Methods for characterizing and treating disorders characterized with altered immune function

Abstract

The present disclosure provides methods for characterizing, prognosing, and treating patients suffering from a heart disorder (e.g., cardiac chronic ischemic heart failure) or inflammatory and fibrotic diseases (e.g., cardiac chronic ischemic heart failure). In particular, the present disclosure provides compositions and methods for companion diagnostics to prognose and identify and monitor treatments of such patients.

Description

[0001] Atty. Docket No. UAZ-44590.601

[0002] METHODS FOR CHARACTERIZING AND TREATING DISORDERS CHARACTERIZED WITH ALTERED IMMUNE FUNCTION

[0003] STATEMENT OF RELATED APPLICATIONS

[0004] This application claims priority to provisional application 63 / 753,163, filed February 3, 2025, which is herein incorporated by reference in its entirety.

[0005] FIELD OF THE DISCLOSURE

[0006] The present disclosure provides methods for characterizing, diagnosing, prognosing, and treating patients suffering from a heart disorder (e.g., cardiac chronic ischemic heart failure), skeletal muscle disorder (volumetric muscle loss), or other inflammatory and fibrotic diseases (e.g., cardiac chronic ischemic heart failure). In particular, the present disclosure provides compositions and methods for companion diagnostics to prognose and identify and monitor treatments of such patients, as well as develop predictive markers for next generation therapeutics. These predictive immune profiles are integrated with computational algorithms to develop a clinically deployable tool that can be used from patient stratification.

[0007] BACKGROUND

[0008] Biologically active platforms that increase the prevalence of alternatively activated M2 macrophage phenotypes in immune competent animal models of chronic ischemic heart failure (mice, rat, swine) have been developed (see, e.g., Lancaster, et al., Ann Thorac Surg 2019 Oct;108(4): 1169-1177; Lancaster, et al., Commun Biol 2023 Nov 25;6(1): 1203). Such biologically active platforms mediate changes in the immune and stromal cell populations that modify disease states by turning off genes associated with inflammation and fibrosis and turn on genes associated with anti-inflammation, extracellular matrix reorganization, wound healing, and tissue repair. Such biologically active platforms are, for example, composed of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and neonatal fibroblasts embedded on a bioresorbable matrix that is implanted at the site of injury, and restores contractile function, increases blood flow, and repairs damaged cardiomyocytes of the chronically infarcted myocardium (see, e.g., Lancaster, et al., Immunome Res. Vol. 20, Issue 1; Lancaster, et al., Commun Biol. 2023;6(1): 1203; Lancaster, et al., Vessel. Plus. 2019, 3, 34; Lancaster, et al., Ann Thorac Surg 2019 Oct;108(4):1169-1177; Lancaster, et al., Commun Biol 2023 Nov 25;6(1): 1203; U. S. Patent Nos. 10,172,976, 11,020,501, and 11,980,698).

[0009] Diagnostic, prognostic, and therapetuic tools that enable patient stratification have beenAtty. Docket No. UAZ-44590.601

[0010] transformative in, for example, treating patients suffering from various forms of cancer.

[0011] However, a similar diagnostic approach does not exist for patients with a heart disorder (e.g., cardiac chronic ischemic heart failure) or other inflammatory and fibrotic diseases. This is principally because in patients with cancer, a clinician can biopsy a patient’s cancer to see what genes are activated by the cancer and target those genes with a therapy. Such a clinician cannot pursue such a therapeutic strategy in patients with heart disease because heart biopsies are potentially risky due to the risk in potentially rupturing the heart. Also, with heart biopsies it is difficult to localize the exact area of the heart needed to biopsy.

[0012] Accordingly, improved techniques for identifying optimal treatment strategies for patients suffering from an inflammatory and fibrotic diseases such as chronic ischemic heart failure are needed.

[0013] The present disclosure address such a need.

[0014] SUMMARY

[0015] The present disclosure provides methods for characterizing, diagnosing, prognosing, and treating patients suffering from a heart disorder (e.g., cardiac chronic ischemic heart failure), skeletal muscle disorder (volumetric muscle loss), or other inflammatory and fibrotic diseases (e.g., cardiac chronic ischemic heart failure). In particular, the present disclosure provides compositions and methods for companion diagnostics to prognose and identify and monitor treatments of such patients, as well as develop predictive markers for next generation therapeutics. These predictive immune profiles are integrated with computational algorithms to develop a clinically deployable tool that can be used from patient stratification.

[0016] In some embodiments, the present disclosure provide methods for uncovering predictive markers in-vivo by correlating functional improvements with circulating biomarkers that can be integrated into a patient specific in-vitro culture to generate computational workflows to drive a clinically deployable tool that can support patient stratification.

[0017] Experiments conducted during the course of developing embodiments for the present disclosure resulted in the development of multivariate analysis in both in vivo and in vitro models of disease, namely a tissue culture assay platform capable of assessing differences in immune cell functions and phenotypes following treatment with immunomodulatory therapeutics in patients, based on predictive preclinical data. This tissue culture platform is capable of using individual patient’s immune cells isolated from peripheral blood to test immunomodulatory therapeutic responses of patients in vitro priorAtty. Docket No. UAZ-44590.601

[0018] to treatment. This platform is capable of identifying novel biomarkers and molecular targets of immune cell responses to immunomodulatory therapies to drive refined diagnostic prototype development for clinical use. Readouts from assays developed using this platform allow for stratification of patients receiving immunomodulatory treatments to select the individuals who will most likely be the best responders. The diagnostic platform developed is applicable for broad use of immunomodulatory treatments.

[0019] Accordingly, the present disclosure provides methods for treating patients suffering from a heart disorder (e.g., cardiac chronic ischemic heart failure) or skeletal muscle disorder comprising first performing a companion diagnostic test of the patient before treatment; and then providing a suitable treatment to the patient according to the results of the companion diagnostic test. The present disclosure further provides methods for developing a predictive signature for stratifying patients receiving novel immunomodulatory treating patients suffering from inflammatory and fibrotic diseases (e.g., cardiac chronic ischemic heart failure) comprising first performing a companion diagnostic test of the patient before treatment; and then providing a suitable treatment to the patient according to the results of the companion diagnostic test. Furthermore, experimental methods are provided for the development of prognostics, which can track patient responses to the treatment. These methods target the immune system and are not tissue specific and methods can also be used to guide the development of future therapeutics for immunomodulatory tissue repair. Patient progress can furthermore be tracked from the data generated here for the use of prognostic prototypes. The conservation of the immune system across other tissues and organs also gives the inventors confidence in the ability for the methods here to be applied to other tissues, such as muscle tissue in large volume skeletal muscle loss.

[0020] In particular, provided herein is a method, comprising: a) obtaining a biological sample from a patient suffering from a disorder (e.g., heart or skeletal muscle disorder) characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with alteredAtty. Docket No. UAZ-44590.601

[0021] immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for the tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

[0022] In certain aspects, the altered immune cell function is increased or decreased immune cell function. In some cases, the method further comprises d) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient. In some embodiments, the method further comprises administering to the patient one or more pharmaceutical agents selected from, for example, ACE inhibitors, β-blockers, ARBs, diuretics, calcium channel blocker, Entresto (sacubitril / valsartan), SGLT2i, GLP-1 RA medicines such as ozempic, nitrates, platelet aggregation inhibitors, cholesterol-lowering medications, ranolazine, and / or ivabradine, or other guideline directed medical therapy (GDMT). This assay platform further services to characterize the immune state of patients already receiving GDMTs.

[0023] In some embodiments, treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient comprises administering to the patient a composition comprising the immunomodulatory agent.

[0024] While not limited to particular therapeutic compositions, in some embodiments, the composition is a bioresorbable matrix, wherein the immunomodulatory agent is embedded within the bioresorbable matrix. In certain aspects, the bioresorbable matrix further comprises human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and neonatal fibroblasts. In some cases, administering to the patient the bioresorbable matrix comprises implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage. For example, in some embodiments, implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage mediates changes in the stromal cell population that repair heart tissue. In some embodiments, implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage results in increased immune cell activity at the site of heart tissue experiencing heart tissue damage. In some cases, implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage restores contractile function, increases blood flow, and repairs damaged cardiomyocytes of the chronically infarcted myocardium.Atty. Docket No. UAZ-44590.601

[0025] In some embodiments, the patient is a human patient (e.g., a human patient suffering from chronic ischemic heart failure, myocardial infarction, or heart failure).

[0026] In certain aspects, the immunomodulatory agent is an immune checkpoint inhibitor (ICI). Examples include but are not limited to an inhibitor of PD-1, PD-L1, CTLA4, LAG3, TIGIT, TIM3, VISTA, ICOS, BTLA, GITR, NKG2A, CD112R, B7-H3, or CD73 (e.g., nivolumab, pembrolizumab, ipilimumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplimab, retifanlimab, dostarlimab, or toripalimab).

[0027] The present disclosure is not limited to particular immune cell activity. Examples include but are not limited to cytokine activity, macrophage activity, B lymphocyte activity, T lymphocyte activity, cytotoxic T-cell lymphocyte (CTL) activity, mast cell activity, monocyte activity, dendritic cell activity, eosinophil activity, natural killer cell activity, basophil activity, and neutrophil activity.

[0028] In some embodiments, the one or more type of immune cell activity is macrophage cellular activity. In some embodiments, the assessment of differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents comprises one or more of the following standard biochemical assays: flow cytometry assessment, nitric oxide assessment, arginase assessment, acetylated LDL uptake assessment, genomic assessment, transcriptomics assessment, mass spectroscopy proteomics assessment, and multiplex ELISA assessment.

[0029] In some embodiments, this platform is made executable by artificial intelligence (AI) and machine learning (ML) computational algorithms that can be employed to analyze and draw conclusion from patient specific immune data using work flows. Model development includes attention-based architectures, such as transformer and Perceiver IO models, to capture complex and high-dimensional relationships among immune features; convolutional neural networks to identify localized and hierarchical immune patterns; and recurrent neural networks, including LSTM and GRU models, to model temporal immune dynamics. State space models are employed to characterize dynamic immune state transitions over time, while forecasting architectures, such as Temporal Fusion Transformers and DeepAR, are used to predict future cardiac trajectories from immune data. These models are benchmarked against interpretable baseline approaches, including regularized linear models, random forests, and gradient boosting methods. Collectively, this multi-model strategy enables robust, generalizable prediction of cardiac function from immune state data and supports translational application across animal models, ex vivo human tissue systems, and patientspecific clinical datasets.Atty. Docket No. UAZ-44590.601

[0030] In some embodiments, the favorable change in measured immune cell activity is a reduction in classically activated Ml macrophage activity and / or an increase in classically activated M2 macrophage activity.

[0031] Also provided is a method, comprising: a) obtaining a biological sample from a patient suffering from a heart disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient. In some embodiments, treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient comprises administering to the patient a composition comprising the immunomodulatory agent.

[0032] Further provided is a method, comprising: a) obtaining a biological sample from a patient suffering from an inflammatory disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immuneAtty. Docket No. UAZ-44590.601

[0033] cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

[0034] Additionally provided is a method, comprising: a) obtaining a biological sample from a patient suffering from a fibrotic disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

[0035] Also provided is a method, comprising: a) obtaining a biological sample from a patient suffering from an inflammatory disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein anAtty. Docket No. UAZ-44590.601

[0036] unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d)treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

[0037] Further embodiments provide a method, comprising: a) obtaining a biological sample from a patient suffering from a fibrotic disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

[0038] Other embodiments provide a method, comprising: a) obtaining a biological sample from a patient suffering from a heart disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein anAtty. Docket No. UAZ-44590.601

[0039] unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; d) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, and optionally e) integrating this data into computational workflow to drive development of a clinically deployable tool that can interpret patient specific immune data.

[0040] In certain embodiments, provided herein is a method, comprising: a) obtaining a biological sample from a patient suffering from an inflammatory disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; d) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, and optionally e) integrating this data into computational workflow to drive development of a clinically deployable tool that can interpret patient specific immune data.

[0041] Additional embodiments provide a method, comprising: a) obtaining a biological sample from a patient suffering from a fibrotic disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from theAtty. Docket No. UAZ-44590.601

[0042] peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent, and optionally e) integrating this data into computational workflow to drive development of a clinically deployable tool that can interpret patient specific immune data.

[0043] Also provided is a system, comprising: a computer processor that receives data from a tissue culture platform configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; and processes the data to generate one of more of a determination of an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function, a prognosis for the subject, a diagnosis for the subject, or a recommended treatment for the subject. In some embodiments, the processing of the data comprises the use of an Al and / or ML algorithm selected from transformer and Perceiver IO models, convolutional neural networks, recurrent neural networks, state space models, and forecasting architectures.

[0044] Additional embodiments are described herein.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows macrophage immune response to various medium. THP-1 cells were incubated in the presence of lOng / ml PMA in RPMI medium for 24h and the medium was then replaced with (a) RPMI medium, (b) DMEM, (c) HDF conditioned medium, (d) cell patch conditioned medium, (e)Atty. Docket No. UAZ-44590.601

[0046] RPMI containing 20 ng / ml IFN-y and 10 pg / ml LPS, or (f) RPMI containing 20 ng / ml IL4 and 20 ng / ml IL 13 and the cells were subsequently cultured for 24h, 48h and 72h.

[0047] FIG. 2 shows RAW264.7 cell expression of CD80, Arginase, and Cd68 after 24

[0048] hour incubation with (Row 1) DMEM complete media, (Row 2) DMEM + INFY + LPS

[0049] (Row 3) DMEM + IL-4 (Row 4) Biologic-conditioned media, (Row 5) unconditioned RPMI + B27 Media.

[0050] FIG. 3 shows THP1 macrophage phenotypes (M0, Ml, M2) expressing HLADR

[0051] and CD209.

[0052] FIG. 4 shows Griess Assessment of RAW264.7 cells.

[0053] FIG. 5 shows Griess Assessment of THP-1 cells.

[0054] FIG. 6 shows induction of activated Ml macrophage phenotype and activated M2 macrophage phenotype.

[0055] FIG. 7 shows an exemplary predictive diagnostic assay workflow: A: Peripheral blood mononuclear Cells (PBMCs) are isolated from CHF patients; B: PBMCs are cultured with an immunomodulatory therapy; C: Cells are evaluated with our biomarker assay panel for functional assessment.

[0056] FIG. 8 shows immunofluorescence staining (A), image quantification (B), and transcriptomic cellular deconvolution (C) of CHF mice compared to CHF mice treated with the biologic therapeutic.

[0057] FIG. 9 shows macrophage assessment in vivo showing a reduction of inflammatory macrophage markers over time, and an increase in anti-inflammatory macrophage markers.

[0058] FIG. 10 shows in vivo confirmation that there are peripheral changes within immune cell populations in splenic tissues after treatment with immunomodulatory biologies

[0059] FIG 11 shows assessment of samples receiving treatment for evaluation of predictive markers with flow cytometry, mass spectroscopy proteomics, and Multiplex ELISAs.

[0060] FIG. 12 shows macrophage phenotype assessment in-vitro: Immunomodulatory differences include known Ml behaviors (A) Nitrite / NO production in RAW 264.7 Cells, and assays detecting M2 target molecules for diagnostics, such as (B) Acetylated LDL

[0061] uptake, and (C) Arginase expression in THP-1 cells. (*P<0.05)

[0062] FIG 13 Shows flow cytometry immune phenotyping of human peripheral blood mononuclear cells which were induced to generate macrophage phenotypes and have been quantified in FIG 15.

[0063] FIG. 14 shows mass spectrometry experiments on secretome PBMC cocultures.

[0064] FIG. 14A shows significant proteins identified. FIG. 14B shows pathways that were

[0065] enriched. FIG 14C focuses on specific biologic and cellular processes that were significantly increased in human immune cells treated with the biologic. FIG 14D-J show specific protein clustered from nonbiased clustering of proteomes with subsequent pathway and individualAtty. Docket No. UAZ-44590.601

[0066] protein hits for each cluster.

[0067] FIG. 15A shows methods for isolating and evaluating CD14+ hPBMC markers. 15B shows Ml like marker expression and FIG. 15C shows M2 like marker expression with statistical significance. These assays have demonstrated the ability to detect donor level variability necessary to develop diagnostic markers.

[0068] FIG. 16 shows markers identification in swine. FIG. 16A shows the experimental workflow. FIG. 16B shows markers tested which have correlated with cardiac MRI. FIG

[0069] 16C also shows Transthoracic echocardiogram in a normal mouse and mouse 3 weeks after infarct showing a dilated left ventricle with Conductance catheter derived pressure / volume (P / V) loops in normal mouse (left) and mouse 3 weeks after a MI (rght) showing a dilated left ventricle with a decrease in the end-systolic P / V relationship or end systolic elastance (Fes). These measurements can be combined with biomarker expression to predict

[0070] functional outcome in human patients.

[0071] DEFINITIONS

[0072] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:

[0073] As used herein, the terms “detect”, “detecting” or “detection” may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition.

[0074] As used herein, the term “subject” refers to any organisms that are screened using the diagnostic methods described herein. Such organisms preferably include, but are not limited to, mammals (e.g., humans).

[0075] The term “diagnosed,” as used herein, refers to the recognition of a disease by its signs and symptoms, or genetic analysis, pathological analysis, histological analysis, and the like.

[0076] As used herein, the term "nucleic acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and

[0077] RNA including, but not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5

[0078] (carboxyhydroxyl-unethyl) uracil, 5-fluorouracil, 5 bromouracil, 5-carboxymethylaminomethyl 2 thiouracil, 5 carboxymethyl¬aminomethyluracil. dihydrouracil, inosine, N6 isopentenyladenine, 1 methyladenine, 1-methylpseudo¬uracil, 1 methylguanine, 1 methylinosine, 2,2-dimethyl¬guanine, 2Atty. Docket No. UAZ-44590.601

[0079] methyladenine, 2 methylguanine, 3-methyl¬cytosine, 5 methylcytosine, N6 methyladenine, 7 methylguanine, 5 methylaminomethyluracil, 5-methoxy-amino¬methyl 2 thiouracil, beta D mannosylqueosine, 5' methoxycarbonylmethyluracil, 5 methoxyuracil, 2 methylthio N6

[0080] isopentenyladenine, uracil 5 oxyacetic acid methylester, uracil 5 oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2 thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4 thiouracil, 5-methyluracil, N-uracil 5 oxyacetic acid methylester, uracil

[0081] 5 oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6 diaminopurine.

[0082] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full length coding

[0083] sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragments are retained. The term also encompasses the coding region of a structural gene and the sequences located

[0084] adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene.

[0085] A genomic form or clone of a gene contains the coding region interrupted with noncoding sequences termed "introns" or "intervening regions" or "intervening

[0086] sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0087] The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the stateAtty. Docket No. UAZ-44590.601

[0088] they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise

[0089] flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or

[0090] polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be

[0091] double-stranded).

[0092] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues (e.g., biopsy samples), cells, and gases. Biological samples include blood products, such as

[0093] plasma, serum and the like. Such examples are not however to be construed as

[0094] limiting the sample types applicable to the present disclosure.

[0095] DETAILED DESCRIPTION

[0096] The present disclosure provides composition, systems, and methods for characterizing, diagnosing, prognosing, and therapeutically treating patients suffering from a heart disorder (e.g,, cardiac chronic ischemic heart failure), skeletal muscle disorder (volumetric muscle loss), or other inflammatory and fibrotic diseases (e.g., cardiac chronic ischemic heart failure). In particular, the present disclosure provides compositions and methods tor companion diagnostics to prognose and identify and monitor treatments of such patients, as well as develop predictive markers for next generation therapeutics. These predictive immune profiles are integrated with computational algorithms to develop a clinically deployable tool that can be used from patient stratification.Atty. Docket No. UAZ-44590.601

[0097] A biologically active platform that increases the prevalence of alternatively activated M2 macrophage phenotypes in immune competent animal models of chronic ischemic heart failure (mice, rat, swine) was utilized. The biologic mediates changes in the stromal cell population that repair tissue (PMID: 31075250, PMID: 38007534). This biologic is composed of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and neonatal fibroblasts on a bioresorbable matrix that is implanted at the site of injury. The biologic restores contractile function, partially reverses adverse left and right ventricular remodeling, increases blood flow, and repairs damaged cardiomyocytes of the chronically infarcted myocardium.

[0098] Platforms to evaluate immunomodulatory therapies targeting macrophages as treatments for chronic heart failure in-vitro were developed to allow for high throughput screening, characterization of new therapeutics, and the development of a diagnostics and prognostics for patients in clinical trials. Several experiments were used to characterize the phenotype and function of endogenous immune cells, namely macrophages. These experiments included whole blood transcriptomics, multiplex ELISAs, mass spectroscopy, flow cytometry, nitric oxide assessment, arginase assessment, and acetylated LDL uptake assessment. These assays were tested on mouse RAW264.7 Cells, human THP-1 cells, mouse, swine, and human whole blood, peripheral blood mononuclear cells, and primary monocyte / macrophages isolated from peripheral blood. To verify macrophage phenotype induction, therapeutic treated cells were compared with known macrophage control phenotypes for statistical significance. These experiments were used to guide the in vitro models of human cell types and are concurrently developed in vivo for comparison and refinement of the prototype.

[0099] The experiments demonstrated the ability to delineate classically activated from alternatively activated macrophages with statistical significance, giving a platform to test macrophages exposed to other therapies. Furthermore, these data support that macrophages exposed to the biologic platform tend to reduce classically activated Ml macrophage phenotypes and elevate behaviors associated with alternatively activated M2 macrophage phenotypes in-vitro, but can vary between assay, cell type, and species.

[0100] The in-vitro methods and results allow for phenotypic assessment of macrophages and allow for characterization of their function in response to treatment that can identify therapeutic candidates for further in-vivo testing and lead to development of a companion diagnostic for patients.Atty. Docket No. UAZ-44590.601

[0101] Accordingly, provided herein are compositions and methods for characterizing, prognosing, and treating a variety of disorders associated with altered immune cells function (e.g., heart failure, heart damage, inflammatory disorders, and fibrotic disorders). In some embodiments, the patient is a human patient suffering from chronic ischemic heart failure, myocardial infarction, or heart failure. In some embodiments, the patient is a human patient suffering from persistent atrial fibrillation. In some embodiments, the patient is suffering from skeletal muscle disorders such as volumetric muscle loss, pulmonary fibrosis (Idiopathic pulmonary fibrosis), chronic inflammation and autoimmune diseases, chronic renal disease. In some embodiments, the platform may be used to better understand immune function as it relates to aging.

[0102] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from a disorder (e.g., heart or skeletal muscle disorder) characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

[0103] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from a heart disorder characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring oneAtty. Docket No. UAZ-44590.601

[0104] or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent.

[0105] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from a heart disorder characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

[0106] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from any type or kind of inflammatory disorder characterized with altered (e.g., increased or decreased) immune cell function,Atty. Docket No. UAZ-44590.601

[0107] wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent.

[0108] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from any type or kind of inflammatory disorder characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be anAtty. Docket No. UAZ-44590.601

[0109] unfavorable therapeutic for treating the patient; and d) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

[0110] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from any type or kind of a fibrotic disorder (e.g., fibrosis) characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

[0111] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from any type or kind of a fibrotic disorder (e.g., fibrosis) characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity inAtty. Docket No. UAZ-44590.601

[0112] comparison to an established norm for baseline immune cell function for tissue characterized altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

[0113] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from any type or kind of fibrotic disorder (e.g., fibrosis) characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; and d) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent.

[0114] In certain embodiments, the present disclosure provides a method, comprising: a) obtaining a biological sample from a patient suffering from any type or kind of inflammatory disorder characterized with altered (e.g., increased or decreased) immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood; b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; c) measuring one or more types of immune cell activity for the immune cells isolatedAtty. Docket No. UAZ-44590.601

[0115] from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent; wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient, wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered (e.g., increased or decreased) immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

[0116] In some embodiments, the present disclosure further comprises administering to the patient one or more guideline directed medical therapy agents. Examples include but are not limited to, ACE inhibitors, β-blockers, ARBs, diuretics, calcium channel blocker, Entresto (sacubitril / valsartan), SGLT2i, GLP-1 RA medicines such as ozempic, nitrates, platelet aggregation inhibitors, cholesterol-lowering medications, ranolazine, ivabradine, or a combination thereof.

[0117] In some embodiments, treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient comprises administering to the patient a composition comprising the immunomodulatory agent.

[0118] In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor (ICI). In some embodiments, the ICI is an inhibitor of PD-1, PD-L1, CTLA4, LAG3, TIGIT, TIM3, VISTA, ICOS, BTLA, GITR, NKG2A, CD112R, B7-H3, or CD73. In some embodiments, the ICI is selected from nivolumab, pembrolizumab, ipilimumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplimab, retifanlimab, dostarlimab, and toripalimab.

[0119] In some embodiments, the one or more type of immune cell activity is selected from cytokine activity, macrophage activity, B lymphocyte activity, T lymphocyte activity, cytotoxic T-cell lymphocyte (CTL) activity, mast cell activity, monocyte activity, dendritic cell activity, eosinophil activity, natural killer cell activity, basophil activity, and neutrophil activity.

[0120] In some embodiments, the one or more type of immune cell activity is M2 macrophage cellular activity.

[0121] In some embodiments, the assessment of differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents comprises one orAtty. Docket No. UAZ-44590.601

[0122] more of the following: flow cytometry assessment, nitric oxide assessment, arginase assessment, acetylated LDL uptake assessment, transcriptomics assessment, proteomics assessment, and multiplex ELISA assessment.

[0123] In some embodiments, the favorable change in measured immune cell activity is a reduction in classically activated Ml macrophage activity. In some embodiments, the favorable change in measured immune cell activity is an increase in classically activated M2 macrophage activity.

[0124] Immunomodulatory agents include, but are not limited to, statins; mTOR inhibitors, such as rapamycin or a rapamycin analog; TGF-P signaling agents; TGF-P receptor agonists; histone deacetylase inhibitors, such as Trichostatin A; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio, Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2), such as Misoprostol; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor (PDE4), such as Rolipram; proteasome inhibitors; kinase inhibitors; G-protein coupled receptor agonists; G-protein coupled receptor antagonists; glucocorticoids: retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB inhibitors, such as TGX-221; autophagy inhibitors, such as 3 -Methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and oxidized ATPs, such as P2X receptor blockers. Immunosuppressants also include IDO, vitamin D3, cyclosporins, such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathiopurine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol; triptolide; OPN-305, OPN-401; Eritoran (E5564); TAK-242; CpnlO; NI-0101; 1A6; AV411; IRS-954 (DV-1079); IMO-3100; CPG-52363; CPG-52364; OPN-305; ATNC05; NI-0101; IMO-8400; Hydroxychloroquine; CU-CPT22; C29; Ortho-vanillin; SSL3 protein; OPN-305; 5 SsnB; Vizantin; (-i-)-N-phenethylnoroxymorphone; VB3323; Monosaccharide 3; (+)-Naltrexone and (+)-naloxone; HT52; HTB2; Compound 4a; CNTO2424; TH1020; INH-ODN; E6446; AT791; CpG ODN 2088; ODN TTAGGG; COV08-0064; 2R9; GpG oligonucleotides; 2-aminopurine; Amlexanox; Bay11-7082; BX795; CH-223191;

[0125] Chloroquine; CLI-095; CU-CPT9a; Cyclosporin A; CTY387; Gefitnib; Glybenclamide; H-89; H-131; Isoliquiritigenin; MCC950; MRT67307; OxPAPC; Parthenolide; Pepinh-MYD; Pepinh-TRIF; Polymyxin B; R406; RU.521; VX-765; YM201636; Z-VAD-FMK; and AHR-Atty. Docket No. UAZ-44590.601

[0126] specific ligands; including but not limited to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD); tryptamine (TA); and 6 formylindolo[3,2 b]carbazole (FICZ). In particular embodiments, the immunosuppressant is FTY720 (also known as fingolimod) (Chung and Harung, Clin.

[0127] Neuropharma col 33: 91-101, 2010), AhR activation by 2-(l’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) or related ligands (Yeste A, et al. Proc. Natl. Acad. Sci. USA 109: 11270-11275, 2012; Quintana F. J., et al Proc. Natl. Acad. Sci. USA 107: 20768-20773, 2010), Trichostatin A (TSA) (Reilly C. M. et al. J. Autoimmun 31: 123-130. 2008). Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, (Lucas J. L., et al. Cell Immunol 257: 97-104, 2009) and / or Rapamycin (Rapa) (Maldonado, R. A., et al Proc. Natl. Acad. Sci. USA 112: E156-165, 2015).

[0128] In some embodiments, the present disclosure provides treatment methods where one or more of the immune modulatory agent or other therapeutic agents are administered.

[0129] In some embodiments, the composition is a bioresorbable matrix, wherein the immunomodulatory agent is embedded within the bioresorbable matrix.

[0130] In some embodiments, the bioresorbable matrix further comprises human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and neonatal fibroblasts.

[0131] In some embodiments, the bioresorbable matrix is a solid or semi solid supports (e.g., scaffold, a gel, a nanoparticle, or a patch).

[0132] In some embodiments, the solid support is a scaffold. In some embodiments, scaffolds comprise a biomaterial support comprising fibroblasts and / or cardiomyocytes.

[0133] The scaffolds described herein are scaffolded of any number of suitable materials. In some embodiments, the scaffold comprises synthetic material. In some embodiments, the scaffold comprises biological material. In some embodiments, the scaffold is a hybrid of synthetic and biological materials.

[0134] Examples of suitable scaffold material include, but are not limited to, one or more of collagen, fibronectin, poly glycolides, polylactides, polypropylene, polyester, silicone, expanded polytetrafluorothylene, Dexon, Vicryl, polycaprolactone, polydioxanone, catgut, silk, nylon, and trimethylene carbonate.

[0135] For certain applications, the scaffold is composed of a polylactide material or a polyglactin 910 material. In some embodiments, the scaffold is derived from human, bovine or porcine tissue.

[0136] In some embodiments, the scaffold is bioabsorbable. In some embodiments, the scaffold is non-bioabsorbable.Atty. Docket No. UAZ-44590.601

[0137] For certain applications, the scaffold further comprises a therapeutic agent such as a drug or biologic. In some embodiments, the therapeutic agent is known to be useful in treating, ameliorating and / or preventing cardiac conditions. Examples include, but are not limited to, angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril, lisinopril, and captopril), angiotensin II (A-II) receptor blockers (e.g., losartan and valsartan), diuretics (e.g., bumetanide, furosemide, and spironolactone), beta blockers, nesiritide, and SGLT2 inhibitors. Additional agents are described herein.

[0138] In some embodiments, the scaffold further comprises cells (e.g., of the same or mixed cell types). In some embodiments, the cells are stem cells (e.g., cardiac stems cells or progenitors thereof) or derived from stem cells or fibroblasts.

[0139] The scaffold can be contacted with the heart or other muscle in any suitable way to promote attachment. The scaffold may be attached to various locations on the heart, including the epicardium, myocardium and endocardium, most preferably the epicardium. Means for attachment include, but arc not limited to, direct adherence between the scaffold and the heart tissue, biological glue, suture, synthetic glue, laser dyes, or hydrogel. A number of commercially available hemostatic agents and sealants include SURGICAL® (oxidized cellulose), ACTIFOAM® (collagen), FIBRX® (light-activated fibrin sealant), BOHEAL® (fibrin sealant), FIBROCAPS® (dry powder fibrin sealant), polysaccharide polymers p-GlcNAc (SYVEC® patch; Marine Polymer Technologies), Polymer 27CK (Protein Polymer Tech.). Medical devices and apparatus for preparing autologous fibrin sealants from 120 ml of a patient's blood in the operating room in one and one-half hour are also known (e.g. Vivostat System).

[0140] In an alternative embodiment of the disclosure utilizing direct adherence, the scaffold is placed directly onto the muscle and the product attaches via natural cellular attachment. In a further alternative embodiment, the scaffold is attached to the heart using surgical glue, preferably biological glue such as a fibrin glue. The use of fibrin glue as a surgical adhesive is well known. Fibrin glue compositions are known (e.g., see U. S. Pat. Nos. 4,414,971;

[0141] 4,627,879 and 5,290,552) and the derived fibrin may be autologous (e.g., see U. S. Pat. No.

[0142] 5,643,192). The glue compositions may also include additional components, such as liposomes containing one or more agent or drug (e.g., see U. S. Pat. Nos. 4,359,049 and 5,605,541) and include via injection (e.g., see U. S. Pat. No. 4,874,368) or by spraying (e.g., see U. S. Pat. Nos. 5,368,563 and 5,759,171). Kits are also available for applying fibrin glue compositions (e.g., see U. S. Pat. No. 5,318,524).Atty. Docket No. UAZ-44590.601

[0143] In another embodiment, a laser dye is applied to the muscle, the scaffold, or both, and activated using a laser of the appropriate wavelength to adhere to the tissues. In alternative embodiments, the laser dye has an activation frequency in a range that does not alter tissue function or integrity. For instance, 800 nm light passes through tissues and red blood cells. Using indocyan green (ICG) as the laser dye, laser wavelengths that pass through tissue may be used. A solution of 5 mg / ml of ICG is painted onto the surface of the three-dimensional stromal tissue (or target site) and the ICG binds to the collagen of the tissue. A 5 ms pulse from a laser emitting light with a peak intensity near 800 nm is used to activate the laser dye, resulting in the denaturation of collagen which fuses elastin of the adjacent tissue to the modified surface.

[0144] In another embodiment, the scaffold is attached to the muscle using a hydrogel. A number of natural and synthetic polymeric materials are sufficient for forming suitable hydrogel compositions. For example, polysaccharides, e.g., alginate, may be crosslinked with divalent cations, polyphosphazenes and polyacrylates are crosslinked ionically or by ultraviolet polymerization (U. S. Pat. No. 5,709,854). Alternatively, a synthetic surgical glue such as 2-octyl cyanoacrylate (" DERMABOND™", Ethicon, Inc., Somerville, NJ.) may be used to attach the three-dimensional stromal tissue.

[0145] In an alternative embodiment of the present invention, the scaffold is secured to the muscle using one or more sutures, including, but not limited to, 5-O, 6-O and 7-O proline sutures (Ethicon Cat. Nos. 8713H, 8714H and 8701H), poliglecaprone, polydioxanone, polyg lactin or other suitable non-biodegradable or biodegradable suture material. When suturing, double armed needles are typically, although not necessarily, used.

[0146] In some embodiments, this platform is made executable by artificial intelligence (AI) and machine learning (ML) computational algorithms that can be employed to analyze and draw conclusion from patient specific immune data using work flows. Model development includes attention-based architectures, such as transformer and Perceiver IO models, to capture complex and high-dimensional relationships among immune features; convolutional neural networks to identify localized and hierarchical immune patterns; and recurrent neural networks, including LSTM and GRU models, to model temporal immune dynamics. State space models are employed to characterize dynamic immune state transitions over time, while forecasting architectures, such as Temporal Fusion Transformers and DeepAR, are used to predict future cardiac trajectories from immune data. These models are benchmarked against interpretable baseline approaches, including regularized linear models, random forests, and gradient boosting methods. Collectively, this multi-model strategy enablesAtty. Docket No. UAZ-44590.601

[0147] robust, generalizable prediction of cardiac function from immune state data and supports translational application across animal models, ex vivo human tissue systems, and patientspecific clinical datasets.

[0148] The present disclosure also provides compositions, kits, or systems, comprising a computer processor configured to perform a method described herein. In some embodiments, the system comprises a computer processor that receives data from a tissue culture platform configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents (e.g., those described herein); and processes the data to generate one of more of a determination of an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function, a prognosis for the subject, a diagnosis for the subject, or a recommended treatment for the subject. In certain cases, the processing of the data comprises the use of an Al and / or ML algorithm described herein.

[0149] EXPERIMENTAL

[0150] The following examples are illustrative, but not limiting, of the compounds, compositions, and methods of the present disclosure. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy, and which are obvious to those skilled in the art are within the spirit and scope of the disclosure. The use of pronouns such as “I”, “we”, and “our”, for example, refer to the inventors.

[0151] Example 1

[0152] This example describes the evaluation of in-vitro models for the translation of diagnostics, prognostics, and therapeutics for immunomodulatory treatments for cardiac repair and / or tissue repair.

[0153] Methods

[0154] Cell Culture- THP-1 Cells

[0155] The Human monocytic THP-1 cell line was obtained from ATCC (#TIB-202) and maintained at 2 x 105cells / ml in RPMI 1640 medium with GlutaMAX (Gibco, 61870036) supplemented with 10 % of heat inactivated fetal bovine serum (Gibco), 10 mM Hepes (Gibco, #15630080), non-essential amino acid (Gibco, #11140050), 1 % Pen / strep and 50 uMAtty. Docket No. UAZ-44590.601

[0156] B-mercaptoethanol (Gibco; 31350-010). THP-1 monocytes were differentiated into macrophages by 24 h incubation with lOng / ml phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) followed by 24 h incubation in RPMI medium. Macrophages were polarized in Ml macrophages by incubation with 20 ng / ml of IFN-y (R& D system, #285-IF) and 10 pg / ml of LPS (Sigma, #8630). Macrophage M2 polarization was conducted by incubation with 20 ng / ml of interleukin 4 (R& D Systems, #204-IL) and 20 ng / ml of interleukin 13 (R& D Systems, #213-ILB). [1,2]

[0157] Cell Culture-RAW264.7 Cells

[0158] Murine RAW264.7 cells (ATCC TIB-71) were thawed and seeded at a concentration of 1x106cells / mL in a T75 flask and sub-cultured to passage 13-15 at 37°C and 5% CO2 for assessment. The cells were cultured in Dulbecco’s Modified Eagle’s Medium that contained 4mM L-glutamine, 4500 mg / L glucose, ImM sodium pyruvate, 1500 mg / L sodium bicarbonate, 10% heat-inactivated FBS, and 1% Penicillin-Streptomycin. The cells were treated with just DMEM growth media to generate a pan-macrophage control group, 20 ng / mL of IFN-y and 10 ng / mL of LPS to generate a classically activated Ml macrophage control group, 20 ng / mL of IL-4 to generate the alternatively activated M2 macrophage control group, biologic-conditioned media was used to generate the biologic treated macrophage group. Biologic conditioned media was collected from the biologic platform 2 days after thawing from cryopreservation in RPMI+B27 and used as treatment. RPMI+B27 alone was used to generate a RPMI+B27 macrophage control media group, relative to biologic conditioned media.

[0159] Immunofluorescence Stain-RAW264.7 cells

[0160] Murine RAW264.7 cells were seeded at a concentration of 5.00x105cells / mL in a 24-well plate with a coverslip on the bottom of the well. The cells were incubated for 24h with their respective activating media (M0, Ml, M2, Biologic Macrophages, RPMI+B27 Macrophages). After incubation, the cells were fixed with 100% ice-cold methanol for 15 minutes. Next, the cells were washed three times with lx PBS and then blocked with 1% BSA in PBS for 1 hour. Then the cells were co-stained with the three primary antibodies: anti-liver Arginase antibody (ab96183, abeam, 1:500), anti-CD80 antibody (AF740, R& D Systems, 1:40), and anti-CD68 antibody (ab53444, abeam, 1:50) diluted in 1% BSA in PBS for 1 hour, covered with foil. Next, the cells were washed three times with lx PBS and then stained for 1 hour with three secondary antibodies: Alexa-fluor 488 (A- 11055, Invitrogen,Atty. Docket No. UAZ-44590.601

[0161] 1:200), Alexa- fluor 546 (A10040, Invitrogen, 1:200), and Alexa-fluor 647 (A78947, Invitrogen, 1:200) diluted in 1% BSA in PBS covered with foil. The cells were washed three times with lx PBS and incubated with DAPI (ab228549, abeam, 1:1000) diluted in 1%BSA in PBS for 15 minutes. The coverslips with then removed and placed onto slides with ProLong Glass Antifade Mountant (P36982, Invitrogen). The slides were imaged with an inverted microscope (Leica, DMI6000).

[0162] Immunofluorescence labeling of THP-1 Cells

[0163] THP-1 monocytes were seeded at 200,000 cells / well in 24-well plates containing a coverslip and were differentiated and polarized as described here. For labeling, cells were fixed with ice-cold methanol for 15 min followed by Ih incubation with 2% BSA-PBS blocking buffer. Cells were then incubated with primary antibody anti-CD80 (Abeam, #ab225674), or anti-MRCl (Sigma, #HPA004114) diluted in 2 % BSA-PBS at 1:1000 dilution for Ih at room temperature. Cells were washed three times and then incubated with secondary antibody Alexa Fluor-546-conjugated anti-rabbit IgG (ThermoFisher, #A10040) for Ih at 1: 1000 dilution at room temperature. After cells were washed three times with PBS, the coverslips were mounted in Dako mounting medium (Agilent) and imaged with an inverted microscope (DMI6000, Leica).

[0164] Macrophage phenotyping by flow cytometry

[0165] Macrophages isolated from cell culture experiments were stained for both membrane specific, as well as intracellular markers. Flow cytometry experiments were validated by utilizing FMO controls to resolve positive and negative unmixed signals. Acquisition was performed on the Cytek Aurora 5 laser spectral cytometer capable of detecting 25 colors that are all spectrally unique. Results were reported as Total Fluorescence Intensity (TFI) = % positive events * Mean Channel Fluorescence. Macrophage markers shown in FIG. 2 were used to gate endogenous monocytic derived macrophage cell populations [3-6],

[0166] Arginase Assay

[0167] Arginase- 1 expressing alternatively activated macrophage suppress chronic inflammation and T-cell responses [7], This protein is known to be more upregulated in M2 macrophages and was used to quantify protein content of in-vitro polarized macrophages described in the cell culture methods below.Atty. Docket No. UAZ-44590.601

[0168] The arginase activity assay (Sigma Aldrich, MAK112) was used in MO, Ml, and M2 macrophages to assess the conversion of L-arginine to urea and L-omithine. Briefly, murine RAW264.7 cells were seeded at a concentration of 1.00x105cells / mL in a T75-flask and cultured with the respective activating media (MO, Ml, M2). Approximately 1x106cells were harvested from the flask and washed with PBS. The cells were then centrifuged at 1,000 x g for 10 minutes at 4°C to remove the PBS. The pellets were then lysed with 100 pl of lysis buffer containing, 0.4% Triton x-100, 10 mM Tris-HCl (pH 7.4), and 0.1% of Halt Protease Inhibitor cocktail (87786, Thermo Fisher) for 10 minutes. The samples were then centrifuged at 13,000 x g for 10 minutes to remove insoluble material. The manufacturer recommends adding up to 40 pL of sample supernatant (for this experiment 16 pL was used to reach an n=3 for each group) to each of two wells in a 96- well plate for a sample and sample blank well. Ultrapure water was added to each well to bring the final volume to 40 pL. 50 pL of prepared 1 mM urea standard working solution and 50 pL of water to separate wells of the plate. 10 pL of 5x substrate buffer (8 pL of Arginine Buffer and 2 pL of Mn solution) was added to each sample wells. The plate was covered with foil, mixed on a horizontal shaker, and then incubated at 37°C for 2 hours. After incubation, 200 pL of Urea reagent (100 pL of Reagent A and 100 pL of Reagent B) were added to every well, with the addition of 10 pL of 5x substrate buffer added to the sample blank wells. The place was incubated for 1 hour at room temperature. Absorbance measurement was done at 430 nm by a microplate reader (Biotek, Epoch). One unit of Arginase is the amount of enzyme that will convert 1.0 pmole of L-arginine to ornithine and urea per minute at pH 9.5 and 37°C. The Arginase activity was determined by the following equation:

[0169] Activity = [(A430)Sample − (A430)Blank / (A430)Standard − (A430)Water] × (1 mM × 50 × 10³) / (V × T)

[0170] Greiss Assay Methods

[0171] The Greiss assay is used to quantify nitric oxide production from cell lysate and is known to be more highly expressed in Ml macrophages. This assay was used to quantify the change in NO activity between Ml and M2 polarized macrophages in-vitro. Nitric oxide production was determined by measuring its stable end product nitrite, using a Griess reagent (Promega Corporation, Madison, WI) according to manufacturer’s protocol. Briefly, 50 pl of supernatant was added to 96- well plate, followed by 50 pl sulphanilamide and 50 pl N-l-napthylethylenediamine dihydrochloride (NED). Absorbance at 540 nm was measured byAtty. Docket No. UAZ-44590.601

[0172] microplate reader and nitrite concentrations were estimated using a standard nitrite curve. For nitric oxide production in conditioned-media-treated MSCs, nitrite in corresponding conditioned media was subtracted from that in MSCs supernatant [8].

[0173] Griess Assay-RAW264.7 cells

[0174] The production of nitric oxide (NO) in murine RAW264.7 cells was detected by measuring the levels of the stable end product nitrite (NO2). Nitrite detection was done by using the Griess Reagent Kit (Thermo Fisher, G7921) following the manufacturers protocol. Briefly, 150 pL of culture supernatant was added to a 96-well plate, mixed with 130 pL of deionized water, and 20 pL of Griess reagent. Absorbance measurement was done at 548 nm by a microplate reader (Biotek, Epoch). Nitrite standards from the kit were used to calculate concentrations of cell culture supernatants. Statistical significance between groups were determined using a one-way ANOVA P<0.05 using SigmaPlot software.

[0175] Griess Assay- h-THP-1 cells

[0176] The production of nitric oxide (NO) in human THP-1 cells was detected by measuring the levels of the stable end product nitrite (NO2). Nitrite detection was done by using the Griess Reagent Kit (Thermo Fisher G7921) following the manufacturers protocol. Briefly, 150 pL of culture supernatant was added to a 96-well plate, mixed with 130 pL of deionized water, and 20 pL of Griess reagent. Absorbance measurement was done at 548 nm by a microplate reader (Biotek, Epoch). Nitrite standards from the kit were used to calculate concentrations of cell culture supernatants. Statistical significance between groups were determined using a one-way ANOVA P<0.05 using SigmaPlot software.

[0177] Blood Collection and PBMC Isolation

[0178] Peripheral blood mononuclear cells (PBMCs) are isolated from human whole blood obtained from donors or biorepositories. Equal volumes of blood and density gradient medium, such as HISTOPAQUE, are combined in a 15 mL tube. Whole blood is carefully layered over the gradient medium, and samples are centrifuged for approximately 30 minutes at 400xg at 4°C without braking. The plasma fraction is aspirated and optionally stored at -80°C. The PBMC layer is collected, diluted to approximately 10 mL with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 2 mM EDTA, and centrifuged at 300xg for 10 minutes at 4°C. Cells are washed sequentially two additional times with 1 mL buffer, followed by treatment with red blood cell lysis solution (e.g., IXAtty. Docket No. UAZ-44590.601

[0179] RBC Lysis from a 10X stock) for 10-15 minutes at room temperature. Cells are washed and centrifuged at 300xg for 10 minutes at 4°C and counted to determine viable cell number.

[0180] CD14+Monocyte Enrichment

[0181] PBMCs may be enriched for classical CD14+CD 16 monocytes using immunomagnetic separation. Cells are pelleted at 300xg for 10 minutes, resuspended at 30 pL per 107total cells in buffer, and incubated with 10 pL Fc receptor blocking reagent per 107cells. Classical monocyte biotinylated antibodies are added at 10 pL per 107cells, and optionally 5 pL of a thrombocyte removal reagent per 107cells is included. After a 5 -minute room temperature incubation, 30 pL buffer and 20 pL anti-biotin magnetic microbeads per 107cells are added, and cells are incubated an additional 5 minutes. Magnetic separation is performed using MS or LS columns, rinsed with appropriate buffer volumes (e.g., 500 pL for MS, 3 mb for LS). Flow-through fractions containing enriched CD14+cells are collected, washed, and optionally subjected to a second round of magnetic separation to increase purity.

[0182] Cryopreservation and Recovery

[0183] PBMCs or enriched CD14+monocytes are pelleted and resuspended in cryopreservation medium at approximately 2×106cells per vial. Cells are cooled in a controlled-rate container (e.g., Mr. Frosty) at -80°C overnight and transferred to liquid nitrogen for long-term storage. For recovery, vials are thawed at 37°C, diluted dropwise into pre- warmed medium, transferred to a 15 mL tube, and further diluted to a final volume of approximately 18 mL. Cells are centrifuged at 300xg for 5-10 minutes, supernatant removed, and cells counted for viability using trypan blue.

[0184] Culture of PBMCs and CD14+Monocytes

[0185] Cells are cultured in RPMI supplemented with 10% fetal bovine serum, 1% penicillinstreptomycin, and, where indicated, human M-CSF at 25-100 ng / mL. PBMCs or CD14+monocytes are seeded at approximately 250,000–1.5×106viable cells per well in 12- or 24-well tissue culture-treated plates, corresponding to a working concentration of 5×105cells / mL. Media are replenished on day 3-4. For polarization, differentiated macrophages are treated on day 6 with pro-inflammatory stimuli (e.g., 10 pg / mL LPS + 20 ng / mL IFN-y) for Ml polarization or anti-inflammatory stimuli (20 ng / mL IL-4 + 20 ng / mL IL-13) for M2 polarization, with a 24-48 hour incubation period prior to downstream analysis. Cell morphology is imaged on days 4, 6, 7, and 8 using a 10× objective.Atty. Docket No. UAZ-44590.601

[0186] Coculture With Biologic Therapeutics

[0187] Fibroblast-derived matrices or composite fibroblast / cardiomyocyte patches are cut to fit 24-well plates. Matrices may be left as acellular controls or seeded with iPSC-derived cardiomyocytes and preconditioned for approximately four days prior to coculture. Coculture groups may include: PBMCs with RPMI + B27, PBMCs + pro-inflammatory stimuli (LPS + IFN-y), PBMCs + anti-inflammatory stimuli (IL-4 + IL- 13), PBMCs + fibroblast patch, or PBMCs + composite fibroblast / cardiomyocyte patch. Media are replenished every other day, and conditioned media are collected at days 1, 2, 4, 6, and 7 for downstream proteomic analysis.

[0188] Flow Cytometric Immunophenotyping

[0189] Harvested cells are detached using TrypLE for approximately 5 minutes, pelleted, and washed with flow staining buffer at 300xg for 5 minutes at 4°C. Cells are incubated with a viability dye (e.g., Fixable Viability Stain 780) for 10 minutes at room temperature and washed. Fc receptor blocking reagent (e.g., TruStain FcX) is added for 10 minutes on ice. Cells are stained with a multiparameter antibody panel, including:

[0190] UV (355 nm): CD3 BUV395, CD4 BUV496, HLA-DR BUV615, CD16 BUV805 Violet (405 nm): CD200R1 BV421, CDllb BV480, CD19 BV750, CD56 BV828

[0191] Blue (488 nm): CD206 RB613, CD163 RB705, CD86 RB780

[0192] Yellow-Green (561 nm): CD209 PE, CD8 RY610, CCR2 RY775

[0193] Red (640 nm): CD80 AF647, CD 14 R718

[0194] Stained cells are washed twice with flow staining buffer, resuspended in preservation buffer, and acquired on a spectral or conventional flow cytometer (e.g., Cytek Aurora), collecting approximately 100,000 events per sample. Data are analyzed to define immune subsets, polarization markers, and viability.

[0195] Secretome Collection and Proteomics

[0196] Conditioned media collected from cultures or cocultures are clarified and subjected to acetone precipitation by adding six times the sample volume of pre-chilled acetone and incubating at -20°C for approximately 1 hour. Pellets are washed with 90% acetone, dried briefly, and resuspended in 50 mM ammonium bicarbonate. Proteins are reduced with 5 mM DTT at 70°C for 30 minutes, alkylated with 15 mM acrylamide for 30 minutes at room temperature, and excess alkylating agent quenched with DTT. Proteins are digested usingAtty. Docket No. UAZ-44590.601

[0197] Lys-C for 2-3 hours followed by trypsin overnight at 37°C while shaking. Digestion is stopped with formic acid and HFBA. Peptides are desalted, dried, and resuspended in 0.1% formic acid, quantified, and analyzed using LC-MS / MS on high-resolution instrumentation with nanoLC separation. Data are acquired using data-dependent acquisition, aligned across samples, and searched against curated human protein databases. Label-free quantification is performed using ion-intensity-based methods, normalized to total ion current. Proteins meeting statistical thresholds are analyzed via hierarchical clustering, principal component analysis, and pathway enrichment analysis using bioinformatics tools such as DAVID and REACTOME.

[0198] Results

[0199] FIG. 1 shows the macrophage immune response to various mediums. THP-1 cells were incubated in the presence of lOng / ml PMA in RPMI medium for 24h and the medium was then replaced with (a) RPMI medium, (b) DMEM, (c) HDF conditioned medium*, (d) cell patch conditioned medium*, (e) RPMI containing 20 ng / ml IFN-y and 10 pg / ml LPS, (f) RPMI containing 20 ng / ml 1L4 and 20 ng / ml 1L13 and the cells were subsequently cultured for 24h, 48h and 72h. mediam were replaced every 24h. Cells were fixed and immunolabeled with anti-CD80, or anti-MRCl antibody. Nuclei were detected with DAPI. HDF conditioned medium was collected from a T75 flask of NHDF cultured in DMEM for 24h. Cell patch conditioned medium was obtained from Avery Therapeutics.

[0200] FIG. 2 shows RAW264.7 cell expression of CD80, Arginase, and Cd68 after 24 hour incubation with (Row 1 ) DMEM complete media, (Row 2) DMEM + INFY + LPS (Row 3) DMEM + IL-4 (Row 4) Biologic-conditioned media, (Row 5) unconditioned RPMI + B27 Media. FIG. 3 shows THP1 macrophage phenotypes (M0, Ml, M2) expressing HLADR and CD209

[0201] Griess Assay-RAW264.7

[0202] Griess Assay: In order to characterize the phenotype of the altered macrophages from the biologic secretome Griess assay was used. The Griess assay is a functional assay that detects nitric oxide production through measuring nitrite, its stable end product [9, 10], Nitrite production is known to increase with LPS stimulation and is a known marker for classically activated Ml macrophage phenotypes [11, 12], The results demonstrated that macrophages activated by biologic conditioned media do not increase nitrite production indicating that the altered phenotype is not associated with classically activated MlAtty. Docket No. UAZ-44590.601

[0203] phenotypes. Furthermore, the data shows a statistical significance between IFN-y and LPS induced macrophages compared to biologic conditioned media treated macrophages by one way analysis of the variance. Data displayed normality by the Shapiro-Wilk test, and all pairwise comparisons were made with the Holm-Sidak method (P<0.05).

[0204] FIG. 4 shows a Griess Assessment of RAW264.7 cells. Nitrite was detected at an absorbance of 548 nm. RAW264.7 cells are immortalized murine macrophages in a MO panmacrophage state. The MO macrophages were cultured in DMEM complete media. The classically activated Ml macrophage phenotype was induced with DMEM complete media containing 20 ng / mL of IFN-y and 10 ng / mL of LPS. The alternatively activated M2 macrophage phenotype was induced with DMEM complete media containing 20 ng / mL of IL-4. The biologic conditioned media macrophages were treated with biologic media collected on day 2 post-cryopreservation. The RPMI+B27 media macrophages were treated with control media not exposed to the biologic.

[0205] Griess Assay- THP-1

[0206] Results demonstrated that macrophages activated by IFN-y and LPS do not increase nitrite production in comparison to other groups. Furthermore, the data shows a statistical significance between THP-1 monocytes and M0 pan-macrophages, and THP-1 and classically activated Ml macrophages by one way analysis of the variance. Data displayed normality by the Shapiro-Wilk test, and all pairwise comparisons were made with the Holm-Sidak method (P<0.05).

[0207] FIG. 5 shows Griess Assessment of THP-1 cells. Nitrite was detected at an absorbance of 548 nm. THP-1 cells are immortalized human monocytes from acute monocytic leukemia. The THP-1 monocytes were cultured with complete RPMI-1640 media. The M0 macrophages were cultured in complete RPMI-1640 media and 10 ng / mL of PMA for 24 hours, then RPMI media alone for the rest of the process. The classically activated Ml macrophage phenotype was induced with complete RPMI-1640 and 10 ng / mL of PMA for 24 hours, then RPMI media containing 20 ng / mL of IFN-y and 10 ng / mL of LPS. The alternatively activated M2 macrophage phenotype was induced with complete RPMI-1640 and 10 ng / mL of PMA for 24 hours, then RPMI media containing 20 ng / mL of IL-4.

[0208] LDL Uptake Assay

[0209] Following RAW264.7 cell culture and macrophage phenotype induction, cells were incubated with Dil conjugated acetylated LDL for 1,2, and 3 hours to assess uptake byAtty. Docket No. UAZ-44590.601

[0210] different phenotypes. Cells were lifted and washed with PBS three times and analyzed for mean fluorescence intensity by flow cytometry on Cytek Aurora instrument. One way ANOVA was performed to assess statistical significance of Acetylated LDL uptake by different phenotypes. Acetylated LDL is reported to be uptaken more readily in the m2 macrophage phenotypes [13, 14],

[0211] FIG. 6 shows that a one hour incubation of Dil-ACLDL with RAW264.7 cells induced to inflammatory and reparative phenotypes display strongest uptake distinction in M2 populations at 1 hour.

[0212] Multiplex Cytokine Release Assays

[0213] Multiplex Cytokine, Chemokine, Growth Factor, and Cardiovascular Biomarker Analysis

[0214] Plasma samples obtained from animal studies, including murine and swine models, as well as conditioned media collected from human tissue culture and coculture experiments, are analyzed using bead-based multiplex immunoassays (Millipore / MILLIPLEX®) to enable simultaneous quantification of immune-related cytokines, chemokines, growth factors, immune modulatory proteins, and cardiovascular biomarkers. All analytes described below are evaluated across all applicable animal models and human experimental systems, enabling cross-species comparison of immune signaling states associated with cardiac injury, repair, and functional improvement.

[0215] Samples are clarified by centrifugation and stored at -80°C until analysis. Prior to assay, plasma and conditioned media samples are thawed on ice and diluted as required to fall within the dynamic range of each assay. Multiplex immunoassays are performed according to manufacturer-recommended protocols for magnetic bead-based platforms, including incubation with antibody-conjugated beads, detection with biotinylated secondary antibodies, and signal amplification using streptavidin-conjugated fluorophores. For analytes requiring activation (e.g., total TGF- ), samples are processed accordingly prior to bead incubation.

[0216] Analytes quantified include cytokines and chemokines associated with immune activation, leukocyte recruitment, and macrophage polarization, including IL- 13, Eotaxin, Eotaxin-2, Eotaxin-3, MCP-1, MCP-2, MCP-3, MCP-4, MDC, MIP-1α, MIP-1β, MIP-1δ, MIP-3a, MIP-3P, MPIF-1, TARC, SDF, C-TACK, I-309, HCC-1, and HCC-4, as well as additional inflammatory mediators such as RANTES. Immune regulatory and checkpoint-associated proteins, including Galectin and MIP-4, are also measured. Growth factor andAtty. Docket No. UAZ-44590.601

[0217] angiogenic signaling are assessed through quantification of hepatocyte growth factor (HGF), while cardiovascular stress and remodeling are evaluated by measurement of NT-proBNP. Immunoregulatory and fibrotic signaling pathways are further interrogated through quantification of transforming growth factor beta isoforms TGF-β1, TGF-β2, and TGF-β3.

[0218] Data acquisition is performed on a Luminex-compatible multiplex detection platform, and analyte concentrations are calculated using five-parameter logistic regression standard curves. Samples are analyzed in technical replicates, and assay performance is monitored using internal quality controls in accordance with established assay guidelines.

[0219] Multiplex immune signaling data generated from plasma and conditioned media are integrated with immune cell phenotyping, transcriptomic, proteomic, and cardiac functional datasets, including echocardiography and cardiac MRI-derived readouts. These combined datasets are used to define inflammatory, reparative, angiogenic, and fibrotic immune states, and to correlate immune biomarker profiles with functional cardiac outcomes across animal models and human ex vivo tissue culture systems.

[0015]

[0220] Imaging

[0221] Cardiac imaging studies in both swine and mice have revealed the ability to develop functional readouts and parameters to correlate biomarker signatures from companion diagnostic to predict improvements in outcomes. This is also not limited to cardiac imaging studies, but can also be used, for example, by using MRI to evaluate skeletal muscle repair in preclinical models (FIG 16).

[0222] In-vivo markers of repair:

[0223] Mouse and swine blood have been profiled for unique reparative immune phonotypes using multiplex ELISA, flow cytometry, transcriptomics and proteomics. These markers are tracked with improvements in cardiac function (FIG 16). Furthermore, mice that have been treated with immunomodulatory therapies have shown the ability to detect significant changes peripherally in immune cell populations (FIG 10).

[0224] Culture of human primary peripheral blood mononuclear cells:

[0225] Culture of primary patient immune cells isolated from whole blood have shown the ability to generate peripheral blood monocular cell and macrophage phenotype. Furthermore, these cultures have demonstrated the ability to detect donor level differences (FIG 13 and 15). Furthermore, coculture of PBMCs with known Ml and M2 polarizers as well as anAtty. Docket No. UAZ-44590.601

[0226] immunomodulatory biologic demonstrate unique proteomes of repair (FIG 14). More specifically, over time these proteomes demonstrated the downregulation and clearance of cell debris and nascent cytoskeleton and intermediate filaments needed for tissue organization and an increase in mature muscle proteins needed to provide improvements in contractile functional that is native and essential to cardiac tissue.

[0227] References for Example 1

[0228] 1. Genin M, Clement F, Fattaccioli A, Raes M, Michiels C. Ml and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer. 2015 Aug 8;15:577. doi: 10.1186 / sl2885-015- 1546-9. PMID: 26253167; PMCID: PMC4545815.

[0229] 2. Park EK, Jung HS, Yang HI, Yoo MC, Kim C, Kim KS. Optimized THP-1 differentiation is required for the detection of responses to weak stimuli. Inflamm Res. 2007 Jan;56(l):45-50. doi: 10.1007 / s00011-007-6115-5. Erratum in: Inflamm Res. 2020 Nov;69(ll): 1157. PMID: 17334670.

[0230] 3. Bertani FR, Mozetic P, Fioramonti M, luliani M, Ribelli G, Pantano F, Santini D, Tonini G, Trombetta M, Businaro L, Selci S, Rainer A. Classification of Ml / M2-polarized human macrophages by label-free hyperspectral reflectance confocal microscopy and multivariate analysis. Sci Rep. 2017 Aug 21;7(1):8965. doi: 10.1038 / s41598-017-08121-8. PMID: 28827726; PMCID: PMC5566322.

[0231] 4. Huismans M, Schloss MJ, Lee IH, Bapat A, Iwamoto Y, Vinegoni C, Paccalet A, Yamazoe M, Grune J, Pabel S, Momin N, Seung H, Kumowski N, Pulous FE, Keller D, Bening C, Green U, Lennerz JK, Mitchell RN, Lewis A, Casadei B, Iborra-Egea O, Bayes-Genis A, Sossalla S, Ong CS, Pierson RN, Aster JC, Rohde D, Wojtkiewicz GR, Weissleder R, Swirski FK, Tellides G, Tolis G Jr, Melnitchouk S, Milan DJ, Ellinor PT, Naxerova K, Nahrendorf M. Recruited macrophages elicit atrial fibrillation. Science. 2023 Jul 14;381(6654):231-239. doi: 10.1126 / science.abq3061. Epub 2023 Jul 13. PMID: 37440641; PMCID: PMC10448807.

[0232] 5. Yu YR, O'Koren EG, Hotten DF, Kan MJ, Kopin D, Nelson ER, Que L, Gunn MD. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues. PLoS One. 2016 Mar 3;ll(3):e0150606. doi: 10.1371 / joumal.pone.0150606. PMID: 26938654; PMCID: PMC4777539.Atty. Docket No. UAZ-44590.601

[0233] 6. Liu L, Stokes JV. Tan W, Pruett SB. An optimized flow cytometry panel for classifying macrophage polarization. J Immunol Methods. 2022 Dec;511:113378. doi:

[0234] 10.1016 / j.jim.2022.113378. Epub 2022 Oct 18. PMID: 36265578.

[0235] 7. Wynn TA, Barron L, Thompson RW, Madala SK, Wilson MS, Cheever AW, Ramalingam T. Quantitative assessment of macrophage functions in repair and fibrosis. Curr Protoc Immunol. 2011 Apr; Chapter 14: Unitl4.22. doi: 10.1002 / 0471142735.iml422s93. PMID: 21462164; PMCID: PMC3109612.

[0236] 8. Schmölz L, Wallert M, Lorkowski S. Optimized incubation regime for nitric oxide measurements in murine macrophages using the Griess assay. J Immunol Methods. 2017 Oct;449:68-70. doi: 10.1016 / j.jim.2017.06.012. Epub 2017 Jul 1. PMID: 28673787.

[0237] 9. Stamler JS. Redox signaling: nitrosy lation and related target interactions of nitric oxide. Cell. 1994 Sep 23;78(6):931 -6. doi: 10.1016 / 0092-8674(94)90269-0. PMID: 7923362.

[0238] 10. Lougheed M, Moore ED, Scriven DR, Steinbrecher UP. Uptake of oxidized LDL by macrophages differs from that of acetyl LDL and leads to expansion of an acidic endolysosomal compartment. Arterioscler Thromb Vase Biol. 1999 Aug;19(8):1881-90. doi: 10.1161 / 01.atv.19.8.1881. PMID: 10446066.

[0239] 11. Lundberg JO, Weitzberg E. Nitric oxide signaling in health and disease. Cell. 2022 Aug 4; 185(16):2853-2878. doi: 10.1016 / j.cell.2022.06.010. PMID: 35931019.

[0240] 12. Leblond MM, Peres EA, Helaine C, Gerault AN, Moulin D, Anfray C, Divoux D, Petit E, Bernaudin M, Valable S. M2 macrophages are more resistant than Ml macrophages following radiation therapy in the context of glioblastoma. Oncotarget. 2017 Aug 7;8(42):72597 -72612. doi: 10.18632 / oncotarget.l9994. PMID: 29069812; PMCID:

[0241] PMC5641155.

[0242] 13. Schmölz L, Wallert M, Lorkowski S. Optimized incubation regime for nitric oxide measurements in murine macrophages using the Griess assay. J Immunol Methods. 2017 Oct;449:68-70. doi: 10.1016 / j.jim.2017.06.012. Epub 2017 Jul 1. PMID: 28673787.

[0243] 14. Ledford KJ, Murphy N, Zeigler F, Bartel RL. Potential beneficial effects of ixmyelocel-T in the treatment of atherosclerotic diseases. Stem Cell Res Ther. 2013 Nov 1;4(6): 135. doi: 10.1186 / scrt346. PMID: 24405662; PMCID: PMC4029553.

[0244] 15. Sapudom J, Karaman S, Mohamed WKE, Garcia-Sabate A, Quartey BC, Teo JCM.

[0245] 3D in vitro M2 macrophage model to mimic modulation of tissue repair. NPJ Regen Med. 2021 Nov 30;6(l):83. doi: 10.1038 / s41536-021-00193-5. PMID: 34848722; PMCID:

[0246] PMC8633361.Atty. Docket No. UAZ-44590.601

[0247] Example 2

[0248] This example describes an evaluation of predictive signatures in chronic ischemic heart failure using in vivo models for the development of diagnostics, prognostics, and therapeutics for tissue repair. Data shown here was generated from spatial transcriptomics digital spatial profiling in myocardial tissue of injured and treated mice. These data identify genes that may be concurrently upregulated in the peripheral circulation after treatment.

[0249] Methods

[0250] Mouse Immune Profiling

[0251] Blood Collection

[0252] Blood is collected from mice either under anesthesia or from awake animals, commonly after echocardiography. The submandibular (submental) facial vein is punctured using a 4-5 mm lancet or 25G needle, and blood is collected into Li-Heparin tubes.

[0253] Necropsy and Tissue Collection

[0254] Mice are anesthetized with 3% isoflurane, the thoracic cavity opened, and the heart exposed. Large- volume cardiac blood is collected into Li-Heparin tubes. Perfusion is performed via the right and left ventricles using PBS, followed by KC1 injection (2 milli eq / kg) to relax the heart. Tissues, including heart, lungs, kidney, liver, spleen, bone marrow, and blood, are collected for histology (FFPE or OCT) or processing / storage.

[0255] Blood Processing

[0256] Blood tubes are centrifuged at 1800 RPM for 5 minutes at room temperature; plasma is collected and stored at -80°C. Remaining blood is transferred to a 15 mL tube, diluted with 7.5 mL DI water, immediately supplemented with 2.5 mL 4X PBS, centrifuged, and the supernatant discarded. Cells are resuspended in 1 mL PBS for downstream applications.

[0257] Spleen Processing

[0258] Spleens are placed in PBS in a 6- well plate and incubated with collagenase type IV (final concentration 1 mg / mL) at 37 °C for 20 minutes. The reaction is stopped with 1 mL 10% FBS medium. Spleens are mashed through a 70 pm strainer, washed with PBS + 2% FBS, centrifuged at 350 g for 5 minutes, and subjected to hypertonic lysis (7.5 mL DI water + 2.5 mL 4X PBS). Cells are washed and resuspended in PBS for downstream applications.Atty. Docket No. UAZ-44590.601

[0259] Flow Cytometry

[0260] Mouse cells are stained using FVS780 viability dye, blocked with TruStain FcX, and incubated with fluorophore-conjugated antibodies as required. Cells are washed twice, resuspended in preservation buffer, and analyzed on a Cytek Aurora.

[0261] RNA Sequencing

[0262] For whole-blood RNA-seq, 0.2 mL mouse blood is collected into DNase / RNase-free tubes containing 0.552 mL Paxgene solution, mixed by inversion, incubated 2-6 hours at room temperature, and stored at -80°C. For PBMC RNA-seq, 1 mL PBMCs are pelleted at 300xg, supernatant removed, and pellets flash-frozen in liquid nitrogen prior to -80°C storage. RNA is subsequently isolated and processed as described above for library preparation and sequencing.

[0263] Mass Spectrometry

[0264] Mouse cells are pelleted at 300xg, lysed in 1 mL R1PA buffer for 10 minutes on ice, centrifuged at 10,000xg for 10 minutes, and protein in the lysate is precipitated for downstream proteomic analysis as described for human samples.

[0265] Cardiac Imaging in Preclinical Models

[0266] Mouse Cardiac Functional Assessment

[0267] Mice are subjected to cardiac functional imaging using echocardiography and, where applicable, MRI to assess structural and functional parameters. Echocardiography is performed under light anesthesia (e.g., 1-2% isoflurane) to maintain stable heart rate and physiologic conditions. Imaging is conducted using high-frequency ultrasound systems equipped with probes suitable for murine hearts.

[0268] Echocardiography includes

[0269] • B-mode (two-dimensional) imaging to visualize cardiac anatomy, chamber dimensions, and wall motion.

[0270] • M-mode (motion-mode) imaging to quantify left ventricular (LV) wall thickness, LV internal diameter during systole and diastole, fractional shortening, and ejection fraction.Atty. Docket No. UAZ-44590.601

[0271] • Doppler echocardiography to evaluate blood flow across cardiac valves, including peak velocity, pressure gradients, and flow patterns.

[0272] • Tissue Doppler imaging (TDI) to assess myocardial motion, including systolic and diastolic velocities of the LV wall, providing insight into contractile and relaxation properties.

[0273] Images are acquired in standard short-axis and long-axis views. Heart rate, LV dimensions, wall thickness, and systolic / diastolic function parameters are recorded and analyzed using software integrated with the imaging system.

[0274] Swine Cardiac Functional Assessment

[0275] For swine models, animals are imaged under sedation or light anesthesia following standard veterinary protocols. Cardiac MRI is performed using clinical or preclinical MRI systems with cardiac gating to synchronize image acquisition with the cardiac cycle.

[0276] Protocols may include:

[0277] • Cine MRI sequences to quantify LV and right ventricular (RV) volumes, ejection fraction, stroke volume, and cardiac output.

[0278] • Late gadolinium enhancement (LGE) to identify areas of fibrosis, infarction, or scar tissue.

[0279] • T1 and T2 mapping sequences to assess tissue characterization, including edema or inflammatory infiltrates.

[0280] Complementary echocardiography may also be performed in swine using B-mode, M-mode, Doppler, and tissue Doppler sequences to provide real-time functional assessment and cross-validation with MRI data.

[0281] Correlation with Immune Profiles

[0282] Cardiac imaging data are correlated with immune cell profiles obtained from blood, spleen, bone marrow, and PBMCs, including both phenotypic (flow cytometry) and functional (RNA-seq, proteomics) readouts. Metrics such as LV ejection fraction, wall motion, diastolic function, stroke volume, and fibrosis burden are compared with immune cell activation states, polarization markers, cytokine secretion, and secretome signatures. These correlations inform ex vivo human tissue culture models, including PBMC and CD14+monocyte coculture with fibroblast or cardiomyocyte matrices, enabling evaluation of functional and molecular responses to human-derived immune cells under controlled conditions.Atty. Docket No. UAZ-44590.601

[0283] Results

[0284] The experiments utilized a biologically active platform composed of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and neonatal fibroblasts on a bioresorbable matrix that increases the prevalence of alternatively activated M2 macrophage phenotypes in immune competent animal models of chronic ischemic heart failure (mice, rat, swine) (see, e.g., Lancaster, et al., Immunome Res. Vol. 20, Issue 1; Lancaster, et al., Commun Biol. 2023;6(1):1203; Lancaster, et al., Vessel. Plus. 2019, 3, 34; Lancaster, et al., Ann Thorac Surg 2019 Oct;108(4):1169-1177; Lancaster, et al., Commun Biol 2023 Nov 25;6(1):1203).

[0285] The biologic restores contractile function, increases blood flow, and repairs damaged cardiomyocytes.

[0286] Diagnostic tools that enable patient stratification have been transformative in cancer treatments. However, a similar diagnostic approach does not exist for patients with chronic ischemic heart failure (CHF).

[0287] Multivariate analysis was conducted using transcriptomics, proteomics, and Multiplex ELISAs to identify biomarker changes in peripheral blood associated with cardiac repair following treatment with the therapeutic.

[0288] In vivo, improvements in cardiac function are correlated with detection of a predictive signature.

[0289] In-vitro, peripheral blood samples from chronic heart failure (CHF) patients are used to perform coculture experiments to identify cell phenotypes via flow cytometry and evaluate biomarkers changes via multivariate analysis.

[0290] The experiments to date have demonstrated the ability to delineate classically activated from alternatively activated macrophage markers with statistical significance in vitro (P<0.05), giving a platform to test blood-derived tissues exposed to other therapies.

[0291] The data indicate that CD45+ cells exposed to the biologic platform tend to reduce inflammatory markers and elevate alternatively activated macrophage markers in vivo.

[0292] FIG. 7 shows the predictive Diagnostic Assay Workflow: A: Peripheral Blood Mononuclear Cells (PBMCs) are isolated from CHF patients; B: PBMCs are cultured with an immunomodulatory therapy; C: Cells are evaluated with the biomarker assay panel for functional assessment.Atty. Docket No. UAZ-44590.601

[0293] FIG. 8 shows immunofluorescence staining (A), image quantification (B), and transcriptomic cellular deconvolution (C) of CHF mice compared to CHF mice treated with the biologic therapeutic.

[0294] FIG. 9 shows macrophage assessment in vivo: transcriptomic studies in CHF mice receiving biologic treatment show a reduction of inflammatory macrophage markers over time, and an increase in anti-inflammatory macrophage markers.

[0295] FIG. 10 shows an assessment of samples receiving treatment for evaluation of predictive markers with flow cytometry, mass spectroscopy proteomics, and Multiplex ELIS As.

[0296] FIG. 11 shows macrophage phenotype assessment in-vitro: immunomodulatory differences include known Ml behaviors (A) Nitrite / NO production in RAW 264.7 Cells, and assays detecting potential M2 target molecules for diagnostics, such as (B) Acetylated LDL uptake, and (C) Arginase expression in THP-1 cells. (*P<0.05)

[0297] Tables 1 and 2 show genes identified in the assays.Atty. Docket No. UAZ-44590.601

[0298] Table 1: Genes that highly correlated with macrophage relative

[0299] abundances and negatively

[0300] correlated with blood progenitor relative abundances

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[0336] Table 2: CD68 and Fcgr3 correlated

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[0362] M2 associated genes

[0363] Pan Macrophage Marker

[0364] Example 3

[0365] This Example describes mass spectrometry experiments on PBMC cocultures. Data analysis involved the steps of loading data, removing unique peptides < 2, removing ANOVAAtty. Docket No. UAZ-44590.601

[0366] P > 0.05, Z-scoring data, generating a heat map, perform overall pathway analysis, generating 4 clusters, and evaluating cluster expression and pathway profiles.

[0367] Results are shown in FIGs. 12A-B. FIG. 12A shows significant proteins identified. FIG. 12B shows pathways that were enriched.

[0368] Example 4

[0369] This Example describes CD14+ hPBMC cell staining. First, CD14+ isolation of monocytes from three donors prior to seeding was performed. Markers were tested on PBMCs to verify viability and marker presence. PBMCs were cultured using MCSF (100 ng / mL) for one week, and then induced with polarizers and markers were tested. FIG. 13A shows the experimental work flow. Table 3 shows flow cytometry markers.

[0370] Table 3: Human and mouse flow cytometry panels

[0371]

[0372] Atty. Docket No. UAZ-44590.601

[0373]

[0374] Results are shown in FIGs. 13B-C. FIG. 13B shows Ml like marker expression and FIG. 13C shows M2 like marker expression.

[0375] Example 5

[0376] This Example describes analysis of marker expression in swine. FIG. 14A shows the experimental workflow. FIG. 14B shows markers tested. Swine plasma and serum isolated from peripheral blood. The same animal is followed across all timepoints, including baseline, post-myocardial infarction (baseline and 4 weeks post MI: pre-patch), and post-patch treatment (7d, 14d, 21d, 28d, 3mo, 6mo).

[0377] All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection withAtty. Docket No. UAZ-44590.601

[0378] specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

Atty. Docket No. UAZ-44590.601We claim1. A method, comprising:a) obtaining a biological sample from a patient suffering from a disorder characterized by altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood;b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

2. The method of claim 1, wherein the altered immune cell function is increased or decreased immune cell function.

3. The method of claim 1 or 2, further comprising:d) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

4. The method of any one of the preceding claims, wherein the disorder is a heart disorder or a skeletal muscle disorder.

5. The method of claim 4, further comprising administering to the patient one or more pharmaceutical agents selected from ACE inhibitors, |3-blockers, ARBs, diuretics, calciumAtty. Docket No. UAZ-44590.601channel blocker, sacubitril / valsartan, SGLT2i, GLP-1 agonists, nitrates, platelet aggregation inhibitors, cholesterol-lowering medications, ranolazine, and ivabradine.

6. The method of claim 4, wherein treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient comprises administering to the patient a composition comprising the immunomodulatory agent.

7. The method of claim 6, wherein the composition is a bioresorbable matrix or implant, wherein the immunomodulatory agent is embedded within the bioresorbable matrix.

8. The method of claim 7, wherein the bioresorbable matrix further comprises human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and human neonatal fibroblasts.

9. The method of claim 8, wherein administering to the patient the bioresorbable matrix comprises implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage.

10. The method of claim 8, wherein implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage mediates changes in the stromal cell population that repair heart tissue.

11. The method of claim 8, wherein implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage results in increased immune cell activity at the site of heart tissue experiencing heart tissue damage.

12. The method of claim 8, wherein implanting the bioresorbable matrix at the site of heart tissue experiencing heart tissue damage restores contractile function, increases blood flow, and repairs damaged cardiomyocytes of the chronically infarcted myocardium.

13. The method of any one of the preceding claims, wherein the patient is a human patient.Atty. Docket No. UAZ-44590.60114. The method of any one of the preceding claims, wherein the patient is a human patient suffering from chronic ischemic heart failure, myocardial infarction, or heart failure.

15. The method of any one of the preceding claims, wherein the immunomodulatory agent is an immune checkpoint inhibitor (ICI).

16. The method of claim 15, wherein the ICI is an inhibitor of PD-1, PD-L1, CTLA4, LAG3, TIGTT, TIM3, VISTA, TCOS, BTLA, GITR, NKG2A, CD112R, B7-H3, or CD73.

17. The method of claim 15 or 16, wherein the ICI is selected from nivolumab, pembrolizumab, ipilimumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplimab, retifanlimab, dostarlimab, and toripalimab.

18. The method of any one of the preceding claims, wherein the one or more type of immune cell activity is selected from cytokine activity, macrophage activity, B lymphocyte activity, T lymphocyte activity, cytotoxic T-cell lymphocyte (CTL) activity, mast cell activity, monocyte activity, dendritic cell activity, eosinophil activity, natural killer cell activity, basophil activity, and neutrophil activity.

19. The method of any one of the preceding claims, wherein the one or more type of immune cell activity is macrophage cellular activity.

20. The method of any one of the preceding claims, wherein the assessment of differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents comprises one or more of the following: flow cytometry assessment, nitric oxide assessment, arginase assessment, acetylated LDL uptake assessment, genomic assessment, transcriptomics assessment, proteomics assessment, and multiplex ELISA assessment.

21. The method of any one of the preceding claims, wherein the favorable change in measured immune cell activity is a reduction in classically activated Ml macrophage activity.

22. The method of any one of the preceding claims, wherein the favorable change in measured immune cell activity is an increase in classically activated M2 macrophage activity.Atty. Docket No. UAZ-44590.60123. A method, comprising:a) obtaining a biological sample from a patient suffering from a disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood;b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; andd) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

24. The method of claim 23, wherein treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient comprises administering to the patient a composition comprising the immunomodulatory agent.

25. A method, comprising:a) obtaining a biological sample from a patient suffering from an inflammatory disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood;Atty. Docket No. UAZ-44590.601b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

26. A method, comprising:a) obtaining a biological sample from a patient suffering from a fibrotic disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood;b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissueAtty. Docket No. UAZ-44590.601characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient.

27. A method, comprising:a) obtaining a biological sample from a patient suffering from an inflammatory disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood:b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; andd) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

28. A method, comprising;a) obtaining a biological sample from a patient suffering from a fibrotic disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood;b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;Atty. Docket No. UAZ-44590.601c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; andd) treating the patient with the immunomodulatory agent if the immunomodulatory agent is determined to be a favorable therapeutic for treating the patient.

29. A method, comprising:a) obtaining a biological sample from a patient suffering from a heart disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood;b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates theAtty. Docket No. UAZ-44590.601immunomodulatory agent to be an unfavorable therapeutic for treating the patient; andd) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent.

30. A method, comprising:a) obtaining a biological sample from a patient suffering from an inflammatory disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood:b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; andd) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent.

31. A method, comprising:Atty. Docket No. UAZ-44590.601a) obtaining a biological sample from a patient suffering from a fibrotic disorder characterized with altered immune cell function, wherein the biological sample comprises immune cells isolated from peripheral blood;b) generating a tissue culture platform with the immune cells isolated from the peripheral blood, wherein the generated tissue culture platform is configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents;c) measuring one or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent,wherein a favorable change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be a favorable therapeutic for treating the patient,wherein an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function for heart tissue characterized with altered immune cell function indicates the immunomodulatory agent to be an unfavorable therapeutic for treating the patient; andd) prognosticating if the immunomodulatory agent would be a favorable or unfavorable treatment based on the measurements of or more types of immune cell activity for the immune cells isolated from the peripheral blood following exposure of the generated live tissue culture platform to an immunomodulatory agent.

32. The method of any one of the preceding claims, wherein the method further comprises the step of integrating the change or lack of change in measured immune cell activity into a computational workflow to drive development of a clinically deployable tool that can interpret patient specific immune data.

33. The method of any one of the preceding claims, wherein one or more steps of the method utilizes artificial intelligence (AI) and machine learning (ML) computational algorithms configured to analyze and draw conclusions from patient specific immune data.Atty. Docket No. UAZ-44590.60134. The method of claim 33, wherein the conclusions are one or more of a diagnosis, a prognosis, and a recommended treatment.

35. The method of any one of claims 33 to 34, wherein the Al and MT algorithms utilize one or more of transformer and Perceiver IO models, convolutional neural networks, recurrent neural networks, state space models, and forecasting architectures.

35. A system, comprisinga computer processor that receives data from a tissue culture platform configured to assess differences in one or more types of immune cell activity following exposure to one or more immunomodulatory agents; andprocesses the data to generate one of more of a determination of an unfavorable change or lack of change in measured immune cell activity in comparison to an established norm for baseline immune cell function, a prognosis for the subject, a diagnosis for the subject, or a recommended treatment for the subject.

36. fhe system of claim 35, wherein the processing of the data comprises the use of an Al and / or MT algorithm selected from transformer and Perceiver IO models, convolutional neural networks, recurrent neural networks, state space models, and forecasting architectures.

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