was calculated by integrating fractional activation curves using scipy. integrate. simpson().

[0388] Spatial RNA Sequencing

[0389] Spatial RNA sequencing reanalysis was performed on data deposited in Gene Expression Omnibus (GSE165857). Filtered count matrices were loaded, including post-MI hearts at Day 1, 3, and 7, and processed in R using Seurat (5.3.0). Each dataset was normalized independently using SCTransform. General QC involved visualizing spot level using both total spot counts and SCT-normalized counts. Dimensionality reduction and clustering were performed per time point. Then a merged Seurat object using all three timepoints was generated. To derive infarct, border, and remote tissue regions, two module scores were computed: (1) Remote cardiomyocyte score -RemoteCMl- marked by elevated healthy cardiomyocyte genes (Myh6, Myh7, Tnnt2, Tnni3, Actn2, Nkx2-5) and a (2) border-zone stress score - BZscorel - marked by elevated stress markers ( Nppa, Xirp2, Ankrdl, Nppb, Dstn). Spots were classified as infarct if RemoteCMl < 0.5, border if BZscorel > the 90th percentile, and Remote if RemoteCMl > the 60th percentile. Gene expression values of ECM genes were exported with selected metadata for statistical analysis and visualized with SpatialDimPlot.

[0390] Enumerated Embodiments

[0391] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance. Attorney Docket No. 046483-7492WO 1(04069)

[0392] Embodiment 1 provides a chimeric IL-4 polypeptide, comprising: interleukin-4 (IL-4); a myocardial infarct targeting agent; and a peptide linker between IL-4 and the targeting agent.

[0393] Embodiment 2 provides the chimeric IL-4 polypeptide of embodiment 1, wherein the myocardial infarct targeting agent is linked to the N-terminus of IL-4.

[0394] Embodiment 3 provides the chimeric IL-4 polypeptide of embodiment 1, wherein the myocardial infarct targeting agent is linked to the C-terminus of IL-4.

[0395] Embodiment 4 provides the chimeric IL-4 polypeptide of any one of embodiments 1-3, wherein the myocardial infarct targeting agent specifically binds an extracellular matrix (ECM) protein expressed in a myocardial infarct.

[0396] Embodiment 5 provides the chimeric IL-4 polypeptide of embodiment 4, wherein the ECM protein is fibronectin, collagen, hyaluronic acid, tenascin, or periostin.

[0397] Embodiment 6 provides the chimeric IL-4 polypeptide of embodiment 5, wherein the myocardial infarct targeting agent specifically binds Fn-EIIIB, Fn-EIIIA, or Type I collagen.

[0398] Embodiment 7 provides the chimeric IL-4 polypeptide of embodiment 6, wherein the myocardial infarct targeting agent specifically binds Fn-EIIIB.

[0399] Embodiment 8 provides the chimeric IL-4 polypeptide of embodiment 6, wherein the myocardial infarct targeting agent specifically binds Fn-EIIIA.

[0400] Embodiment 9 provides the chimeric IL-4 polypeptide of embodiment 6, wherein the myocardial infarct targeting agent specifically binds Type I collagen.

[0401] Embodiment 10 provides the chimeric IL-4 polypeptide of any one of embodiments 1-9, wherein the myocardial infarct targeting agent is an antibody or antigen binding fragment thereof.

[0402] Embodiment 11 provides the chimeric IL-4 polypeptide of embodiment 10, wherein the myocardial infarct targeting agent is a nanobody.

[0403] Embodiment 12 provides the chimeric IL-4 polypeptide of embodiment 11, wherein the nanobody comprises the amino acid sequence of SEQ ID NO: 1.

[0404] Embodiment 13 provides the chimeric IL-4 polypeptide of any one of embodiments 1-9, wherein the myocardial infarct targeting agent is a protein that specifically binds an ECM protein.

[0405] Embodiment 14 provides the chimeric IL-4 polypeptide of embodiment 13, wherein the ECM protein is a collagen. Attorney Docket No. 046483-7492WO 1(04069)

[0406] Embodiment 15 provides the chimeric IL-4 polypeptide of embodiment 13, wherein the protein is lumican.

[0407] Embodiment 16 provides the chimeric IL-4 polypeptide of embodiment 15, wherein the lumican comprises the amino acid sequence of SEQ ID NO: 61.

[0408] Embodiment 17 provides the chimeric IL-4 polypeptide of any one of embodiments 1-16, further comprising human serum albumin (HSA) linked to the myocardial infarct targeting agent.

[0409] Embodiment 18 provides the chimeric IL-4 polypeptide of embodiment 17, wherein the chimeric IL-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 65.

[0410] Embodiment 19 provides the chimeric IL-4 polypeptide of any one of embodiments 1-18, further comprising an IL-4 masking moiety and a cardiovascular inflammatory protease-sensitive linker between IL-4, wherein IL-4 activity is unmasked following cleavage of the cardiovascular inflammatory protease-sensitive linker and release of the IL-4 masking moiety.

[0411] Embodiment 20 provides the chimeric IL-4 polypeptide of embodiment 19, wherein the IL-4 masking moiety comprises an IL-4 receptor alpha- 1 (IL-4Ral) extracellular domain (ECD).

[0412] Embodiment 21 provides the chimeric IL-4 polypeptide of embodiment 20, wherein the IL-4Ral ECD comprising the amino acid sequence of SEQ ID NO: 6.

[0413] Embodiment 22 provides the chimeric IL-4 polypeptide of embodiment 20, wherein the IL-4Ral ECD is a mutant IL-4Ral ECD with reduced binding to IL-4.

[0414] Embodiment 23 provides the chimeric IL-4 polypeptide of embodiment 22, wherein the mutant IL-4Ral ECD comprises one or more alanine substitutions relative to a corresponding wild-type IL-4Ral ECD.

[0415] Embodiment 24 provides the chimeric IL-4 polypeptide of embodiment 23, wherein the mutant IL-4Ral ECD comprises one or more mutations of the mature wild-type IL-4Ral polypeptide at any one of amino acid residues 13-15, 39, 41-43, 66-70, 72-74, 91-94, 125-129, 183, or a combination thereof.

[0416] Embodiment 25 provides the chimeric IL-4 polypeptide of embodiment 24, wherein the mutant IL-4Ral ECD comprises one or more mutations selected from the group consisting of L39A, F41A, L42A, L43A, D72A, and Y74A.

[0417] Embodiment 26 provides the chimeric IL-4 polypeptide of any one of embodiments 22-24, wherein the binding affinity of the mutant IL-4Rcd ECD to the IL-4 is between about 25 nm to about 250 nm. Attorney Docket No. 046483-7492WO 1(04069)

[0418] Embodiment 27 provides the chimeric IL-4 polypeptide of embodiment 22-24, wherein the binding affinity of the mutant IL-4Ral ECD to the IL-4 is between about 50 nm to about 150 nm.

[0419] Embodiment 28 provides the chimeric IL-4 polypeptide of any one of embodiments 19-27, wherein the cardiovascular inflammatory protease-sensitive linker is an MMP-2 or MMP-9 cleavable peptide linker.

[0420] Embodiment 29 provides the chimeric IL-4 polypeptide of embodiment 28, wherein the MMP-2 or MMP-9 cleavable linker comprises an amino acid sequence set forth in any one of SEQ IDNOs: 10-47.

[0421] Embodiment 30 provides the chimeric IL-4 polypeptide of any one of embodiments 1-29, wherein the peptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 48-58.

[0422] Embodiment 31 provides an immune cell expressing or treated with the chimeric IL-4 polypeptide of any one of embodiments 1-30.

[0423] Embodiment 32 provides the immune cell of embodiment 31, wherein the cell is a bone marrow derived macrophage (BMDM).

[0424] Embodiment 33 provides a nucleic acid composition encoding the encoding the chimeric IL-4 polypeptide of any one of embodiments 1-30.

[0425] Embodiment 34 provides a vector comprising the nucleic acid of embodiment 33.

[0426] Embodiment 35 provides an expression vector comprising the nucleic acid of embodiment 34.

[0427] Embodiment 36 provides an mRNA encoding the chimeric IL-4 polypeptide of any one of embodiments 1-30.

[0428] Embodiment 37 provides a lipid nanoparticle (LNP) composition comprising the mRNA of embodiment 36.

[0429] Embodiment 38 provides a pharmaceutical composition, comprising: the chimeric IL-4 polypeptide of any one of embodiments 1-30, the immune cell of embodiment 31 or 32, or the LNP composition of embodiment 37; and pharmaceutically acceptable carrier.

[0430] Embodiment 39 provides a method for treating a myocardial infarction, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of embodiment 38. Attorney Docket No. 046483-7492WO 1(04069)

[0431] Embodiment 40 provides the method of embodiment 39, wherein the pharmaceutical composition is administered intravenously.

[0432] Embodiment 41 provides the method of embodiment 39, wherein the pharmaceutical composition is administered via intracoronary or intramyocardial delivery.

[0433] Embodiment 42 provides the method of any one of embodiments 39-41, wherein the pharmaceutical composition is administered between 2-4 hours after a myocardial infarction.

[0434] Embodiment 43 provides the method of embodiment 42, wherein the pharmaceutical composition is further administered on at least 5-7 consecutive days following the myocardial infarction.

[0435] Embodiment 44 provides a method for treating or reducing the severity of heart failure, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of embodiment 38.

[0436] Embodiment 45 provides the method of embodiment 44, wherein the pharmaceutical composition is administered intravenously.

[0437] Embodiment 46 provides the method of embodiment 44, wherein the pharmaceutical composition is administered via intracoronary or intramyocardial delivery.

[0438] Embodiment 47 provides the method of any one of embodiments 44-46, wherein the pharmaceutical composition is administered between 2-4 hours after a myocardial infarction.

[0439] Embodiment 48 provides the method of any one of embodiments 44-47, wherein the pharmaceutical composition is further administered on at least 5-7 consecutive days following a myocardial infarction.

[0440] Embodiment 49 provides the method of any one of embodiments 44-48, wherein the pharmaceutical composition is administered at least once a week following the myocardial infarction.

[0441] Embodiment 50 provides the method of any one of embodiments 44-48, wherein the pharmaceutical composition is administered at least once every two weeks following the myocardial infarction.

[0442] Embodiment 51 provides the method of any one of embodiments 44-48, wherein the pharmaceutical composition is administered at least once a month following the myocardial infarction. Attorney Docket No. 046483-7492WO 1(04069)

[0443] Embodiment 52 provides a method for treating or reducing the severity of heart failure, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of embodiment 38.

[0444] Embodiment 53 provides the method of embodiment 52, wherein the pharmaceutical composition comprises the immune cell of claim 31 or 32.

[0445] Embodiment 54 provides a method for treating ischemia / reperfusion injury, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of embodiment 38.

[0446] Embodiment 55 provides the method of embodiment 54, wherein the ischemia / reperfusion injury is a cardiac ischemia / reperfusion injury selected from the group consisting of acute cardiac ischemia, chronic cardiac ischemia, chronic coronary disease and acute coronary syndrome.

[0447] Embodiment 56 provides the method of embodiment 54, wherein the ischemia / reperfusion injury is caused by stroke, surgery, organ transplantation, ovarian torsion, or small bowel obstruction.

[0448] Embodiment 57 provides the method of any one of embodiments 54-56, wherein the pharmaceutical composition is administered intravenously.

[0449] Embodiment 58 provides the method of any one of embodiments 54-56, wherein the pharmaceutical composition is administered via intracoronary or intramyocardial delivery.

[0450] Embodiment 59 provides the method of any one of embodiments 54-58, wherein the pharmaceutical composition is administered between 2-4 hours after a myocardial infarction.

[0451] Embodiment 60 provides the method of any one of embodiments 54-59, wherein the pharmaceutical composition is further administered on at least 5-7 consecutive days following the myocardial infarction.

[0452] Embodiment 61 provides a method of performing cardiovascular surgery in a subject in need thereof, comprising performing a surgical procedure and administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 38.

[0453] Embodiment 62 provides the method of embodiment 61, wherein the surgical procedure is an angioplasty or stent placement.

[0454] Embodiment 63 provides the method of embodiment 61 or 62, wherein the pharmaceutical composition is administered between 2-6 hours after the myocardial infarction. Attorney Docket No. 046483-7492WO 1(04069)

[0455] Embodiment 64 provides a method for producing a chimeric IL-4 polypeptide, comprising culturing a host cell containing a nucleic acid encoding the chimeric IL -4 polypeptide of any one of embodiments 1-30 in a culture medium under conditions such that the chimeric IL-4 polypeptide is expressed.

[0456] Embodiment 65 provides the method of embodiment 64, wherein the chimeric IL-4 polypeptide is recovered and / or purified from the culture medium or the host cell.

[0457] Other Embodiments

[0458] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0459] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attorney Docket No. 046483-7492WO 1(04069)CLAIMSWhat is claimed:

1. A chimeric IL-4 polypeptide, comprising:interleukin-4 (IL-4);a myocardial infarct targeting agent; anda peptide linker between IL-4 and the targeting agent.

2. The chimeric IL-4 polypeptide of claim 1, wherein the myocardial infarct targeting agent is linked to the N-terminus of IL-4.

3. The chimeric IL-4 polypeptide of claim 1, wherein the myocardial infarct targeting agent is linked to the C-terminus of IL-4.

4. The chimeric IL-4 polypeptide of any one of claims 1-3, wherein the myocardial infarct targeting agent specifically binds an extracellular matrix (ECM) protein expressed in a myocardial infarct.

5. The chimeric IL-4 polypeptide of claim 4, wherein the ECM protein is fibronectin, collagen, hyaluronic acid, tenascin, or periostin.

6. The chimeric IL-4 polypeptide of claim 5, wherein the myocardial infarct targeting agent specifically binds Fn-EIIIB, Fn-EIIIA, or Type I collagen.

7. The chimeric IL-4 polypeptide of claim 6, wherein the myocardial infarct targeting agent specifically binds Fn-EIIIB.

8. The chimeric IL-4 polypeptide of claim 6, wherein the myocardial infarct targeting agent specifically binds Fn-EIIIA.

9. The chimeric IL-4 polypeptide of claim 6, wherein the myocardial infarct targeting agent specifically binds Type I collagen.Attorney Docket No. 046483-7492WO 1(04069)10. The chimeric IL-4 polypeptide of any one of claims 1-9, wherein the myocardial infarct targeting agent is an antibody or antigen binding fragment thereof.

11. The chimeric IL-4 polypeptide of claim 10, wherein the myocardial infarct targeting agent is a nanobody.

12. The chimeric IL-4 polypeptide of claim 11, wherein the nanobody comprises the amino acid sequence of SEQ ID NO: 1.

13. The chimeric IL-4 polypeptide of any one of claims 1-9, wherein the myocardial infarct targeting agent is a protein that specifically binds an ECM protein.

14. The chimeric IL-4 polypeptide of claim 13, wherein the ECM protein is a collagen.

15. The chimeric IL-4 polypeptide of claim 13, wherein the protein is lumican.

16. The chimeric IL-4 polypeptide of claim 15, wherein the lumican comprises the amino acid sequence of SEQ ID NO: 61.

17. The chimeric IL-4 polypeptide of any one of claims 1-16, further comprising human serum albumin (HSA) fused to the myocardial infarct targeting agent.

18. The chimeric IL-4 polypeptide of claim 17, wherein the chimeric IL-4 polypeptide comprises the amino acid sequence of SEQ ID NO: 65.

19. The chimeric IL-4 polypeptide of any one of claims 1-18, further comprising an IL-4 masking moiety and a cardiovascular inflammatory protease-sensitive linker between IL-4, wherein IL-4 activity is unmasked following cleavage of the cardiovascular inflammatory protease-sensitive linker and release of the IL-4 masking moiety.

20. The chimeric IL-4 polypeptide of claim 19, wherein the IL-4 masking moiety comprises an IL-4 receptor alpha- 1 (IL-4Ral) extracellular domain (ECD).

21. The chimeric IL-4 polypeptide of claim 20, wherein the IL-4Ral ECD comprising the amino acid sequence of SEQ ID NO: 6.Attorney Docket No. 046483-7492WO 1(04069)22. The chimeric IL-4 polypeptide of claim 20, wherein the TL-4Ral ECD is a mutant TL-4Ral ECD with reduced binding to IL-4.

23. The chimeric IL-4 polypeptide of claim 22, wherein the mutant IL-4Ral ECD comprises one or more alanine substitutions relative to a corresponding wild-type IL-4Ral ECD.

24. The chimeric IL-4 polypeptide of claim 23, wherein the mutant IL-4Ral ECD comprises one or more mutations of the mature wild-type IL-4Ral polypeptide at any one of amino acid residues 13-15, 39, 41-43, 66-70, 72-74, 91-94, 125-129, 183, or a combination thereof.

25. The chimeric IL-4 polypeptide of claim 24, wherein the mutant IL-4Ral ECD comprises one or more mutations selected from the group consisting of L39A, F41 A, L42A, L43A, D72A, and Y74A.

26. The chimeric IL-4 polypeptide of any one of claims 22-24, wherein the binding affinity of the mutant IL-4Ral ECD to the IL-4 is between about 25 nm to about 250 nm.

27. The chimeric IL-4 polypeptide of claim 22-24, wherein the binding affinity of the mutant IL-4Rotl ECD to the IL-4 is between about 50 nm to about 150 nm.

28. The chimeric IL-4 polypeptide of any one of claims 19-27, wherein the cardiovascular inflammatory protease-sensitive linker is an MMP-2 or MMP-9 cleavable peptide linker.

29. The chimeric IL-4 polypeptide of claim 28, wherein the MMP-2 or MMP-9 cleavable linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10-47.

30. The chimeric IL-4 polypeptide of any one of claims 1-29, wherein the peptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 48-58.

31. An immune cell expressing or treated with the chimeric IL-4 polypeptide of any one of claims 1-30.

32. The immune cell of claim 31, wherein the cell is a bone marrow derived macrophage (BMDM).Attorney Docket No. 046483-7492WO 1(04069)33. A nucleic acid encoding the encoding the chimeric IL-4 polypeptide of any one of claims 1-30.

34. A vector comprising the nucleic acid of claim 33.

35. An expression vector comprising the nucleic acid of claim 33.

36. An mRNA encoding the chimeric IL-4 polypeptide of any one of claims 1-30.

37. A lipid nanoparticle (LNP) composition comprising the mRNA of claim 36.

38. A pharmaceutical composition, comprising:the chimeric IL-4 polypeptide of any one of claims 1-30,the immune cell of claim 31 or 32, orthe LNP composition of claim 36; anda pharmaceutically acceptable carrier.

39. A method for treating a myocardial infarction, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 38.

40. The method of claim 39, wherein the pharmaceutical composition is administered intravenously.

41. The method of claim 39, wherein the pharmaceutical composition is administered via intracoronary or intramyocardial delivery.

42. The method of any one of claims 39-41, wherein the pharmaceutical composition is administered between 2-4 hours after a myocardial infarction.

43. The method of claim 42, wherein the pharmaceutical composition is further administered on at least 5-7 consecutive days following the myocardial infarction.

44. A method for treating or reducing the severity of heart failure, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 38.Attorney Docket No. 046483-7492WO 1(04069)45. The method of claim 44, wherein the pharmaceutical composition is administered intravenously.

46. The method of claim 44, wherein the pharmaceutical composition is administered via intracoronary or intramyocardial delivery.

47. The method of any one of claims 44-46, wherein the pharmaceutical composition is administered between 0.5-4 hours after a myocardial infarction.

48. The method of any one of claims 44-47, wherein the pharmaceutical composition is further administered on at least 5-7 consecutive days following a myocardial infarction.

49. The method of any one of claims 44-48, wherein the pharmaceutical composition is administered at least once a week following the myocardial infarction.

50. The method of any one of claims 44-48, wherein the pharmaceutical composition is administered at least once every two weeks following the myocardial infarction.

51. The method of any one of claims 44-48, wherein the pharmaceutical composition is administered at least once a month following the myocardial infarction.

52. A method for treating or reducing the severity of heart failure, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 38.

53. The method of claim 52, wherein the pharmaceutical composition comprises the immune cell of claim 31 or 32.

54. A method for treating ischemia / reperfusion injury, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 38.

55. The method of claim 54, wherein the ischemia / reperfusion injury is a cardiac ischemia / reperfusion injury selected from the group consisting of acute cardiac ischemia, chronic cardiac ischemia, chronic coronary disease and acute coronary syndrome.Attorney Docket No. 046483-7492WO 1(04069)56. The method of claim 54, wherein the ischemia / reperfusion injury is caused by stroke, surgery, organ transplantation, ovarian torsion, or small bowel obstruction.

57. The method of any one of claims 54-56, wherein the pharmaceutical composition is administered intravenously.

58. The method of any one of claims 54-56, wherein the pharmaceutical composition is administered via intracoronary or intramyocardial delivery.

59. The method of any one of claims 54-58, wherein the pharmaceutical composition is administered between 2-4 hours after a myocardial infarction.

60. The method of any one of claims 54-59, wherein the pharmaceutical composition is further administered on at least 5-7 consecutive days following the myocardial infarction.

61. A method of performing cardiovascular surgery in a subject in need thereof, comprising performing a surgical procedure and administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 38.

62. The method of claim 61, wherein the surgical procedure is an angioplasty or stent placement.

63. The method of claim 61 or 62, wherein the pharmaceutical composition is administered between 2-6 hours after the myocardial infarction.

64. A method for producing a chimeric IL-4 polypeptide, comprising culturing a host cell containing a nucleic acid encoding the chimeric IL-4 polypeptide of any one of claims 1-30 in a culture medium under conditions such that the chimeric IL-4 polypeptide is expressed.

65. The method of claim 64, wherein the chimeric IL-4 polypeptide is recovered and / or purified from the culture medium or the host cell.

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