Modified immune cells for fibrosis and inflammation

Abstract

The present disclosure pertains to immune cells comprising one or more nucleic acids encoding one or more exogenous fibrolytic agents, one or more exogenous anti-inflammatory agents, one or more exogenous regenerative agents, and / or one or more exogenous efferocytic agents and methods of using such immune cells.

Description

Attorney Docket No.2012851-0634 MODIFIED IMMUNE CELLS FOR FIBROSIS AND INFLAMMATION RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 563,228, filed on March 8, 2024, and U.S. Provisional Patent Application No. 63 / 697,419, filed September 20, 2024, the entire contents of which are hereby incorporated by reference in their entirety. BACKGROUND

[0002] Fibrosis is a serious health problem characterized by the development of excess fibrous connective tissue often due, at least in part, to reparative and / or reactive processes, such as in response to an injury. Fibrosis can occur in various organs including the lung, liver, heart, kidney, pancreas, skin, and brain. Currently available therapies for fibrotic diseases, disorders, and conditions have limited efficacy.

[0003] Inflammatory diseases are a significant cause of morbidity and mortality in humans. There are various side effects associated with currently available treatments for inflammation, such as adrenal suppression, weakening of bones, muscle wasting, peptic ulcers, hypokalemia, and immune system suppression.

[0004] Therefore, a need exists for the development of new therapeutic modalities optimized to reduce and treat fibrosis and inflammation. SUMMARY OF THE INVENTION

[0005] The present disclosure pertains to immune cells comprising one or more exogenous fibrolytic agents, one or more exogenous anti-inflammatory agents, one or more exogenous regenerative agents, and / or one or more exogenous efferocytic agents and methods of using such immune cells. In part, the present disclosure encompasses the recognition that the administration of specifically modified immune cells is surprisingly effective in treating one or more symptoms of fibrosis and / or inflammation. Page 1 of 118 12583381v1Attorney Docket No.2012851-0634

[0006] In one aspect, the disclosure provides modified immune cells comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, (ii) at least one exogenous anti-inflammatory agent, (iii) at least one exogenous regenerative agent, and / or (iv) at least one exogenous efferocytic agent. In some embodiments, at least one exogenous fibrolytic agent is or comprises a Relaxin polypeptide, Decorin polypeptide, dominant negative TGF-beta Receptor 2 (DN TGFβR2) polypeptide, soluble TGFβ receptor, and / or TGFβ switch receptor. In some embodiments, at least one exogenous anti-inflammatory agent comprises or is a cytokine or a cytokine receptor. In some embodiments, a cytokine comprises or is IL-10, IL-22, and / or IL- 27. In some embodiments, a cytokine receptor comprises or is IL17A switch receptor.

[0007] In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous anti- inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous anti- inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous fibrolytic agent comprises a dominant negative (DN) TGFβR2 polypeptide and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrolytic agent comprises a DN TGFβR2 polypeptide and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrolytic agent comprises a DN TGFβR2 polypeptide and at least one exogenous anti- inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrolytic agent comprises a DN TGFβR2 polypeptide and at least one exogenous anti-inflammatory agent Page 2 of 118 12583381v1Attorney Docket No.2012851-0634 comprises an IL-17A switch receptor. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor and at least one exogenous anti-inflammatory agent comprises an IL- 17A switch receptor. In some embodiments, at least one exogenous fibrolytic agent comprises a MMP13 polypeptide and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrolytic agent comprises a MMP13 polypeptide and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrolytic agent comprises a MMP13 polypeptide and at least one exogenous anti- inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrolytic agent comprises a MMP13 polypeptide and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor.

[0008] In some embodiments, at least one exogenous regenerative agent is or comprises Fibrotic Growth Factor 21 (FGF21), Fibrotic Growth Factor 19 (FGF19), Vascular Endothelial Growth Factor α (VEGF-α), and / or Hepatocyte Growth factor (HGF). In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous regenerative agent comprises VEGF-α. In some Page 3 of 118 12583381v1Attorney Docket No.2012851-0634 embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous fibrolytic agent comprises a Decorin polypeptide and at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous fibrolytic agent comprises a DN TGFβR2 polypeptide and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous fibrolytic agent comprises a DN TGFβR2 polypeptide and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous fibrolytic agent comprises a DN TGFβR2 polypeptide and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor rand at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous fibrolytic agent comprises a TGFβ switch receptor rand at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-10 and at least one exogenous regenerative Page 4 of 118 12583381v1Attorney Docket No.2012851-0634 agent comprises FGF21. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-10 and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-10 and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-10 and at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-22.and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-22 and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-22 and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-22 and at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-27.and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-27 and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-27 and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous anti-inflammatory agent comprises IL-27 and at least one exogenous regenerative agent comprises HGF. In some embodiments, at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous anti- inflammatory agent comprises an IL-17A switch receptor and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous anti- inflammatory agent comprises an IL-17A switch receptor and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous anti- inflammatory agent comprises an IL-17A switch receptor and at least one exogenous regenerative agent comprises HGF.

[0009] In some embodiments, at least one exogenous efferocytic agent is or comprises a T-cell immunoglobulin and mucin domain containing 4 (TIM4) polypeptide. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous fibrolytic agent comprises a Relaxin polypeptide. In some embodiments, at least one exogenous Page 5 of 118 12583381v1Attorney Docket No.2012851-0634 efferocytic agent comprises a TIM4 polypeptide and at least one exogenous fibrolytic agent comprises a Decorin polypeptide. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous fibrolytic agent comprises a DN TGFβR2 polypeptide. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous fibrolytic agent comprises a TGFβ switch receptor. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous anti- inflammatory agent comprises IL-27. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous regenerative agent comprises FGF21. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous regenerative agent comprises FGF19. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous regenerative agent comprises VEGF-α. In some embodiments, at least one exogenous efferocytic agent comprises a TIM4 polypeptide and at least one exogenous regenerative agent comprises HGF.

[0010] In some embodiments, at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent are tethered to the immune cell or secreted from the immune cell.

[0011] In some embodiments, one or more nucleic acid sequences comprise one or more liver specific promoters or cirrhosis specific promoters. In some embodiments, one or more nucleic acid sequences comprise a CX3CR1 promoter, an insulin-like growth factor 1 (IGF1), or a CD11B promoter.

[0012] In some embodiments, a modified immune cell comprises a macrophage, monocyte, or dendritic cell. In some embodiments, a macrophage is derived from a monocyte or a precursor immune cell. In some embodiments, a precursor immune cell comprises or is a hematopoietic Page 6 of 118 12583381v1Attorney Docket No.2012851-0634 stem cell, myeloid progenitor, myeloblast, monoblast, promonocyte, or an intermediate thereof. In some embodiments, a macrophage is a G-MCSF derived macrophage or an M-CSF derived macrophage.

[0013] In some embodiments, one or more nucleic acid sequences described herein encode one or more polypeptides comprising one or more amino acid sequences of any one of SEQ ID Nos: 1-5 or 13-18.

[0014] In another aspect, the disclosure provides pharmaceutical compositions comprising a modified immune cell of any aspect or embodiment described herein. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises a delivery vehicle. In some embodiments, a delivery vehicle described herein is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles).

[0015] In another aspect, the disclosure provides nucleic acid constructs comprising one or more nucleic acid sequences encoding least one exogenous fibrolytic agent of any aspect or embodiment described herein, at least one exogenous anti-inflammatory agent of any aspect or embodiment described herein, and / or at least one exogenous regenerative agent of any aspect or embodiment described herein, and / or at least one efferocytic agent of any aspect or embodiment described herein.

[0016] In another aspect, the disclosure provides pharmaceutical compositions comprising a nucleic acid construct of any aspect or embodiment described herein. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0017] In another aspect, the disclosure provides methods of treating or preventing fibrosis or inflammation in a subject, comprising delivering to a subject a therapeutically effective amount of a pharmaceutical composition of any aspect or embodiment described herein.

[0018] In some embodiments, fibrosis comprises or is a liver, lung, heart, vasculature, kidney, pancreas, skin, gastrointestinal, bone marrow, hematopoietic tissue, nervous system, and / or eye fibrotic disease, disorder, or condition. In some embodiments, a liver fibrotic disease, disorder, or condition comprises a fatty liver disease, disorder, or condition. In some embodiments, a fatty liver disease, disorder, or condition comprises non-alcoholic fatty liver disease (NAFL) or Page 7 of 118 12583381v1Attorney Docket No.2012851-0634 alcoholic liver disease. In some embodiments, NAFL comprises non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In some embodiments, alcoholic liver disease comprises alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH). In some embodiments, a subject has one or more of cirrhosis, liver damage, hepatocarcinoma, steatosis, an increased risk of liver failure, an increased risk of death, and / or Hepatitis C infection (HCV).

[0019] In some embodiments, inflammation comprises or is a liver, gastrointestinal tract, lung, skin, cardiovascular system, nervous system, kidney, pancreas, joint, eye, and / or an endocrine system inflammatory disease, disorder, or condition.

[0020] In some embodiments, methods described herein reduce activation of hepatic stellate cells. In some embodiments, methods described herein improve liver regeneration and / or liver resolution. In some embodiments, methods described herein balance pro-fibrotic and anti- fibrotic macrophage populations.

[0021] In another aspect, the disclosure provides methods of modifying an immune cell, comprising delivering to an immune cell a nucleic acid construct comprising one or more nucleic acid sequences encoding at least one exogenous anti-fibrotic agent of any aspect or embodiment described herein, at least one exogenous anti-inflammatory agent of any aspect or embodiment described herein, at least one exogenous regenerative agent of any aspect or embodiment described herein and / or at least one exogenous efferocytic agent of any aspect or embodiment described herein, thereby forming a modified immune cell.

[0022] In some embodiments, delivering comprises electroporation or transfection with mRNA, DNA, or chemically modified mRNA. In some embodiments, delivering comprises administration of one or more lipid particles (e.g., a lipid nanoparticle or LNP). In some embodiments, lipid particles comprise cationic lipids, PEG-modified lipids, sterols, non-cationic lipids, or a combination thereof. In some embodiments, a cationic lipid comprises MC3. In some embodiments, delivering comprises administration of one or more MC3-lipid nanoparticles. In some embodiments, delivering comprises transduction with an adeno-associated viral (AAV) vector, an adenoviral vector, or a retroviral vector. In some embodiments, a retroviral vector comprises a lentiviral vector or a gammaretroviral vector. In some embodiments, delivering comprises transduction with a viral vector (e.g., a lentiviral vector) and at least one Vpx protein. Page 8 of 118 12583381v1Attorney Docket No.2012851-0634 In some embodiments, an immune cell described herein is one or both of electroporated or transfected with at least one Vpx mRNA prior to and / or concurrently with, transfection with the viral vector. In some embodiments, a lentiviral vector is packaged with a Vpx protein. In some embodiments, an adenoviral vector comprises an Ad2 vector or an Ad5 vector. In some embodiments, an Ad5 vector comprises an Ad5f35 adenoviral vector.

[0023] In another aspect, the disclosure provides methods of producing a modified immune cell comprising administering to a subject a composition comprising: (a) one or more nucleic acid constructs comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) at least one exogenous regenerative agent, and (b) a delivery vehicle. In some embodiments, following administration of a composition, one or more nucleic acid constructs are translated in an immune cell to produce a modified immune cell comprising (i) the at least one exogenous fibrolytic agent, (ii) the at least one exogenous anti-inflammatory agent, and / or (iii) the at least one exogenous regenerative agent. In some embodiments, a modified immune cell comprises a macrophage, monocyte, or dendritic cell in the subject. In some embodiments, a delivery vehicle is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles), a liposome, a polymer, an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, or any combination thereof.

[0024] In another aspect, the disclosure provides modified immune cells comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and the at least one efferocytic agent comprises a TIM4 polypeptide. In some embodiments, a modified immune cell comprises a macrophage, monocyte, or dendritic cell. In some embodiments, a macrophage is derived from a monocyte or a precursor immune cell. In some embodiments, a precursor immune cell comprises or is a hematopoietic stem cell, myeloid progenitor, myeloblast, monoblast, promonocyte, or an intermediate thereof.

[0025] In another aspect, the disclosure provides a pharmaceutical composition comprising a modified immune cell of any aspect or embodiment described herein. In some embodiments, a macrophage is a G-MCSF derived macrophage or an M-CSF derived macrophage. Page 9 of 118 12583381v1Attorney Docket No.2012851-0634

[0026] In another aspect, the disclosure provides a method of modifying an immune cell, comprising delivering to the immune cell a nucleic acid construct comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and the at least one efferocytic agent comprises a TIM4 polypeptide. In some embodiments, an immune cell comprises a macrophage, monocyte, or dendritic cell. In some embodiments, a macrophage is derived from a monocyte or a precursor immune cell. In some embodiments, a precursor immune cell comprises or is a hematopoietic stem cell, myeloid progenitor, myeloblast, monoblast, promonocyte, or an intermediate thereof. In some embodiments, delivering comprises electroporation or transfection with mRNA, DNA, or chemically modified mRNA (e.g., packaged in a liposome). In some embodiments, delivering comprises transduction with an adeno-associated viral (AAV) vector, an adenoviral vector, or a retroviral vector. In some embodiments, a retroviral vector comprises a lentiviral vector or a gammaretroviral vector. In some embodiments, a lentiviral vector is packaged with a Vpx protein. In some embodiments, Vpx protein is delivered to the immune cell either before, concurrently with, or subsequently to the nucleic acid construct. In some embodiments, an adenoviral vector comprises an Ad2 vector or an Ad5 vector. In some embodiments, an Ad5 vector comprises an Ad5f35 adenoviral vector. In some embodiments, delivering comprises a delivery vehicle. In some embodiments, a delivery vehicle described herein is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles).

[0027] In another aspect, the disclosure provides a nucleic acid construct comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent. In some embodiments, a nucleotide sequence encoding at least one exogenous fibrolytic agent encodes a polypeptide comprising a Relaxin polypeptide and wherein the nucleotide sequence encoding the at least one efferocytic agent encodes a polypeptide comprising a TIM4 polypeptide. In some embodiments, a nucleic acid construct described herein further comprises a nucleotide sequence encoding a cleavage peptide, wherein the cleavage peptide comprises one or more of a porcine teschovirus-l (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, a foot-and-mouth disease virus (F2A) peptide, and / or a variant thereof. Page 10 of 118 12583381v1Attorney Docket No.2012851-0634

[0028] In another aspect, the disclosure provides a method of producing a modified immune cell, the method comprising administering to a subject a composition comprising: (a) one or more nucleic acid constructs comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent, and (b) a delivery vehicle. In some embodiments, following administration of a composition described herein, one or more nucleic acid constructs are translated in an immune cell to produce the modified immune cell comprising (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent. In some embodiments, a modified immune cell comprises a macrophage, monocyte, or dendritic cell in the subject. In some embodiments, at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and the at least one efferocytic agent comprises a TIM4 polypeptide. In some embodiments, a delivery vehicle is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles), a liposome, a polymer, an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings are for illustration purposes only, not for limitation.

[0030] Figure 1 is a series of schematics of timepoints for generating and validating anti-fibrotic engineered macrophages described herein in Example 1.

[0031] Figure 2 is a schematic of timepoints of supernatant collection from macrophages to evaluate payload expression over time in vitro described herein in Example 1.

[0032] Figures 3A-3C are a series of graphs showing recovery and viability of macrophages on Day 5 (Figure 3A), frozen macrophages immediately post-thaw (Figure 3B), and post-thaw macrophages cultured in vitro for 48 hr (Figure 3C).

[0033] Figures 4A- 4D are a series of graphs showing levels of IL-10 (Figure 4A), FGF21 (Figure 4B), Relaxin (Figure 4C), and Decorin (Figure 4D) in supernatant of engineered macrophages on Day 5.

[0034] Figures 5A-5E are a series of schematics of transmembrane receptors and graphs showing viability and expression levels of said transmembrane proteins in engineered Page 11 of 118 12583381v1Attorney Docket No.2012851-0634 macrophages. Figure 5A is a schematic of dominant negative TGFβR2. Figure 5B is a schematic of IL17A switch receptor. Figure 5C shows viability of engineered macrophages expressing synthetic transmembrane receptors dominant negative TGFβR2 and IL17A switch receptor. Figure 5D shows flow cytometry detection of dominant negative TGFβR2 expression. Figure 5E shows flow cytometry detection of IL17A switch receptor expression.

[0035] Figures 6A- 6B are a series of graphs showing expression levels (Figure 6A) and rate of production of IL-10 on Days 7, 9, 13, and 14 (Figure 6B).

[0036] Figures 7A-7B are schematics showing timepoints for generating and validating anti- fibrotic engineered murine macrophages (Figure 7A) and timepoints of IVIS®imaging to monitor payload expression over time in vivo (Figure 7B).

[0037] Figures 8A- 8B are a series of graphs showing recovery and viability of murine macrophages on Day 5 (Figure 8A) and frozen murine macrophages immediately post-thaw (Figure 8B).

[0038] Figures 9A-9B are a graph and image showing luciferase signal in vivo. Figure 9A shows quantification of luciferase signal in mice from Day 1 to Day 13 following intravenous injection of engineered macrophages. Figure 9B shows IVIS®images of signal on Day 1 and Day 13 from data plotted in Figure 9A.

[0039] Figure 10 is a schematic of timepoints of CCl4administration to mice for generating a CCl4-induced mouse model of liver fibrosis and treatment of mice with engineered macrophages on Day 28.

[0040] Figures 11A- 11B are a graph and image showing detection of IL-10 and Relaxin in engineered macrophages. Figure 11A shows ELISA assay detection of IL-10 levels of engineered macrophages transduced with IL-10-P2A-Relaxin compared to untransduced cells. Figure 11B shows detection of Relaxin using non-denaturing western blot analysis.

[0041] Figures 12A- 12B are a series of graphs showing mouse bodyweight during the course of CCl4administration (Figure 12A) and at study endpoint on Day 43 (Figure 12B).

[0042] Figures 13A-13B are a graph and image showing collagen content of tissue sections of a CCl4-induced liver fibrosis mouse model treated with engineered macrophages transduced with IL-10-P2A-Relaxin. Figure 13A shows quantification of collagen as percentage of area in Page 12 of 118 12583381v1Attorney Docket No.2012851-0634 histological images (n=9 per group), where the average signal from Corn Oil / Vehicle group is defined as 100% reduction, and the average signal from CCl4 / Vehicle group is defined as 0% reduction. Figure 13B shows representative Masson’s Trichrome images from data plotted in Figure 13A.

[0043] Figure 14 shows a schematic of timepoints for generating a bleomycin-induced model of pulmonary fibrosis in mice and treatment with engineered macrophages on Day 1.

[0044] Figures 15A-15C are a series of graphs showing viability and expression levels of transmembrane proteins in engineered macrophages. Figure 15A shows viability of engineered macrophages expressing synthetic transmembrane receptors dominant negative TGFβR2 and IL17A switch receptor. Figure 15B shows ELISA assay detection of IL-10 production by engineered macrophages to be administered to CCl4-induced fibrotic mice. Figure 15C shows flow cytometry detection of dominant negative TGFβR2 expression.

[0045] Figures 16A- 16B are a series of graphs showing mouse bodyweight during the course of the study post-bleomycin administration (Figure 16A) and at study endpoint on Day 21 (Figure 16B).

[0046] Figures 17A-17B are a series of graphs showing hydroxyproline (“HYP”) measurement of a bleomycin-induced pulmonary fibrosis mouse model treated with engineered macrophages transduced with IL-10-P2A-DN- TGFβR2 (Figure 17A) or IL-10-P2A-Relaxin (Figure 17B).

[0047] Figure 18 shows a schematic of timepoints for evaluating viability and TIM4 expression of engineered macrophages via flow cytometry.

[0048] Figure 19 shows flow cytometry detection of TIM4 expression and viability of macrophages that were either untransduced (UTD) or transduced with a TIM4 construct described herein (TIM4 Mɸ) in Example 6.

[0049] Figure 20 shows a schematic of timepoints for an efferocytosis assay of engineered macrophages.

[0050] Figure 21 is a graph showing change in efferocytosis signal (AU) for macrophages that were UTD or transduced either with a lentivirus control (miRFP670) or a TIM4 construct described herein in Example 6. Page 13 of 118 12583381v1Attorney Docket No.2012851-0634

[0051] Figure 22 shows a schematic of timepoints for generating and validating anti-fibrotic engineered macrophages described herein in Example 6.

[0052] Figures 23A-23C are a series of graphs showing viability and expression levels of engineered macrophages described herein. Figure 23A shows viability and flow cytometry detection of Cd11b expression of macrophages engineered with either a TIM4 construct or a TIM4-P2A-Relaxin construct described herein in Example 6. Figure 23B shows flow cytometry detection of TIM4 expression at 5 Days post-transduction of macrophages using methods described herein. Figure 23C shows flow cytometry detection of Tim4 expression at 7 Days post-transduction of macrophages using methods described herein.

[0053] Figures 24A-24B are a series of graphs demonstrating supernatant measurements of Relaxin. Figure 24A shows ELISA measurements for Relaxin from supernatant collected from macrophages transduced with either a TIM4 construct or a TIM4-P2A-Relaxin construct described in Example 6. Figure 24B shows time course ELISA measurements for Relaxin from supernatant collect from macrophages transduced with either aTIM4 construct or a TIM4-P2A- Relaxin construct described in Example 6.

[0054] Figure 25 shows a schematic of timepoints for generating a choline-deficient, L-amino acid-defined high fat diet (CDAHFD)-induced model of liver fibrosis in mice and treatment with engineered macrophages described herein on Week 4.

[0055] Figure 26A-26B are an immunoblot and graph showing TIM4 expression in mouse livers following CDAHFD-induced model of liver fibrosis in mice. Figure 26A is an immunoblot of TIM4 expression in mouse livers following CDAHFD. Livers (n = 3) from each of the following groups were analyzed for TIM4 protein content after study completion: PBS (Chow / Vehicle; Healthy Liver), Ctr Mɸ (CDAHFD / Ctrl Mɸ), and TIM4 Mɸ (CDAHFD / TIM4 Mɸ). Figure 26B shows quantification of immunoblots in Figure 26A, with TIM4 protein levels normalized to expression of housekeeping protein GAPDH in each lane.

[0056] Figure 27 is a graph showing in situ analysis of macrophage efferocytosis in mouse livers following CDAHFD-induced model of liver fibrosis in mice treated with untransduced macrophages (CDAHFD / Ctrl Mɸ) or engineered macrophages transduced with either a TIM4 construct (CDAHFD / Mɸ TIM4) or a TIM4 +Relaxin construct (CDAHFD / Mɸ TIM4 + Relaxin).Page 14 of 118 12583381v1Attorney Docket No.2012851-0634

[0057] Figures 28A-28B are a series of graphs showing Sirius Red measurement (Figure 28A) and immunohistochemistry (IHC) images (Figure 28B) of a CDAHFD-induced model of liver fibrosis in mice treated with engineered macrophages transduced with either a TIM4 construct or TIM4-P2A-Relaxin construct described herein in Example 7. These mice were compared to controls including mice fed a normal chow diet (Healthy Liver), CDAHFD-induced mice that were not treated with macrophages (Untreated), or CDAHFD-induced mice that were treated with UTD macrophages (Control Mɸ).

[0058] Figures 29A-29B are a series of graphs showing Collagen type 1 alpha 1 (Col1a1) measurements (Figure 29A) and IHC images (Figure 29B) of a CDAHFD-induced model of liver fibrosis in mice treated with engineered macrophages transduced with either a TIM4 construct or TIM4-P2A-Relaxin construct described herein in Example 7. These mice were compared to controls including mice fed a normal chow diet (“Healthy Liver”), CDAHFD- induced mice that were untreated (“Untreated”), or CDAHFD-induced mice that were transduced with a control vector (“Control Mɸ”).

[0059] Figures 30A-30B are a series of graphs showing alpha-smooth muscle actin (α-SMA) measurement (Figure 30A) and IHC images (Figure 30B) of a CDAHFD-induced model of liver fibrosis in mice treated with engineered macrophages transduced with either a TIM4 construct or TIM4-P2A-Relaxin construct as described herein in Example 7. These mice were compared to controls including mice fed a normal chow diet (“Healthy Liver”), CDAHFD- induced mice that were untreated (“Untreated”), or CDAHFD-induced mice that were transduced with a control vector (“Control Mɸ”).

[0060] Figures 31A-31B are a series of graphs showing Osteopontin (Opn; also interchangeably referred herein as “secreted phosphoprotein 1” and “SPP1”) measurement (Figure 31A) and IHC images (Figure 31B) of a CDAHFD-induced model of liver fibrosis in mice treated with engineered macrophages transduced with either a TIM4 construct or TIM4-P2A-Relaxin construct described herein in Example 7. These mice were compared to controls including mice fed a normal chow diet (“Healthy Liver”), CDAHFD-induced mice that were untreated (“Untreated”), or CDAHFD-induced mice that were transduced with a control vector (“Control Mɸ”). Page 15 of 118 12583381v1Attorney Docket No.2012851-0634

[0061] Figure 32 shows a schematic of timepoints for generation and formulation of engineered human macrophages overexpressing TIM4. On Day 0, primary human monocytes were thawed and either left untransduced (UTD; Control Mɸ) or transduced with lentivirus encoding human TIM4 (hu. TIM4 Mɸ). Cells were differentiated for 5 Days in the presence of GM-CSF. Cells were collected on 5, analyzed for viability and TIM4 expression, and formulated for in vivo

[0062] Figures 33A-33B are graphs showing expression and viability levels of engineered human macrophages overexpressing TIM4. Figure 33A shows flow cytometry detection of TIM4 expression. Figure 33B shows viability of engineered human macrophages expressing TIM4.

[0063] Figure 34 is a series of schematics of timepoints for generating and administering anti- fibrotic engineered human macrophages in an NSG™ liver fibrosis mouse model described herein in Example 8.

[0064] Figures 35A-35B are a graph and image showing collagen content of tissue sections of a CDAHFD-induced liver fibrosis NSG™ mouse model treated with engineered human macrophages transduced with TIM4 described herein in Example 8. Figure 35A shows quantification of collagen as percentage of area in histological images. Figure 35B shows representative Masson’s Trichrome images from data plotted in Figure 35A. These mice were compared to controls including NSG™ mice fed a normal chow diet (Healthy Liver), NSG™ CDAHFD-induced mice that were not treated with macrophages (Untreated), or NSG™ CDAHFD-induced mice that were treated with UTD macrophages (Control Mɸ).

[0065] Figures 36A-36B are a series of graphs showing Picrosirius Red measurement (Figure 36A) and IHC images (Figure 36B) of a CDAHFD-induced model of liver fibrosis in NSG™ mice treated with engineered human macrophages transduced with TIM4 described herein in Example 8. These mice were compared to controls including NSG™ mice fed a normal chow diet (Healthy Liver), NSG™ CDAHFD-induced mice that were not treated with macrophages (Untreated), or NSG™ CDAHFD-induced mice that were treated with UTD macrophages (Control Mɸ).

[0066] Figures 37A-37B are a series of graphs showing Collagen type 1 alpha 1 (Col1a1) measurements (Figure 37A) and IHC images (Figure 37B) of a CDAHFD-induced model of Page 16 of 118 12583381v1Attorney Docket No.2012851-0634 liver fibrosis in NSG™ mice treated with engineered human macrophages transduced with a TIM4 described herein in Example 8. These mice were compared to controls including NSG™ mice fed a normal chow diet (“Healthy Liver”), NSG™ CDAHFD-induced mice that were untreated (“Untreated”), or NSG™ CDAHFD-induced mice that were transduced with a control vector (“Control Mɸ”).

[0067] Figures 38A-38B are a series of graphs showing alpha-smooth muscle actin (α-SMA) measurement (Figure 38A) and IHC images (Figure 38B) of a CDAHFD-induced model of liver fibrosis in NSG™ mice treated with engineered human macrophages transduced with a TIM4 construct as described herein in Example 8. These mice were compared to controls including mice fed a normal chow diet (“Healthy Liver”), NSG™ CDAHFD-induced mice that were untreated (“Untreated”), or NSG™ CDAHFD-induced mice that were transduced with a control vector (“Control Mɸ”).

[0068] Figures 39A-39B are a series of graphs showing Osteopontin (Opn; also interchangeably referred herein as “secreted phosphoprotein 1” and “SPP1”) measurement (Figure 39A) and IHC images (Figure 39B) of a CDAHFD-induced model of liver fibrosis in NSG™ mice treated with engineered human macrophages transduced a TIM4 construct described herein in Example 8. These NSG™ mice were compared to controls including NSG™ mice fed a normal chow diet (“Healthy Liver”), NSG™ CDAHFD-induced mice that were untreated (“Untreated”), or NSG™ CDAHFD-induced mice that were transduced with a control vector (“Control Mɸ”).

[0069] Figure 40 is a graph showing in situ analysis of macrophage efferocytosis in mouse livers from NSG™ CDAHFD-induced mice treated with engineered macrophages transduced with a TIM4 construct. These NSG™ mice were compared to NSG™ CDAHFD-induced mice that were untreated (“Untreated”), or NSG™ CDAHFD-induced mice that were transduced with a control vector (“Control Mɸ”).

[0070] Figure 41 shows a schematic of timepoints for generating a model CDAHFD-induced model of advanced liver fibrosis in mice and treatment with engineered macrophages described herein on Week 8.

[0071] Figures 42A-42D are a series of graphs showing choline-deficient, L-amino acid-defined high fat diet (CDAHFD)-induced advanced liver fibrosis impact in body weight (Figure 42A), liver weight (Figure 42B), liver-to-body weight ratio (Figure 42C), and blood glucose levels Page 17 of 118 12583381v1Attorney Docket No.2012851-0634 measured after a 4 hour fast (Figure 42D), and impact of treatment with UTD macrophages (Control Mɸ), TIM4 macrophages (TIM4 Mɸ), or TIM4 + Relaxin macrophages (TIM4 + Rel.Mɸ) on these metrics.

[0072] Figures 43A-43B are a series of graphs showing Picrosirius Red measurement (Figure 43A) and IHC images (Figure 43B) of a CDAHFD-induced model of advanced liver fibrosis in mice treated with engineered macrophages transduced with TIM4 or TIM4 + Relaxin described herein in Example 9. These mice were compared to controls including mice fed a normal chow diet (Healthy Liver), CDAHFD-induced mice that were not treated with macrophages (Untreated), or CDAHFD-induced mice that were treated with UTD macrophages (Control Mɸ).

[0073] Figure 44 shows a schematic of timepoints for generating a model CDAHFD-induced model of liver fibrosis in mice and treatment with LNP-TIM4 beginning on Week 4. Mice were treated intravenously (“IV”) with TIM4-LNP (1 mg / kg dose) or vehicle control. Mice received 3 total doses, spaced 3-4 Days apart.

[0074] Figure 45 shows a graph of mouse body weight throughout the course of study described herein in Example 10. Error bars depict mean ± SEM. Green vertical line indicates first IV LNP- TIM4 / vehicle treatment.

[0075] Figures 46A-46F shows a series of graphs depicting serum toxicity markers ALP (Figure 46A), AST (Figure 46B), ALT (Figure 46C), bile acids (Figure 46D), triglycerides (Figure 46E), or creatine (Figure 46F) at study endpoint for CDAHFD-induced mice treated with LNP-TIM4. These mice were compared to controls including mice fed a normal chow diet (“chow vehicle”) and CDAHFD-induced mice that were transduced with a control vector (“CDAHFD Vehicle”).

[0076] Figures 47A-47C shows a series of graphs depicting quantification of histological markers of fibrosis, collagen (Figure 47A), αSMA (Figure 47B) and Osteopontin (Figure 47C) at study endpoint for CDAHFD-induced mice treated with LNP-TIM4. These mice were compared to controls including mice fed a normal chow diet (“chow vehicle”) and CDAHFD- induced mice that were transduced with a control vector (“CDAHFD Vehicle”). DEFINITIONS Page 18 of 118 12583381v1Attorney Docket No.2012851-0634

[0077] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0078] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0079] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0080] Activation: As used herein, the term “activation” refers to the state of a cell, for example a monocyte, macrophage, or dendritic cell that has been sufficiently stimulated to induce detectable cellular proliferation or has been stimulated to exert its effector function. Activation can also be associated with induced cytokine production, cytokine secretion, phagocytosis, cell signaling (e.g., gene expression changes), target cell killing, metabolic changes, production of inflammatory mediators, proliferation, epigenetic reprogramming, phenotypic switching of macrophages (e.g., M1 polarization), suppression of pro-tumor or M2 macrophages, phenotypic switching of pro-tumor or M2 macrophages, and / or antigen processing and presentation.

[0081] Activated monocytes / macrophages / dendritic cells: As used herein, the term “activated monocytes / macrophages / dendritic cells” refers to, among other things, monocyte / macrophage / dendritic cells that are undergoing cell division or exerting effector function. The term “activated monocytes / macrophages / dendritic cells” refers to, among others thing, cells that are performing an effector function or exerting any activity not seen in the resting state, including phagocytosis, cytokine secretion, proliferation, gene expression changes, metabolic changes, production of inflammatory mediators, proliferation, epigenetic Page 19 of 118 12583381v1Attorney Docket No.2012851-0634 reprogramming, phenotypic switching of macrophages (e.g., M1 polarization), suppression of pro-tumor or M2 macrophages, phenotypic switching of pro-tumor or M2 macrophages, and other functions.

[0082] Agent: As used herein, the term “agent” (or “biological agent” or “therapeutic agent”), refers to a molecule that may be expressed, released, secreted or delivered to a target by a modified immune cell described herein. An agent includes, but is not limited to, a nucleic acid, an antibiotic, an antibody or fragments thereof, an antibody agent or fragments thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, a microsome, a derivative or a variation thereof, a formulation or composition including one or more thereof, and any combinations thereof. An agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell. An agent may diffuse or be transported into a cell, where it may act intracellularly. For example, an agent may be an exogenous fibrolytic agent as described herein. An agent may be an exogenous anti- inflammatory agent as described herein. An agent may be an exogenous regenerative agent as described herein. An agent may be an exogenous efferocytic agent as described herein.

[0083] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprising two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain comprises at least four domains (each about 110 amino acids long) – an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain comprises two domains – an amino-terminal variable (VL) domain, followed by a carboxy- terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody Page 20 of 118 12583381v1Attorney Docket No.2012851-0634 tetramers comprise two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and a tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. Affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and / or utilized include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation. In some embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and / or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal. In some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art. Moreover, the term “antibody”, as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody utilized in accordance with methods and compositions described herein is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies Page 21 of 118 12583381v1Attorney Docket No.2012851-0634 (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].

[0084] Antibody fragment: As used herein, the term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments and human and humanized versions thereof.

[0085] Antibody heavy chain: As used herein, the term “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0086] Antibody light chain: As used herein, the term “antibody light chain” refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0087] Antigen: As used herein, the term “antigen” or “Ag” refers to a molecule that is capable of provoking an immune response. This immune response may involve either antibody production, the activation of specific immunologically-competent cells, or both. A skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA that comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response encodes an “antigen” as Page 22 of 118 12583381v1Attorney Docket No.2012851-0634 that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0088] Autologous: As used herein, the term “autologous” refers to any material derived from an individual to which it is later to be re-introduced into the same individual.

[0089] Allogeneic: As used herein, the term “allogeneic” refers to a graft (e.g., a population of cells) derived from a different animal of the same species.

[0090] Xenogeneic: As used herein, the term “xenogeneic” refers to a graft (e.g., a population of cells) derived from an animal of a different species.

[0091] Conservative sequence modifications: As used herein, the term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter at least one property or characteristic of a particular protein, for example, the binding characteristics of an antibody, containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues Page 23 of 118 12583381v1Attorney Docket No.2012851-0634 from the same side chain family and the altered antibody can be tested for the ability to bind antigens using the functional assays described herein.

[0092] Effective amount: As used herein, “effective amount” and “therapeutically effective amount” are interchangeable, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0093] Effector function: As used herein, “effector function” or “effector activity” refers to a specific activity carried out by an immune cell in response to stimulation of the immune cell. For example, an effector function of macrophages to engulf and digest cellular debris, foreign substances, microbes, cancer cells and other unhealthy cells by phagocytosis.

[0094] Encoding: As used herein, “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0095] Endogenous: As used herein “endogenous” refers to any material from or produced inside a particular organism, cell, tissue or system.

[0096] Exogenous: As used herein, the term “exogenous” refers to any material introduced from or produced outside a particular organism, cell, tissue or system.

[0097] Expand: As used herein, the term “expand” refers to increasing in number, as in an increase in the number of monocytes / macrophages. In one embodiment, monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, monocytes, macrophages, or Page 24 of 118 12583381v1Attorney Docket No.2012851-0634 dendritic cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term “ex vivo,” as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0098] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to generation of any gene product from a nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0099] Expression vector: As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses (e.g., Ad5f35) that incorporate the recombinant polynucleotide.

[0100] Fibrosis: As used herein, the term “fibrosis” refers to formation of fibrous tissue as a reparative and / or reactive process, rather than as a normal constituent of a cell, tissue, or organ. Fibrosis is characterized by fibroblast accumulation and collagen deposition in excess of normal deposition in any particular tissue. Fibrotic diseases, disorders, and conditions include, but are not limited to, fibrosis arising from wound healing, systemic and local scleroderma, atherosclerosis, restenosis, pulmonary inflammation, idiopathic pulmonary fibrosis, interstitial lung disease, liver cirrhosis, fibrosis as a result of chronic hepatitis B or C infection, kidney disease (e.g., glomerulonephritis), heart disease resulting from scar tissue, keloids and hypertrophic scars, and eye diseases (e.g., macular degeneration, retinal retinopathy, and vitreal retinopathy). Fibrotic diseases, disorders, and conditions can be hepatic-related, such as Page 25 of 118 12583381v1Attorney Docket No.2012851-0634 diseases, disorders, and conditions resulting from accumulation of cholesterol and / or triglycerides within hepatocytes, which may result in a pro-inflammatory response that leads to liver fibrosis and / or cirrhosis. Hepatic disorders having a fibrotic component include, but are not limited to, non-alcoholic fatty liver disease (NAFL) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)) or alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)). Fibrotic diseases, disorders, and conditions can include mechanical trauma, biliary obstruction, autoimmune hepatitis, iron overload, Hepatitis B infection (HBV), and / or Hepatitis C infection (HCV).

[0101] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that Page 26 of 118 12583381v1Attorney Docket No.2012851-0634 position. The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0102] Identity: As used herein, the term “identity” refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0103] Substantial identity: As used herein, the term “substantial identity” refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. In the context of a CDR, reference to “substantial identity” typically refers to a CDR having an amino acid sequence at Page 27 of 118 12583381v1Attorney Docket No.2012851-0634 least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to that of a reference CDR.

[0104] Inflammation: As used herein, the term “inflammation” refers to a cellular, physiological, and / or immunological response to noxious stimuli including, but not limited to, injuries, immunologic reactions, infections, altered endogenous substances, defective endogenous pathways, and / or foreign substances. Inflammation can be systemic or local. Inflammation can be acute or chronic. Exemplary inflammatory conditions, diseases, and disorders include, but are not limited, to a liver, a gastrointestinal tract inflammatory, a lung, a skin a cardiovascular system, a nervous system, a kidney, a pancreas, a joint, an eye, and / or an endocrine system inflammatory condition, disease, or disorder. An inflammatory disease, disorder, or condition can be associated with a toxin, an insult (e.g., an environmental hazard (e.g., asbestos, coal dust, and / or polycyclic aromatic hydrocarbons) or cigarette smoking), and / or a medical treatment (e.g., surgery). An inflammatory disease, disorder, or condition can be or include an autoimmune disorder.

[0105] Immunoglobulin: As used herein, the term “immunoglobulin” or “Ig,” refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as a BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0106] Isolated: As used herein, the term “isolated” refers to something altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist Page 28 of 118 12583381v1Attorney Docket No.2012851-0634 in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0107] Lentivirus: As used herein, the term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of a host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0108] Modified: As used herein, the term “modified” refers to a changed state or structure of a molecule or cell of the disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0109] Modulating: As used herein the term “modulating,” refers to mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0110] Operably linked: As used herein, the term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0111] Polynucleotide: As used herein, the term “polynucleotide” refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric Page 29 of 118 12583381v1Attorney Docket No.2012851-0634 “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0112] Polypeptide: As used herein, the term “polypeptide” refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In Page 30 of 118 12583381v1Attorney Docket No.2012851-0634 some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0113] Protein: As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some Page 31 of 118 12583381v1Attorney Docket No.2012851-0634 embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0114] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0115] Substantially purified: As used herein, the term “substantially purified”, for example as applied to a cell, refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro. Page 32 of 118 12583381v1Attorney Docket No.2012851-0634

[0116] Therapeutic: As used herein, the term “therapeutic” refers to a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0117] Transfected: As used herein, the term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0118] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to partial or complete alleviation, amelioration, delay of onset of, inhibition, prevention, relief, and / or reduction in incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with fibrosis and / or inflammation). In some embodiments, treatment may be administered to a subject who does not exhibit signs or features of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or features of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits established, severe, and / or late-stage signs of the disease, disorder, or condition.

[0119] Vector: As used herein, the term “vector” refers to a composition of matter that comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0120] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience Page 33 of 118 12583381v1Attorney Docket No.2012851-0634 and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. DETAILED DESCRIPTION Immune Cells

[0121] The present disclosure, among other things, provides modified immune cells (e.g., macrophages, monocytes, or dendritic cells) comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one efferocytic agent (e.g., a polypeptide described herein). Modified immune cells (e.g., macrophages, monocytes, or dendritic cells) can comprise or express at least one exogenous fibrolytic agent (e.g., a polypeptide described herein) at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein, at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein).

[0122] As used herein, the term “immune cell,” refers to a cell that is involved in an immune response, e.g., promotion of an immune response. Examples of immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans’ cells, natural killer (NK) cells, T- lymphocytes, or B-lymphocytes. A source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0123] In some embodiments, provided methods and / or compositions include a population of immune cells. In some embodiments, a population of immune cells as described herein Page 34 of 118 12583381v1Attorney Docket No.2012851-0634 comprises monocytes, macrophages, dendritic cells, and / or precursors thereof. In some embodiments, a population of immune cells comprises a purified population of monocytes, macrophages, or dendritic cells, or a cell line.

[0124] In some embodiments, an immune cell is activated, e.g., an immune cell exhibits increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation, e.g., relative to an inactive cell. In some embodiments, an activated immune cell exhibits changes in gene expression, e.g., an induction of pro- inflammatory gene expression (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., relative to an inactive cell. In certain embodiments, activated immune cells are undergoing cell division.

[0125] In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. Immune cells may be autologous or sourced from allogeneic or universal donors. Cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In certain embodiments, cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to a skilled artisan, such as Ficoll separation. In some embodiments, cells from circulating blood of a subject are obtained by apheresis or leukapheresis. Cells collected by apheresis may be washed to remove a plasma fraction and resuspended in a variety of buffers (e.g., phosphate buffered saline (PBS)) or culture media. In some embodiments, enrichment of immune cells (e.g., monocytes) comprises plastic adherence. In some embodiments, following enrichment, differentiation of immune cells (e.g., monocytes) comprises stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and / or red blood cells), such as a leukapheresis composition (e.g., a leukopak) is used for enrichment. In some embodiments, a leukapheresis composition (e.g., a leukopak) comprises a sample from a healthy human donor. In certain embodiments, apheresis of immune cells (e.g., monocytes) is followed by mobilization with GM-CSF. In certain embodiments, selection of immune cells (e.g., monocytes) comprises CD14 positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). Page 35 of 118 12583381v1Attorney Docket No.2012851-0634

[0126] In some embodiments, an immune cell precursor (e.g., precursors to macrophages, monocytes, or dendritic cells) is used in compositions and methods described herein. Immune cell precursors may be differentiated in vivo or ex vivo into immune cells. Non-limiting examples of precursor immune cells include hematopoietic stem cells, myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof. For example, induced pluripotent stem cells may be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells (iPSCs) may be derived from normal human tissue, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs could be differentiated toward a myeloid lineage (e.g., a monocyte, macrophage, dendritic cell, or precursor thereof).

[0127] Immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein can be isolated from peripheral blood, for example, by lysing red blood cells and depleting lymphocytes and red blood cells, such as by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells can be isolated from umbilical cord tissue. A specific subpopulation of immune cells can be further isolated by positive or negative selection techniques. In some embodiments, immune cells can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20. In some embodiments, enrichment of an immune cell population, for example, by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. By way of non-limiting example, cell selection can also comprise negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells.

[0128] During isolation of a desired population of immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein by positive or negative selection, immune cell concentration and surface (e.g., particles, such as beads) can be varied. It may be desirable to significantly decrease volume in which beads and cells are mixed together to ensure maximum contact area of cells and beads. Macrophages Page 36 of 118 12583381v1Attorney Docket No.2012851-0634

[0129] The present disclosure, among other things, provides macrophages comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein) at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein). Macrophages are immune cells specialized for detection, phagocytosis, and destruction of target cells, such as pathogens or tumor cells, as well as for initiation, maintenance, and resolution of tissue repair processes. Macrophages are potent contributors to tissue repair, including through the production of numerous growth factors including, for example, PDGF, IGF-1, TGF-β1, and VEGF-α. In some embodiments, macrophages comprise or express Resolvins (e.g., Resolvin Ds and / or Resolvin Es). In some embodiments, macrophages comprise or express Osteopontin. In some embodiments, macrophages exhibiting an M2 or M0 phenotype are particularly advantageous when used in methods and / or compositions encompassed by the present disclosure.

[0130] In some embodiments, a macrophage comprises or is an undifferentiated or M0 macrophage. In certain embodiments, a macrophage comprises or expresses one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71, or CCR5. Exposure to various stimuli can induce M0 macrophages to polarize into several distinct populations, which may be identified by macrophage phenotype markers, cytokine production, and / or chemokine secretion.

[0131] In some embodiments, a macrophage comprises or is a polarized macrophage. Under classical conditions of activation, M0 macrophages can be exposed to pro-inflammatory signals, such as LPS, IFNγ, and GM-CSF, and polarize into M1 macrophages. Generally, M1 macrophages are associated with pro-inflammatory immune responses, such as Th1 and Th17 T cell responses. Exposure to other stimuli can polarize macrophages into a diverse group of “alternatively activated” or M2 macrophages.

[0132] In some embodiments, a macrophage comprises or is an M1 macrophage. In some embodiments, a macrophage expresses one or more markers of M1 macrophages (e.g., one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, a IFIT family member, or an ISG family member). Page 37 of 118 12583381v1Attorney Docket No.2012851-0634

[0133] In some embodiments, a macrophage comprises or is an M2 macrophage (e.g., an M2A, M2B, M2C, or M2D macrophage). An M2A macrophage can be induced by IL-4, IL-13, and / or fungal infection. An M2B macrophage can be induced by IL-1R ligands, an immune complex, and / or LPS. An M2C macrophage can be induced by IL-10 and / or TGFβ. An M2D macrophage can be induced by IL-6 and / or adenosine. In some embodiments, a macrophage expresses one or more markers of M2 macrophages (e.g., one, two, or three of CD206, CD163, or CD209).

[0134] In some embodiments, a macrophage comprises at least one upregulated M2 marker and / or at least one downregulated M1 marker. In some embodiments, at least one M2 marker (e.g., CD206, CD163, and / or CD209) is upregulated in a macrophage. In some embodiments, at least one M1 marker (e.g., HLA DR, CD86, CD80, PD-L1, CD83, CD69, MHC I, CD64, CD32, CD16, IL1R, a IFIT family member, and / or an ISG family member) is downregulated in a macrophage. Monocytes

[0135] The present disclosure, among other things, provides monocytes comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein). Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen, and generally do not proliferate when in a steady state. Monocytes can vary in size significantly in the range of about 10-30 μm in diameter. A ratio of nucleus to cytoplasm for a monocyte can range from about 2:1 to about 1:1. Typically, monocytes comprise chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues, such as during an wound repair or infection. Monocytes can produce inflammatory and / or anti-inflammatory cytokines, take up cells and / or toxic molecules, and differentiate into dendritic cells or macrophages.

[0136] In some embodiments, a monocyte comprises or expresses one or more phenotypic markers. Exemplarily phenotypic markers for human monocyte cells include, but are not limited to, CD9, CD11b, CD11c, CDw12, CD13, CD15, CDw17, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84. CD85, CD86, Page 38 of 118 12583381v1Attorney Docket No.2012851-0634 CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, CD115, CD116, CD119, CDwl2lb, CDw123, CD127, CDw128, CDw131, CD147, CD155, CD156a, CD157, CD162 CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131a1, CD2132, CDw210, CD226, CD281, CD282, CD284, and CD286. Exemplarily phenotypic markers for mouse monocyte cells include, but are not limited to, CD11a, CD11b, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, CD115, CD116, Cdw131, CD281, CD282, CD284, CD286, F4 / 80, and CD49b. In certain embodiments monocytes comprises one, two, or three of CD11b, CD14, or CD16. In certain embodiments, monocytes comprises CD14+ CD16- monocytes, CD14+ CD16+ monocytes, or CD14- CD16+ monocytes.

[0137] In some embodiments, a monocyte differentiates into a macrophage. In some embodiments, a monocyte differentiates into a dendritic cell (DC). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages can be induced by macrophage colony stimulating factor (M-CSF). Differentiation of monocytes into DCs can be induced by granulocyte-macrophage colony stimulating factor (GM-CSF) in combination with IL-4. Dendritic Cells

[0138] The present disclosure, among other things, provides dendritic cells (DCs) comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein). DCs are bone marrow-derived, specialized antigen presenting cells that are involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells may be found in both lymphoid and non-lymphoid organs and are generally thought to arise from lymphoid or myeloid lineages.

[0139] In some embodiments, a DC comprises or expresses one or more phenotypic markers. Exemplarily phenotypic markers for DCs include, but are not limited to, CD11c, CD83, CD1a, CD1c, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286 and partially CD206, CD207, CD208 and CD209. Page 39 of 118 12583381v1Attorney Docket No.2012851-0634

[0140] Immature DCs can be characterized by a high capacity for antigen capture, but relatively low T cell stimulatory capability. Inflammatory mediators promote DC maturation. In some embodiments, a DC comprises or is an immature DC. In other embodiments, a DC comprises or is a mature DC. Modified Immune Cell

[0141] In some embodiments, a modified immune cell, for example, a modified macrophage, monocyte, or dendritic cell, is generated by expressing at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein). The present disclosure also provides immune cells comprising a nucleic acid sequence (e.g., an isolated nucleic acid sequence) encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein). In some embodiments, an immune cell is a monocyte, macrophage or a dendritic cell that expresses at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent. Exogenous Fibrolytic Agents

[0142] The term “exogenous fibrolytic agent,” as used herein, refers to an exogenous molecule that may be expressed, released, secreted, or delivered, for example, to affect a fibrotic target via modified immune cells described herein (e.g., monocytes, macrophages and / or dendritic cells). A fibrotic target may cause or result from the formation of fibrous tissue and include any biochemical, cellular, or physiological protein associated with fibrosis. A fibrotic target may be associated with any fibrotic condition, disease, or disorder (e.g., a fibrotic condition, disease, or disorder described herein). An exogenous fibrolytic agent can include, but is not limited to, a polypeptide, a fusion protein, a nucleic acid, an antibiotic, an antibody or fragment thereof, an Page 40 of 118 12583381v1Attorney Docket No.2012851-0634 antibody agent or fragment thereof, a growth factor, a cytokine, an enzyme, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, and / or a microsome, or any derivative or variant of any of the foregoing.

[0143] In some embodiments, an exogenous fibrolytic agent comprises or is a Relaxin polypeptide, Decorin polypeptide, dominant negative TGF-beta Receptor 2 (DN TGFβR2) polypeptide, soluble TGFβ receptor, and / or TGFβ switch receptor. In some embodiments, an exogenous fibrolytic agent comprises or is a Relaxin polypeptide. In some embodiments, an exogenous fibrolytic agent comprises or is a Decorin polypeptide. In some embodiments, an exogenous fibrolytic agent comprises or is a DN TGFβR2 polypeptide. In some embodiments, an exogenous fibrolytic agent comprises or is a soluble TGFβ receptor. In some embodiments, an exogenous fibrolytic agent comprises or is a TGFβ switch receptor.

[0144] In some embodiments, an exogenous fibrolytic agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell). In some embodiments, an exogenous fibrolytic agent is secreted from an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell).

[0145] In some embodiments, an exogenous fibrolytic agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by fusion to one or more glycosylphosphatidylinositol (GPI)-anchors, one or more transmembrane receptors or ligands, or one or more hinge domains and / or one or more transmembrane domains. In some embodiments, a hinge domain comprises or is a CD8a, an IgG4, or a CD28 hinge domain. In some embodiments, a transmembrane domain comprises or is a CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3-zeta, FcR γ, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR- 2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain. In some embodiments, an exogenous fibrolytic agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by a short linker (e.g., a glycine-rich and / or serine-rich linker). In some embodiments, a linker comprises Page 41 of 118 12583381v1Attorney Docket No.2012851-0634 (GGGGS)n where n = 1, 2, 3, 4, or 5, or permuted versions thereof, e.g., (SGGGG)n where n = 1, 2, 3, 4, or 5.

[0146] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent decreases or prevents activation of hepatic stellate cells, e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent improves tissue regeneration and / or resolution (e.g., liver tissue regeneration and / or resolution), e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent balances pro- fibrotic and anti-fibrotic immune cell populations (e.g., macrophage populations), e.g., relative to an unmodified immune cell of the same type.

[0147] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent decreases or prevents fibrosis (e.g., liver fibrosis), e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent decreases or prevents tissue injury (e.g., liver tissue injury), e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent decreases a level of alanine transaminase (ALT) and / or aspartate transaminase (AST), e.g., relative to an unmodified immune cell of the same type.

[0148] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent decreases or prevents hepatocyte inflammation (e.g., resulting in a decreased level of NF-kB, IL- lβ, IL-2, MCP-1, and / or MIP-1), e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent results in an Page 42 of 118 12583381v1Attorney Docket No.2012851-0634 increased a level or activity of an anti-inflammatory agent (e.g., IL-10, IL-2, and / or CCL18), e.g., relative to an unmodified immune cell of the same type.

[0149] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous fibrolytic agent decreases or prevents steatosis (e.g., liver steatosis), e.g., relative to an unmodified immune cell of the same type. Exogenous Anti-Inflammatory Agents

[0150] The term “exogenous anti-inflammatory agent,” as used herein, refers to an exogenous molecule that may be expressed, released, secreted, or delivered, for example, to an inflammatory target via modified immune cells described herein (e.g., monocytes, macrophages and / or dendritic cells). An inflammatory target may cause or result from a biochemical, cellular, or physiological response to noxious stimuli including, but not limited to, injuries, immunologic reactions, infections, altered endogenous substances, defective endogenous pathways, and / or foreign substances. In some embodiments, an inflammatory target is or comprises a protein. An exogenous anti-inflammatory agent can include, but is not limited to, a polypeptide, a fusion protein, a nucleic acid, an antibiotic, an antibody or fragment thereof, an antibody agent or fragment thereof, a growth factor, a cytokine, an enzyme, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, and / or a microsome, or any derivative or variant of any of the foregoing.

[0151] In some embodiments, an exogenous anti-inflammatory agent comprises or is a cytokine. In some embodiments, a cytokine comprises or is IL-10, IL-22, and / or IL-27. In some embodiments, an exogenous anti-inflammatory agent comprises or is a cytokine receptor. In some embodiments, a cytokine receptor comprises or is a IL17A switch receptor.

[0152] In some embodiments, an exogenous anti-inflammatory agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell). In some embodiments, an exogenous anti-inflammatory agent is secreted from an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell). Page 43 of 118 12583381v1Attorney Docket No.2012851-0634

[0153] In some embodiments, an exogenous anti-inflammatory agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by fusion to one or more GPI-anchors, one or more transmembrane receptors or ligands, or one or more hinge domains and / or one or more transmembrane domains. In some embodiments, a hinge domain comprises or is a CD8a, an IgG4, or a CD28 hinge domain. In some embodiments, a transmembrane domain comprises or is a CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3-zeta, FcR γ, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain. In some embodiments, an exogenous anti-inflammatory agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by a short linker (e.g., a glycine-rich and / or serine-rich linker). In some embodiments, a linker comprises (GGGGS)n where n = 1, 2, 3, 4, or 5, or permuted versions thereof, e.g., (SGGGG)n where n = 1, 2, 3, 4, or 5.

[0154] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous anti-inflammatory agent decreases a level of expression of one or more inflammatory agents (e.g., TNFα, IL-6, IL- 1β, MCP-1, CCL2, and / or MIP-1), e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous anti-inflammatory agent decreases inflammatory signaling (e.g., NF-kB signaling), e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous anti-inflammatory agent increases a level of expression of an anti-inflammatory agent (e.g., IL-10, IL-2, and / or CCL18), e.g., relative to an unmodified immune cell of the same type.

[0155] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous anti-inflammatory agent decreases or prevents oxidative stress, e.g., relative to an unmodified immune cell of the Page 44 of 118 12583381v1Attorney Docket No.2012851-0634 same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous anti-inflammatory agent decreases or prevents tissue inflammation (e.g., liver tissue inflammation), e.g., relative to an unmodified immune cell of the same type. In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous anti-inflammatory agent decreases activation of inflammatory effector cells (e.g., hepatic stellate cells), e.g., relative to an unmodified immune cell of the same type. Exogenous Regenerative Agents

[0156] The term “exogenous regenerative agent,”, as used herein, refers to an exogenous molecule that may be expressed, released, secreted, or delivered, for example, to a regenerative target via modified immune cells described herein (e.g., monocytes, macrophages and / or dendritic cells) to promote cellular regrowth. In some embodiments, a regenerative target is or comprises a protein. An exogenous regenerative agent can include, but is not limited to, a polypeptide, a fusion protein, a nucleic acid, an antibiotic, an antibody or fragment thereof, an antibody agent or fragment thereof, a growth factor, a cytokine, an enzyme, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, and / or a microsome, or any derivative or variant of any of the foregoing.

[0157] In some embodiments, an exogenous regenerative agent comprises or is Fibrotic Growth Factor 21 (FGF21), Fibrotic Growth Factor 19 (FGF19), Vascular Endothelial Growth Factor α (VEGF-α), and / or Hepatocyte Growth factor (HGF).

[0158] In some embodiments, an exogenous regenerative agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell). In some embodiments, an exogenous regenerative agent is secreted from an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell).

[0159] In some embodiments, an exogenous regenerative agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by fusion to one or more glycosylphosphatidylinositol (GPI)-anchors, one or more transmembrane receptors or ligands, or one or more hinge domains and / or one or more transmembrane domains. In some embodiments, Page 45 of 118 12583381v1Attorney Docket No.2012851-0634 a hinge domain comprises or is a CD8a, an IgG4, or a CD28 hinge domain. In some embodiments, a transmembrane domain comprises or is a CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3-zeta, FcR γ, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR- 2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain. In some embodiments, an exogenous regenerative agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by a short linker (e.g., a glycine-rich and / or serine-rich linker). In some embodiments, a linker comprises (GGGGS)n where n = 1, 2, 3, 4, or 5, or permuted versions thereof, e.g., (SGGGG)n where n = 1, 2, 3, 4, or 5.

[0160] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous regenerative agent improves tissue regeneration and / or resolution (e.g., liver tissue regeneration and / or resolution), e.g., relative to an unmodified immune cell of the same type. Exogenous Efferocytic Agents

[0161] The term “exogenous efferocytic agent,” as used herein, refers to an exogenous molecule that may be expressed, released, secreted, or delivered, for example, to an efferocytic target via modified immune cells described herein (e.g., monocytes, macrophages and / or dendritic cells) to promote efferocytosis. In some embodiments, an efferocytic target is or comprises a protein. An exogenous regenerative agent can include, but is not limited to, a polypeptide, a fusion protein, a nucleic acid, an antibiotic, an antibody or fragment thereof, an antibody agent or fragment thereof, a growth factor, a cytokine, an enzyme, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, and / or a microsome, or any derivative or variant of any of the foregoing.

[0162] In some embodiments, an exogenous efferocytic agent comprises or is a T-cell immunoglobulin and mucin domain containing 4 (TIM4) polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a MERTK polypeptide. In some embodiments, an Page 46 of 118 12583381v1Attorney Docket No.2012851-0634 exogenous efferocytic agent comprises or is an Axl polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a Tyro3 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is an SRA-1 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a CD36 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a LOX1 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a TIM1 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a TIM3 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a LXR polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a CD14 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a Stabilin-1 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a Stabilin-2 polypeptide. In some embodiments, an exogenous efferocytic agent comprises or is a CD300f polypeptide.

[0163] In some embodiments, an exogenous efferocytic agent is tethered to and / or expressed by an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell). In some embodiments, an exogenous efferocytic agent is secreted from an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell).

[0164] In some embodiments, an exogenous efferocytic agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by fusion to one or more glycosylphosphatidylinositol (GPI)-anchors, one or more transmembrane receptors or ligands, or one or more hinge domains and / or one or more transmembrane domains. In some embodiments, a hinge domain comprises or is a CD8a, an IgG4, or a CD28 hinge domain. In some embodiments, a transmembrane domain comprises or is a CD8a, CD64, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphA1, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41BB, CD28, OX40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, CD3-zeta, FcR γ, V / I / LxYxxL / V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR- 2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, CD36, or Syk transmembrane domain. In some embodiments, an exogenous efferocytic agent is tethered to an immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) by a short linker (e.g., a glycine-rich and / or serine-rich linker). In some embodiments, a linker Page 47 of 118 12583381v1Attorney Docket No.2012851-0634 comprises (GGGGS)n where n = 1, 2, 3, 4, or 5, or permuted versions thereof, e.g., (SGGGG)n where n = 1, 2, 3, 4, or 5.

[0165] In some embodiments, a modified immune cell described herein (e.g., a macrophage, monocyte, or dendritic cell) expressing or comprising at least one exogenous efferocytic agent improves efferocytosis e.g., relative to an unmodified immune cell of the same type. Switch Receptors

[0166] The term “switch receptor” or “chimeric switch receptor”, as used herein, refers to an artificial chimeric protein comprising an extracellular domain from a first receptor and an intracellular domain from a second (i.e., different) receptor, such that the receptor can convert one signal into another signal. In some embodiments, a switch receptor of the present disclosure converts a pro-inflammatory signal to an anti-inflammatory signal.

[0167] In some embodiments, a chimeric switch receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain. In some embodiments, an extracellular domain is derived from a first receptor and an intracellular domain is derived from a second receptor. In some embodiments, a first receptor is a cytokine receptor (i.e., a first cytokine receptor). In some embodiments a chimeric switch receptor comprising an extracellular domain from a first cytokine receptor binds a cytokine as described herein. In some embodiments, a first receptor binds an antigen as described herein. In some embodiments, a second receptor is a cytokine receptor (i.e., a second cytokine receptor). In some embodiments, binding of a ligand to the extracellular portion of the chimeric switch receptor results in the intracellular domain from the second cytokine receptor producing a signal substantially similar to a signal that would result from binding of a ligand to the naturally occurring (i.e., full) second cytokine receptor as described herein. By way of non-limiting example, in some embodiments, in a chimeric switch receptor comprising the extracellular domain of an IFN-γ and an intracellular domain of IL10, binding of IFN-γ to the extracellular portion of the chimeric switch receptor results in a signal being produced substantially similar to the signal that would be produced if IL10 and bound to a full IL10 receptor. In some embodiments, a transmembrane domain is derived from a first receptor or a second receptor.

[0168] In some embodiments, a first cytokine receptor is a receptor for an anti-inflammatory cytokine (i.e., anti-inflammatory cytokine receptor). In some embodiments, a second cytokine Page 48 of 118 12583381v1Attorney Docket No.2012851-0634 receptor is an anti-inflammatory cytokine receptor. In some embodiments, a first cytokine receptor is a pro-inflammatory cytokine receptor and a second cytokine receptor is an anti- inflammatory cytokine receptor.

[0169] In some embodiments, chimeric switch receptors of the present disclosure are membrane- bound. In some embodiments, chimeric switch receptors of the present disclosure are not membrane-bound. By way of non-limiting example, several potential embodiments of switch receptors are described in International publication number WO2024 / 006281, filed June 27, 2023, the disclosure of which is hereby incorporated in its entirety. Nucleic Acid Constructs

[0170] The present disclosure, among other things, provides nucleic acid molecules comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein). An immune cell can comprise a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein).

[0171] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene Page 49 of 118 12583381v1Attorney Docket No.2012851-0634 produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0172] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0173] As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0174] Nucleic acid molecules encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein) can be a DNA molecule, an RNA molecule, or a combination thereof. In some Page 50 of 118 12583381v1Attorney Docket No.2012851-0634 embodiments, a nucleic acid molecule comprises or is a messenger RNA (mRNA) transcript encoding at least one exogenous fibrolytic agent. In some embodiments, a nucleic acid molecule comprises or is an mRNA transcript encoding at least one exogenous anti-inflammatory agent. In some embodiments, a nucleic acid molecule comprises or is a DNA construct encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent.

[0175] In some embodiments, all or a fragment of at least one exogenous fibrolytic agent, described herein is encoded by a codon optimized nucleic acid molecule, e.g., for expression in a cell (e.g., a mammalian cell). In some embodiments, all or a fragment of at least one exogenous anti-inflammatory agent, described herein is encoded by a codon optimized nucleic acid molecule, e.g., for expression in a cell (e.g., a mammalian cell). A variety of codon optimization methods are known in the art, e.g., as disclosed in US Patent Nos.5,786,464 and 6,114,148, each of which is hereby incorporated by reference in its entirety.

[0176] In some embodiments, a vector comprises a nucleic acid molecule encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti- inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein). In some embodiments, a vector comprises a plasmid, viral vector, phagemid, retrotransposon (e.g. piggyback or sleeping beauty), site directed insertion vector (e.g. CRISPR / Cas systems (e.g., CRISPR / Cas systems comprising one or more of Cas9, Cas12a, or C2c2), Zn finger nucleases, and / or TALEN for insertion of a template donor DNA comprising a nucleic acid sequence encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein) and / or at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein) and / or at least one exogenous regenerative agent (e.g., a polypeptide described herein) and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein), suicide expression vector, or any other vector known in the art. Vectors can be suitable for replication and integration in eukaryotes. Vectors can include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0177] A vector can comprise an origin of replication, a promoter sequence (e.g., a constitutive or inducible promoter), and / or convenient restriction endonuclease sites (See, e.g., WO Page 51 of 118 12583381v1Attorney Docket No.2012851-0634 01 / 96584; WO 01 / 29058; and U.S. Pat. No.6,326,193, each of which are hereby incorporated by reference in their entirety). A vector can also include, e.g., a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator (e.g., a Bovine Growth Hormone (BGH) polyadenylation signal), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and / or ColE1), elements to allow selection (e.g., an ampicillin resistance gene and / or zeocin marker), and / or reporter genes (e.g., luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein).

[0178] Expression of nucleic acids described herein may be achieved by operably linking a nucleic acid encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein) and / or at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein) and / or at least one exogenous regenerative agent (e.g., a polypeptide described herein) and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein) to a promoter in an expression vector. Expression of nucleic acids described herein may be achieved by operably linking a nucleic acid encoding at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein) and / or at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein) and / or at least one exogenous regenerative agent (e.g., a polypeptide described herein) and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein) to a promoter in an expression vector. Exemplary promoters (e.g., constitutive promoters) include, but are not limited to, an elongation factor-1α promoter (EF-1α) promoter, immediate early cytomegalovirus (CMV) promoter, ubiquitin C promoter, phosphoglycerokinase (PGK) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. A vector can also comprise additional promoter elements, e.g., enhancers, to regulate the frequency of transcriptional initiation.

[0179] A vector can comprise one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein) and / or at least one exogenous Page 52 of 118 12583381v1Attorney Docket No.2012851-0634 anti-inflammatory agent (e.g., a polypeptide described herein) and / or at least one exogenous regenerative agent (e.g., a polypeptide described herein) and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein), operably linked to one or more tissue specific promoters. In some embodiments, a vector described herein comprises one or more liver specific promoters. In some embodiments, a vector described herein comprises one or more cirrhosis specific promoters. In some embodiments, a vector described herein comprises a CX3CR1 promoter. In some embodiments, a vector described herein comprises an insulin-like growth factor 1 (IGF1). In some embodiments, a vector described herein comprises a CD11B promoter. In some embodiments, a vector (or combination of vectors) may comprise nucleic acid sequences encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fibrolytic agents, anti-inflammatory agents, regenerative agents, and / or efferocytic agents.

[0180] In some embodiments, a vector comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein) and / or at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein) and / or at least one exogenous regenerative agent (e.g., a polypeptide described herein) and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein) comprises or is a viral vector . Viral vector technology is well known in the art and is described, e.g., in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY). Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno- associated viral vectors, or retroviral vectors (e.g., a lentiviral vector or a gammaretroviral vector). In some embodiments, a vector comprises a lentiviral vector (e.g., as described in US Patent No.9,149,519 or International Publication No. WO 2017 / 044487, each of which is hereby incorporated by reference in its entirety).

[0181] In some embodiments, a viral vector comprises an adenoviral vector. Adenoviruses are a large family of viruses containing double stranded DNA. They replicate within the nucleus of a host cell, using the host’s cell machinery to synthesize viral RNA, DNA and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, to accommodate large transgenes, and to code for proteins without integrating into the host cell genome. In some embodiments, an adenoviral vector comprises an Ad2 vector or an Ad5 vector (e.g., Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector). Page 53 of 118 12583381v1Attorney Docket No.2012851-0634

[0182] In some embodiments, a viral vector is an adeno-associated virus (AAV) vector. AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., P.N.A.S. U.S.A., 89(13):6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(4):699-708 (2012)). Methods for generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Pat. Nos.5,139,941 and 4,797,368.

[0183] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as variants thereof. Generally, any AAV serotype may be used to deliver at least one exogenous fibrolytic agent (e.g., a polypeptide described herein) and / or at least one exogenous anti- inflammatory agent (e.g., a polypeptide described herein). In some embodiments, an AAV serotype has a tropism for a particular tissue.

[0184] In some embodiments, CRISPR / Cas9 system (or other Cas systems including Cas12a, Cas13, etc) has recently been shown to facilitate high levels of precise genome editing using adeno associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR).

[0185] In some embodiments, a vector comprises a gammaretroviral vector (e.g., as described in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses.2011 Jun; 3(6): 677–713, which is hereby incorporated by reference in its entirety). Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom.

[0186] In some embodiments, a vector comprises two or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent. In some embodiments, two or more nucleic acid sequences encoding at least two agents (e.g., two or more exogenous fibrolytic agents, two or more exogenous anti-inflammatory agents, two or more exogenous regenerative agents, and / or two or more exogenous efferocytic agents, or a combination of any of Page 54 of 118 12583381v1Attorney Docket No.2012851-0634 the foregoing) are encoded by a single nucleic molecule, e.g., in same frame and as a single polypeptide chain. In some embodiments, two or more agents are separated by one or more cleavage peptide sites (e.g., an auto-cleavage site or a substrate for an intracellular protease). In certain embodiments, a cleavage peptide comprises a porcine teschovirus-l (P2A) peptide, Thosea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, foot-and-mouth disease virus (F2A) peptide, or a variant thereof. Pharmaceutical Compositions

[0187] The present disclosure, among other things, provides pharmaceutical compositions comprising immune cells as described herein (e.g., macrophages, monocytes, or dendritic cells) and / or delivery vehicle comprising one or more nucleic acids as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0188] When “a therapeutically effective amount, “an immunologically effective amount,” “an anti-immune response effective amount,” or “an immune response-inhibiting effective amount” is indicated, a precise amount of a pharmaceutical composition comprising immune cells as described herein (e.g., macrophages, monocytes, or dendritic cells) can be determined by a physician with consideration of individual differences in age, weight, immune response, and condition of the patient (subject).

[0189] Pharmaceutical compositions comprising immune cells as described herein (e.g., macrophages, monocytes, or dendritic cells) may comprise buffers including neutral buffered saline or phosphate buffered saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextrans, or mannitol; proteins, polypeptides, or amino acids (e.g., glycine); antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, a pharmaceutical composition is substantially free of contaminants, e.g., there are no detectable levels of a contaminant (e.g., an endotoxin).

[0190] Pharmaceutical compositions described herein may be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. Quantity and frequency of administration will be determined by such factors as condition of a patient, and type Page 55 of 118 12583381v1Attorney Docket No.2012851-0634 and severity of a patient’s disease, disorder, or condition, although appropriate dosages may be determined by clinical trials.

[0191] Pharmaceutical compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusible solutions. Pharmaceutical compositions described herein can be formulated for administration intravenously, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, transarterially, or intraperitoneally.

[0192] In some embodiments, a pharmaceutical composition described herein is formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, a pharmaceutical composition described herein is formulated for intravenous infusion or injection. In some embodiments, a pharmaceutical composition described herein is formulated for intramuscular or subcutaneous injection. Pharmaceutical compositions described herein can be formulated for administered by using infusion techniques that are commonly known in immunotherapy (See, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988, which is hereby incorporated by reference in its entirety).

[0193] As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, and intrasternal injection and infusion.

[0194] Pharmaceutical compositions comprising immune cells as described herein may be administered at a dosage of about 104to about 109cells / kg body weight (e.g., about 105to about 106cells / kg body weight), including all integer values within those ranges). In some embodiments, a dose of immune cells as described herein (e.g., macrophages, monocytes, or dendritic cells) comprises at least about 1 x 106, about 1.1 x 106, about 2 x 106, about 3.6 x 106, about 5 x 106, about 1 x 107, about 1.8 x 107, about 2 x 107, about 5 x 107, about 1 x 108, about 2 x 108, about 5 x 108, about 1 x 109, about 2 x 109, or about 5 x 109cells. Pharmaceutical Page 56 of 118 12583381v1Attorney Docket No.2012851-0634 compositions described herein may also be administered multiple times at a certain dosage. An optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art by monitoring a patient for signs of a disease, disorder, or condition and adjusting treatment accordingly.

[0195] It may be desired to administer pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein to a subject and then subsequently redraw blood (or have apheresis performed), activate collected immune cells, and reinfuse a subject with activated immune cells. This process can be performed multiple times, e.g., every few weeks. Immune cells (e.g., macrophages, monocytes, or dendritic cells) can be activated from blood draws of from about 10 cc to about 400 cc. In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are activated from blood draws of about 20 cc, about 30 cc, about 40 cc, about 50 cc, about 60 cc, about 70 cc, about 80 cc, about 90 cc, or about 100 cc. Without being bound by theory, methods comprising multiple blood draw and reinfusions as described herein may select for certain immune cell populations.

[0196] In some embodiments, pharmaceutical compositions comprising immune cells as described herein (e.g., macrophages, monocytes, or dendritic cells) are administered in combination with (e.g., before, simultaneously, or following) a second therapy. For example, a second therapy can include, but is not limited to farnesoid X receptor agonist, a stearoyl CoA desaturase inhibitor, a CCR2 / CCR5 chemokine antagonist, a PPAR agonist, a caspase inhibitor, a galectin-3 inhibitor, an acetyl CoA carboxylase inhibitor, a lysyl oxidase-2 (LOX-2) antagonist, TGFβ antagonist, a GLP1 agonist, a thyroid hormone receptor-β (THR-β) agonist, a recombinant FGF molecule, and / or TIMP antagonist. In some embodiments, a GLP-1 agonist is semaglutide. In some embodiments, a GLP-1 agonist is tirzepatide. In some embodiments, a THR-β agonist is resmetirom. In some embodiments, a recombinant FGF molecule is efruxifermin. A dosage of any aforementioned therapy to be administered to a subject will vary with a disease, disorder, or condition being treated and based on a specific subject. Scaling of dosages for human administration can be performed according to art-accepted practices. Methods of Treatment Page 57 of 118 12583381v1Attorney Docket No.2012851-0634

[0197] The present disclosure, among other things, provides methods of treating a disease, disorder, or condition (e.g., a disease, disorder, or condition described herein) in a subject comprising delivering a pharmaceutical composition comprising immune cells as described herein (e.g., macrophages, monocytes, or dendritic cells). In some embodiments, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease, disorder, or condition.

[0198] A subject to be treated with methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a subject having, or at risk of having, a disease, disorder, or condition described herein). In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) may be autologous, allogeneic, or xenogeneic with respect to a subject. Pharmaceutical compositions as described herein can be administered to a subject in accordance with a dosage regimen described herein, alone or in combination with one or more therapeutic agents, procedures, or modalities.

[0199] Administration of pharmaceutical compositions described herein may be carried out in any convenient manner (e.g., injection, ingestion, transfusion, inhalation, implantation, or transplantation). In some embodiments, pharmaceutical compositions described herein is administered by injection or infusion. Pharmaceutical compositions described herein may be administered to a subject transarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, or intraperitoneally. In some embodiments, a pharmaceutical composition described herein is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, a pharmaceutical composition described herein is administered by intravenous infusion or injection. In some embodiments, a pharmaceutical composition described herein is administered by intramuscular or subcutaneous injection. Pharmaceutical compositions described herein may be injected directly into a site of inflammation, a local disease site, a lymph node, an organ, a tumor, or site of infection in a subject. Fibrosis

[0200] Modified immune cells and / or delivery vehicle comprising one or more nucleic acids as described herein described herein can be administered to improve one or more symptoms of or Page 58 of 118 12583381v1Attorney Docket No.2012851-0634 reduce fibrosis in a subject, e.g., to treat or prevent a fibrotic disease, disorder, or condition in a subject. Methods can include administering modified immune cells described herein to a subject in need thereof, in an amount sufficient to decrease one or more symptoms of or prevent fibrosis in the subject. Modified immune cells described herein can be administered to improve tissue repair, e.g., in a subject with a fibrotic disease, disorder, or condition. In some embodiments, a subject has fibrosis or has been diagnosed with a fibrotic disease, disorder, or condition. In some embodiments, a subject has not received prior treatment with modified immune cells described herein (e.g., a naïve subject).

[0201] In some embodiments, modified immune cells described herein are for use as a medicament in treating (e.g., reversing, reducing, ameliorating, or preventing) fibrosis in a subject (e.g., a subject with a fibrotic disease, disorder, or condition). In some embodiments, modified immune cells described herein are for use in the manufacture of a medicament for treating (e.g., reversing, reducing, or ameliorating one or more symptoms of, or preventing) fibrosis in a subject (e.g., a subject with a fibrotic disease, disorder, or condition).

[0202] In some embodiments, administration of modified immune cells described herein to a subject delays onset of or reduces one or more of: the formation or deposition of tissue fibrosis; the size, cellularity, composition, or cellular content of a fibrotic lesion; the collagen and / or hydroxyproline content of a fibrotic lesion; expression or activity of a fibrogenic protein; fibrosis associated with an inflammatory response; and / or weight loss associated with fibrosis as compared to a subject that did not receive such administration. In some embodiments, reducing fibrosis increases survival of a subject as compared to a subject that did not receive such administration.

[0203] Exemplary fibrotic diseases, disorders, or conditions include systemic diseases, disorders, or conditions (e.g., systemic sclerosis, multifocal fibrosclerosis, sclerodermatous chronic graft- versus-host disease, nephrogenic systemic fibrosis, or scleroderma) and organ-specific diseases, disorders, or conditions (e.g., liver, lung, heart, kidney, pancreas, skin, and / or nervous system fibrosis). In some embodiments, a fibrotic disease, disorder, or condition comprises or is a hyperproliferative fibrotic disease, e.g., a non-cancerous fibrotic disease.

[0204] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a liver, lung, heart, vasculature, kidney, pancreas, skin, gastrointestinal, bone marrow, hematopoietic Page 59 of 118 12583381v1Attorney Docket No.2012851-0634 tissue, nervous system, and / or eye fibrotic disease, disorder, or condition. In some embodiments, a fibrotic disease, disorder, or condition affects tissue comprising tendon, cartilage, skin (e.g., skin epidermis and / or endodermis), cardiac tissue, vascular tissue (e.g., artery and / or vein), pancreatic tissue, lung tissue, kidney tissue, uterine tissue, ovarian tissue, neural tissue, testicular tissue, peritoneal tissue, colon, small intestine, biliary tract, gut, bone marrow, hematopoietic tissue, and / or eye tissue (e.g., retinal tissue).

[0205] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a liver fibrotic disease, disorder, or condition. Modified immune cells described herein can be administered to improve liver function, e.g., in a subject with a liver fibrotic disease, disorder, or condition. In some embodiments, a liver fibrotic disease, disorder, or condition comprises or is a fatty liver disease, disorder, or condition. In some embodiments, a fatty liver disease, disorder, or condition comprises or is non-alcoholic fatty liver disease (NAFL) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)) or alcoholic liver disease (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)). In some embodiments, a subject has cirrhosis, hepatocarcinoma, an increased risk of liver failure, an increased risk of death, metabolic syndrome, type 2 diabetes, Hepatitis B infection (HBV), mechanical trauma, biliary obstruction, autoimmune hepatitis, iron overload, Hepatitis B infection (HBV), and / or Hepatitis C infection (HCV).

[0206] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a lung fibrotic disease, disorder, or condition. Modified immune cells described herein can be administered to improve lung function, e.g., in a subject with a lung fibrotic disease, disorder, or condition. In some embodiments, a lung fibrotic disease, disorder, or condition comprises or is pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonitis (UIP), interstitial lung disease, cryptogenic fibrosing alveolitis (CFA), bronchiectasis, and / or scleroderma lung disease. In some embodiments, lung fibrosis is associated with an occupational hazard, an environmental pollutant, cigarette smoking, an autoimmune connective tissue disorders (e.g., rheumatoid arthritis, scleroderma, or systemic lupus erythematosus (SLE)), a connective tissue disorder (e.g., sarcoidosis), and / or or an infectious disease.

[0207] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a kidney fibrotic disease, disorder, or condition. Modified immune cells described herein can be Page 60 of 118 12583381v1Attorney Docket No.2012851-0634 administered to improve kidney function, e.g., in a subject with a kidney fibrotic disease, disorder, or condition. In some embodiments, a kidney fibrotic disease, disorder, or condition comprises or is renal fibrosis (e.g., chronic kidney fibrosis), nephropathies associated with fibrosis (e.g., diabetic nephropathy), lupus, scleroderma of the kidney, glomerular nephritis, focal segmental glomerular sclerosis, IgA nephropathyrenal fibrosis associated with human chronic kidney disease (CKD), chronic progressive nephropathy (CPN), tubulointerstitial fibrosis, ureteral obstruction, chronic uremia, chronic interstitial nephritis, radiation nephropathy, glomerulosclerosis, progressive glomerulonephrosis (PGN), endothelial / thrombotic microangiopathy injury, and / or HIV-associated nephropathy. In some embodiments, kidney fibrosis is associated with exposure to a toxin, an irritant, or a chemotherapeutic agent.

[0208] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a heart fibrotic disease, disorder, or condition. Modified immune cells described herein can be administered to improve heart function, e.g., in a subject with a heart fibrotic disease, disorder, or condition. In some embodiments, a heart fibrotic disease, disorder, or condition comprises or is myocardial fibrosis. In some embodiments, myocardial fibrosis is associated with radiation myocarditis, surgical procedure complications (e.g., myocardial post-operative fibrosis), infectious diseases (e.g., Chagas disease), granulomatous, metabolic storage disorders (e.g., cardiomyopathy or hemochromatosis), developmental disorders (e.g., endocardial fibroelastosis), arteriosclerotic, and / or exposure to toxins or irritants (e.g., drug-induced cardiomyopathy, drug- induced cardiotoxicity, or alcoholic cardiomyopathy). In some embodiments, myocardial fibrosis is associated with myocardial sarcoidosis, myocardial infarction, and / or congestive heart failure.

[0209] In some embodiments, a fibrotic disease, disorder, or condition comprises or is an eye fibrotic disease, disorder, or condition. Modified immune cells described herein can be administered to improve eye function, e.g., in a subject with an eye fibrotic disease, disorder, or condition. In some embodiments, an eye fibrotic disease, disorder, or condition comprises or is glaucoma, macular degeneration (e.g., age-related macular degeneration), macular edema (e.g., diabetic macular edema), retinopathy (e.g., diabetic retinopathy), and / or dry eye disease.

[0210] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a skin fibrotic disease, disorder, or condition. Modified immune cells described herein can be Page 61 of 118 12583381v1Attorney Docket No.2012851-0634 administered to improve skin function, e.g., in a subject with a skin fibrotic disease, disorder, or condition. In some embodiments, a skin fibrotic disease, disorder, or condition comprises or is skin fibrosis (e.g., hypertrophic scarring and / or keloid scars), scleroderma of the skin, and / or nephrogenic systemic fibrosis.

[0211] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a gastrointestinal tract fibrotic disease, disorder, or condition. Modified immune cells described herein can be administered to improve gastrointestinal tract function, e.g., in a subject with a gastrointestinal tract fibrotic disease, disorder, or condition. In some embodiments, a gastrointestinal tract fibrotic disease, disorder, or condition comprises or is radiation-induced gut fibrosis, fibrosis associated with a foregut inflammatory disorder (e.g., Barrett’s esophagus or chronic gastritis), and / or fibrosis associated with a hindgut inflammatory disorder (e.g., inflammatory bowel disease, ulcerative colitis, or Crohn’s disease).

[0212] In some embodiments, a fibrotic disease, disorder, or condition comprises or is a chronic fibrotic disease, disorder, or condition. In some embodiments, a fibrotic disease, disorder, or condition is associated with an inflammatory disease, disorder, or condition. In some embodiments, a fibrotic disease, disorder, or condition comprises or is osteomyelitis (e.g., chronic osteomyelitis). In some embodiments, a fibrotic disease, disorder, or condition comprises or is an amyloidosis (e.g., amyloidosis associated with chronic osteomyelitis). Inflammation

[0213] Modified immune cells and / or delivery vehicle comprising one or more nucleic acids as described herein described herein can be administered to improve or reduce inflammation in a subject, e.g., to treat or prevent an inflammatory disease, disorder, or condition in a subject (e.g., one or more symptoms of an inflammatory disease, disorder, or condition). Methods can include administering modified immune cells described herein to a subject in need thereof, in an amount sufficient to decrease or prevent inflammation in the subject. In some embodiments, a subject has inflammation or has been diagnosed with an inflammatory disease, disorder, or condition. In some embodiments, a subject has not received prior treatment with modified immune cells described herein (e.g., a naïve subject). Page 62 of 118 12583381v1Attorney Docket No.2012851-0634

[0214] In some embodiments, modified immune cells described herein are for use as a medicament in treating (e.g., reversing, reducing, ameliorating, or preventing) inflammation in a subject (e.g., a subject with an inflammatory disease, disorder, or condition). In some embodiments, modified immune cells described herein are for use in the manufacture of a medicament for treating (e.g., reversing, reducing, or ameliorating one or more symptoms of, or preventing) inflammation in a subject (e.g., a subject with an inflammatory disease, disorder, or condition).

[0215] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a systemic disease, disorder, or condition. In some embodiments, an inflammatory disease, disorder, or condition comprises or is a local disease, disorder, or condition. In some embodiments, an inflammatory disease, disorder, or condition comprises or is an acute disease, disorder, or condition. In some embodiments, an inflammatory disease, disorder, or condition comprises or is a chronic disease, disorder, or condition.

[0216] Exemplary inflammatory diseases, disorders, or conditions include, but are not limited to, systemic diseases, disorders, or conditions (e.g., chronic systemic inflammation, Behçet disease, sarcoidosis, systemic lupus erythematosus, juvenile idiopathic arthritis, scleroderma, Sjögren syndrome, and / or sepsis) and organ-specific diseases, disorders, or conditions (e.g., liver, lung, heart, gut, kidney, and / or pancreas inflammation). In some embodiments, an inflammatory disease, disorder, or condition comprises or is a liver, gastrointestinal tract, lung, skin, cardiovascular system, nervous system, kidney, pancreas, joint, eye, and / or an endocrine system inflammatory disease, disorder, or condition. In some embodiments, an inflammatory disease, disorder, or condition comprises or is an autoimmune disorder.

[0217] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a liver inflammatory disease, disorder, or condition. In some embodiments, a liver inflammatory disease, disorder, or condition comprises or is a fatty liver disease, disorder, or condition (e.g., NAFLD (e.g., NASH) or AFLD (e.g., ASH)). In certain embodiments, a liver inflammatory disease, disorder, or condition comprises or is cirrhosis (e.g., primary biliary cirrhosis or primary sclerosing cholangitis), hepatitis (e.g., hepatitis from a viral infection, an autoimmune response, a drug treatment, a toxin, and / or an environmental agent), and / or biliary atresia. Page 63 of 118 12583381v1Attorney Docket No.2012851-0634

[0218] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a gastrointestinal tract inflammatory disease, disorder, or condition. In certain embodiments, a gastrointestinal tract inflammatory disease, disorder, or condition comprises or is colitis (e.g., ulcerative colitis, pseudomembranous colitis, microscopic colitis, indeterminatal colitis, ischemic colitis, radiation colitis, and / or collagenous colitis), Crohn's disease, leaky gut syndrome, irritable bowel syndrome (IBS), Barrett’s esophagus, intestinal inflammation, chronic gastritis, distal proctitis, enteritis, enterocolitis, gastritis, gastroenteritis, cholangitis, ileitis, constipation, diarrhea; indigestion or non-ulcer dyspepsia, diverticulosis, and / or polyps.

[0219] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a lung inflammatory disease, disorder, or condition. In some embodiments, the lung inflammatory disease, disorder, or condition comprises or is asthma, COPD, adult respiratory distress syndrome (ARDS), bronchitis (e.g., chronic bronchitis), bronchiolitis, pulmonary inflammation, pulmonary fibrosis, cystic fibrosis, pneumonitis (e.g., hypersensitivity pneumonitis, usual interstitial pneumonitis (UIP), pneumonia, extrinsic allergic alveolitis, asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, lung sarcoidosis, and / or pleuritis.

[0220] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a skin inflammatory disease, disorder, or condition. In some embodiments, a skin inflammatory disease, disorder, or condition comprises or is psoriasis, eczema, dermatitis (e. g., eczematous dermatitides, topic or seborrheic dermatitis, allergic or irritant contact dermatitis, eczema craquelee, photoallergic dermatitis, phototoxicdermatitis, phytophotodermatitis, radiation dermatitis, or stasis dermatitis), an ulcer, ichthyoses, epidermolysis bullosae, a hypertrophic scar, a keloid, inflammatory dermatosis, photoaging, cutaneous atrophy, inflammatory dermatosis, dermatomyositis; pemphigus.

[0221] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a cardiovascular system inflammatory disease, disorder, or condition. In some embodiments, a cardiovascular system inflammatory disease, disorder, or condition comprises or is atherosclerosis, coronary infarct damage, peripheral vascular disease, myocarditis, vasculitis, revascularization of stenosis, myocarditis, pericarditis, vascular disease associated with Type II diabetes, endocarditis, a cholesterol-related metabolic disorder, oxygen free radical injury, and / or ischemia. Page 64 of 118 12583381v1Attorney Docket No.2012851-0634

[0222] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a nervous system inflammatory disease, disorder, or condition. In some embodiments, a nervous system inflammatory disease, disorder, or condition comprises or is a neurodegenerative disease (e.g., Alzheimer's disease or dementia), multiple sclerosis, encephalitis (e.g., encephalitis with inflammatory edema), depression, attention deficit disorder (ADD), Parkinson's disease, schizophrenia, and / or a neuropathy (e.g., peripheral neuropathy).

[0223] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a kidney inflammatory disease, disorder, or condition. In some embodiments, a kidney inflammatory disease, disorder, or condition comprises or is a nephritis, nephritis secondary to Wegener's disease, acute renal failure secondary to acute nephritis, post-obstructive syndrome, tubular ischemia, pyelonephritis glomerulosclerosis, membranous neuropathy, and / or renal arteriosclerosis. In some embodiments, nephritis comprises or is glomerulonephritis, interstitial nephritis, and / or lupus nephritis.

[0224] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a pancreas inflammatory disease, disorder, or condition. In some embodiments, a pancreas inflammatory disease, disorder, or condition comprises or is pancreatitis (e.g., acute or chronic pancreatitis).

[0225] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a joint inflammatory disease, disorder, or condition. In some embodiments, a joint inflammatory disease, disorder, or condition comprises or is rheumatoid arthritis, rheumatoid spondylitis, juvenile rheumatoid arthritis, osteoarthritis, gouty arthritis, psoriatic arthritis, lupus-associated arthritis, ankylosing spondylitis, spondyloarthrosis, degenerative arthritis, and / or synovitis.

[0226] In some embodiments, an inflammatory disease, disorder, or condition comprises or is an eye inflammatory disease, disorder, or condition. In some embodiments, an eye inflammatory disease, disorder, or condition comprises or is uveitis, iritis, optic neuritis, conjunctivitis, scleritis, iritis, keratoconjunctivitis sicca, blepharitis, age-related macular degeneration (AMD), dry eye syndrome, optic nerve damage, diabetic retinopathy, corneal inflammation, and / or retinal inflammation.

[0227] In some embodiments, an inflammatory disease, disorder, or condition comprises or is an endocrine system disease, disorder, or condition. In some embodiments, an endocrine system Page 65 of 118 12583381v1Attorney Docket No.2012851-0634 inflammatory disease, disorder, or condition comprises or is autoimmune thyroiditis (Hashimoto's disease), adrenal cortex inflammation (e.g., acute adrenal cortex inflammation or chronic adrenal cortex inflammation), and / or Type I diabetes mellitus.

[0228] In some embodiments, an inflammatory disease, disorder, or condition comprises or is an autoimmune disease, disorder, or condition. In some embodiments, an autoimmune disease, disorder, or condition comprises or is arthritis (e.g., rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-associated arthritis, and / or ankylosing spondylitis), autoimmune thyroiditis, scleroderma, lupus, systemic lupus erythematosus (SLE), HIV, Sjogren's syndrome, vasculitis, multiple sclerosis, dermatitis (e.g., atopic dermatitis or eczematous dermatitis), myasthenia gravis, IBD, Crohn's disease, colitis, an acute inflammatory condition (e.g., endotoxemia, septicaemia, or toxic shock syndrome), a transplant rejection, allergy (e.g., asthma, allergic rhinitis, eczema, allergic contact dermatitis, or allergic conjunctivitis), Wegener's gramilornatosis, angiitis, polymyalgia rheumatica (PMR), tendonitis, bursitis, an immediate hypersensitivity reaction (e.g., asthma, hay fever, cutaneous allergies, or acute anaphylaxis), acute disseminated encephalomyelitis, Sorgen's disease, Addison's disease, heart disease, osteoporosis, alopecia universalis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, pernicious anemia, autoimmune hepatitis, Bullous pemphigoid, endometriosis, Goodpasture's syndrome, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphea, neuromyotonia, temporal arteritis, vasculitis, and / or vitiligo.

[0229] In some embodiments, an inflammatory disease, disorder, or condition comprises or is a wound. In certain embodiment, a wound comprises or is an acute wound, a chronic wound, an open wound, a closed wound, an infected wound, an external wound, an internal wound, and / or a hemorrhage.

[0230] In some embodiments, an inflammatory disease, disorder, or condition comprises or is ischemia. In some embodiments, ischemia comprises or is cardiac ischemia, bowel ischemia, brain ischemia (e.g., stroke), limb ischemia, mesenteric ischemia, cutaneous ischemia. In some embodiments, an inflammatory disease, disorder, or condition comprises or is ischemia reperfusion injury, post-perfusion syndrome, and / or transient ischemia of an organ (e.g., gastrointestinal tract, bladder, or heart). Page 66 of 118 12583381v1Attorney Docket No.2012851-0634

[0231] In some embodiments, an inflammatory disease, disorder, or condition comprises or is sepsis. In some embodiments, species comprise or is sepsis resulting from pneumonia, abdominal infection, kidney infection, and / or bloodstream infection. In some embodiments, sepsis comprises or is sepsis syndrome, gram positive sepsis, gram negative sepsis, culture negative sepsis, fungal sepsis, and / or urosepsis.

[0232] In some embodiments, an inflammatory disease, disorder, or condition comprises an infectious disease. In some embodiments, an infectious disease comprises or is malaria, pneumonia, African trypanosomiasis, tuberculosis, HIV, human cytomegalovirus (HCMV), herpes virus infection, influenza (e.g., influenzavirus A, influenzavirus B, or influenzavirus C), Epstein-Barr Virus infection, Chagas disease, bacterial, trichinosis, or fungal myocarditis, meningitis, legionella, hepatitis (e.g., chronic active hepatitis), pneumonia, Clostridium difficile infection, small intestine bacterial overgrowth (SIBO), Dengue hemorrhagic fever, lyme disease, meningococcemia, necrotizing fascilitis, necrotizing enterocolitis, leprosy, streptococcal myositis, infectious colitis, mycoses (e.g., Candida albicans infection), Vancomycin-resistant enterococci (VRE) infection, pneumonia epiglottitis, peritonitis, hemolytic uremic syndromic, toxic shock syndrome, pneumocystis carinii, Campylobacter jejuni infection, Helicobacter pylori infection, viral encephalitis, septic arthritis, gas gangrene, pelvic inflammatory disease, Mycobacterium avium-intracellulare infection, orchitis, and / or virus-associated hemophagocytic syndrome (VAHS). Methods of Immune Cell Modification

[0233] The present disclosure, among other things, provides methods for modifying an immune cell (e.g., a monocyte, macrophage, or dendritic cell) comprising delivering a nucleic acid construct comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein), at least one exogenous anti-inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent (e.g., a polypeptide described herein), and / or at least one exogenous efferocytic agent (e.g. a polypeptide described herein) Delivery Methods Page 67 of 118 12583381v1Attorney Docket No.2012851-0634

[0234] A nucleic acid construct comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a polypeptide described herein),at least one exogenous anti- inflammatory agent (e.g., a polypeptide described herein), at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent (e.g., a polypeptide described herein), can be introduced into an immune cell (e.g., a monocyte, macrophage, or dendritic cell) by physical, chemical, or biological methods. In some embodiments, the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a monocyte, macrophage, or dendritic cell) ex vivo. In some embodiments, the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a monocyte, macrophage, or dendritic cell) in a subject (i.e., in vivo). For example, in some embodiments, certain provided methods of modifying immune cells (e.g., macrophages, monocytes, and / or dendritic cells) in a subject include administering / delivering compositions comprising one or more one or more nucleic acid constructs described herein and a delivery vehicle to the subject.

[0235] Physical methods for introducing a nucleic acid construct as described herein into an immune cell (e.g., a monocyte, macrophage, or dendritic cell) can comprise electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or a combination thereof. A nucleic acid construct can be introduced into immune cells using commercially available methods, including electroporation (Amaxa Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.) Gene Pulser II® (BioRad, Denver, Colo.), or Multiporator® (Eppendort, Hamburg Germany)). A nucleic acid construct can also be introduced into immune cells using mRNA transfection, e.g., cationic liposome-mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems, such as “gene guns” (See, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001), which is hereby incorporated by reference in its entirety).

[0236] Biological methods for introducing a nucleic acid construct as described herein into an immune cell (e.g., a monocyte, macrophage, or dendritic cell) include use of DNA and RNA vectors. In one embodiment, a vector comprises a plasmid vector, a viral vector, a transposon, a retrotransposon (e.g., piggyback, sleeping beauty), a site directed insertion vector (e.g., CRISPR, Page 68 of 118 12583381v1Attorney Docket No.2012851-0634 Zn finger nucleases, TALEN), suicide expression vector, or another vector known in the art. Viral vectors, and especially retroviral vectors, have become widely used for inserting genes into mammalian cells (e.g., human cells). Viral vectors can also be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses (e.g., Adf535), or adeno-associated viruses (See, e.g., U.S. Patent Nos.5,350,674 and 5,585,362, which are hereby incorporated by reference in their entirety). Retroviral vectors, such as lentivirus, are suitable tools to achieve long-term gene transfer that allow for long-term, stable integration of a transgene and its propagation in daughter cells.

[0237] Immune cells described herein (e.g., macrophages, monocytes, or dendritic cells) can be refractory to lentiviral transduction because of expression of a restriction factor, SAMHD1, which depletes nucleotide triphosphates available for reverse transcription. For example, SAMHD1 can restrict replication of human immunodeficiency virus type 1 (HIV-1) by depleting an intracellular pool of deoxynucleoside triphosphates. Viral protein X (Vpx), an accessory protein associated with simian immunodeficiency virus (SIV) and HIV-2, induces degradation of SAMHD1. In some embodiments, delivery of (a) a viral vector comprising one or more nucleic acid sequences encoding at (i) at least one exogenous fibrolytic agent described herein, and / or (ii) at least one exogenous anti-inflammatory agent described herein and (b) at least one Vpx protein can increase transfection of immune cells describes herein (e.g., macrophages, monocytes, or dendritic cells), e.g., relative to the same type of immune cell comprising (i) at least one exogenous fibrolytic agent, and / or (ii) at least one exogenous anti-inflammatory agent and not delivered at least one Vpx protein.

[0238] In some embodiments, Vpx lentivirus can lead to genomic integration of (i) at least one exogenous fibrolytic agent, (ii) at least one exogenous anti-inflammatory agent, (iii) at least one exogenous regenerative agent, and / or (iv) at least one exogenous efferocytic agent, thereby enabling long-term, permanent expression of one or more of (i)-(iv). Vpx-lentivirus transduced macrophages are not phenotypically impacted by viral transduction – a finding that enables the production of modified macrophages as described herein with phenotypic plasticity. While other viral vectors (such as Ad5f35) induce an M1, pro-inflammatory phenotype, Vpx-lentivirus does not have an impact on the M1 / M2 phenotype – enabling an M0 modified macrophage product that may not have pro-inflammatory functions / toxicities at baseline. Vpx-lentivirus transduced Page 69 of 118 12583381v1Attorney Docket No.2012851-0634 macrophages retain phenotypic plasticity and may be further polarized to an M1 or M2 phenotype with cytokines, agonists, peptides, culture media, and other factors. Vpx-lentivirus transduced macrophages can be exposed to pro-inflammatory signals (e.g., one or more pro- inflammatory cytokines, e.g., one or more of LPS, IFNa, IFNb, IFNγ, CpG, CD40L, GM-CSF, TNFa, IL-6, or a STING ligand (STING-L)) and polarize into M1 macrophages. Vpx-lentivirus transduced macrophages can be exposed to immunosuppressive signals (e.g., one or more immunosuppressive cytokines (e.g., one or more of IL-4, IL-10, IL-13, or TGFb) and / or one or both of at least one prostaglandin or at least corticosteroid) and polarize into M2 macrophages.

[0239] In some embodiments, a lentiviral vector is packaged with a Vpx protein (e.g., as described in International Publication No. WO 2017 / 044487, which is hereby incorporated by reference in its entirety). In some embodiments, Vpx comprises a virion-associated protein (e.g., an accessory protein for viral replication). In some embodiments, a Vpx protein is encoded by human immunodeficiency virus type 2 (HIV-2). In some embodiments, a Vpx protein is encoded by simian immunodeficiency virus (SIV). In some embodiments, an immune cell as described herein (e.g., a monocyte, macrophage, or dendritic cell) is transfected with a lentiviral vector packaged with a Vpx protein. In some embodiments, Vpx inhibits at least one antiviral factor of an immune cell as described herein (e.g., a monocyte, macrophage, or dendritic cell). In some embodiments, a lentiviral vector packaged with a Vpx protein exhibits increased transfection efficiency of an immune cell as described herein (e.g., a monocyte, macrophage, or dendritic cell), e.g., relative to a lentiviral vector not packaged with a Vpx protein. In some embodiments, an immune cell as described herein (e.g., a monocyte, macrophage, or dendritic cell) is one or both of electroporated or transfected with at least one VPX mRNA prior to transfection with a viral vector (e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., Ad5f35 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector)).

[0240] Chemical means for introducing a nucleic acid construct as described herein into an immune cell (e.g., a monocyte, macrophage, or dendritic cell) include colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes). Page 70 of 118 12583381v1Attorney Docket No.2012851-0634

[0241] An exemplary system for delivery of a nucleic acid construct as described herein is a lipid-based system. A nucleic acid construct as described herein may be encapsulated in an aqueous interior of a liposome, interspersed within a lipid bilayer, attached to a liposome via a linking molecule, attached to a lipid nanoparticle (LNP) via a linking molecule, entrapped in a liposome, entrapped in an LNP, complexed with a liposome, complexed with an LNP, dispersed in a solution or suspension comprising a lipid, mixed with a lipid, complexed with a micelle, or otherwise associated with a lipid. Lipids for use in methods described herein may be naturally occurring or synthetic lipids. Lipids can also be obtained from commercial sources. For example, dimyristyl phosphatidylcholine can be obtained from Sigma (St. Louis, MO); dicetyl phosphate can be obtained from K & K Laboratories (Plainview, NY); cholesterol can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20ºC. In some embodiments, a lipid-based system may comprise one or more lipids that facilitate targeting of the composition to a desired cell type or cell types (e.g., monocytes, macrophages, or dendritic cells). In some embodiments, a delivery vehicle allows a composition to be preferentially taken up (e.g. endocytosed, phagocytosed) by an immune cell (e.g., monocyte, macrophage, or dendritic cell) relative to a composition that does not comprise the delivery vehicle. Targeting Moieties

[0242] In some embodiments, a delivery vehicle may comprise one or more targeting moieties. In some embodiments, a targeting moiety may facilitate passive targeting of a composition to a desired target. In some embodiments, a targeting moiety may facilitate active targeting of a composition to a desired target.

[0243] In some embodiments, a targeting moiety comprises or is one of more of an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody) or any fragment thereof, for example an scFv, an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule, for example, to bind to one or more of CD14, CD11b, Page 71 of 118 12583381v1Attorney Docket No.2012851-0634 CD163, CD206, CD33, and / or CD209. In some embodiments, a targeting moiety may be or comprise a small molecule.

[0244] In some embodiments, a targeting moiety comprises or is a particular lipid or combination of hydrophobic entities, for example, present in or forming an exterior surface of a liposome or lipid nanoparticle (e.g., for targeting to a particular cell type or cell types). Nucleic Acid Molecules

[0245] In some embodiments of the present disclosure, one or more nucleic acid molecules are or comprise DNA. In some embodiments of the present disclosure, one or more nucleic acid molecules are or comprise messenger RNA (mRNA). In some embodiments, mRNA according to the present disclosure may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. A modified mRNA according to the present disclosure can thus include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, a step of modifying an mRNA comprises causing the mRNA to include a modified nucleotide, an alteration to the 5’ or 3’ untranslated region (UTR), a cap structure, and / or a poly(A) tail.

[0246] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent described herein) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-O-(1- thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which comprises replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

[0247] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one Page 72 of 118 12583381v1Attorney Docket No.2012851-0634 exogenous regenerative agent, and / or at least one exogenous efferocytic agent described herein) may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'- triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O- alkyloligoribonucleotide, 2'-deoxy-2'-C-alkyloligoribonucleotide (2'-O-methylcytidine 5'- triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azidotriphosphates (2'- azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0248] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent described herein) comprise modified nucleotide comprising pseudouridine (PsU), 5-methoxyuridine (5moU), 5- methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or combinations thereof.

[0249] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent described herein) may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide.

[0250] Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5’) end, and a “tail” on the C-terminal (3’) end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0251] Thus, in some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent described herein) include a 5’ cap structure. A 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two Page 73 of 118 12583381v1Attorney Docket No.2012851-0634 terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’ triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. In some embodiments, a cap comprises a Cap0 structure. A cap0 structures lack a 2'-O-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, a cap comprises an AGCap1 structure. An AGCap1 structures has a 2'-O-methyl residue at base 2. In some embodiments, a cap comprises a Cap2 structure. Cap2 structures have a 2'-O-methyl residue attached to both bases 2 and 3. In some embodiments, a cap structure comprises AGCap1, m6AGCap1, or Anti-Reverse Cap Analog (ARCA). In some embodiments, a modified mRNA of the present disclosure comprises an m6AGCap1 and modified nucleotides comprising pseudouridine (PsU).

[0252] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent described herein) include a 3’ poly(A) tail structure. A poly(A) tail on the 3' terminus of mRNA typically includes about 10 to 400 adenosine nucleotides (e.g., about 100 to 400 adenosine nucleotides, about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3’ poly(C) tail structure. A suitable poly(C) tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). A poly(C) tail may be added to a poly(A) tail or may be a substitute for the poly(A) tail.

[0253] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent described herein) include a 5’ and / or 3’ untranslated region. In some embodiments, a 5’ untranslated region includes one or more elements that affect an mRNA’s stability or translation, for example, an iron responsive element. In some embodiments, a 5’ untranslated region may be between about 50 and 500 nucleotides in length. Page 74 of 118 12583381v1Attorney Docket No.2012851-0634

[0254] In some embodiments, a 3’ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3’ untranslated region may be between 50 and 500 nucleotides in length or longer.

[0255] One of skill in the art would recognize that for disclosed amino acid sequences there are several nucleotide sequences capable of generating that amino acid sequence. While certain exemplary nucleotide sequences are disclosed, it is specifically contemplated that each nucleotide sequence giving rise to a particular amino acid sequence may be useful in certain embodiments. Administration of Additional Payloads

[0256] In some embodiments, methods of the present disclosure comprise one or more steps of treating an immune cell (e.g., a macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell. In some embodiments, methods of the present disclosure comprise one or more steps of administering to a subject an additional payload for modulating an immune cell (e.g., a macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell. In some embodiments, a composition comprises one or more additional payloads. In some embodiments, a composition comprises one or more additional payloads in the same delivery vehicle as one or more nucleic acid molecules. In some embodiments, a composition comprises one or more additional payloads in a different delivery vehicle than the one used with one or more nucleic acid molecules.

[0257] In some embodiments, methods of the present disclosure comprise a step of treating an immune cell (e.g., a macrophage, monocyte, or dendritic cell) with a modulator of a pathway activated by in vitro transcribed mRNA. In some embodiments, an additional payload may be or comprise a modulator of a pathway activated by in vitro transcribed mRNA. In vitro transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7 and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA- activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation- associated protein 5 (MDA5) and 2ʹ-5ʹ-oligoadenylate synthase (OAS)). Signaling through these different pathways results in inflammation associated with type 1 interferon (IFN), tumor Page 75 of 118 12583381v1Attorney Docket No.2012851-0634 necrosis factor (TNF), interleukin-6 (IL-6), IL-12 and the activation of cascades of transcriptional programs. Overall, these create a pro-inflammatory microenvironment poised for inducing specific immune responses. Moreover, downstream effects such as slow-down of translation by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, enhanced RNA degradation by ribonuclease L (RNaseL), and overexpression and inhibition of replication of self-amplifying mRNA are of relevance for the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0258] In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL, RNase T2 or RNase1 inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL inhibitor. In some embodiments, an RNaseL inhibitor comprises sunitinib. In some embodiments, an RNaseL inhibitor comprises ABCE1.

[0259] In some embodiments, treating an immune cell (e.g., a macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases mRNA stability in a modified immune cell relative to mRNA stability in a modified immune cell of the same type that was not treated with an RNaseL inhibitor. In some embodiments, treating an immune cell (e.g., a macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases expression of at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent in a modified immune cell relative to a modified immune cell of the same type that was not treated with an RNaseL inhibitor. In some embodiments, treating an immune cell (e.g., a macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases effector activity in a modified immune cell relative to effector activity in a modified immune cell of the same type that was not treated with an RNaseL inhibitor.

[0260] In some embodiments, administering to a subject an RNaseL inhibitor increases mRNA stability in a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) relative to mRNA stability in a modified immune cell of the same type in a subject that that was not administered an RNaseL inhibitor. In some embodiments, administering to a subject an RNaseL inhibitor increases expression of at least one exogenous fibrolytic agent, at least one exogenous Page 76 of 118 12583381v1Attorney Docket No.2012851-0634 anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent in a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) relative to a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor. In some embodiments, administering to a subject an RNaseL inhibitor increases effector activity in a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) relative to effector activity in a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor.

[0261] In some embodiments of the present disclosure, a step of treating an immune cell (e.g., a macrophage, monocyte, or dendritic cell) occurs before a step of delivering an mRNA to the immune cell. In some embodiments of the present disclosure, a step of administering an additional payload to a subject occurs before a step of administering a composition comprising an mRNA to the subject.

[0262] In some embodiments, methods of the present disclosure comprise a step of culturing an immune cell (e.g., a macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein. In some embodiments, methods of the present disclosure comprise a step of administering to a subject a cytokine or immune stimulating recombinant protein. In some embodiments, a cytokine comprises IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, a CD40 agonist, a 4-1BB ligand, recombinant 4-1BB, a CD19 agonist, a TLR agonist (e.g., TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 or TLR-9), TGF-β (e.g., TGF-β1, TGF- β2, or TGF-β3), a glucocorticoid, an immune complex, interleukin-1 alpha (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), Leukemia inhibitory factor (LIF), oncostatin M (OSM), TNF-β, CD154, lymphotoxin beta (LT-β), an A proliferation-inducing ligand (APRIL), CD70, CD153, glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L (CD252), TALL-1 (Tumor necrosis factor ligand superfamily member 13B - TNFSF13B), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), TNF-related activation-induced cytokine (TRANCE), erythropoietin (Epo), thyroid peroxidase precursor (Tpo), FMS-related tyrosine kinase 3 ligand (FLT-3L), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite Page 77 of 118 12583381v1Attorney Docket No.2012851-0634 surface protein (MSP), a Nucleotide-binding oligomerization domain-containing protein (NOD) ligand (e.g., NOD1, NOD2, or NOD1 / 2 agonists), a RIG-I-like receptor (RLR) ligand (e.g., 5'ppp-dsRNA, 3p-hpRNA, Poly(I:C), or Poly(dA:dT)), a C-type lectin receptor (CLR) ligand (e.g., curdlan, β-glucan, HKCA, laminarin, pustulan, scleroglucan, WGP dispersible, WGP soluble, zymosan, zymosan depleted, furfurman, b-GlcCer, GlcC14C18, HKMT, TDB, TDB- HS15, or TDM), a cyclic dinucleotide sensor ligand (e.g., C-Gas agonist or stimulator of interferon gene (STING) ligand), an inflammasome inducer (e.g., alum, ATP, CPPD crystals, hemozoin, MSU crystals, Nano-SiO2, Nigericin, or TDB), an aryl hydrocarbon (AhR) ligand (e.g., FICZ, indirubin, ITE, or L-kynurenine), an alpha-protein kinase 1 (ALPK1) ligand, a multi-PRR ligand, an NFKB / NFAT activator (e.g., concavalin A, ionomycin, PHA-P, or PMA) or combinations thereof. In some embodiments, a cytokine comprises IFN-β.

[0263] In some embodiments of the present disclosure, a step of culturing an immune cell (e.g., a macrophage, monocyte, or dendritic cell) occurs after a step of delivering an mRNA to the immune cell. In some embodiments of the present disclosure, a step of administering to a subject a cytokine or immune stimulating recombinant protein occurs after a step of administering a composition comprising an mRNA to the subject.

[0264] In some embodiments, culturing a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases the viability of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases protein expression in the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases longevity of protein expression relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases effector activity of the modified immune cell relative to a modified immune Page 78 of 118 12583381v1Attorney Docket No.2012851-0634 cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases pro-inflammatory (M1) polarization of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.

[0265] In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases the viability of a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases expression of at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent in a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases longevity of expression of at least one exogenous fibrolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous efferocytic agent in a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases effector activity of a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases pro- inflammatory (M1) polarization of a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. Page 79 of 118 12583381v1Attorney Docket No.2012851-0634 Assays

[0266] A variety of assays may be performed to confirm presence of a nucleic acid construct as described herein in an immune cell (e.g., a monocyte, macrophage, or dendritic cell). For example, such assays include molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots). Other assays of the present disclosure, include, for example, fluorescence- activated cell sorting (FACS), immunofluorescent microscopy, MSD cytokine analysis, mass spectrometry (MS), RNA-Seq and functional assays.

[0267] A variety of assays may be performed to determine various characteristics of a modified immune cell (e.g., a macrophage, monocyte, or dendritic cell), such as, but not limited to, immune cell viability, nucleic acid expression, nucleic acid longevity, protein expression, protein longevity, effector activity, and pro-inflammatory (M1) polarization. For example, such assays include flow cytometry, quantitative PCR, and in vitro functional assays such as cytokine / chemokine secretion, phagocytosis, efferocytosis assays, and specific lysis assays of target tumor cells.

[0268] All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0269] The disclosure is further illustrated by the following example. An example is provided for illustrative purposes only. It is not to be construed as limiting the scope or content of the disclosure in any way. Page 80 of 118 12583381v1Attorney Docket No.2012851-0634 EXAMPLES

[0270] The following examples are provided so as to describe to the skilled artisan how to make and use methods and compositions described herein, and are not intended to limit the scope of the present disclosure. Example 1: Methods for generation and validation of viable anti-fibrotic engineered macrophages

[0271] This Example describes generation and validation of anti-fibrotic engineered murine macrophages. As shown in Figure 1, primary hematopoietic stem cells (HSCs) were isolated from bone marrow of wild-type C57BL / 6 (Bl6) mice on Day 0. Cells were either untransduced or transduced with ecotropic retrovirus comprising an exemplary payload from Table 1. Table 1: Exemplified Payload Constructs PayloadPage 81 of 118 12583381v1Attorney Docket No.2012851-0634

[0272] Transduction efficiency of HSCs was enhanced by culturing on RetroNectin®-coated vessels. HSCs were cultured with 10 ng / mL M-CSF to stimulate differentiation into bone marrow-derived macrophages. Cell culture media was replaced with fresh media containing M- CSF (final concentration 10 ng / mL) on Day 2 and Day 4 of the study. On Day 5, differentiated BMDMs were collected and analyzed using an NC200 cell counter. Aliquots of cells from each culture were saved as frozen stocks in 90% FBS + 10% DMSO in cryotubes to determine tolerance to freeze-thaw.

[0273] To evaluate payload expression over time in vitro, supernatant was collected on Day 1, Day 7, Day 9, Day 13, and Day 14 of the study and analyzed using ELISA (Figure 2). Cell culture media was exchanged and collected supernatant was replaced with fresh media containing M-CSF. BMDMs were analyzed on Day 5 for cell recovery, viability, and expression of transduced payload sequence using an NC200. Transduced HSCs differentiated into healthy BMDMs on Day 5 and had comparable recovery and viability to standard untransduced macrophages (Figure 3A). Moreover, both untransduced and engineered macrophages successfully expanded from the seeded quality of HSCs (>20-fold expansion) and were highly viable (>90%).

[0274] BMDMs that were stored as frozen stocks were assessed for recovery and viability immediately post thaw (Figure 3B) and after 48 hr incubation (Figure 3C). All BMDM conditions had high recovery and viability immediately following thaw and maintained viability for 48 hr in vitro.

[0275] Together, these results suggest engineered anti-fibrotic engineered macrophages are highly viable for use for in vivo and in vitro study as well as that frozen stocks are viable immediately post-thaw (akin to what would be administered in in vivo studies) and can maintain viability for at least 48 hr in vitro. Example 2: Engineered macrophages can secrete diverse therapeutic proteins and can express therapeutic transmembrane proteins Page 82 of 118 12583381v1Attorney Docket No.2012851-0634

[0276] This Example describes the characterization of engineered anti-fibrotic engineered macrophages to secrete therapeutic payloads using ELISA assays. This Example also describes the characterization of engineered macrophages to express therapeutic transmembrane proteins.

[0277] HSCs were produced and analyzed as described in Example 1. HSCs were either untransduced or transduced with retrovirus encoding an exemplary payload from Table 1 and analyzed for production of immunosuppressive cytokine IL-10. High levels of IL-10 were produced by engineered macrophages expressing an IL-10-P2A-MMP13 payload, IL-10-P2A- Relaxin payload, or IL-10-P2A-FGF21 payload, compared to little or no IL-10 production in untransduced cells (Figure 4A).

[0278] High levels of FGF21 were produced by engineered macrophages expressing IL-10-P2A- FGF21, whereas no expression was detected from untransduced cells or from cells expressing IL-10-P2A-MMP13 or IL-10-P2A-Relaxin (Figure 4B). These results demonstrated the ability to detect two payloads from the same construct (IL-10 and FGF21 from IL-10-P2A-FGF21).

[0279] High levels of Relaxin were produced by engineered macrophages expressing a Relaxin construct (Figure 4C), whereas no expression was detected from untransduced cells.

[0280] High levels of Decorin were produced by engineered macrophages expressing an IL-10- P2A-Decorin construct, whereas no Decorin expression was detected from untransduced cells or cells expressing an anti-HER2 construct (Figure 4D).

[0281] Together, these results demonstrate that secreted payloads can be produced at high concentrations. These results also suggest that multiple proteins can be co-expressed by a single cell product.

[0282] Engineered macrophages were also assessed for viability when expressing transmembrane receptors proteins, DN TGFβR2 and IL17AR. Cells expressing DN TGFβR2 (Figure 5A) and IL17AR (Figure 5B) were highly viable (Figure 5C), and flow cytometry analysis demonstrated high surface expression of each receptor compared to untransduced cells. (Figure 5D-5E).

[0283] As shown in Figure 6A, cells transduced with IL-10-P2A-GFP were analyzed with ELISA and results demonstrated that high levels of IL-10 were detected in Day 7 supernatant of engineered macrophages, unlike the supernatant of untransduced cells. Moreover, ELISA assay Page 83 of 118 12583381v1Attorney Docket No.2012851-0634 results showed that engineered macrophages sustained IL-10 production for at least 10 weeks in vitro (Figure 6B).

[0284] Together, these results demonstrate that secreted payloads can be produced at high concentrations. These results also suggest that multiple proteins can be co-expressed by a single cell product. Example 3: Engineered macrophages generated for in vivo use are viable and express encoded payloads in vivo

[0285] This Example describes methods of evaluating payload expression in vivo. This Example also describes viability characterization and payload expression of engineered macrophages in vivo.

[0286] As shown in Figures 7A-7B, BMDMs were generated and frozen as described in Example 1. HSCs were transduced with retrovirus encoding a Luciferase-P2A-GFP construct or an IL-10-P2A-Luciferase construct. Transduced HSCs were differentiated into healthy BMDMs on Day 5. Recovery and viability on Day 5 were analyzed using an NC200. BMDMs expressing Luciferase-P2A-GFP or IL-10-P2A-Luciferase successfully expanded from the seeded quantity of HSCs (12 to 20-fold expansion) and were highly viable (Figure 8A), as were BMDMs that were stored as frozen stocks and assessed for viability immediately post thaw (Figure 8B).

[0287] To monitor trafficking and payload expression in vivo, frozen BMDMs were thawed and evaluated for cell count and viability. A total of 5 x 106cells were administered via intravenous (“i.v.”) injection to Balb / c mice (Figure 7B).

[0288] On Day 1, Day 7, Day 10, and Day 13 after BMDM administration, IVIS®imaging was performed to monitor luciferase expression (Figure 7B). Engineered macrophages produced detectable luciferase signal in mice for at least 13 Days following administration (Figure 9A- 9B). Without wishing to be bound by theory, this data suggests IL-10 may improve persistence of engineered macrophages. These results confirm that BMDMs can generate functional signals in vivo after being transduced as HSCs with retrovirus, frozen as BMDMs on Day 5, and then administered by intravenous injection after freeze-thaw. Page 84 of 118 12583381v1Attorney Docket No.2012851-0634 Example 4: Methods for evaluating anti-fibrotic efficacy of engineered macrophages in liver fibrosis mouse model

[0289] This Example describes the characterization of engineered anti-fibrotic macrophages for use in a carbon tetrachloride (CCl4)-induced model of liver fibrosis. Anti-fibrotic engineered murine macrophages were generated as described in Example 1. HSCs were either untransduced or transduced with retrovirus encoding an exemplary construct listed in Table 1.

[0290] As shown in Figure 10, for 28 Days prior to BMDM injection, male Bl6 mice received an intraperitoneal injection of freshly prepared CCl4diluted 1:3 in corn oil (1 mL / kg) (n= 9 per group). On Day 28 after initiation of CCl4challenge, mice were administered BMDMs (5 x 106cells) via intravenous injection. On Day 42, mice were sacrificed, and livers were collected for histological analysis.

[0291] To first validate that transduced BMDMs were expressing payloads, ELISA and western blot analysis was used to confirm payload expression. High levels of IL-10 production was detected by ELISA assay in cells expressing IL-10-P2A-Relaxin (Figure 11A). Supernatant from cell culture was further analyzed using non-denaturing western blot detection. As shown in Figure 11B, production of Relaxin was specific to engineered macrophages expressing IL-10- P2A-Relaxin, whereas no expression was detected from untransduced cells or from BMDMs expressing other anti-fibrotic payloads.

[0292] To determine whether engineered macrophages were well-tolerated by mice challenged with CCl4, mouse bodyweight weight was measured twice a week throughout the 42-day study. Compared to the control group, mice challenged with CCl4exhibited reduced and fluctuating bodyweight, indicative of successful CCl4administration (Figure 12A). In addition, CCl4- challenged mice that received vehicle, UTD, or IL-10-P2A-Relaxin treatment displayed similar bodyweights on Day 43, suggesting that engineered macrophages were well-tolerated (Figure 12B).

[0293] Fibrotic liver tissue collected from mice was also analyzed using histological analysis of collagen content to determine whether administration of engineered macrophages expressing therapeutic payloads affected CCl4-induced fibrosis. To conduct histological analysis, middle Page 85 of 118 12583381v1Attorney Docket No.2012851-0634 sections of left later lobes of livers were formalin-fixed and paraffin-embedded, then sectioned. Masson’s Trichrome Stain was used identify total collagen content. Collagen content was quantified using HALO analysis software.

[0294] Collagen content in tissue sections from control mice (vehicle treatment), showed significant increase in collagen staining, indicating successful fibrosis induction (Figures 13A- 13B). While untransduced macrophages induced a 55% reduction in collagen staining, engineering macrophages expressing IL-10-P2A-Relaxin induced greater than 100% reduction in collagen staining. These results suggest a single dose of macrophages producing Relaxin and IL- 10 significantly reduce CCl4induced liver fibrosis. Example 5: Evaluating anti-fibrotic efficacy of engineered macrophages in a bleomycin- induced model of pulmonary fibrosis

[0295] This Example describes the characterization of treating a bleomycin-induced pulmonary fibrosis mouse model with engineered macrophages.

[0296] Anti-fibrotic engineered murine macrophages were generated as described in Example 1. HSCs were either untransduced or transduced with retrovirus encoding an IL-10-P2A-Relaxin or IL-10-P2A-DN-TGFβR2 construct, as listed in Table 1.

[0297] As shown in Figure 14, to generate a murine pulmonary fibrosis model, Taconic Bl6 mice (6-7 weeks old), received 1 U / kg of bleomycin (“BLM”) intranasally (n=10-15 per group). The following day on Day 1, mice were treated with engineered macrophages (5 x 106cells per mouse) or vehicle control by intravenous administration. On Day 21, mice were sacrificed to assess hydroproxyproline content in lungs.

[0298] BMDMs expressing IL-10-P2A-Relaxin or IL-10-P2A-DN-TGF βR2 successfully expanded from the seeded quantity of HSCs (12 to 20-fold expansion) and were highly viable (Figure 15A). To validate that transduced BMDMs were expressing payloads, ELISA and flow cytometry analysis was used to confirm payload expression. High levels of IL-10 production was detected by ELISA assay in cells expressing IL-10-P2A-Relaxin and IL-10-P2A-TGFβR2 DN (Figure 15B) as compared to untransduced cells. As shown in Figure 15C, flow cytometry Page 86 of 118 12583381v1Attorney Docket No.2012851-0634 analysis detected dominant negative TGFβR2 at surface of cells transduced with IL-10-P2A- TGFβR2 DN, confirming proper payload expression.

[0299] To determine whether engineered macrophages were well-tolerated by mice challenged with CCl4, mouse bodyweight weight was measured twice a week throughout the 42-day study. Compared to the control (herein “Sham”) group, mice challenged with BLM exhibited an expected 10-15% reduction in bodyweight followed by a rebound, indicative of successful BLM challenge (Figure 16A). In addition, CCl4-challenged mice that received vehicle, UTD, or IL- 10-P2A-Relaxin or IL-10-P2A-TGFβR2 DN treatment displayed similar bodyweights on Day 21, suggesting that engineered macrophages were well-tolerated (Figure 16B).

[0300] Fibrotic tissue was quantified by measuring hydroxyproline (herein “HYP”) concentration from homogenized left lung tissue samples. Compared to the Sham group, BLM challenge (untreated) mice induced a significant increase in HYP, indicating successful fibrosis induction. While mice treated with untransduced BMDMs exhibited a decrease in median HYP values, no statistically significant change was observed (Figure 17A). However, engineered macrophages expressing IL-10-P2A-TGFβR2 DN significantly induced greater than 90% reduction in HYP, as shown in Figure 17A. In addition, engineered macrophages expressing IL- 10-P2A-Relaxin induced a 44% reduction in average HYP, which was statistically different compared to untransduced cells (Figure 17B). Together, these results indicate a single dose of engineered macrophages comprising IL-10-P2A-TGFβR2 or IL-10-P2A-Relaxin reduce bleomycin-induced pulmonary fibrosis. Example 6: Assessment of anti-fibrotic engineered macrophages overexpressing TIM4 or TIM4 + Relaxin

[0301] This Example describes, inter alia, the generation of engineered macrophages that overexpress TIM4 or TIM4 and Relaxin as well as the effect of such engineered macrophages on several measures of fibrosis in a murine model as well as effect on overall efferocytic capacity.

[0302] Primary human macrophages were thawed and either not transduced or transduced with VPX-lentiviral particles encoding full-length human TIM4. After 7 Days post-transduction, engineered and untransduced macrophages were collected and analyzed for production of TIM4 Page 87 of 118 12583381v1Attorney Docket No.2012851-0634 via flow cytometry (Figure 18). High levels of TIM4 were produced by macrophages engineered to express TIM4 compared to the untransduced (UTD) control (Figure 19).

[0303] Next, efferocytic capacity of macrophages engineered to overexpress TIM4 was tested using an efferocytosis assay (Figure 19). First, primary human macrophages were thawed and either left UTD or transduced with VPX-lentiviral particles encoding full-length human TIM4 or a lentivirus control (miRFP670). Jurkat cells were labeled with pHrodo, a pH-sensitive dye whose signal increases in acidic environments such as during efferocytosis. Apoptosis was induced in Jurkat cells using staurosporine. Macrophages were mixed with the apoptotic, pHrodo-labeled Jurkat cells and were monitored using live cell imaging. Successful engulfment of the apoptotic target Jurkat cells induces an increase in pHrodo fluorescent signal.

[0304] UTD macrophages did not strongly efferocytose. Transduction with the lentivirus control did not improve efferocytosis. However, macrophages engineered to overexpress TIM4 resulted in enhanced efferocytosis when compared to both UTD and lentivirus control transduced macrophages (Figure 21).

[0305] Together, these results demonstrate that secreted TIM4 can be produced at high concentrations by engineered macrophages described herein. These results also show that macrophages engineered to express TIM4 had enhanced efferocytic capacity.

[0306] Next, as shown in Figure 22, engineered anti-fibrotic murine macrophages were generated. First primary HSCs were isolated from bone marrow of BI6 mice and then transduced with an ecotropic retrovirus encoding TIM4 or TIM4 + Relaxin (TIM4-P2A-Relaxin). Transduction efficiency of HSCs was enhanced by culturing on RectroNectin-coated vessels. HSCs were cultured with fresh media containing 10 ng / mL M-CSF to simulate differentiation into engineered bone marrow-derived macrophages (BMDM). The cell culture was supplemented at Days 2 and 4 with fresh M-CSF. At Day 5, differentiated engineered BMDM were collected and saved in frozen stocks containing 90% FBS with 10% DMSO.

[0307] Engineered macrophages were collected and analyzed at Day 5 using flow cytometry to check for viability and expression of CD11b with F4 / 80 (Figure 23A) and TIM4 (Figure 23B). The majority of macrophages engineered to express either TIM4 or TIM4 + Relaxin were positive for CD11b and F4 / 80 expression (Figure 23A). Both sets of engineered macrophages Page 88 of 118 12583381v1Attorney Docket No.2012851-0634 also demonstrated expression of TIM4. Macrophages engineered to express TIM4 + Relaxin had notably higher expression than macrophages engineered to express TIM4 alone.

[0308] Engineered macrophages were also collected and analyzed at day 7 using flow cytometry to evaluate expression of TIM4 (Figure 23C). Expression of TIM4 by the engineered macrophages increased from Day 5 to Day 7.

[0309] Next, macrophages engineered to express TMI4 or TIM4 + Relaxin were evaluated for production of Relaxin. Macrophages were produced and engineered using methods described herein and were cultured out to 14 Days. On Days 5, 7, and 14, supernatant of engineered macrophages was collected and measured for Relaxin via ELISA. Fresh media was replaced in cell culture after collection. On day 14, high levels of Relaxin were produced by engineered macrophages expressing TIM4 + Relaxin, while engineered macrophages expressing TIM4 construct had no detectable levels of Relaxin (Figure 24A).

[0310] Time course analysis of supernatant collected at Days 5, 7, and 14 from engineered macrophages expressing TIM4 + Relaxin was also performed to assess the rate of Relaxin production. The rate of Relaxin production was calculated as the concentration of Relaxin measured divided by the time elapsed since the previous media change. While the rate of Relaxin production decreased between Days 3 and 6, it ultimately stabilized from Day 6 onward (Figure 24B).

[0311] Together, these results demonstrate that secreted TIM4 was produced by macrophages engineered to express TIM4 and at even higher levels by macrophages engineered to express TIM4 + Relaxin. Engineered macrophages expressing TIM4 + Relaxin secreted high levels of Relaxin and at a stabilized rate up to at least 14 Days. Example 7: Methods for evaluating anti-fibrotic efficacy of engineered macrophages overexpressing TIM4 or TIM4 + Relaxin in a liver fibrosis mouse model

[0312] This Example describes the characterization of engineered anti-fibrotic macrophages for use in a choline-deficient, L-amino acid defined, high fat diet (CDAHFD) model of liver fibrosis. Anti-fibrotic engineered murine macrophages were generated as described in Example 6. Bone marrow-derived macrophages (BMDM) were either transduced with retrovirus encoding TIM4 Page 89 of 118 12583381v1Attorney Docket No.2012851-0634 or TIM4 + Relaxin, or were untransduced (UTD) as a control (referred to herein as “Control Mɸ”).

[0313] As shown in Figure 25, for 28 Days prior to BMDM injection, male Bl6 mice were either fed normal chow and remained untreated (referred to herein as “Healthy Liver”) or a fibrosis- inducing choline-deficient, L-amino acid defined, high fat diet (CDAHFD) and either remain untreated (referred to herein as “Untreated”) or were treated with the aforementioned engineered macrophages. CDAHFD (also known as “HF-CDAA”) model was followed as described in, for example, Shi, H. et al., “Loss of TIM4-Dependent Efferocytosis in Kupffer Cells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis,” bioRxiv 2024.01.30.578023 (2024), which is incorporated herein by reference in its entirety. On Day 28, after initiation of diet, mice were administered engineered macrophages (5 x 106cells) or vehicle control via intravenous injection. On Day 42, mice were sacrificed, and livers were collected for histological analysis.

[0314] Immunoblots of TIM4 expression in mouse livers were performed to confirm CDAHFD- induced liver fibrosis reduced intrahepatic levels of TIM4. Whereas healthy mice (treated with PBS) expressed high levels of TIM4, mice fed CDAHFD exhibited reduced levels of TIM4 protein, despite treatment with control macrophages (“Ctr Mɸ”). Treatment with engineered TIM4 macrophages rescued TIM4 expression in CDAHFD-fed mice (Figures 26A-26B). These results confirmed that engineered macrophages overexpressing TIM4 help restore TIM4 expression, as intended.

[0315] Next, in situ analysis of efferocytic capacity of engineered macrophages was performed by quantifying histology sections as described in, for example, Ampomah et al., “Macrophages use apoptotic cell-derived methionine and DNMT3A during efferocytosis to promote tissue resolution,” Nat Metab 4, 444-457 (2022), which is incorporated herein by reference in its entirety. Macrophages were identified using the pan-macrophage marker Mac2. Apoptotic cells were identified using a cleaved caspase3 (cl-Casp3) marker. Apoptotic cells that spatially associated with macrophages were considered to be undergoing efferocytosis. The number of apoptotic cells that were associated with macrophages was normalized to the number of apoptotic cells that were free-standing away from macrophages. Larger ratios indicated an increasing amount of efferocytosis. As shown in Figure 27, treatment with either TIM4 or TIM4+Relaxin macrophages increased in situ efferocytosis. Page 90 of 118 12583381v1Attorney Docket No.2012851-0634

[0316] Next, fibrotic liver tissue collected from mice was analyzed using histological analysis of collagen content to determine whether administration of engineered macrophages expressing TIM4 or TIM4 + Relaxin affected CDAHFD-induced fibrosis. To conduct histological analysis, middle sections of left later lobes of livers were formalin-fixed and paraffin-embedded, then sectioned. Picosirius red (also referred to herein as “Sirius Red”) staining (Figure 28A-28B) and Collagen type 1 alpha 1 (Col1a1) staining (Figures 29A-29B) were used to identify total collagen content. Collagen content was quantified using HALO analysis software.

[0317] Collagen content as measured by Sirius Red in tissue sections from CDAHFD mice treated with vehicle (untreated) showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 28A-28B). Mice treated with UTD macrophages (“Control Mɸ”) did not achieve a statistically significant reduction in Sirius Red staining with approximately a 16% reduction in Sirius Red area. Engineered macrophages expressing TIM4 alone induced a significant 49% reduction in collagen staining. In comparison, engineered macrophages co-expressing both TIM4 and Relaxin induced a significant 70% reduction.

[0318] Collagen content as measured by Col1A1 in tissue sections from untreated CDAHFD mice showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 29A-29B). Mice treated with UTD macrophages (“Control Mɸ”) did not achieve a statistically significant reduction in Col1A1 staining with approximately a 5% reduction in Col1A1 area. Engineered macrophages expressing TIM4 alone induced a significant 30% reduction. In comparison, engineered macrophages co-expressing both TIM4 and Relaxin induced a significant 36% reduction.

[0319] Further histological analysis was conducted on fibrotic liver tissue collected from mice to assess the cytoskeletal and intracellular components, as well as to determine whether administration of engineered macrophages expressing TIM4 or TIM4 + Relaxin affected CDAHFD-induced activation of fibroblasts and inflammation. Middle sections of left later lobes of livers were formalin-fixed and paraffin-embedded, then sectioned. Alpha-smooth muscle acting (“⍺-SMA”) staining (Figures 30A-30B) and Osteopontin (“Opn”) staining (Figures 31A- 31B) were used as a pro-inflammatory and pro-fibrotic biomarker, associated with liver fibrosis. OPN content was quantified using HALO analysis software. Page 91 of 118 12583381v1Attorney Docket No.2012851-0634

[0320] ⍺-SMA content in tissue sections from untreated CDAHFD mice showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 30A-30B). Mice treated with UTD macrophages (Control Mɸ) did not achieve a statistically significant reduction in ⍺-SMA staining with approximately a 18% reduction in ⍺-SMA. Engineered macrophages expressing TIM4 alone induced a significant 48% reduction. In comparison, engineered macrophages co-expressing both TIM4 and Relaxin induced a significant 56% reduction.

[0321] Osteopontin content in tissue sections from untreated CDAHFD mice showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 31A-31B). Mice treated with UTD macrophages (Control Mɸ) did not achieve a statistically significant reduction in Opn staining with approximately a 17% reduction in Opn. Engineered macrophages expressing TIM4 alone induced a significant 43% reduction. In comparison, engineered macrophages co-expressing both TIM4 and Relaxin induced a significant 64% reduction.

[0322] The results disclosed herein demonstrate that engineered macrophages expressing TIM4 or both TIM4 and Relaxin achieved a significant reduction in fibrotic area. Even at low levels of 15-20% transduction, TIM4 improved the anti-fibrotic effect of engineered macrophages relative to UTD macrophages. These results also support increasing macrophage efferocytosis capacity as an anti-fibrotic strategy (e.g., via other efferocytosis receptors, synthetic efferocytosis receptors, and / or other signaling molecules that upregulate endogenous efferocytosis machinery). Unlike proteins such as Relaxin, TIM4 has no counterpart that could be systemically administered as a standalone protein therapy; therefore, efficacy of TIM4 necessitates a cell- based vehicle to leverage its functions. These results also demonstrate that engineered macrophages can indeed leverage co-expression of TIM4 and Relaxin for additive anti-fibrotic effects. Example 8: Assessment of anti-fibrotic engineered human macrophages overexpressing TIM4 or TIM4 + Relaxin in a liver fibrosis mouse model

[0323] This Example describes the characterization of anti-fibrotic engineered human macrophages for use in a choline-deficient, L-amino acid defined, high fat diet (CDAHFD) Page 92 of 118 12583381v1Attorney Docket No.2012851-0634 model of liver fibrosis applied to NSG™ mice, to enable treatment with human TIM4 or human TIM4 + human Relaxin without rejection by the mouse innate immune system.

[0324] On Day 0, primary human monocytes were thawed and either left untransduced (UTD; Control Mɸ) or transduced with lentivirus encoding human TIM4 (hu. TIM4 Mɸ). Cells were differentiated for five Days in the presence of GM-CSF. On Day 5, cells were collected and analyzed for viability and TIM4 expression and formulated for in vivo injection (Figure 32).

[0325] TIM4 expression was analyzed by flow cytometry. TIM4 expression was detected on the surface of engineered cells five Days after transduction (Figure 33A). Cell viability was also measured by an NC200 cell counter, and both groups (Control Mɸ and hu. TIM4 Mɸ) had acceptable viability (e.g., >80%) prior to formulation for in vivo injection (Figure 33B).

[0326] The CDAHFD model described in Example 7 was applied to immunodeficient NSG™ mice. Nine-week-old female NSG™ mice were fed chow or a fibrosis-inducing CDAHFD. On Day 28, after initiation of diet, mice were administered engineered human macrophages (5 x 106cells) or vehicle control via intravenous injection. On Day 42, mice were sacrificed, and livers were collected for histological analysis (Figure 34).

[0327] Next, fibrotic liver tissue collected from mice was analyzed using histological analysis of collagen content to determine whether administration of engineered human macrophages expressing TIM4 affected CDAHFD-induced fibrosis. To conduct histological analysis, middle sections of left later lobes of livers were formalin-fixed and paraffin-embedded, then sectioned. Masson’s Trichome staining (Figures 35A-35B), Picosirius red (also referred to herein as “Sirius Red”) staining (Figure 36A-36B) and Collagen type 1 alpha 1 (Col1a1) staining (Figures 37A- 37B) were used to identify total collagen content. Collagen content was quantified using HALO analysis software.

[0328] Collagen content was measured by Masson’s Trichome staining in tissue sections from NSG™ CDAHFD mice treated with vehicle (untreated) showed significant increase in collagen staining, indicating successful fibrosis induction (Figures 35A-35B). While untransduced macrophages induced a 29% reduction in collagen staining, engineered human macrophages expressing TIM4 induced 58% reduction in collagen staining. Page 93 of 118 12583381v1Attorney Docket No.2012851-0634

[0329] Collagen content as measured by Sirius Red in tissue sections from NSG™ CDAHFD mice treated with vehicle (untreated) showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 36A-36B). NSG™ mice treated with UTD macrophages (“Control Mɸ”) did not reduce Sirius Red staining. Engineered human macrophages expressing TIM4 induced a significant 47% reduction in collagen staining.

[0330] Collagen content as measured by Col1A1 in tissue sections from untreated NSG™ CDAHFD mice showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 37A-37B). NSG™ mice treated with UTD macrophages (“Control Mɸ”) did not achieve a statistically significant reduction in Col1A1 staining with approximately a 12% reduction in Col1A1 stained area. In comparison, engineered human macrophages expressing TIM4 alone induced a significant 44% reduction.

[0331] Further histological analysis was conducted on fibrotic liver tissue collected from mice to assess the cytoskeletal and intracellular components, as well as to determine whether administration of engineered human macrophages expressing TIM4 affected CDAHFD-induced activation of fibroblasts and inflammation. Middle sections of left later lobes of livers were formalin-fixed and paraffin-embedded, then sectioned. Alpha-smooth muscle acting (“⍺-SMA”) staining (Figures 38A-38B) and Osteopontin (“Opn”) staining (Figures 39A-39B) were used as a pro-inflammatory and pro-fibrotic biomarker, associated with liver fibrosis. OPN content was quantified using HALO analysis software.

[0332] ⍺-SMA content in tissue sections from untreated NSG™ CDAHFD mice showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 38A-38B). NSG™ mice treated with UTD macrophages (Control Mɸ) did not achieve a statistically significant reduction in ⍺-SMA staining with approximately a 5% reduction in ⍺-SMA. In comparison, engineered human macrophages expressing TIM4 induced a significant 46% reduction.

[0333] Osteopontin content in tissue sections from untreated NSG™ CDAHFD mice showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 39A-39B). NSG™ mice treated with UTD macrophages (Control Mɸ) did not reduce Opn staining. Engineered human macrophages expressing TIM4 induced a significant 41% reduction. Page 94 of 118 12583381v1Attorney Docket No.2012851-0634

[0334] Finally, in situ analysis of efferocytic capacity of engineered macrophages was performed as described in Example 7. As shown in Figure 40, treatment with engineered human TIM4 macrophages increased in situ efferocytosis.

[0335] Together, these results disclosed herein demonstrate that engineered human macrophages expressing TIM4 achieved a significant reduction in fibrotic area. TIM4 improved the anti- fibrotic effect of engineered human macrophages relative to UTD macrophages. These results also support increasing macrophage efferocytosis capacity as an anti-fibrotic strategy (e.g., via other efferocytosis receptors, synthetic efferocytosis receptors, and / or other signaling molecules that upregulate endogenous efferocytosis machinery). Example 9: Methods for evaluating anti-fibrotic efficacy of engineered macrophages overexpressing TIM4 or TIM4 + Relaxin in an advanced liver fibrosis mouse model

[0336] This Example describes the characterization of engineered anti-fibrotic macrophages for use in a choline-deficient, L-amino acid defined, high fat diet (CDAHFD) model of advanced liver fibrosis, where the CDAHFD model was followed for an extended duration of disease induction.

[0337] Anti-fibrotic engineered murine macrophages were generated as described in Example 6. Engineered macrophages were either left untransduced (UTD; Control Mɸ), transduced to express murine TIM4 (TIM4 Mɸ; Timd4), or transduced to co-express murine TIM4 and Relaxin. Cryopreserved D5 BMDM were evaluated for efficacy in a 16-week model of advanced liver fibrosis induced by a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD).

[0338] For 28 Days, C57BL6 / J mice were either fed normal chow and remained untreated (referred to herein as “Healthy Liver”) or a fibrosis-inducing choline-deficient, L-amino acid defined, high fat diet (CDAHFD) and either remain untreated (referred to herein as “Untreated”) or were treated with the aforementioned engineered macrophages. On Day 28, mice began receiving biweekly engineered macrophages (5 x 106cells) or vehicle control via intravenous injection (Figure 41).

[0339] After 16 weeks, mice were sacrificed, and livers were collected for histological analysis. As shown in Figures 42A-42C, engineered macrophages were well-tolerated by mice. Relative Page 95 of 118 12583381v1Attorney Docket No.2012851-0634 to healthy mice, CDAHFD induced a reduction in body weight (Figure 42A), increase in liver weight (Figure 42B), and increase in liver-to-body weight ratio (Figure 42C), as expected for this advanced liver fibrosis model. Treatment with macrophages did not exacerbate these changes, suggesting treatment was well-tolerated. As shown in Figure 42D, blood glucose levels were measured after a 4-hour fast, and CDAHFD-fed mice exhibited reduced fasting blood glucose levels compared to healthy controls. In contrast, blood glucose levels in macrophage- treated mice were closer to healthy controls compared to untreated, CDAHFD mice, indicating a restorative effect.

[0340] Next, fibrotic liver tissue collected from mice was analyzed using histological analysis of collagen content to determine whether administration of engineered macrophages expressing TIM4 or TIM4 + Relaxin affected CDAHFD-induced advanced fibrosis. To conduct histological analysis, middle sections of left later lobes of livers were formalin-fixed and paraffin-embedded, then sectioned. Picosirius red (also referred to herein as “Sirius Red”) staining was used to identify total collagen content. Collagen content was quantified using HALO analysis software.

[0341] Collagen content as measured by Sirius Red in tissue sections from CDAHFD mice treated with vehicle (untreated) showed a significant increase in staining, indicating successful activation of fibroblasts by the fibrotic model (Figures 43A-43B). Mice treated with UTD macrophages (“Control Mɸ”) did not significantly reduce Sirius Red staining. Engineered macrophages expressing TIM4 alone induced a significant 34% reduction in collagen staining. In comparison, engineered macrophages co-expressing both TIM4 + Relaxin induced a significant 45% reduction.

[0342] Together, these results disclosed herein demonstrate that engineered macrophages expressing TIM4 or both TIM4 + Relaxin achieved a significant reduction in fibrotic area in an advanced liver fibrosis model. These results also demonstrate that engineered macrophages can indeed leverage co-expression of TIM4 + Relaxin for additive anti-fibrotic effects in an advanced fibrosis model. Example 10: Assessment of LNP-TIM4 in a liver fibrosis mouse model Page 96 of 118 12583381v1Attorney Docket No.2012851-0634

[0343] This Example describes, inter alia, characterization of lipid nanoparticle (“LNP”) delivery of mRNA encoding TIM4 as well as the effect of such on several measures of fibrosis in a murine model as well as effect on overall efferocytic capacity.

[0344] Briefly, MC3-lipid nanoparticles were used to encapsulate mRNA encoding murine TIM4 (LNP-TIM4). For 28 Days, C57BL6 / J mice were either fed normal chow and remained untreated (referred to herein as “Healthy Liver”) or a fibrosis-inducing choline-deficient, L- amino acid defined, high fat diet (CDAHFD), as described in Example 7, and either remained untreated (referred to herein as “Untreated”) or were treated with the aforementioned LNP- TIM4. On Day 28, mice were treated intravenously with LNP-TIM4 or vehicle control. Mice received 3 single doses, spaced 3-4 Days apart (Figure 44).4 Days following the final LNP dose, mice were sacrificed for LNP-TIM4 tolerance and efficacy analysis.

[0345] As shown in Figure 45, mouse body weight was measured throughout the study. Mice on CDAHFD that received LNP-TIM4 treatment displayed similar bodyweight to those receiving vehicle, indicating that LNP-TIM4 was well-tolerated.

[0346] Next, systemic biomarkers of liver toxicity were measured to further characterize safety and tolerance of LNP-TIM4. In mice fed CDAHFD, LNP-TIM4 did not significantly alter serum toxicity markers ALP (Figure 46A), AST (Figure 46B), ALT (Figure 46C), bile acids (Figure 46D), triglycerides (Figure 46E), or creatine (Figure 46F). Together, these results suggested that LNP-TIM4 was well-tolerated.

[0347] Next, fibrotic liver tissue collected from mice was analyzed using histological analysis of collagen content to determine whether administration of LNP-TIM4 affected CDAHFD-induced fibrosis. Histological analysis was performed as described in Example 7. Masson’s Trichome staining was used to identify total collagen content. As shown in Figure 47A, treatment with LNP-TIM4 lead to a numerical, but not statistically significant, decrease in collagen.

[0348] Further histological analysis was conducted on fibrotic liver tissue collected, and alpha- smooth muscle acting (“⍺-SMA”) staining (Figure 47B) and Osteopontin (“Opn”) staining (Figure 47C) were used as a pro-inflammatory and pro-fibrotic biomarker, associated with liver fibrosis, as described in Example 7. As shown in Figure 47C, treatment with LNP-TIM4 led to a numerical, but not statistically significant, reduction in ⍺-SMA staining. In contrast, LNP- TIM4 treatment significantly reduced Opn. Page 97 of 118 12583381v1Attorney Docket No.2012851-0634

[0349] Together, these results demonstrate that LNP-TIM4 appear safe and well-tolerated by mice and indicates a potential for anti-fibrotic efficacy. Dosing regimens and LNP formulations will be optimized to improve anti-fibrotic efficacy in future experiments. Page 98 of 118 12583381v1Attorney Docket No.2012851-0634 Example 11: Sequence Tables

[0350] The following tables provide exemplary amino acid sequences as described herein. Table 2: Exemplary amino acid sequences Payload Amino Acid Sequence (SEQ ID NO) A Q K M V A V A Q K G I S A Q K T R L N Ng 12583381v1Attorney Docket No.2012851-0634 Payload Amino Acid Sequence (SEQ ID NO) A Q K L I V P I K A Q K L Y E A P MPage 100 of 118 12583381v1Attorney Docket No.2012851-0634 Table 3: Exemplary amino acid sequences Payload Amino Acid Sequence (SEQ ID NO) Q T S E K R T S Q I Q T D K Q T T R L N APage 101 of 118 12583381v1Attorney Docket No.2012851-0634 Payload Amino Acid Sequence (SEQ ID NO) Q T L N H Y D N Q T F L G L E L A LTable 4: Exemplary amino acid sequences Page 102 of 118 12583381v1Attorney Docket No.2012851-0634 Payload Amino Acid Sequence (SEQ ID NO) E E D N E E S V P K G A G P H G T S A12583381v1Attorney Docket No.2012851-0634 Payload Amino Acid Sequence (SEQ ID NO) G A T Q L E L GPage 104 of 118 12583381v1Attorney Docket No.2012851-0634 Table 5: Exemplary amino acid sequences Payload Amino Acid Sequence (SEQ ID NO) A Q L N L E R E C P P P VPage 105 of 118 12583381v1Attorney Docket No.2012851-0634 Payload Amino Acid Sequence (SEQ ID NO) LMVLLFLAFLLRGKVTGANCLQRHKRPDNTEDSDSVLNDMSHGRDD G V R D F CTable 6: Exemplified amino acid sequences Payload Amino Acid Sequence (SEQ ID NO) I G S I D Q L S E L12583381v1Attorney Docket No.2012851-0634 Payload Amino Acid Sequence (SEQ ID NO) NKVLEVQSRTNFIKRIIILDTVENIHGCESLPNFISRYSDGNIANFKPLHF V G Y P D L F L G E V F E F S EPage 107 of 118 12583381v1Attorney Docket No.2012851-0634 EQUIVALENTS

[0351] It is to be appreciated by those skilled in the art that various alterations, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of the present disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawing are by way of example only and any invention described in the present disclosure if further described in detail by the claims that follow.

[0352] Those skilled in the art will appreciate typical standards of deviation or error attributable to values obtained in assays or other processes as described herein. The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference in their entireties. Page 108 of 118 12583381v1

Claims

Attorney Docket No.2012851-0634 CLAIMS 1. A modified immune cell comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) at least one exogenous regenerative agent.

2. The modified immune cell of claim 1, wherein the at least one exogenous fibrolytic agent comprises a Relaxin polypeptide, Decorin polypeptide, dominant negative TGF-beta Receptor 2 (DN TGFβR2) polypeptide, soluble TGFβ receptor, and / or TGFβ switch receptor.

3. The modified immune cell of claim 1 or 2, wherein the at least one exogenous anti- inflammatory agent comprises or is one or both of a cytokine or cytokine receptor.

4. The modified immune cell of claim 3, wherein: (i) the cytokine comprises or is IL-10, IL-22, IL-27, and / or (ii) the cytokine receptor comprises or is IL17A switch receptor.

5. The modified immune cell of any one of claims 1-4, wherein the at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and the at least one exogenous anti- inflammatory agent comprises IL-10.

6. The modified immune cell of any one of claims 1-5, wherein the at least one exogenous regenerative agent is or comprises Fibrotic Growth Factor 21 (FGF21), Fibrotic Growth Factor 19 (FGF19), Vascular Endothelial Growth Factor α (VEGF-α), and / or Hepatocyte Growth factor (HGF).

7. The modified immune cell of any one of claims 1-6, wherein the at least one exogenous fibrolytic agent, the at least one exogenous anti-inflammatory agent, and / or the at least one exogenous regenerative agent are tethered to the immune cell or secreted from the immune cell. Page 109 of 118 12583381v1Attorney Docket No.2012851-0634 8. The modified immune cell of any one of claims 1-7, wherein the one or more nucleic acid sequences comprise one or more liver specific promoters or cirrhosis specific promoters.

9. The modified immune cell of any one of claims 1-8, wherein the one or more nucleic acid sequences comprise a CX3CR1 promoter, an insulin-like growth factor 1 (IGF1), or a CD11B promoter.

10. The modified immune cell of any one of claims 1-9, wherein the modified immune cell comprises a macrophage, monocyte, or dendritic cell.

11. The modified immune cell of claim 10, wherein the macrophage is derived from a monocyte or a precursor immune cell.

12. The modified immune cell of claim 11, wherein the precursor immune cell comprises or is a hematopoietic stem cell, myeloid progenitor, myeloblast, monoblast, promonocyte, or an intermediate thereof.

13. The modified immune cell of any one of claims 10-12, wherein the macrophage is a G-MCSF derived macrophage or an M-CSF derived macrophage.

14. The modified immune cell of any one of claims 1-13, wherein the one or more nucleic acid sequences encode one or more polypeptides comprising one or more amino acid sequences of any one of SEQ ID NOs: 1-5 or 13-18.

15. A pharmaceutical composition comprising a modified immune cell of any one of claims 1-14.

16. The pharmaceutical composition of claim 15, comprising a pharmaceutically acceptable carrier. Page 110 of 118 12583381v1Attorney Docket No.2012851-0634 17. A nucleic acid construct comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) at least one exogenous regenerative agent.

18. A pharmaceutical composition comprising the nucleic acid construct of claim 17.

19. The pharmaceutical composition of claim 18, comprising a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 18, comprising a delivery vehicle.

21. The pharmaceutical composition of claim 20, wherein the delivery vehicle is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles).

22. A method for treating or preventing fibrosis or inflammation in a subject, comprising delivering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 15, 16, 18, 19, 20, or 21.

23. The method of claim 22, wherein the fibrosis comprises or is a liver, lung, heart, vasculature, kidney, pancreas, skin, gastrointestinal, bone marrow, hematopoietic tissue, nervous system, and / or an eye fibrotic disease, disorder, or condition.

24. The method of claim 23, wherein the liver fibrotic disease, disorder, or condition comprises a fatty liver disease, disorder, or condition.

25. The method of claim 24, wherein the fatty liver disease, disorder, or condition comprises non-alcoholic fatty liver disease (NAFL) or alcoholic liver disease.

26. The method of claim 25, wherein the NAFL comprises non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Page 111 of 118 12583381v1Attorney Docket No.2012851-0634 27. The method of claim 26, wherein the alcoholic liver disease comprises alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH).

28. The method of any one of claims 22-27, wherein the subject has one or more of cirrhosis, liver damage, hepatocarcinoma, steatosis, an increased risk of liver failure, an increased risk of death, and / or Hepatitis C infection (HCV).

29. The method of any one of claims 22-28, wherein the inflammation comprises or is a liver, gastrointestinal tract, lung, skin, cardiovascular system, nervous system, kidney, pancreas, joint, eye, and / or an endocrine system inflammatory disease, disorder, or condition.

30. The method of any one of claims 22-29, wherein the method reduces activation of hepatic stellate cells.

31. The method of any one of claims 22-30, wherein the method improves liver regeneration and / or liver resolution.

32. The method of any one of claims 22-31, wherein the method balances pro-fibrotic and anti-fibrotic macrophage populations in the subject.

33. A method of modifying an immune cell, comprising delivering to the immune cell a nucleic acid construct comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) at least one exogenous regenerative agent, thereby forming a modified immune cell.

34. The method of claim 33, wherein the delivering comprises electroporation or transfection with mRNA, DNA, or chemically modified mRNA. Page 112 of 118 12583381v1Attorney Docket No.2012851-0634 35. The method of claim 33, wherein the delivering comprises transduction with an adeno-associated viral (AAV) vector, an adenoviral vector, or a retroviral vector.

36. The method of claim 33, wherein the delivering comprises a delivery vehicle.

37. The method of claim 36, wherein the delivery vehicle is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles).

38. The method of claim 35, wherein the retroviral vector comprises a lentiviral vector or a gammaretroviral vector.

39. The method of claim 38, wherein the lentiviral vector is packaged with a Vpx protein.

40. The method of claim 39, wherein a Vpx protein is delivered to the immune cell either before, concurrently with, or subsequently to the nucleic acid construct.

41. The method of claim 40, wherein the adenoviral vector comprises an Ad2 vector or an Ad5 vector.

42. The method of claim 41, wherein the Ad5 vector comprises an Ad5f35 adenoviral vector.

43. A method of producing a modified immune cell, the method comprising administering to a subject a composition comprising: (a) one or more nucleic acid constructs comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, (ii) at least one exogenous anti- inflammatory agent, and / or (iii) at least one exogenous regenerative agent, and (b) a delivery vehicle; wherein following administration of the composition, the one or more nucleic acid constructs are translated in an immune cell to produce the modified immune cell comprising (i) Page 113 of 118 12583381v1Attorney Docket No.2012851-0634 the at least one exogenous fibrolytic agent, (ii) the at least one exogenous anti-inflammatory agent, and / or (iii) the at least one exogenous regenerative agent, wherein the modified immune cell comprises a macrophage, monocyte, or dendritic cell in the subject.

44. The method of claim 43, wherein the delivery vehicle is or comprises a lipid nanoparticle, a liposome, a polymer, an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, or any combination thereof.

45. A modified immune cell comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent.

46. The modified immune cell of claim 45, wherein the at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and the at least one efferocytic agent comprises a TIM4 polypeptide.

47. The modified immune cell of claim 45 or claim 46, wherein the modified immune cell comprises a macrophage, monocyte, or dendritic cell.

48. The modified immune cell of claim 47, wherein the macrophage is derived from a monocyte or a precursor immune cell.

49. The modified immune cell of claim 48, wherein the precursor immune cell comprises or is a hematopoietic stem cell, myeloid progenitor, myeloblast, monoblast, promonocyte, or an intermediate thereof.

50. A pharmaceutical composition comprising a modified immune cell of any one of claims 45-49. Page 114 of 118 12583381v1Attorney Docket No.2012851-0634 51. The modified immune cell of any one of claims 45-50, wherein the macrophage is a G-MCSF derived macrophage or an M-CSF derived macrophage.

52. A method of modifying an immune cell, comprising delivering to the immune cell a nucleic acid construct comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent.

53. The method of claim 52, wherein the at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and the at least one efferocytic agent comprises a TIM4 polypeptide.

54. The method of claim 52 or claim 53, wherein the immune cell comprises a macrophage, monocyte, or dendritic cell.

55. The method of claim 54, wherein the macrophage is derived from a monocyte or a precursor immune cell.

56. The method of claim 55, wherein the precursor immune cell comprises or is a hematopoietic stem cell, myeloid progenitor, myeloblast, monoblast, promonocyte, or an intermediate thereof.

57. The method of any one of claims 52-56, wherein the delivering comprises electroporation or transfection with mRNA, DNA, or chemically modified mRNA (e.g., packaged in a liposome).

58. The method of any one of claims 52-56, wherein the delivering comprises transduction with an adeno-associated viral (AAV) vector, an adenoviral vector, or a retroviral vector. Page 115 of 118 12583381v1Attorney Docket No.2012851-0634 59. The method of any one of claims 52-56, wherein the delivering comprises a delivery vehicle.

60. The method of claim 59, wherein the delivery vehicle is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles).

61. The method of claim 58, wherein the retroviral vector comprises a lentiviral vector or a gammaretroviral vector.

62. The method of claim 61, wherein the lentiviral vector is packaged with a Vpx protein.

63. The method of claim 62, wherein a Vpx protein is delivered to the immune cell either before, concurrently with, or subsequently to the nucleic acid construct.

64. The method of claim 58, wherein the adenoviral vector comprises an Ad2 vector or an Ad5 vector.

65. The method of claim 64, wherein the Ad5 vector comprises an Ad5f35 adenoviral vector.

66. A nucleic acid construct comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent.

67. The nucleic acid construct of claim 66, wherein the nucleotide sequence encoding at least one exogenous fibrolytic agent encodes a polypeptide comprising a Relaxin polypeptide and wherein the nucleotide sequence encoding the at least one efferocytic agent encodes a polypeptide comprising a TIM4 polypeptide.

68. The nucleic acid construct of claim 66 or claim 67, further comprising a nucleotide sequence encoding a cleavage peptide, wherein the cleavage peptide comprises one or more of a Page 116 of 118 12583381v1Attorney Docket No.2012851-0634 porcine teschovirus-l (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, a foot-and-mouth disease virus (F2A) peptide, and / or a variant thereof.

69. A method of producing a modified immune cell, the method comprising administering to a subject a composition comprising: (a) one or more nucleic acid constructs comprising one or more nucleic acid sequences encoding: (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent, and (b) a delivery vehicle; wherein following administration of the composition, the one or more nucleic acid constructs are translated in an immune cell to produce the modified immune cell comprising (i) at least one exogenous fibrolytic agent, and (ii) at least one exogenous efferocytic agent, wherein the modified immune cell comprises a macrophage, monocyte, or dendritic cell in the subject.

70. The method of claim 69, wherein the at least one exogenous fibrolytic agent comprises a Relaxin polypeptide and the at least one efferocytic agent comprises a TIM4 polypeptide.

71. The method of claim 69 or claim 70, wherein the delivery vehicle is or comprises a lipid nanoparticle (e.g., MC3-lipid nanoparticles), a liposome, a polymer, an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, or any combination thereof. Page 117 of 118 12583381v1

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