Modified immune cells for fibrosis and inflammation

Abstract

The present disclosure relates 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

[0001] Related applications This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 563,228, filed March 8, 2024, and U.S. Provisional Patent Application No. 63 / 697,419, filed September 20, 2024, the entire contents of which are incorporated herein by reference. Background Technology

[0002] Fibrosis is a serious health problem characterized by the formation of excessive fibrous connective tissue, usually at least in part due to repair and / or reactive processes, such as in response to injury. Fibrosis can occur in a variety of organs, including the lungs, liver, heart, kidneys, pancreas, skin, and brain. Current treatments available for fibrotic diseases, conditions, and lesions have limited efficacy.

[0003] Inflammatory diseases are a major cause of morbidity and mortality in humans. Many side effects are associated with current treatments for inflammation, such as adrenal suppression, bone weakening, muscle atrophy, peptic ulcers, hypokalemia, and immunosuppression.

[0004] Therefore, there is a need to develop new, optimized treatments to reduce and treat fibrosis and inflammation. Summary of the Invention

[0005] This disclosure relates to immune cells, including 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, as well as methods of using such immune cells. In part, this disclosure covers the understanding that the administration of specifically modified immune cells is surprisingly effective in treating one or more symptoms of fibrosis and / or inflammation.

[0006] In one aspect, this disclosure provides modified immune cells comprising encoding one or more nucleic acid sequences of: (i) at least one exogenous fibrinolytic agent, (ii) at least one exogenous anti-inflammatory agent, (iii) at least one exogenous regenerative agent, and / or (iv) at least one exogenous cytotoxic agent. In some embodiments, the at least one exogenous fibrinolytic agent is or includes a relaxin polypeptide, a decorin polypeptide, a dominant-negative TGF-β receptor 2 (DNTGFβR2) polypeptide, a soluble TGFβ receptor, and / or a TGFβ switch receptor. In some embodiments, the at least one exogenous anti-inflammatory agent comprises or is a cytokine or a cytokine receptor. In some embodiments, the cytokine comprises or is IL-10, IL-22, and / or IL-27. In some embodiments, the cytokine receptor comprises or is an IL-17A switch receptor.

[0007] In some embodiments, at least one exogenous fibrinolytic agent comprises a relaxin peptide, and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrinolytic agent comprises a relaxin peptide, and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrinolytic agent comprises a relaxin peptide, and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrinolytic agent comprises a relaxin peptide, and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous fibrinolytic agent comprises a core proteoglycan peptide, and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrinolytic agent comprises a core proteoglycan peptide, and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrinolytic agent comprises a core proteoglycan peptide, and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrinolytic agent comprises a core proteoglycan peptide, and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous fibrinolytic agent comprises a dominant-negative (DN) TGFβR2 peptide, and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrinolytic agent comprises a DN TGFβR2 peptide, and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrinolytic agent comprises a DN TGFβR2 peptide, and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrinolytic agent comprises a DN TGFβR2 peptide, and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous fibrinolytic agent comprises a soluble TGFβ receptor, and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrinolytic agent comprises a soluble TGFβ receptor, and at least one exogenous anti-inflammatory agent comprises IL-22. In some embodiments, at least one exogenous fibrinolytic agent comprises a soluble TGFβ receptor, and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous fibrinolytic 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 fibrinolytic agent comprises a TGFβ switch receptor, and at least one exogenous anti-inflammatory agent comprises IL-10. In some embodiments, at least one exogenous fibrinolytic agent comprises a TGFβ switch receptor, and at least one exogenous anti-inflammatory agent comprises IL-22.In some embodiments, at least one exogenous fibrinolytic agent includes a TGFβ switch receptor, and at least one exogenous anti-inflammatory agent includes IL-27. In some embodiments, at least one exogenous fibrinolytic agent includes a TGFβ switch receptor, and at least one exogenous anti-inflammatory agent includes an IL-17A switch receptor. In some embodiments, at least one exogenous fibrinolytic agent includes an MMP13 peptide, and at least one exogenous anti-inflammatory agent includes IL-10. In some embodiments, at least one exogenous fibrinolytic agent includes an MMP13 peptide, and at least one exogenous anti-inflammatory agent includes IL-22. In some embodiments, at least one exogenous fibrinolytic agent includes an MMP13 peptide, and at least one exogenous anti-inflammatory agent includes IL-27. In some embodiments, at least one exogenous fibrinolytic agent includes an MMP13 peptide, and at least one exogenous anti-inflammatory agent includes an IL-17A switch receptor.

[0008] In some embodiments, at least one exogenous regenerative agent is or includes fibroblast growth factor 21 (FGF21), fibroblast 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 includes a relaxin peptide, and at least one exogenous regenerative agent includes FGF21. In some embodiments, at least one exogenous fibrolytic agent includes a relaxin peptide, and at least one exogenous regenerative agent includes FGF19. In some embodiments, at least one exogenous fibrolytic agent includes a relaxin peptide, and at least one exogenous regenerative agent includes VEGF-α. In some embodiments, at least one exogenous fibrolytic agent includes a relaxin peptide, and at least one exogenous regenerative agent includes HGF. In some embodiments, at least one exogenous fibrolytic agent includes a core proteoglycan peptide, and at least one exogenous regenerative agent includes FGF21. In some embodiments, at least one exogenous fibrinolytic agent comprises a core proteoglycan peptide, and at least one exogenous regenerator comprises FGF19. In some embodiments, at least one exogenous fibrinolytic agent comprises a core proteoglycan peptide, and at least one exogenous regenerator comprises VEGF-α. In some embodiments, at least one exogenous fibrinolytic agent comprises a core proteoglycan peptide, and at least one exogenous regenerator comprises HGF. In some embodiments, at least one exogenous fibrinolytic agent comprises a DN TGFβR2 peptide, and at least one exogenous regenerator comprises FGF21. In some embodiments, at least one exogenous fibrinolytic agent comprises a DNTGFβR2 peptide, and at least one exogenous regenerator comprises FGF19. In some embodiments, at least one exogenous fibrinolytic agent comprises a DN TGFβR2 peptide, and at least one exogenous regenerator comprises VEGF-α. In some embodiments, at least one exogenous fibrinolytic agent comprises a soluble TGFβ receptor, and at least one exogenous regenerator comprises HGF. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor, and at least one exogenous regenerator comprises FGF21. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor, and at least one exogenous regenerator comprises FGF19. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor, and at least one exogenous regenerator comprises VEGF-α. In some embodiments, at least one exogenous fibrolytic agent comprises a soluble TGFβ receptor, and at least one exogenous regenerator comprises HGF.In some embodiments, at least one exogenous fibrolytic agent includes a TGFβ switch receptor, and at least one exogenous regenerator includes FGF21. In some embodiments, at least one exogenous fibrolytic agent includes a TGFβ switch receptor, and at least one exogenous regenerator includes FGF19. In some embodiments, at least one exogenous fibrolytic agent includes a TGFβ switch receptor, and at least one exogenous regenerator includes VEGF-α. In some embodiments, at least one exogenous fibrolytic agent includes a TGFβ switch receptor, and at least one exogenous regenerator includes HGF. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-10, and at least one exogenous regenerator includes FGF21. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-10, and at least one exogenous regenerator includes FGF19. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-10, and at least one exogenous regenerator includes VEGF-α. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-10, and at least one exogenous regenerator includes HGF. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-22, and at least one exogenous regenerative agent includes FGF21. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-22, and at least one exogenous regenerative agent includes FGF19. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-22, and at least one exogenous regenerative agent includes VEGF-α. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-22, and at least one exogenous regenerative agent includes HGF. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-27, and at least one exogenous regenerative agent includes FGF21. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-27, and at least one exogenous regenerative agent includes FGF19. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-27, and at least one exogenous regenerative agent includes VEGF-α. In some embodiments, at least one exogenous anti-inflammatory agent includes IL-27, and at least one exogenous regenerative agent includes HGF. In some embodiments, at least one exogenous anti-inflammatory agent includes an IL-17A switch receptor, and at least one exogenous regenerative agent includes FGF21. In some embodiments, at least one exogenous anti-inflammatory agent includes an IL-17A switch receptor, and at least one exogenous regenerative agent includes FGF19. In some embodiments, at least one exogenous anti-inflammatory agent includes an IL-17A switch receptor, and at least one exogenous regenerative agent includes VEGF-α. In some embodiments, at least one exogenous anti-inflammatory agent includes an IL-17A switch receptor, and at least one exogenous regenerative agent includes HGF.

[0009] In some embodiments, at least one exogenous embalming agent is or includes a peptide containing T-cell immunoglobulin and mucin domain 4 (TIM4). In some embodiments, at least one exogenous embalming agent includes a TIM4 peptide, and at least one exogenous fibrinolytic agent includes a relaxin peptide. In some embodiments, at least one exogenous embalming agent includes a TIM4 peptide, and at least one exogenous fibrinolytic agent includes a core proteoglycan peptide. In some embodiments, at least one exogenous embalming agent includes a TIM4 peptide, and at least one exogenous fibrinolytic agent includes a DN TGFβR2 peptide. In some embodiments, at least one exogenous embalming agent includes a TIM4 peptide, and at least one exogenous fibrinolytic agent includes a soluble TGFβ receptor. In some embodiments, at least one exogenous embalming agent includes a TIM4 peptide, and at least one exogenous fibrinolytic agent includes a TGFβ switch receptor. In some embodiments, at least one exogenous embalming agent includes a TIM4 peptide, and at least one exogenous anti-inflammatory agent includes IL-22. In some embodiments, at least one exogenous embalming agent comprises a TIM4 peptide, and at least one exogenous anti-inflammatory agent comprises IL-27. In some embodiments, at least one exogenous embalming agent comprises a TIM4 peptide, and at least one exogenous anti-inflammatory agent comprises an IL-17A switch receptor. In some embodiments, at least one exogenous embalming agent comprises a TIM4 peptide, and at least one exogenous regenerator comprises FGF21. In some embodiments, at least one exogenous embalming agent comprises a TIM4 peptide, and at least one exogenous regenerator comprises FGF19. In some embodiments, at least one exogenous embalming agent comprises a TIM4 peptide, and at least one exogenous regenerator comprises VEGF-α. In some embodiments, at least one exogenous embalming agent comprises a TIM4 peptide, and at least one exogenous regenerator comprises HGF.

[0010] In some embodiments, at least one exogenous fibrinolytic agent, at least one exogenous anti-inflammatory agent, at least one exogenous regenerative agent, and / or at least one exogenous necrolysis agent are tethered to or secreted from immune cells.

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

[0012] In some embodiments, the modified immune cells include macrophages, monocytes, or dendritic cells. In some embodiments, the macrophages are derived from monocytes or precursor immune cells. In some embodiments, the precursor immune cells include hematopoietic stem cells, myeloid progenitor cells, myeloid cells, monocytes, premonocytes, or intermediates thereof. In some embodiments, the macrophages are G-MCSF-derived macrophages or M-CSF-derived macrophages.

[0013] In some embodiments, one or more nucleic acid sequences described herein encode one or more polypeptides comprising one or more amino acid sequences including any one of SEQ ID No: 1-5 or 13-18.

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

[0015] On the other hand, this disclosure provides nucleic acid constructs comprising encoding one or more of the following nucleic acid sequences: at 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 endocytogen of any aspect or embodiment described herein.

[0016] On the other hand, this disclosure provides pharmaceutical compositions comprising nucleic acid constructs of any aspect or embodiment described herein. In some embodiments, the pharmaceutical compositions comprise pharmaceutically acceptable carriers.

[0017] On the other hand, this disclosure provides methods for treating or preventing fibrosis or inflammation in a subject, including delivering to the subject a therapeutically effective amount of a pharmaceutical composition of any aspect or embodiment described herein.

[0018] In some embodiments, fibrosis includes, or is defined as, fibrotic diseases, conditions, or illnesses affecting the liver, lungs, heart, vascular system, kidneys, pancreas, skin, gastrointestinal tract, bone marrow, hematopoietic tissue, nervous system, and / or eyes. In some embodiments, liver fibrotic diseases, conditions, or illnesses include fatty liver disease, conditions, or illnesses. In some embodiments, fatty liver disease, conditions, or illnesses include non-alcoholic fatty liver disease (NAFLD) or alcoholic liver disease. In some embodiments, NAFLD includes non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In some embodiments, alcoholic liver disease includes alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH). In some embodiments, the subject has one or more of the following: cirrhosis, liver injury, liver cancer, steatosis, increased risk of liver failure, increased risk of death, and / or hepatitis C infection (HCV).

[0019] In some implementations, inflammation includes inflammatory diseases, conditions, or illnesses of the liver, gastrointestinal tract, lungs, skin, cardiovascular system, nervous system, kidneys, pancreas, joints, eyes, and / or endocrine system.

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

[0021] On the other hand, this disclosure provides a method for modifying immune cells, comprising delivering a nucleic acid construct to immune cells, the nucleic acid construct comprising encoding one or more nucleic acid sequences of: at least one exogenous antifibrotic 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 cytotoxic agent of any aspect or embodiment described herein, thereby forming modified immune cells.

[0022] In some embodiments, delivery includes electroporation or transfection with mRNA, DNA, or chemically modified mRNA. In some embodiments, delivery includes the application of one or more lipid particles (e.g., lipid nanoparticles or LNPs). In some embodiments, the lipid particles include cationic lipids, PEG-modified lipids, sterols, non-cationic lipids, or combinations thereof. In some embodiments, the cationic lipids include MC3. In some embodiments, delivery includes the application of one or more MC3-lipid nanoparticles. In some embodiments, delivery includes transduction with an adeno-associated virus (AAV) vector, an adenovirus vector, or a retroviral vector. In some embodiments, the retroviral vector includes a lentiviral vector or a gamma retroviral vector. In some embodiments, delivery includes transduction with a viral vector (e.g., a lentiviral vector) and at least one Vpx protein. In some embodiments, the immune cells described herein are subjected to one or both of the following: electroporation or transfection with at least one Vpx mRNA before and / or simultaneously with transfection with a viral vector. In some embodiments, the lentiviral vector is packaged with a Vpx protein. In some embodiments, the adenoviral vector includes an Ad2 vector or an Ad5 vector. In some implementations, the Ad5 vector includes the Ad5f35 adenovirus vector.

[0023] In another aspect, this disclosure provides a method for generating modified immune cells, comprising administering to a subject a composition comprising: (a) one or more nucleic acid constructs including sequences encoding one or more nucleic acid sequences of: (i) at least one exogenous fibrinolytic agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) at least one exogenous regenerative agent; and (b) a delivery solvent. In some embodiments, after administration of the composition, the one or more nucleic acid constructs are translated in immune cells to generate modified immune cells comprising (i) the at least one exogenous fibrinolytic agent, (ii) the at least one exogenous anti-inflammatory agent, and / or (iii) the at least one exogenous regenerative agent. In some embodiments, the modified immune cells comprise macrophages, monocytes, or dendritic cells in the subject. In some embodiments, the delivery solvent is or comprises lipid nanoparticles (e.g., MC3-lipid nanoparticles), liposomes, polymers, adeno-associated virus (AAV) vectors, adenovirus vectors, retroviral vectors, or any combination thereof.

[0024] In another aspect, this disclosure provides modified immune cells comprising encoding one or more nucleic acid sequences of: (i) at least one exogenous fibrinolytic agent, and (ii) at least one exogenous cytotoxic agent. In some embodiments, the at least one exogenous fibrinolytic agent comprises a relaxin polypeptide, and the at least one cytotoxic agent comprises a TIM4 polypeptide. In some embodiments, the modified immune cells comprise macrophages, monocytes, or dendritic cells. In some embodiments, macrophages are derived from monocytes or precursor immune cells. In some embodiments, precursor immune cells comprise hematopoietic stem cells, myeloid progenitor cells, myeloid cells, monocytes, promonocytes, or intermediates thereof.

[0025] In another aspect, this disclosure provides pharmaceutical compositions comprising modified immune cells of any aspect or embodiment described herein. In some embodiments, the macrophages are G-MCSF-derived macrophages or M-CSF-derived macrophages.

[0026] In another aspect, this disclosure provides a method for modifying immune cells, including delivering a nucleic acid construct to immune cells, the nucleic acid construct comprising encoding one or more nucleic acid sequences: (i) at least one exogenous fibrinolytic agent, and (ii) at least one exogenous cytotoxic agent. In some embodiments, the at least one exogenous fibrinolytic agent comprises a relaxin polypeptide, and the at least one cytotoxic agent comprises a TIM4 polypeptide. In some embodiments, the immune cells comprise macrophages, monocytes, or dendritic cells. In some embodiments, the macrophages are derived from monocytes or precursor immune cells. In some embodiments, the precursor immune cells comprise hematopoietic stem cells, myeloid progenitor cells, myeloid cells, monocytes, premonocytes, or intermediates thereof. In some embodiments, delivery comprises electroporation or transfection with mRNA, DNA, or chemically modified mRNA (e.g., packaged in liposomes). In some embodiments, delivery comprises transduction using an adeno-associated virus (AAV) vector, an adenovirus vector, or a retroviral vector. In some embodiments, the retroviral vector comprises a lentiviral vector or a gamma retroviral vector. In some embodiments, the lentiviral vector is packaged with the Vpx protein. In some embodiments, the Vpx protein is delivered to immune cells before, simultaneously with, or after the nucleic acid construct. In some embodiments, the adenoviral vector includes an Ad2 vector or an Ad5 vector. In some embodiments, the Ad5 vector includes an Ad5f35 adenoviral vector. In some embodiments, delivery includes a delivery solvent. In some embodiments, the delivery solvent described herein is or includes lipid nanoparticles (e.g., MC3-lipid nanoparticles).

[0027] On the other hand, this disclosure provides nucleic acid constructs comprising encoding one or more nucleic acid sequences of: (i) at least one exogenous fibrinolytic agent, and (ii) at least one exogenous cytotoxic agent. In some embodiments, the nucleotide sequence encoding at least one exogenous fibrinolytic agent encodes a polypeptide comprising a relaxin polypeptide, and wherein the nucleotide sequence encoding at least one cytotoxic agent encodes a polypeptide comprising a TIM4 polypeptide. In some embodiments, the nucleic acid constructs described herein further comprise a nucleotide sequence encoding a cleavage peptide, wherein the cleavage peptide comprises one or more of porcine chezinvirus-1 (P2A) peptide, Thoseea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, foot-and-mouth disease virus (F2A) peptide, and / or variants thereof.

[0028] In another aspect, this disclosure provides a method for generating modified immune cells, the method comprising administering to a subject a composition comprising: (a) one or more nucleic acid constructs including sequences encoding one or more nucleic acid sequences: (i) at least one exogenous fibrinolytic agent, and (ii) at least one exogenous cytotoxic agent; and (b) a delivery solvent. In some embodiments, after administration of the composition described herein, one or more nucleic acid constructs are translated in immune cells to generate modified immune cells comprising (i) at least one exogenous fibrinolytic agent, and (ii) at least one exogenous cytotoxic agent. In some embodiments, the modified immune cells comprise macrophages, monocytes, or dendritic cells in the subject. In some embodiments, the at least one exogenous fibrinolytic agent comprises a relaxin polypeptide, and the at least one cytotoxic agent comprises a TIM4 polypeptide. In some embodiments, the delivery solvent is or comprises lipid nanoparticles (e.g., MC3-lipid nanoparticles), liposomes, polymers, adeno-associated virus (AAV) vectors, adenovirus vectors, retroviral vectors, or any combination thereof. Attached Figure Description

[0029] The accompanying drawings are for illustrative purposes only and are not intended to be limiting.

[0030] Figure 1 These are a series of schematic diagrams showing the time points used to generate and validate the anti-fibrotic engineered macrophages described in Example 1 of this document.

[0031] Figure 2 This is a schematic diagram of the time points used in Example 1 of this document to evaluate the expression of in vitro payload over time and the collection of supernatant from macrophages.

[0032] Figures 3A-3C It is a series of graphs showing macrophages on day 5 ( Figure 3A ), immediately after thawing frozen macrophages Figure 3B), and thawed macrophages cultured in vitro for 48 hours ( Figure 3C The recovery rate and vitality of ).

[0033] Figures 4A-4D This is a series of graphs showing the levels of IL-10 in the supernatant of engineered macrophages on day 5. Figure 4A ), FGF21 ( Figure 4B ), relaxin ( Figure 4C ) and core proteoglycans ( Figure 4D ) level.

[0034] Figures 5A-5E These are a series of schematic diagrams of transmembrane receptors and graphs showing the activity and expression levels of the transmembrane proteins in engineered macrophages. Figure 5A This is a schematic diagram of dominant and negative TGFβR2. Figure 5B This is a schematic diagram of the IL17A switch receptor. Figure 5C This demonstrates the viability of engineered macrophages expressing synthetic transmembrane receptors dominant and negative TGFβR2 and IL17A switch receptors. Figure 5D Flow cytometry analysis showing dominant and negative TGFβR2 expression. Figure 5E Flow cytometry analysis of IL17A switch receptor expression.

[0035] Figures 6A-6B It is a series of graphs showing the expression levels of IL-10. Figure 6A ) and the production rates on days 7, 9, 13, and 14 ( Figure 6B ).

[0036] Figures 7A-7B This is a schematic diagram showing the time points used to generate and validate anti-fibrotic engineered mouse macrophages. Figure 7A ) and IVIS for monitoring in vivo payload expression over time. ® The time point of imaging ( Figure 7B ).

[0037] Figures 8A-8B It is a series of graphs showing mouse macrophages on day 5 ( Figure 8A ) and immediately after thawing frozen mouse macrophages ( Figure 8B The recovery rate and vitality of ).

[0038] Figures 9A-9B These are diagrams and images showing the luciferase signal in the body. Figure 9A This study demonstrates the quantification of luciferase signaling in mice from day 1 to day 13 following intravenous injection of engineered macrophages. Figure 9B Showing from Figure 9A IVIS of the signals on day 1 and day 13 of the data plotted in the image. ® image.

[0039] Figure 10 This is a schematic diagram showing the time points of administering CCl4 to mice to generate a CCl4-induced liver fibrosis mouse model and treating the mice with engineered macrophages on day 28.

[0040] Figure 11A-11B These are graphs and images showing the detection of IL-10 and relaxin in engineered macrophages. Figure 11A This shows the IL-10 levels in engineered macrophages transduced with IL-10-P2A-chassotin compared to untransduced cells, as measured by ELISA. Figure 11B The results show that relaxin was detected using non-denatured protein blot analysis.

[0041] Figure 12A-12B It is a series of graphs showing the CCl4 application period ( Figure 12A ) and the study endpoint on day 43 ( Figure 12B The body weight of the mice.

[0042] Figures 13A-13B These are figures and images showing collagen content in tissue sections of a mouse model of CCl4-induced liver fibrosis treated with IL-10-P2A-relaxin-transduced engineered macrophages. Figure 13A The text shows the quantification of collagen as a percentage of area in histological images (n=9 per group), where the mean signal in the corn oil / solvent group was defined as a 100% reduction and the mean signal in the CCl4 / solvent group was defined as a 0% reduction. Figure 13B Showing from Figure 13A A representative Masson tricolor image of the data plotted in the image.

[0043] Figure 14 This diagram illustrates the time points used to generate a bleomycin-induced pulmonary fibrosis model in mice and the treatment with engineered macrophages on day 1.

[0044] Figures 15A-15C It is a series of figures showing the activity and expression levels of transmembrane proteins in engineered macrophages. Figure 15A This demonstrates the viability of engineered macrophages expressing synthetic transmembrane receptors dominant and negative TGFβR2 and IL17A switch receptors. Figure 15B The experiment showed that ELISA assays were used to detect IL-10 production in engineered macrophages of CCl4-induced fibrotic mice. Figure 15C Flow cytometry analysis showing dominant and negative TGFβR2 expression.

[0045] Figures 16A-16B It is a series of graphs showing the progress of the study after bleomycin administration ( Figure 16A ) and the study endpoint on day 21 ( Figure 16B The body weight of the mice.

[0046] Figures 17A-17B It is a series of graphs showing the effects of IL-10-P2A-DN-TGFβR2 ( Figure 17A ) or IL-10-P2A-relaxin ( Figure 17B Hydroxyproline (“HYP”) measurement in a bleomycin-induced pulmonary fibrosis mouse model treated with transduced engineered macrophages.

[0047] Figure 18 This diagram illustrates the time points used to assess the viability and TIM4 expression of engineered macrophages via flow cytometry.

[0048] Figure 19 Flow cytometry analysis of TIM4 expression and viability in macrophages that were not transduced (UTD) or transduced with the TIM4 construct (TIM4 Mɸ) described herein, as shown in Example 6.

[0049] Figure 20 This diagram shows the time points for the cytotoxicity assay of engineered macrophages.

[0050] Figure 21 This is a graph showing changes in the endocytic signal (AU) of macrophages transduced with UTD or the lentivirus control (miRFP670) or the TIM4 construct described herein in Example 6.

[0051] Figure 22 This diagram shows a timeline for generating and validating anti-fibrotic engineered macrophages as described in Example 6 of this document.

[0052] Figures 23A-23C This is a series of figures showing the viability and expression levels of the engineered macrophages described in this article. Figure 23A The viability and Cd11b expression of macrophages engineered with the TIM4 construct or the TIM4-P2A-relaxin construct described in Example 6 of this document were shown by flow cytometry. Figure 23B The flow cytometry analysis shows TIM4 expression in macrophages 5 days after transduction using the methods described in this paper. Figure 23C Flow cytometry analysis of Tim4 expression in macrophages 7 days after transduction using the methods described herein.

[0053] Figures 24A-24B It is a series of graphs demonstrating the measurement of relaxin in the supernatant. Figure 24A This shows an ELISA measurement of relaxin in the supernatant collected from macrophages transduced with the TIM4 construct or the TIM4-P2A-chalasin construct described in Example 6. Figure 24BThe time-course ELISA measurement of relaxin in the supernatant collected from macrophages transduced with the TIM4 construct or the TIM4-P2A-chalasin construct described in Example 6 is displayed.

[0054] Figure 25 This diagram illustrates the time points at which a choline-deficient, L-amino acid-restricted high-fat diet (CDAHFD)-induced liver fibrosis model was generated in mice and treated with engineered macrophages as described in this paper at week 4.

[0055] Figures 26A-26B This is an immunoblot and image showing TIM4 expression in the liver of mice after a CDAHFD-induced liver fibrosis model in mice. Figure 26A This is an immunoblot of TIM4 expression in mouse livers after CDAHFD. After the study was completed, the TIM4 protein content in each of the following groups of livers (n=3) was analyzed: PBS (feed / solvent; healthy liver), Ctr Mɸ (CDAHFD / Ctrl Mɸ), and TIM4 Mɸ (CDAHFD / TIM4 Mɸ). Figure 26B show Figure 26A Quantitative analysis of immunoblotting was performed, and the TIM4 protein level was normalized relative to the expression of the housekeeping protein GAPDH in each lane.

[0056] Figure 27 This figure shows an in situ analysis of macrophage burial in the liver of a mouse model of CDAHFD-induced liver fibrosis after treatment with untransduced macrophages (CDAHFD / Ctrl Mɸ) or engineered macrophages transduced with the TIM4 construct (CDAHFD / Mɸ TIM4) or the TIM4+chalasin construct (CDAHFD / Mɸ TIM4+chalasin).

[0057] Figures 28A-28B This is a series of figures showing Sirius red measurements in a mouse model of CDAHFD-induced liver fibrosis treated with engineered macrophages transduced with the TIM4 construct or the TIM4-P2A-chalcogenin construct described in Example 7 of this document. Figure 28A ) and immunohistochemical (IHC) images ( Figure 28B These mice were compared with controls, which included mice fed a normal diet (healthy livers), CDAHFD-induced mice not treated with macrophages (untreated), or CDAHFD-induced mice treated with UTD macrophages (control Mɸ).

[0058] Figures 29A-29BThis is a series of figures showing the measurement of type 1 collagen α1 (Col1a1) in a CDAHFD-induced liver fibrosis model in mice using engineered macrophages transduced with the TIM4 construct or the TIM4-P2A-chalasin construct described in Example 7 of this document. Figure 29A ) and IHC images ( Figure 29B These mice were compared to controls, which included mice fed a normal diet (“healthy liver”), untreated CDAHFD-induced mice (“untreated”), or CDAHFD-induced mice transduced with a control vector (“control Mɸ”).

[0059] Figures 30A-30B This is a series of figures showing the measurement of α-smooth muscle actin (α-SMA) in a CDAHFD-induced liver fibrosis model in mice treated with engineered macrophages transduced with the TIM4 construct or the TIM4-P2A-chalasin construct described in Example 7 of this document. Figure 30A ) and IHC images ( Figure 30B These mice were compared to controls, which included mice fed a normal diet (“healthy liver”), untreated CDAHFD-induced mice (“untreated”), or CDAHFD-induced mice transduced with a control vector (“control Mɸ”).

[0060] Figures 31A-31B This is a series of figures showing osteopontin (Opn; also referred to herein as “secreted phosphoprotein 1” and “SPP1” interchangeably) measurements in a CDAHFD-induced liver fibrosis model in mice treated with engineered macrophages transduced with the TIM4 construct or the TIM4-P2A-chalcogenin construct described in Example 7 of this document. Figure 31A ) and IHC images ( Figure 31B These mice were compared to controls, which included mice fed a normal diet (“healthy liver”), untreated CDAHFD-induced mice (“untreated”), or CDAHFD-induced mice transduced with a control vector (“control Mɸ”).

[0061] Figure 32 This diagram illustrates the timelines used to generate and formulate engineered human macrophages overexpressing TIM4. On day 0, primary human monocytes were thawed and kept either untransduced (UTD; control Mɸ) or transduced with a lentivirus encoding human TIM4 (hu. TIM4 Mɸ). Cells were differentiated for 5 days in the presence of GM-CSF. On day 5, cells were collected, viability and TIM4 expression were analyzed, and cells were formulated for in vivo injection.

[0062] Figures 33A-33BThis is a graph showing the expression and activity levels of engineered human macrophages that overexpress TIM4. Figure 33A Flow cytometry analysis showing TIM4 expression. Figure 33B This demonstrates the activity of engineered human macrophages expressing TIM4.

[0063] Figure 34 It is used for the NSG described in Example 8 of this document. TM A series of schematic diagrams showing the time points at which anti-fibrotic engineered human macrophages were generated and administered in a mouse model of liver fibrosis.

[0064] Figures 35A-35B It is an NSG that shows CDAHFD-induced liver fibrosis. TM Figures and images of collagen content in tissue sections of a mouse model treated with TIM4-transduced engineered human macrophages as described in Example 8 of this document. Figure 35A This displays the quantification of collagen as a percentage of area in histological images. Figure 35B Showing from Figure 35A Representative Masson tricolor images of the data were plotted. These mice were compared to controls, including NSG mice fed a normal diet. TM Mice (healthy livers), NSG not treated with macrophages TM CDAHFD-induced mice (untreated) or NSG treated with UTD macrophages TM CDAHFD-induced mice (control Mɸ).

[0065] Figures 36A-36B It is a series of graphs showing NSG TM A CDAHFD-induced liver fibrosis model in mice was measured using picric Sirius red (BSRS) from engineered human macrophages transduced with TIM4 as described in Example 8 of this paper. Figure 36A ) and IHC images ( Figure 36B These mice were compared to a control group, which included NSG mice fed a normal diet. TM Mice (healthy livers), NSG not treated with macrophages TM CDAHFD-induced mice (untreated) or NSG treated with UTD macrophages TM CDAHFD-induced mice (control Mɸ).

[0066] Figures 37A-37B It is a series of graphs showing NSG TM A CDAHFD-induced liver fibrosis model in mice was measured using type 1 collagen α1 (Col1a1) treated with TIM4-transduced engineered human macrophages as described in Example 8 of this paper. Figure 37A ) and IHC images ( Figure 37B These mice were compared to a control group, which included NSG mice fed a normal diet. TM Mice ("healthy livers"), untreated NSG TM CDAHFD-induced mice (“untreated”) or NSG transduced with a control vector TM CDAHFD-induced mice (“control Mɸ”).

[0067] Figures 38A-38B It is a series of graphs showing NSG TM A CDAHFD-induced liver fibrosis model in mice was measured using α-smooth muscle actin (α-SMA) from engineered human macrophages transduced with the TIM4 construct described in Example 8 of this paper. Figure 38A ) and IHC images ( Figure 38B These mice were compared to controls, which included mice fed a normal diet (“healthy livers”) and untreated NSG. TM CDAHFD-induced mice (“untreated”) or NSG transduced with a control vector TM CDAHFD-induced mice (“control Mɸ”).

[0068] Figures 39A-39B It is a series of graphs showing NSG TM A CDAHFD-induced liver fibrosis model in mice was measured using osteopontin (Opn; also referred to herein as “secreted phosphoprotein 1” and “SPP1” interchangeably) in engineered human macrophages transduced with the TIM4 construct described in Example 8 of this document. Figure 39A ) and IHC images ( Figure 39B ). These NSGs TM Mice were compared with controls, including NSG mice fed a normal diet. TM Mice ("healthy livers"), untreated NSG TM CDAHFD-induced mice (“untreated”) or NSG transduced with a control vector TM CDAHFD-induced mice (“control Mɸ”).

[0069] Figure 40 It is displayed from NSG TM A figure showing in situ analysis of macrophage burial in the liver of CDAHFD-induced mice treated with engineered macrophages transduced with a TIM4 construct. These NSGs... TM Mice and untreated NSG TM CDAHFD-induced mice (“untreated”) or NSG transduced with a control vector TMThe CDAHFD-induced mice (“control Mɸ”) were compared.

[0070] Figure 41 This diagram shows a time point of treatment with engineered macrophages described in this paper at week 8, used to generate a CDAHFD-induced model of advanced liver fibrosis in mice.

[0071] Figures 42A-42D This is a series of graphs showing the effect of choline deficiency, L-amino acid-restricted high-fat diet (CDAHFD)-induced advanced liver fibrosis on body weight ( Figure 42A ), liver weight ( Figure 42B ), liver to body weight ratio ( Figure 42C ) and blood glucose levels measured after 4 hours of fasting ( Figure 42D The effects of treatment with UTD macrophages (control Mɸ), TIM4 macrophages (TIM4 Mɸ), or TIM4+ relaxin macrophages (TIM4+Rel.Mɸ) on these indicators were investigated.

[0072] Figures 43A-43B This is a series of figures showing the picric Sirius red assay in a mouse model of CDAHFD-induced advanced liver fibrosis treated with engineered macrophages transduced with TIM4 or TIM4+ relaxin as described in Example 9 of this document. Figure 43A ) and IHC images ( Figure 43B These mice were compared with controls, which included mice fed a normal diet (healthy livers), CDAHFD-induced mice not treated with macrophages (untreated), or CDAHFD-induced mice treated with UTD macrophages (control Mɸ).

[0073] Figure 44 This diagram illustrates the time points used to generate a CDAHFD-induced liver fibrosis model in mice and to begin treatment with LNP-TIM4 at week 4. Mice were treated with TIM4-LNP (1 mg / kg dose) intravenously (“IV”) or as a solvent control. Mice received a total of 3 doses, spaced 3–4 days apart.

[0074] Figure 45 A graph showing mouse body weight throughout the study described in Example 10 of this article. Error bars represent mean ± SEM. The green vertical line indicates the first IV LNP-TIM4 / solvent treatment.

[0075] Figures 46A-46F A series of figures are shown depicting the serum toxicity marker ALP (Alanine Alpha P) in CDAHFD-induced mice treated with LNP-TIM4 at the study endpoint. Figure 46A ), AST ( Figure 46B ), ALT Figure 46C ), bile acids ( Figure 46D ), triglycerides ( Figure 46E ), or creatine ( Figure 46F These mice were compared to controls, which included mice fed a normal diet (“feed solvent”) and CDAHFD-induced mice transduced with a control vector (“CDAHFD solvent”).

[0076] Figures 47A-47C A series of figures are shown depicting collagen, a histological marker of fibrosis, at the study endpoint in CDAHFD-induced mice treated with LNP-TIM4. Figure 47A ), αSMA ( Figure 47B ) and osteopontin ( Figure 47C Quantification of the results. These mice were compared with controls, which included mice fed a normal diet (“feed solvent”) and CDAHFD-induced mice transduced with a control vector (“CDAHFD solvent”).

[0077] definition To facilitate understanding of this invention, certain terms are first defined below. Additional definitions for the following terms and others are set forth throughout this specification. Publications and other references cited herein to describe the background of the invention and provide additional details on its practice are incorporated herein by reference.

[0078] The articles “a” and “an” are used in this text to refer to one or more grammatical objects (i.e., at least one). For example, “element” means one or more elements.

[0079] Approximately or about As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to the reference value. In some embodiments, the term "approximately" or "about" refers to a value falling within a range of 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 of the reference value, unless otherwise stated or otherwise clearly apparent from the context (except where such figures exceed 100% of the possible value).

[0080] activationAs used herein, the term “activation” refers to a state in which cells (e.g., monocytes, macrophages, or dendritic cells) have been adequately stimulated to induce detectable cell proliferation or to perform their effector functions. Activation can also be associated with induced cytokine production, cytokine secretion, phagocytosis, cell signaling (e.g., changes in gene expression), target cell killing, metabolic changes, production of inflammatory mediators, proliferation, epigenetic reprogramming, macrophage phenotypic switching (e.g., M1 polarization), inhibition 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 monocyte / macrophage / dendritic cell" refers, among other things, a monocyte / macrophage / dendritic cell that is undergoing cell division or performing effector functions. The term "activated monocyte / macrophage / dendritic cell" also refers, among other things, a cell that is performing effector functions or exhibiting any activity not observed in the resting state, including phagocytosis, cytokine secretion, proliferation, changes in gene expression, metabolic changes, production of inflammatory mediators, epigenetic reprogramming, macrophage phenotypic switching (e.g., M1 polarization), suppression of pro-tumorigenic or M2 macrophages, and other functions.

[0082] Agent As used herein, the term "particle" (or "biological agent" or "therapeutic agent") refers to a molecule that can be expressed, released, secreted, or delivered to a target by modified immune cells as described herein. Particles include, but are not limited to, nucleic acids, antibiotics, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, microsomes, derivatives or variants thereof, formulations or compositions including one or more of these, and any combination thereof. Particles can bind to any cellular part, such as receptors, antigenic determinants, or other binding sites presented on a target or target cell. Particles can diffuse or translocate into cells and can act intracellularly therein. For example, a particle can be an exogenous fibrolytic agent as described herein. A particle can be an exogenous anti-inflammatory agent as described herein. A particle can be an exogenous regenerative agent as described herein. A particle can be an exogenous cytotoxic agent as described herein.

[0083] AntibodyAs used herein, the term "antibody" refers to a polypeptide comprising typical immunoglobulin sequence elements sufficient to confer specific binding to a specific target antigen. As is known in the art, naturally occurring complete antibodies are tetrameric substances of approximately 150 kDa, comprising two identical heavy-chain polypeptides (approximately 50 kDa each) and two identical light-chain polypeptides (approximately 25 kDa each), associated with each other in a structure commonly referred to as a "Y-shape." Each heavy chain comprises at least four domains (each approximately 110 amino acids long)—an amino-terminal variable (VH) domain (located at the tip of the Y-shape), followed by three constant domains: CH1, CH2, and a carboxyl-terminal CH3 (located at the base of the Y-stem). A short region called the "switch" connects the heavy-chain variable and constant domains. A "hinge region" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region link the two heavy-chain polypeptides together in the complete antibody. Each light chain comprises two domains—an amino-terminal variable (VL) domain followed by a carboxyl-terminal constant (CL) domain, separated from each other by another “transition region.” A complete antibody tetramer consists of two heavy-chain-light-chain dimers, where the heavy and light chains are linked together by a single disulfide bond; two additional disulfide bonds link the heavy-chain hinge regions together, causing the dimers to link together and form a tetramer. Naturally occurring antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by “immunoglobulin folding,” formed by two β-sheets (e.g., 3-, 4-, or 5-chain sheets) stacked on top of each other in compressed antiparallel β-barrels. Each variable domain comprises three hypervariable loops called “complementarity-determining regions” (CDR1, CDR2, and CDR3) and four relatively invariant “framework” regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR region forms a β-sheet that provides a structural framework for the domain, and the CDR loop regions from both the heavy and light chains aggregate in three-dimensional space, creating a single hypervariable antigen-binding site at the tip of the Y structure. The Fc region of a naturally occurring antibody binds elements of the complement system, as well as receptors on effector cells, including, for example, effector cells mediating cytotoxicity. The affinity and / or other binding properties of the Fc region to Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, the antibody produced and / or utilized includes a glycosylated Fc domain, including a modified or engineered glycosylated Fc domain. In some embodiments, any polypeptide or polypeptide complex including a sufficient sequence of immunoglobulin domains as found in natural antibodies may be referred to as and / or used as an "antibody," regardless of whether such polypeptides are naturally occurring (e.g., generated by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is monoclonal.In some embodiments, the antibody has a constant region sequence specific to mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence element is humanized, primate-based, chimeric, etc., as known in the art. Furthermore, as used herein, the term "antibody" in appropriate embodiments (unless otherwise stated or clear from the context) may refer to any construct or form known or developed in the art for utilizing the structural and functional characteristics of an antibody in an alternative presentation. For example, in some embodiments, the antibody utilized according to the methods and compositions described herein is selected from, but not limited to, the following forms: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masking antibodies (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs"). TM "); single-chain or tandem biantibodies (TandAb®); VHH; Anticalins®; Nanobodies® microantibodies; BiTE®; ankylosing repeats or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®, Centryrins®; and KALBITOR®. In some embodiments, the antibody may lack the covalent modifications (e.g., glycan attachment) that it would have naturally. In some embodiments, the antibody may contain covalent modifications (e.g., glycan attachment, payload [e.g., detectable portion, therapeutic portion, catalytic portion, etc.] or other side groups [e.g., polyethylene glycol, etc.]).

[0084] antibody fragments As used herein, the term "antibody fragment" refers to a portion of a complete antibody and to the antigenic determination variable region of the complete 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 their human and humanized versions.

[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 native conformation.

[0086] Antibody light chainAs 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 native conformation.

[0087] antigen As used herein, the term "antigen" or "Ag" refers to a molecule capable of evoking an immune response. This immune response may involve antibody production, activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can act as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA comprising a nucleotide sequence or a portion thereof encoding a protein that evokes an immune response encodes an "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen is not necessarily encoded solely by the full-length nucleotide sequence of a gene. It is apparent that this 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 evoke a desired immune response. Furthermore, those skilled in the art will understand that an antigen is not necessarily encoded by a "gene." It is apparent that antigens can be generated synthetically or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0088] self As used in this article, the term “self” refers to any material that originates from an organism and will later be reintroduced into the same organism.

[0089] Allogeneic As used in this article, the term "allogeneic" refers to a graft (e.g., a cell population) derived from different animals of the same species.

[0090] Alien As used in this article, the term "heterogeneous" refers to a graft (e.g., a cell population) derived from an animal of a different species.

[0091] Conservative sequence modificationAs used herein, the term "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter at least one property or characteristic (e.g., antibody binding characteristics) of a specific protein comprising an amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies adapted to various embodiments using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are identified 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the antibody CDR region can be replaced with other amino acid residues from the same side chain family, and the modified antibody's ability to bind antigens can be tested using the functional assays described herein.

[0092] Effective amount As used herein, the terms "effective amount" and "therapeutic effective amount" are used interchangeably to refer to the amount of a compound, formulation, material, or composition described herein that effectively achieves a specific biological outcome or provides a therapeutic or preventative benefit. Such outcomes may include, but are not limited to, antitumor activity determined by any means appropriate in the art.

[0093] Effector function As used herein, "effector function" or "effector activity" refers to the specific activities performed by an immune cell in response to stimulation by that immune cell. For example, macrophages exhibit effector function by phagocytizing and digesting cell debris, foreign bodies, microorganisms, cancer cells, and other unhealthy cells.

[0094] codingAs used herein, “encoding” refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (e.g., gene, cDNA, or mRNA) for use as a template in biological processes to synthesize other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing) and the non-coding strand (used as a template for gene or cDNA transcription) can be referred to as the protein or other product encoding that gene or cDNA.

[0095] endogenous As used in this article, “endogenous” means any material that is derived from or produced within a specific organism, cell, tissue, or system.

[0096] exogenous As used herein, the term “exogenous” means any material introduced from or produced outside of a specific organism, cell, tissue, or system.

[0097] Amplification As used herein, the term "amplification" refers to an increase in quantity, such as an increase in the number of monocytes / macrophages. In one embodiment, the number of monocytes, macrophages, or dendritic cells expanded in vitro is increased relative to the number initially present in the culture. In another embodiment, the number of monocytes, macrophages, or dendritic cells expanded in vitro is increased relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells that have been removed from a living organism (e.g., a human) and proliferated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

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

[0099] expression carrierAs used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide including an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be supplied by a host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as viscera, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses (e.g., Ad5f35), incorporating the recombinant polynucleotide.

[0100] fibrosis As used herein, the term "fibrosis" refers to the formation of fibrous tissue as a reparative and / or reactive process (rather than as a normal component of cells, tissues, or organs). Fibrosis is characterized by the accumulation of fibroblasts and collagen deposition exceeding the normal deposition in any particular tissue. Fibrotic diseases, conditions, and conditions include, but are not limited to, fibrosis resulting from wound healing, systemic and localized scleroderma, atherosclerosis, restenosis, lung inflammation, idiopathic pulmonary fibrosis, interstitial lung disease, cirrhosis, fibrosis resulting from chronic hepatitis B or C infection, kidney diseases (e.g., glomerulonephritis), heart diseases resulting from scar tissue, keloids, and hypertrophic scars, and eye diseases (e.g., macular degeneration, retinopathy, and vitreoretinopathy). Fibrotic diseases, conditions, and conditions can also be liver-related, such as those resulting from the accumulation of cholesterol and / or triglycerides within hepatocytes, which may lead to a pro-inflammatory response that causes liver fibrosis and / or cirrhosis. Liver diseases with fibrotic components include, but are not limited to, non-alcoholic fatty liver disease (NAFLD) (e.g., non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)) or alcoholic liver diseases (e.g., alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH)). Fibrotic diseases, conditions, and illnesses may include mechanical trauma, biliary obstruction, autoimmune hepatitis, iron overload, hepatitis B infection (HBV), and / or hepatitis C infection (HCV).

[0101] homologyAs used herein, the term "homology" refers to the overall relevance between polymer molecules, such as between nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered "homological" 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, polymer molecules are considered "homological" 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 relevant chemical properties at corresponding positions). As those skilled in the art will understand, various algorithms are available for comparing sequences to determine their degree of homology, including allowing a gap of a specified length in one sequence relative to another when considering which residues in different sequences "correspond" to each other. The percentage of homology between two nucleic acid sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In some embodiments, the length of the 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. 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, 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, the molecules are similar at that position. The percentage of homology between 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] identityAs used herein, the term "identity" refers to the subunit sequence identity between two polymer molecules, and more particularly between two amino acid molecules (e.g., two polypeptide molecules). Two amino acid sequences are identical when they have the same residues at the same positions; for example, if one position in each of two polypeptide molecules is occupied by arginine, then they are identical at that position. The degree of identity, or similarity, of two amino acid sequences having the same residues at the same positions in alignment is typically expressed as a percentage. Identity between two amino acid sequences is a direct function of the number of matched or identical positions; for example, if half the positions in two sequences (e.g., five positions out of ten amino acid lengths in a polymer) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., nine out of ten) are matched or identical, then the two amino acid sequences are 90% identical.

[0103] Substantive identity As used herein, the term "substantially identical" refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered "substantially identical" if they contain the same residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can 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 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 corresponding residues are identical in the relevant residue segment. In some embodiments, the relevant segment is a complete sequence. In some embodiments, the relevant segment 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 "substantially identical" generally means that the CDR has an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the reference CDR.

[0104] inflammationAs used herein, the term "inflammation" refers to a cellular, physiological, and / or immune response to a harmful stimulus, including but not limited to injury, immune response, infection, altered endogenous substances, defective endogenous pathways, and / or foreign substances. Inflammation can be systemic or localized. Inflammation can be acute or chronic. Exemplary inflammatory conditions, diseases, and illnesses include, but are not limited to, inflammatory conditions, diseases, and illnesses of the liver, gastrointestinal tract, lungs, skin, cardiovascular system, nervous system, kidneys, pancreas, joints, eyes, and / or endocrine system. Inflammatory diseases, conditions, or illnesses may be associated with toxins, trauma (e.g., environmental hazards such as asbestos, coal dust, and / or polycyclic aromatic hydrocarbons) or smoking) and / or medical treatments (e.g., surgery). Inflammatory diseases, conditions, or illnesses may be or include autoimmune diseases.

[0105] Immunoglobulins 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 called BCRs (B cell receptors) or antigen receptors. This class of proteins includes five members: IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody found in bodily fluid secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions from the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most subjects. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in the defense against bacteria and viruses. IgD is an immunoglobulin without a known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by inducing the release of mediators from mast cells and basophils upon exposure to allergens.

[0106] Separate As used herein, the term "isolated" refers to something altered or removed from its native state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," but the same nucleic acid or peptide that is partially or completely separated from its native coexisting material is "isolated." Isolated nucleic acids or proteins may exist in substantially purified forms or may exist in non-native environments such as host cells.

[0107] Lentiviral As used herein, the term "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses because they are capable of infecting non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are examples of lentiviruses. Lentiviral-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0108] Modified As used herein, the term "modified" refers to the altered state or structure of molecules or cells disclosed herein. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by introducing nucleic acids.

[0109] adjust As used herein, the term "modulation" refers to a detectable increase or decrease in response level in a subject compared to response level in a subject without treatment or the compound, and / or compared to response level in otherwise identical but untreated subjects. This term encompasses perturbations and / or influences on natural signals or responses that mediate a beneficial therapeutic response in a subject (preferably a human).

[0110] Operable connection As used herein, the term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, when a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operably linked to that coding sequence. Typically, the operably linked DNA sequences are contiguous and located in the same reading frame when necessary for binding two protein-coding regions.

[0111] Polynucleotides As used herein, the term "polynucleotide" refers to a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, the terms nucleic acid and polynucleotide are used interchangeably herein. It is common knowledge to those skilled in the art that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant methods, i.e., using conventional cloning techniques and PCR. TM Nucleic acid sequences are cloned from recombinant libraries or cell genomes, and synthesized.

[0112] polypeptideAs used herein, the term "peptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, the peptide has a naturally occurring amino acid sequence. In some embodiments, the peptide has a non-naturally occurring amino acid sequence. In some embodiments, the peptide has an engineered amino acid sequence, i.e., it is designed and / or generated through artificial means. In some embodiments, the peptide may include natural amino acids, non-natural amino acids, or both, or consist of them. In some embodiments, the peptide may include only natural amino acids or only non-natural amino acids, or consist of them. In some embodiments, the peptide may include D-amino acids, L-amino acids, or both. In some embodiments, the peptide may include only D-amino acids. In some embodiments, the peptide may include only L-amino acids. In some embodiments, the peptide may include one or more side groups or other modifications, such as modifications or attachments to one or more amino acid side chains at the N-terminus, C-terminus, or any combination thereof. In some embodiments, such side groups or modifications may be selected from acetylation, amidation, esterification, methylation, polyethylene glycolation, etc., including combinations thereof. In some embodiments, the peptide may be cyclic, and / or may include a cyclic portion. In some embodiments, the polypeptide is not cyclic and / or does not include any cyclic portion. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or include pinned polypeptides. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to a polypeptide that shares a relevant activity or structure and is therefore considered a member of the same class or family of polypeptides. For each class, exemplary polypeptides within that class are provided in this specification and / or will be known to those skilled in the art, whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides of a polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of that class (and with all polypeptides within that class in some embodiments), share common sequence motifs (e.g., characteristic sequence elements), and / or share common activities (in some embodiments, at comparable levels or within specified ranges). For example, in some embodiments, the member polypeptide shows an overall degree of sequence homology or identity of at least about 30-40% with respect to the reference polypeptide, and is typically 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, which in some embodiments may be or include a characteristic sequence element) that shows a very high sequence identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99%.Such conserved regions typically encompass at least 3-4 and usually up to 20 or more amino acids; in some embodiments, the conserved region encompasses at least a segment of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids. In some embodiments, the useful polypeptide may include or consist of fragments of the parent polypeptide. In some embodiments, the useful polypeptide may include or consist of multiple fragments, each found in the same parent polypeptide, but arranged spatially with respect to each other differently from those seen in the polypeptide of interest (e.g., fragments directly linked in the parent may be spatially separated in the polypeptide of interest, and vice versa, and / or fragments may be present in the polypeptide of interest in a different order than in the parent), such 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 together by peptide bonds). Proteins may include portions other than amino acids (e.g., glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those skilled in the art will understand that a "protein" can be a complete polypeptide chain produced by a cell (with or without a signal sequence), or it can be a characteristic portion thereof. Those skilled in the art will understand that proteins may sometimes include more than one polypeptide chain, for example, linked by one or more disulfide bonds or otherwise associated. A polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein may include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides 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 length. In some embodiments, a protein is an antibody, an antibody fragment, its biologically active portion, and / or a characteristic portion thereof.

[0114] SubjectsAs used herein, the term "subject" refers to an organism, such as a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, gerbil, cat, dog). In some embodiments, the human subject is an adult, adolescent, or pediatric subject. In some embodiments, the subject has a disease, symptom, or condition, such as a disease, symptom, or condition that can be treated as provided herein, such as cancer or tumor listed herein. In some embodiments, the subject is susceptible to a disease, symptom, or condition; in some embodiments, susceptible subjects have a predisposition to develop a disease, symptom, or condition and / or show an increased risk (compared to the average risk observed in a reference subject or population). In some embodiments, the subject displays one or more symptoms of a disease, symptom, or condition. In some embodiments, the subject does not display specific symptoms (e.g., clinical manifestations of a disease) or characteristics of a disease, symptom, or condition. In some embodiments, the subject does not display any symptoms or characteristics of a disease, symptom, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is receiving and / or has received diagnostic and / or therapeutic treatments.

[0115] Essentially purified As used herein, the term "substantially purified," for example when applied to cells, refers to cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been isolated from other cell types normally associated with their natural state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term refers only to cells that have been isolated from cells naturally associated with their natural state. In some embodiments, cells are cultured in vitro. In other embodiments, cells are not cultured in vitro.

[0116] Therapeutic As used herein, the term "therapeutic" refers to treatment and / or prevention. Therapeutic effects are achieved through the suppression, relief, or eradication of a disease state.

[0117] Transfected As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0118] treatAs used herein, the term "treat" (or "treatment") refers to the partial or complete reduction, improvement, delay of onset, suppression, prevention, relief, and / or decrease in the incidence and / or severity of one or more symptoms or features of a disease, condition, and / or illness (e.g., a disease, condition, and / or illness 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, condition, and / or illness (e.g., it may be preventative). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or features of a disease, condition, and / or illness, for example, to reduce the risk of developing a pathology associated with the disease, condition, and / or illness. In some embodiments, treatment may be administered to a subject who exhibits established, severe, and / or late signs of a disease, condition, or illness.

[0119] carrier As used herein, the term "vector" refers to a composition comprising isolated nucleic acids and capable of delivering the isolated nucleic acids into the cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0120] Throughout this disclosure, various aspects of the invention can be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the scope description should be considered as specifically disclosing all possible sub-scopes and the individual values ​​within those scopes. For example, a scope description such as 1 to 6 should be considered as specifically disclosing sub-scopes, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within those scopes, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope. Detailed Implementation

[0121] immune cells Among other things, this disclosure provides modified immune cells (e.g., macrophages, monocytes, or dendritic cells) that include encoding one or more nucleic acid sequences of the following: at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). Modified immune cells (e.g., macrophages, monocytes, or dendritic cells) may include or express at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein).

[0122] As used herein, the term "immune cell" refers to cells that participate in an immune response (e.g., promote 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. Immune cells (e.g., macrophages, monocytes, or dendritic cells) may be obtained from the subject.

[0123] In some embodiments, the provided methods and / or compositions include a population of immune cells. In some embodiments, the immune cell population as described herein includes monocytes, macrophages, dendritic cells, and / or their precursors. In some embodiments, the immune cell population includes a purified population or cell line of monocytes, macrophages, or dendritic cells.

[0124] In some embodiments, the immune cells are activated, for example, exhibiting increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and / or antigen presentation, relative to inactive cells. In some embodiments, activated immune cells exhibit changes in gene expression, such as 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), relative to inactive cells. In some embodiments, activated immune cells are undergoing cell division.

[0125] In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are obtained from (e.g., isolated from) the subject. Immune cells can be autologous or derived from allogeneic or universal donors. Cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In some embodiments, cells can be obtained from blood units collected from the subject using any number of isolation techniques known to those skilled in the art (e.g., Ficoll isolation). In some embodiments, cells from the subject's circulating blood are obtained via apheresis or leukocyte apheresis. Cells collected via apheresis may be washed to remove plasma fractions and resuspended in various buffers (e.g., phosphate-buffered saline (PBS)) or culture media. In some embodiments, enrichment of immune cells (e.g., monocytes) includes plastic adhesion. In some embodiments, differentiation of immune cells (e.g., monocytes) after enrichment includes stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and / or erythrocytes), such as a leukocyte apheresis composition (e.g., leukopak), is used for enrichment. In some embodiments, the leukocyte apheresis composition (e.g., leukopak) comprises a sample from a healthy human donor. In some embodiments, the apheresis of immune cells (e.g., monocytes) is followed by mobilization with GM-CSF. In some embodiments, the selection of immune cells (e.g., monocytes) includes CD14-positive selection using microbeads (e.g., MACS® MicroBeads on the CliniMACS Prodigy device).

[0126] In some embodiments, immune cell precursors (e.g., precursors of macrophages, monocytes, or dendritic cells) are used in the compositions and methods described herein. Immune cell precursors can differentiate into immune cells in vivo or in vitro. Non-limiting examples of precursor immune cells include hematopoietic stem cells, myeloid progenitor cells, myeloid cells, monocytes, premonocytes, or intermediates thereof. For example, induced pluripotent stem cells can be used to generate monocytes, macrophages, and / or dendritic cells. Induced pluripotent stem cells (iPSCs) can be derived from normal human tissues, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs can differentiate into myeloid cells (e.g., monocytes, macrophages, dendritic cells, or precursors thereof).

[0127] Immune cells such as those described herein (e.g., macrophages, monocytes, or dendritic cells) can be isolated from peripheral blood, for example by lysing erythrocytes and depleting lymphocytes and erythrocytes, for example by PERCOLL TMGradient centrifugation. Alternatively, immune cells can be isolated from umbilical cord tissue. Specific subsets of immune cells can be further isolated using positive or negative selection techniques. In some embodiments, cells expressing specific antigens (including, but not limited to, CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20) can be removed from the immune cells. In some embodiments, enrichment of the immune cell population, for example by negative selection, can be accomplished using a combination of antibodies targeting surface markers specific to the negatively selected cells. By way of non-limiting examples, cell selection may also include negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells.

[0128] During the separation of desired immune cell populations (e.g., macrophages, monocytes, or dendritic cells) as described herein through positive or negative selection, immune cell concentrations and surface areas (e.g., granules, such as beads) can vary. It may be desirable to significantly reduce the volume of beads and cells mixed together to ensure maximum contact area between cells and beads.

[0129] macrophages Among other things, this disclosure provides macrophages that include encoding one or more nucleic acid sequences of the following: at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). Macrophages are immune cells specifically designed to detect, engulf, and destroy target cells (e.g., pathogens or tumor cells) and to initiate, maintain, and resolve tissue repair processes. Macrophages are effective contributors to tissue repair, including by producing a variety of growth factors, including, for example, PDGF, IGF-1, TGF-β1, and VEGF-α. In some embodiments, macrophages include or express resolvins (e.g., resolvin D and / or resolvin E). In some embodiments, macrophages include or express osteopontin. In some embodiments, macrophages exhibiting the M2 or M0 phenotype are particularly advantageous when used in the methods and / or compositions covered by this disclosure.

[0130] In some embodiments, macrophages include either undifferentiated macrophages or M0 macrophages. In some embodiments, macrophages include or express 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 can be identified by macrophage phenotypic markers, cytokine production, and / or chemokine secretion.

[0131] In some implementations, macrophages include or are polarized macrophages. Under classic activation conditions, M0 macrophages can be exposed to pro-inflammatory signals such as LPS, IFNγ, and GM-CSF and polarize into M1 macrophages. Typically, 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 diverse populations of "alternative activated" or M2 macrophages.

[0132] In some embodiments, the macrophages include or are M1 macrophages. In some embodiments, the macrophages express one or more markers of M1 macrophages (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen of the following: CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD16, IL1R, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen of the following: IFIT family members or ISG family members).

[0133] In some embodiments, the macrophages include or are M2 macrophages (e.g., M2A, M2B, M2C, or M2D macrophages). M2A macrophages may be induced by IL-4, IL-13, and / or fungal infection. M2B macrophages may be induced by IL-1R ligands, immune complexes, and / or LPS. M2C macrophages may be induced by IL-10 and / or TGFβ. M2D macrophages may be induced by IL-6 and / or adenosine. In some embodiments, the macrophages express one or more markers of M2 macrophages (e.g., one, two, or three of CD206, CD163, or CD209).

[0134] In some embodiments, macrophages include 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 macrophages. In some embodiments, at least one M1 marker (e.g., HLA DR, CD86, CD80, PD-L1, CD83, CD69, MHC I, CD64, CD32, CD16, IL1R, IFIT family members, and / or ISG family members) is downregulated in macrophages.

[0135] Monocytes Among other things, this disclosure provides monocytes comprising encoding one or more nucleic acid sequences of the following: at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). Monocytes are pluripotent cells circulating in the blood, bone marrow, and spleen, and typically do not proliferate at homeostasis. The size of monocytes can vary significantly in diameter from about 10 to 30 μm. The nucleocytoplasmic ratio of monocytes can range from about 2:1 to about 1:1. Typically, monocytes include chemokine receptors and pathogen recognition receptors, which mediate migration from the blood to tissues, such as during wound healing or infection. Monocytes can produce inflammatory and / or anti-inflammatory cytokines, take up cellular and / or toxic molecules, and differentiate into dendritic cells or macrophages.

[0136] In some implementations, monocytes include or express one or more phenotypic markers. Exemplary phenotypic markers for human monocytes 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, CD87, CD89, CD91, CDw92, CD93, CD11b, CD11c, CD12, CD13, CD15, CD15, CD16, CD17, CD18, CD19b, CD11c, CD19c, CD19b, CD19c ... 93, 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. Exemplary phenotypic markers for mouse monocytes 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 some embodiments, monocytes include one, two, or three of CD11b, CD14, or CD16. In some embodiments, monocytes include CD14+CD16- monocytes, CD14+CD16+ monocytes, or CD14-CD16+ monocytes.

[0137] In some embodiments, monocytes differentiate into macrophages. In some embodiments, monocytes differentiate into dendritic cells (DCs). Monocytes can differentiate into macrophages or DCs using 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 a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4.

[0138] dendritic cells Among other things, this disclosure provides dendritic cells (DCs) comprising encoding one or more of the following nucleic acid sequences: at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). DCs are specialized antigen-presenting cells derived from the bone marrow, involved in initiating immune responses and maintaining the immune system's tolerance to self-antigens. Dendritic cells can be found in lymphatic and non-lymphatic organs and are generally considered to originate from lymphatic or myeloid lineages.

[0139] In some implementations, DC includes or expresses one or more phenotypic markers. Exemplary phenotypic markers of DC include, but are not limited to, CD11c, CD83, CD1a, CD1c, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286, and portions of CD206, CD207, CD208, and CD209.

[0140] Immature dendritic cells (DCs) are characterized by high antigen capture capacity but relatively low T-cell stimulation capacity. Inflammatory mediators promote DC maturation. In some embodiments, DCs include or are immature DCs. In other embodiments, DCs include or are mature DCs.

[0141] Modified immune cells In some embodiments, modified immune cells, such as modified macrophages, monocytes, or dendritic cells, are generated by expressing at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). This disclosure also provides immune cells comprising nucleic acid sequences (e.g., isolated nucleic acid sequences) encoding at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). In some embodiments, the immune cells are monocytes, macrophages, or dendritic cells expressing 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 cytotoxic agent.

[0142] Exogenous fiber pyrolysis agent As used herein, the term "exogenous fibrolytic agent" refers to an exogenous molecule that can be expressed, released, secreted, or delivered (e.g., via modified immune cells as described herein, such as monocytes, macrophages, and / or dendritic cells) to affect fibrotic targets. Fibrotic targets can cause or originate from the formation of fibrotic tissue and include any biochemical, cellular, or physiological proteins associated with fibrosis. Fibrotic targets can be associated with any fibrotic condition, disease, or symptom (e.g., the fibrotic condition, disease, or symptom described herein). Exogenous fibrolytic agents may include, but are not limited to, peptides, fusion proteins, nucleic acids, antibiotics, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, and / or microsomes, or any derivatives or variants of any of the foregoing.

[0143] In some embodiments, the exogenous fibrinolytic agent includes a relaxin peptide, a core proteoglycan peptide, a dominant-negative TGF-β receptor 2 (DN TGFβR2) peptide, a soluble TGFβ receptor, and / or a TGFβ switch receptor. In some embodiments, the exogenous fibrinolytic agent includes a relaxin peptide. In some embodiments, the exogenous fibrinolytic agent includes a core proteoglycan peptide. In some embodiments, the exogenous fibrinolytic agent includes a DNTGFβR2 peptide. In some embodiments, the exogenous fibrinolytic agent includes a soluble TGFβ receptor. In some embodiments, the exogenous fibrinolytic agent includes a TGFβ switch receptor.

[0144] In some embodiments, the exogenous fibrinolytic agent is retained on the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells). In some embodiments, the exogenous fibrinolytic agent is secreted from the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells).

[0145] In some embodiments, the exogenous fibrinolytic agent is tethered to the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein by means of fusion with 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, the hinge domain includes, or may be, a CD8a, IgG4, or CD28 hinge domain. In some embodiments, the transmembrane domain includes, or is not limited to, 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-ζ, 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 domains. In some embodiments, the exogenous fibrinolytic agent is tethered to the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein via short linkers (e.g., glycine-rich and / or serine-rich linkers). In some embodiments, the linker comprises (GGGGS)n, where n = 1, 2, 3, 4, or 5, or alternative versions thereof, such as (SGGGG)n, where n = 1, 2, 3, 4, or 5.

[0146] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrolytic agent, as described herein, reduce or prevent the activation of hepatic stellate cells, for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrolytic agent, as described herein, improve tissue regeneration and / or regression (e.g., liver tissue regeneration and / or regression), for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrolytic agent, as described herein, balance pro-fibrotic and anti-fibrotic immune cell populations (e.g., macrophage populations), for example, relative to unmodified immune cells of the same type.

[0147] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrinolytic agent, as described herein, reduce or prevent fibrosis (e.g., liver fibrosis), for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrinolytic agent, as described herein, reduce or prevent tissue damage (e.g., liver tissue damage), for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrinolytic agent, as described herein, lower the levels of alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST), for example, relative to unmodified immune cells of the same type.

[0148] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrinolytic agent, as described herein, reduce or prevent hepatocellular inflammation (e.g., leading to decreased levels of NF-κB, IL-1β, IL-2, MCP-1, and / or MIP-1), for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrinolytic agent, as described herein, result in increased levels or activity of anti-inflammatory agents (e.g., IL-10, IL-2, and / or CCL18), for example, relative to unmodified immune cells of the same type.

[0149] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous fibrolytic agent, as described herein, reduce or prevent steatosis (e.g., hepatic steatosis), for example, relative to unmodified immune cells of the same type.

[0150] Exogenous anti-inflammatory agents As used herein, the term "exogenous anti-inflammatory agent" refers to an exogenous molecule that can be expressed, released, secreted, or delivered (e.g., via modified immune cells (e.g., monocytes, macrophages, and / or dendritic cells) as described herein) to an inflammatory target. Inflammatory targets can elicit or originate from biochemical, cellular, or physiological responses to harmful stimuli, including but not limited to injury, immune responses, infection, altered endogenous substances, defective endogenous pathways, and / or exogenous substances. In some embodiments, inflammatory targets are or include proteins. Exogenous anti-inflammatory agents can include, but are not limited to, peptides, fusion proteins, nucleic acids, antibiotics, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, and / or microsomes, or any derivatives or variants of any of the foregoing.

[0151] In some embodiments, the exogenous anti-inflammatory agent includes or is a cytokine. In some embodiments, the cytokine includes or is IL-10, IL-22, and / or IL-27. In some embodiments, the exogenous anti-inflammatory agent includes or is a cytokine receptor. In some embodiments, the cytokine receptor includes or is an IL-17A on / off receptor.

[0152] In some embodiments, the exogenous anti-inflammatory agent is retained on the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells). In some embodiments, the exogenous anti-inflammatory agent is secreted from the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells).

[0153] In some embodiments, the exogenous anti-inflammatory agent is tethered to immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein by means of fusion with 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, the hinge domain includes, or may be, a CD8a, IgG4, or CD28 hinge domain. In some embodiments, the transmembrane domain includes, or is not limited to, 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-ζ, 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 domains. In some embodiments, the exogenous anti-inflammatory agent is tethered to the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein via short linkers (e.g., glycine-rich and / or serine-rich linkers). In some embodiments, the linker comprises (GGGGS)n, where n = 1, 2, 3, 4, or 5, or alternative versions thereof, such as (SGGGG)n, where n = 1, 2, 3, 4, or 5.

[0154] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous anti-inflammatory agent, as described herein, reduce the expression levels of one or more inflammatory agents (e.g., TNFα, IL-6, IL-1β, MCP-1, CCL2, and / or MIP-1), for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous anti-inflammatory agent, as described herein, reduce inflammatory signaling (e.g., NF-κB signaling), for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous anti-inflammatory agent, as described herein, increase the expression levels of anti-inflammatory agents (e.g., IL-10, IL-2, and / or CCL18), for example, relative to unmodified immune cells of the same type.

[0155] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous anti-inflammatory agent, as described herein, reduce or prevent oxidative stress, for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous anti-inflammatory agent, as described herein, reduce or prevent tissue inflammation (e.g., liver tissue inflammation), for example, relative to unmodified immune cells of the same type. In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous anti-inflammatory agent, as described herein, reduce the activation of inflammatory effector cells (e.g., hepatic stellate cells), for example, relative to unmodified immune cells of the same type.

[0156] Exogenous regenerator As used herein, the term "exogenous regenerant" refers to an exogenous molecule that can be expressed, released, secreted, or delivered (e.g., via modified immune cells (e.g., monocytes, macrophages, and / or dendritic cells) as described herein) to a regeneration target to promote cell regeneration. In some embodiments, the regeneration target is or includes proteins. Exogenous regenerants may include, but are not limited to, peptides, fusion proteins, nucleic acids, antibiotics, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, and / or microsomes, or any derivatives or variants of any of the foregoing.

[0157] In some embodiments, the exogenous regenerative agent includes fibroblast growth factor 21 (FGF21), fibroblast growth factor 19 (FGF19), vascular endothelial growth factor α (VEGF-α), and / or hepatocyte growth factor (HGF).

[0158] In some embodiments, the exogenous regenerative agent is retained on the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells). In some embodiments, the exogenous regenerative agent is secreted from the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells).

[0159] In some embodiments, the exogenous regenerator is retained in the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein by means of fusion with 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, the hinge domain includes, or may be, a CD8a, IgG4, or CD28 hinge domain. In some embodiments, the transmembrane domain includes, or is not limited to, 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-ζ, 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 domains. In some embodiments, the exogenous regenerator is tethered to the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein via short linkers (e.g., glycine-rich and / or serine-rich linkers). In some embodiments, the linker comprises (GGGGS)n, where n = 1, 2, 3, 4, or 5, or alternative versions thereof, such as (SGGGG)n, where n = 1, 2, 3, 4, or 5.

[0160] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous regenerative agent, as described herein, improve tissue regeneration and / or regression (e.g., liver tissue regeneration and / or regression), for example, relative to unmodified immune cells of the same type.

[0161] Exogenous burial agents As used herein, the term "exogenous necrolysis agent" refers to an exogenous molecule that can be expressed, released, secreted, or delivered (e.g., via modified immune cells (e.g., monocytes, macrophages, and / or dendritic cells) as described herein) to a necrolysis target to promote necrolysis. In some embodiments, the necrolysis target is or includes proteins. Exogenous regenerative agents may include, but are not limited to, peptides, fusion proteins, nucleic acids, antibiotics, antibodies or fragments thereof, antibody agents or fragments thereof, growth factors, cytokines, enzymes, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, and / or microsomes, or any derivatives or variants of any of the foregoing substances.

[0162] In some embodiments, the exogenous embalming agent includes or contains a peptide containing T-cell immunoglobulin and mucin domain 4 (TIM4). In some embodiments, the exogenous embalming agent includes or contains a MERTK peptide. In some embodiments, the exogenous embalming agent includes or contains an Axl peptide. In some embodiments, the exogenous embalming agent includes or contains a Tyro3 peptide. In some embodiments, the exogenous embalming agent includes or contains an SRA-1 peptide. In some embodiments, the exogenous embalming agent includes or contains a CD36 peptide. In some embodiments, the exogenous embalming agent includes or contains a LOX1 peptide. In some embodiments, the exogenous embalming agent includes or contains a TIM1 peptide. In some embodiments, the exogenous embalming agent includes or contains a TIM3 peptide. In some embodiments, the exogenous embalming agent includes or contains an LXR peptide. In some embodiments, the exogenous embalming agent includes or contains a CD14 peptide. In some embodiments, the exogenous embalming agent includes or contains a Stabilin-1 peptide. In some embodiments, the exogenous embalming agent includes or contains a Stabilin-2 peptide. In some implementations, the exogenous necrolysis agent includes or may include the CD300f polypeptide.

[0163] In some embodiments, the exogenous cytotoxic agent is retained in and / or expressed by the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells). In some embodiments, the exogenous cytotoxic agent is secreted from the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells).

[0164] In some embodiments, the exogenous cytotoxic agent is retained on immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein by means of fusion with 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, the hinge domain includes, or may be, a CD8a, IgG4, or CD28 hinge domain. In some embodiments, the transmembrane domain includes, or is not limited to, 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-ζ, 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 domains. In some embodiments, the exogenous cytotoxic agent is ligated to the immune cells (e.g., macrophages, monocytes, or dendritic cells) described herein via short linkers (e.g., glycine-rich and / or serine-rich linkers). In some embodiments, the linker comprises (GGGGS)n, where n = 1, 2, 3, 4, or 5, or alternative versions thereof, such as (SGGGG)n, where n = 1, 2, 3, 4, or 5.

[0165] In some embodiments, modified immune cells (e.g., macrophages, monocytes, or dendritic cells) expressing or including at least one exogenous cytotoxic agent, as described herein, improve cytotoxicity, for example, relative to unmodified immune cells of the same type.

[0166] Switch receptor As used herein, the term "switch receptor" or "chimeric switch receptor" 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. In some embodiments, the switch receptor of this disclosure converts a pro-inflammatory signal into an anti-inflammatory signal.

[0167] In some embodiments, the chimeric switch receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain is derived from a first receptor and the intracellular domain is derived from a second receptor. In some embodiments, the first receptor is a cytokine receptor (i.e., a first cytokine receptor). In some embodiments, the chimeric switch receptor, including an extracellular domain derived from a first cytokine receptor, binds cytokines as described herein. In some embodiments, the first receptor binds antigens as described herein. In some embodiments, the second receptor is a cytokine receptor (i.e., a second cytokine receptor). In some embodiments, the binding of a ligand to the extracellular portion of the chimeric switch receptor results in the intracellular domain of the second cytokine receptor producing a signal substantially similar to the signal that would result from the binding of the ligand to a naturally occurring (i.e., intact) second cytokine receptor as described herein. By way of non-limiting examples, in some embodiments, in a chimeric switch receptor comprising an extracellular domain of IFN-γ and an intracellular domain of IL-10, the binding of IFN-γ to the extracellular portion of the chimeric switch receptor results in a signal substantially similar to the signal produced by the binding of IL-10 to the intact IL-10 receptor. In some embodiments, the transmembrane domain is derived from a first or second receptor.

[0168] In some embodiments, the first cytokine receptor is a receptor for an anti-inflammatory cytokine (i.e., an anti-inflammatory cytokine receptor). In some embodiments, the second cytokine receptor is an anti-inflammatory cytokine receptor. In some embodiments, the first cytokine receptor is a pro-inflammatory cytokine receptor and the second cytokine receptor is an anti-inflammatory cytokine receptor.

[0169] In some embodiments, the chimeric switch receptor of this disclosure is membrane-bound. In some embodiments, the chimeric switch receptor of this disclosure is not membrane-bound. Several potential embodiments of the switch receptor are described by way of non-limiting example in International Publication No. WO2024 / 006281, filed June 27, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0170] Nucleic acid constructs Among other things, this disclosure provides nucleic acid molecules that include encoding one or more of the following nucleic acid sequences: at least one exogenous fibrinolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). Immune cells may include nucleic acid molecules (e.g., exogenous nucleic acid molecules) encoding: at least one exogenous fibrinolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein).

[0171] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. A phrase encoding a protein or RNA nucleotide sequence may also include introns, provided that the nucleotide sequence encoding the protein may contain introns in some versions. The term "encodes" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide (e.g., gene, cDNA, or mRNA), namely, serving as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties during biological processes. Thus, if the transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system, then the gene, cDNA, or RNA encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be referred to as the protein or other product encoding the gene or cDNA.

[0172] The terms "operably linked" or "transcriptional control" refer to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the heterologous nucleic acid sequence. For example, when a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operably linked to that coding sequence. The operably linked DNA sequences can be contiguous to each other and, for example, located in the same reading frame when necessary for binding two protein-coding regions.

[0173] As used herein, the terms "fragment" or "part" refer to a structure that includes a discrete portion of the whole but lacks one or more parts seen in the overall structure. In some embodiments, a fragment consists of such discrete portions. In some embodiments, a fragment consists of, or includes, a characteristic structural element or part seen in the whole. In some embodiments, the nucleotide fragment comprises, or is composed 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) seen in the whole nucleotide. In some embodiments, the nucleotide fragment comprises, or is composed 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 monomeric units (e.g., residues) seen in the whole nucleotide. The whole material or entity may be referred to as the whole “parent” in some embodiments.

[0174] The nucleic acid molecule encoding at least one exogenous fibrolytic agent (e.g., the peptide described herein), at least one exogenous anti-inflammatory agent (e.g., the peptide described herein), at least one exogenous regenerative agent (e.g., the peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., the peptide described herein), can be a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, the nucleic acid molecule includes or encodes a messenger RNA (mRNA) transcript encoding at least one exogenous fibrolytic agent. In some embodiments, the nucleic acid molecule includes or encodes an mRNA transcript encoding at least one exogenous anti-inflammatory agent. In some embodiments, the nucleic acid molecule includes or encodes 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 cytotoxic 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, for example, for expression in cells (e.g., mammalian cells). 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, for example, for expression in cells (e.g., mammalian cells). Various codon optimization methods are known in the art, such as those disclosed in U.S. Patent Nos. 5,786,464 and 6,114,148, each of which is incorporated herein by reference in its entirety.

[0176] In some embodiments, the vector includes a nucleic acid molecule encoding at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein). In some embodiments, the vector includes plasmids, viral vectors, phage particles, retrotransposons (e.g., piggyback or sleeping beauty), site-directed insertion vectors (e.g., CRISPR / Cas systems including one or more of Cas9, Cas12a, or C2c2), Zn finger nucleases, and / or TALENs for inserting template donor DNA (which includes 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 regenerant (e.g., a polypeptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a polypeptide described herein)), suicide expression vectors, or any other vector known in the art. The vector may be adapted for replication and integration in eukaryotes. Vectors may include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0177] Vectors may include a replication origin, a promoter sequence (e.g., a constitutive or inducible promoter), and / or a convenient restriction endonuclease site (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193, each of which is incorporated herein by reference in its entirety). Vectors may also include, for example, a secretion-promoting signal sequence, a polyadenylation signal and a transcription terminator (e.g., bovine growth hormone (BGH) polyadenylation signal), elements that allow episome replication and replication in prokaryotes (e.g., SV40 origin and / or ColE1), selectable elements (e.g., ampicillin resistance genes and / or zeocin markers), and / or reporter genes (e.g., luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein).

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

[0179] The vector may include one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., the peptide described herein) and / or at least one exogenous anti-inflammatory agent (e.g., the peptide described herein) and / or at least one exogenous regenerative agent (e.g., the peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., the peptide described herein), operably linked to one or more tissue-specific promoters. In some embodiments, the vector described herein includes one or more liver-specific promoters. In some embodiments, the vector described herein includes one or more cirrhosis-specific promoters. In some embodiments, the vector described herein includes the CX3CR1 promoter. In some embodiments, the vector described herein includes insulin-like growth factor 1 (IGF1). In some embodiments, the vector described herein includes the CD11B promoter. In some embodiments, the vector (or combination of vectors) may include nucleic acid sequences encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fibrolytic agents, anti-inflammatory agents, regenerative agents, and / or cytotoxic agents.

[0180] In some embodiments, the vector comprising one or more nucleic acid sequences encoding at least one exogenous fibrinolytic agent (e.g., the peptide described herein) and / or at least one exogenous anti-inflammatory agent (e.g., the peptide described herein) and / or at least one exogenous regenerative agent (e.g., the peptide described herein) and / or at least one exogenous cytotoxic agent (e.g., the peptide described herein) includes, for example, a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Volumes 1-4, Cold SpringHarbor Press, NY. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, or retroviral vectors (e.g., lentiviral vectors or gamma retroviral vectors). In some embodiments, the vector includes a lentiviral vector (e.g., as described in U.S. Patent No. 9,149,519 or International Publication No. WO 2017 / 044487, each of which is incorporated herein by reference in its entirety).

[0181] In some embodiments, the viral vector includes an adenovirus vector. Adenoviruses are a large family of viruses containing double-stranded DNA. They replicate within the host cell nucleus, using host cell mechanisms to synthesize viral RNA, DNA, and proteins. Adenoviruses are known in the art to influence replicating and non-replicating cells, accommodate large transgenes, and encode proteins without integrating into the host cell genome. In some embodiments, the adenovirus vector includes an Ad2 vector or an Ad5 vector (e.g., an Ad5f35 adenovirus vector, such as a helper-dependent Ad5F35 adenovirus vector).

[0182] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. AAV systems are generally well known in the art (see, for example, Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., PNASUSA, 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. Patent 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 their variants. Generally, any AAV serotype can be used to deliver at least one exogenous fibrolytic agent (e.g., the peptide described herein) and / or at least one exogenous anti-inflammatory agent (e.g., the peptide described herein). In some embodiments, the AAV serotype exhibits tissue tropism.

[0184] In some implementations, the CRISPR / Cas9 system (or other Cas systems including Cas12a, Cas13, etc.) has recently demonstrated the ability to use adeno-associated virus (AAV) vectors as donor template DNA during homologous recombination (HR) to facilitate high levels of precise genome editing.

[0185] In some embodiments, the vector includes gamma retroviral vectors (e.g., as described in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 Jun; 3(6): 677-713, the entirety of which is incorporated herein by reference). Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen focal formation virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom.

[0186] In some embodiments, the vector comprises two or more nucleic acid sequences encoding at least one exogenous fibrinolytic 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 cytotoxic agent. In some embodiments, two or more nucleic acid sequences encoding at least two agents (e.g., two or more exogenous fibrinolytic agents, two or more exogenous anti-inflammatory agents, two or more exogenous regenerative agents, and / or two or more exogenous cytotoxic agents, or any combination thereof) are encoded by a single nucleic acid molecule, for example, within the same reading frame and as a single polypeptide chain. In some embodiments, the two or more agents are separated by one or more cleavage peptide sites (e.g., self-cleavage sites or substrates of intracellular proteases). In some embodiments, the cleavage peptides include porcine cephalovirus-1 (P2A) peptide, Thoseea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, foot-and-mouth disease virus (F2A) peptide, or variants thereof.

[0187] Pharmaceutical Composition Among other things, this disclosure provides pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein and / or delivery solvents 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 the instructions specify “therapeutic effective amount,” “immune effective amount,” “anti-immune response effective amount,” or “immune response suppression effective amount,” the precise amount of a pharmaceutical composition including immune cells (such as macrophages, monocytes, or dendritic cells) as described herein may be determined by the physician taking into account individual differences in the patient’s (subject’s) age, weight, immune response, and condition.

[0189] Pharmaceutical compositions comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein may include buffers, including neutral buffered saline or phosphate buffered saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextran, or mannitol; proteins, peptides, or amino acids (e.g., glycine); antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the pharmaceutical composition is substantially free of contaminants, such as the absence of detectable levels of contaminants (e.g., endotoxins).

[0190] The pharmaceutical compositions described herein may be administered in a manner suitable for treating or preventing a disease, condition, or illness. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, condition, or illness, although appropriate dosage may be determined through clinical trials.

[0191] The pharmaceutical compositions described herein can be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusionable solutions. The pharmaceutical compositions described herein can be formulated for intravenous, subcutaneous, intradermal, intratumoral, intramedullary, intramuscular, transarterial, or intraperitoneal administration.

[0192] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous infusion or injection. In some embodiments, the pharmaceutical compositions described herein are formulated for intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be formulated for administration using infusion techniques commonly used in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, the entirety of which is incorporated herein by reference).

[0193] As used herein, the terms “parenteral administration” and “extraterrestrial administration” refer to administration methods that are not intravenous or localized, usually by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, and intrasternal injections and infusions.

[0194] Pharmaceutical compositions including immune cells as described herein can be used in approximately 10 4 To about 10 9 Administer a dose of cells / kg body weight (e.g., approximately 10). 5 To about 10 6 (cells / kg body weight), including all integer values ​​within these ranges. In some embodiments, the dose of immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein comprises at least about 1 x 10⁻⁶ cells / kg body weight. 6 Approximately 1.1 x 10 6 Approximately 2 x 10 6 Approximately 3.6 x 10 6 Approximately 5 x 10 6 Approximately 1 x 10 7 Approximately 1.8 x 10 7 Approximately 2 x 10 7 Approximately 5 x 10 7 Approximately 1 x 10 8 Approximately 2 x 10 8 Approximately 5 x 10 8 Approximately 1 x 10 9 Approximately 2 x 10 9 Or approximately 5 x 10 9 Individual cells. The pharmaceutical compositions described herein can also be administered in multiple doses. The optimal dose and treatment regimen for a specific patient can be readily determined by those skilled in the art by monitoring the signs of the patient's disease, symptoms, or condition and adjusting the treatment accordingly.

[0195] It may be desirable to administer a pharmaceutical composition comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein to a subject, followed by a subsequent blood draw (or apheresis) to activate the collected immune cells, and then re-infuse the activated immune cells to the subject. This process may be performed multiple times, for example, every few weeks. Immune cells (e.g., macrophages, monocytes, or dendritic cells) may be activated from a blood draw of approximately 10 cc to approximately 400 cc. In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are activated from blood draws of approximately 20 cc, approximately 30 cc, approximately 40 cc, approximately 50 cc, approximately 60 cc, approximately 70 cc, approximately 80 cc, approximately 90 cc, or approximately 100 cc. Without being bound by theory, methods including multiple blood draws and re-infusions as described herein may select certain populations of immune cells.

[0196] In some embodiments, a pharmaceutical composition comprising immune cells (e.g., macrophages, monocytes, or dendritic cells) as described herein is administered in combination with a second therapy (e.g., before, simultaneously with, or after). For example, the second therapy may include, but is not limited to, farnesol X receptor agonists, stearoyl-CoA desaturase inhibitors, CCR2 / CCR5 chemokine antagonists, PPAR agonists, caspase inhibitors, galactohexine-3 inhibitors, acetyl-CoA carboxylase inhibitors, lysyl oxidase-2 (LOX-2) antagonists, TGFβ antagonists, GLP1 agonists, thyroid hormone receptor-β (THR-β) agonists, recombinant FGF molecules, and / or TIMP antagonists. In some embodiments, the GLP-1 agonist is semaglutide. In some embodiments, the GLP-1 agonist is telposide. In some embodiments, the THR-β agonist is resmetiro. In some embodiments, the recombinant FGF molecule is efruxifermin. The dosage of any of the aforementioned therapies to be administered to a subject will vary depending on the disease, condition, or illness being treated and on a subject-specific basis. Scaling of dosages for human administration may be performed in accordance with recognized practices in the art.

[0197] Treatment Among other things, this disclosure provides methods for treating a disease, condition, or illness (such as those described herein) in a subject, including delivering a pharmaceutical composition comprising immune cells (such as macrophages, monocytes, or dendritic cells) as described herein. In some embodiments, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a subject suffering from a disease, condition, or illness.

[0198] Subjects treated with the methods described herein may be mammals, such as primates, or humans (e.g., subjects who have or are at risk of having the diseases, conditions, or illnesses described herein). In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) may be autologous, allogeneic, or xenogeneic relative to the subject. The pharmaceutical compositions described herein may be administered to the subject alone or in combination with one or more therapeutic agents, procedures, or modalities according to the dosing regimens described herein.

[0199] The pharmaceutical compositions described herein can be administered in any convenient manner (e.g., injection, ingestion, infusion, inhalation, implantation, or transplantation). In some embodiments, the pharmaceutical compositions described herein are administered by injection or infusion. The pharmaceutical compositions described herein can be administered to a subject via artery, subcutaneous, intravenous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, or intraperitoneal route. In some embodiments, the pharmaceutical compositions described herein are administered parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). In some embodiments, the pharmaceutical compositions described herein are administered by intravenous infusion or injection. In some embodiments, the pharmaceutical compositions described herein are administered by intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be injected directly into the subject at sites of inflammation, local disease, lymph nodes, organs, tumors, or infections.

[0200] fibrosis The modified immune cells described herein and / or delivery solvents comprising one or more nucleic acids may be administered to improve one or more fibrotic symptoms or reduce fibrosis in a subject, for example, to treat or prevent fibrotic disease, condition, or illness in the subject. Methods may include administering to a subject in need an amount of the modified immune cells described herein sufficient to reduce one or more symptoms of fibrosis or prevent fibrosis in the subject. The modified immune cells described herein may be administered to improve tissue repair, for example, in a subject with a fibrotic disease, condition, or illness. In some embodiments, the subject has fibrosis or has been diagnosed with a fibrotic disease, condition, or illness. In some embodiments, the subject has not received prior treatment with the modified immune cells described herein (e.g., treatment-naïve subject).

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

[0202] In some embodiments, administration of the modified immune cells described herein to subjects, compared to subjects who have not received such administration, delays the onset of or reduces one or more of the following: the formation or deposition of tissue fibrosis; the size, cellularity, composition, or cellular content of fibrotic lesions; the collagen and / or hydroxyproline content of fibrotic lesions; the expression or activity of fibrotic proteins; fibrosis associated with an inflammatory response; and / or weight loss associated with fibrosis. In some embodiments, reduction in fibrosis increases subject survival compared to subjects who have not received such administration.

[0203] Exemplary fibrotic diseases, conditions, or illnesses include systemic diseases, conditions, or illnesses (e.g., systemic sclerosis, multifocal fibrosis, scleroderma-like chronic graft-versus-host disease, renal systemic fibrosis, or scleroderma) and organ-specific diseases, conditions, or illnesses (e.g., fibrosis of the liver, lungs, heart, kidneys, pancreas, skin, and / or nervous system). In some embodiments, fibrotic diseases, conditions, or illnesses include or are hyperproliferative fibrotic diseases, such as non-cancerous fibrotic diseases.

[0204] In some embodiments, fibrotic diseases, conditions, or illnesses include, or are present in, the liver, lungs, heart, vascular system, kidneys, pancreas, skin, gastrointestinal tract, bone marrow, hematopoietic tissue, nervous system, and / or eye. In some embodiments, fibrotic diseases, conditions, or illnesses affect tissues including tendons, cartilage, skin (e.g., epidermis and / or endothelium), heart tissue, vascular tissue (e.g., arteries and / or veins), pancreatic tissue, lung tissue, kidney tissue, uterine tissue, ovarian tissue, nerve tissue, testicular tissue, peritoneum, colon, small intestine, bile ducts, intestines, bone marrow, hematopoietic tissue, and / or eye tissue (e.g., retinal tissue).

[0205] In some embodiments, the fibrotic disease, condition, or illness includes or is a liver fibrotic disease, condition, or illness. The modified immune cells described herein may be applied to improve liver function, for example, in subjects with a liver fibrotic disease, condition, or illness. In some embodiments, the liver fibrotic disease, condition, or illness includes or is a fatty liver disease, condition, or illness. In some embodiments, the fatty liver disease, condition, or illness includes 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, the subject has cirrhosis, liver cancer, increased risk of liver failure, 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, fibrotic diseases, conditions, or illnesses include or are pulmonary fibrotic diseases, conditions, or illnesses. The modified immune cells described herein may be applied to improve lung function, for example, in subjects with pulmonary fibrotic diseases, conditions, or illnesses. In some embodiments, pulmonary fibrotic diseases, conditions, or illnesses include or are pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), common interstitial pneumonia (UIP), interstitial lung disease, cryptogenic fibrotic alveolitis (CFA), bronchiectasis, and / or scleroderma. In some embodiments, pulmonary fibrosis is associated with occupational hazards, environmental pollutants, smoking, autoimmune connective tissue diseases (e.g., rheumatoid arthritis, scleroderma, or systemic lupus erythematosus (SLE)), connective tissue diseases (e.g., sarcoidosis), and / or infectious diseases.

[0207] In some embodiments, fibrotic diseases, conditions, or illnesses include or are renal fibrotic diseases, conditions, or illnesses. The modified immune cells described herein may be administered to improve renal function, for example, in subjects with renal fibrotic diseases, conditions, or illnesses. In some embodiments, renal fibrotic diseases, conditions, or illnesses include or are renal fibrosis (e.g., chronic renal fibrosis), fibrosis-related nephropathy (e.g., diabetic nephropathy), lupus, nephroscleroderma, glomerulonephritis, focal segmental glomerulosclerosis, IgA nephropathy associated with human chronic kidney disease (CKD), chronic progressive nephropathy (CPN), tubulointerstitial fibrosis, ureteral obstruction, chronic uremia, chronic interstitial nephritis, radiation nephropathy, glomerulosclerosis, progressive glomerulonephritis (PGN), endothelial / thrombotic microangiopathy, and / or HIV-associated nephropathy. In some embodiments, renal fibrosis is associated with exposure to toxins, irritants, or chemotherapeutic agents.

[0208] In some embodiments, the fibrotic disease, condition, or illness includes or is a cardiac fibrotic disease, condition, or illness. The modified immune cells described herein may be applied to improve cardiac function, for example, in subjects with a cardiac fibrotic disease, condition, or illness. In some embodiments, the cardiac fibrotic disease, condition, or illness includes or is myocardial fibrosis. In some embodiments, myocardial fibrosis is associated with radiation myocarditis, surgical complications (e.g., post-myocardial fibrosis), infectious diseases (e.g., Chagus disease), granulomatous metabolic storage diseases (e.g., cardiomyopathy or hemochromatosis), developmental abnormalities (e.g., endocardial fibroelastosis), arteriosclerosis, 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 sarcoidosis, myocardial infarction, and / or congestive heart failure.

[0209] In some embodiments, the fibrotic disease, condition, or illness includes or may include ocular fibrotic diseases, conditions, or illnesses. The modified immune cells described herein may be applied to improve eye function, for example, in subjects suffering from ocular fibrotic diseases, conditions, or illnesses. In some embodiments, the ocular fibrotic disease, condition, or illness includes or may include 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, fibrotic diseases, conditions, or illnesses include or are skin fibrotic diseases, conditions, or illnesses. The modified immune cells described herein may be applied to improve skin function, for example, in subjects suffering from skin fibrotic diseases, conditions, or illnesses. In some embodiments, skin fibrotic diseases, conditions, or illnesses include or are skin fibrosis (e.g., hypertrophic scars and / or keloids), scleroderma, and / or renal systemic fibrosis.

[0211] In some embodiments, fibrotic diseases, conditions, or illnesses include or are gastrointestinal fibrotic diseases, conditions, or illnesses. The modified immune cells described herein may be administered to improve gastrointestinal function, for example, in subjects suffering from gastrointestinal fibrotic diseases, conditions, or illnesses. In some embodiments, gastrointestinal fibrotic diseases, conditions, or illnesses include or are radiation-induced intestinal fibrosis, fibrosis associated with foregut inflammatory conditions (e.g., Barrett's esophagus or chronic gastritis), and / or fibrosis associated with hindgut inflammatory conditions (e.g., inflammatory bowel disease, ulcerative colitis, or Crohn's disease).

[0212] In some embodiments, the fibrotic disease, condition, or illness includes or is chronic fibrotic disease, condition, or illness. In some embodiments, the fibrotic disease, condition, or illness is associated with an inflammatory disease, condition, or illness. In some embodiments, the fibrotic disease, condition, or illness includes or is osteomyelitis (e.g., chronic osteomyelitis). In some embodiments, the fibrotic disease, condition, or illness includes or is amyloidosis (e.g., amyloidosis associated with chronic osteomyelitis).

[0213] inflammation The modified immune cells described herein and / or delivery solvents comprising one or more nucleic acids may be administered to improve or reduce inflammation in a subject, for example, to treat or prevent an inflammatory disease, condition, or illness (e.g., one or more symptoms of an inflammatory disease, condition, or illness) in the subject. Methods may include administering to a subject in need an amount of the modified immune cells described herein sufficient to reduce or prevent inflammation in the subject. In some embodiments, the subject has inflammation or has been diagnosed with an inflammatory disease, condition, or illness. In some embodiments, the subject has not received prior treatment with the modified immune cells described herein (e.g., treatment-naïve subject).

[0214] In some embodiments, the modified immune cells described herein are used as a medicament for treating (e.g., reversing, reducing, alleviating, or preventing) inflammation in a subject (e.g., a subject suffering from an inflammatory disease, condition, or illness). In some embodiments, the modified immune cells described herein are used to manufacture a medicament for treating (e.g., reversing, reducing, or alleviating one or more symptoms, or preventing) inflammation in a subject (e.g., a subject suffering from an inflammatory disease, condition, or illness).

[0215] In some embodiments, the inflammatory disease, condition, or illness includes or is a systemic disease, condition, or illness. In some embodiments, the inflammatory disease, condition, or illness includes or is a localized disease, condition, or illness. In some embodiments, the inflammatory disease, condition, or illness includes or is an acute disease, condition, or illness. In some embodiments, the inflammatory disease, condition, or illness includes or is a chronic disease, condition, or illness.

[0216] Exemplary inflammatory diseases, conditions, or illnesses include, but are not limited to, systemic diseases, conditions, or illnesses (e.g., chronic systemic inflammation, Behçet's disease, sarcoidosis, systemic lupus erythematosus, juvenile idiopathic arthritis, scleroderma, Sjögren's syndrome, and / or sepsis) and organ-specific diseases, conditions, or illnesses (e.g., inflammation of the liver, lungs, heart, intestines, kidneys, and / or pancreas). In some embodiments, inflammatory diseases, conditions, or illnesses include, or may include, inflammatory diseases, conditions, or illnesses of the liver, gastrointestinal tract, lungs, skin, cardiovascular system, nervous system, kidneys, pancreas, joints, eyes, and / or endocrine system. In some embodiments, inflammatory diseases, conditions, or illnesses include, or may include, autoimmune diseases.

[0217] In some embodiments, the inflammatory disease, condition, or illness includes or is a liver inflammatory disease, condition, or illness. In some embodiments, the liver inflammatory disease, condition, or illness includes or is a fatty liver disease, condition, or illness (e.g., NAFLD (e.g., NASH) or AFLD (e.g., ASH)). In some embodiments, the liver inflammatory disease, condition, or illness includes or is cirrhosis (e.g., primary biliary cirrhosis or primary sclerosing cholangitis), hepatitis (e.g., hepatitis resulting from viral infection, autoimmune response, drug treatment, toxins, and / or environmental factors), and / or biliary atresia.

[0218] In some embodiments, the inflammatory disease, condition, or symptom includes or is a gastrointestinal inflammatory disease, condition, or symptom. In some embodiments, the gastrointestinal inflammatory disease, condition, or symptom includes or is colitis (e.g., ulcerative colitis, pseudomembranous colitis, endoscopic colitis, undetermined 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; dyspepsia or non-ulcerative dyspepsia, diverticulosis, and / or polyps.

[0219] In some embodiments, the inflammatory disease, condition, or illness includes or is a lung inflammatory disease, condition, or illness. In some embodiments, the lung inflammatory disease, condition, or illness includes or is asthma, COPD, adult respiratory distress syndrome (ARDS), bronchitis (e.g., chronic bronchitis), bronchiolitis, lung inflammation, pulmonary fibrosis, cystic fibrosis, pneumonitis (e.g., allergic pneumonia, common interstitial pneumonia (UIP), acute pneumonia), extrinsic allergic alveolitis, asbestosis, silicosis, bronchiectasis, beryllium poisoning, talcosis, pneumoconiosis, sarcoidosis, and / or pleurisy.

[0220] In some embodiments, the inflammatory disease, condition, or lesion includes or is a skin inflammatory disease, condition, or lesion. In some embodiments, the skin inflammatory disease, condition, or lesion includes or is psoriasis, eczema, dermatitis (e.g., eczematous dermatitis, atopic or seborrheic dermatitis, allergic or irritant contact dermatitis, fissuring eczema, photosensitive dermatitis, phototoxic dermatitis, phytophotodermatitis, radiation dermatitis, or stasis dermatitis), ulcers, ichthyosis, epidermolysis bullosa, hypertrophic scars, keloids, inflammatory skin diseases, photoaging, skin atrophy, dermatomyositis; pemphigus.

[0221] In some embodiments, the inflammatory disease, condition, or illness includes, or is a cardiovascular inflammatory disease, condition, or illness. In some embodiments, the cardiovascular inflammatory disease, condition, or illness includes, or is a atherosclerosis, coronary artery infarction, peripheral vascular disease, myocarditis, vasculitis, revascularization of stenosis, myocarditis, pericarditis, vascular disease associated with type II diabetes, endocarditis, cholesterol-related metabolic disorders, oxygen free radical damage, and / or ischemia.

[0222] In some embodiments, the inflammatory disease, condition, or illness includes or is a neurological inflammatory disease, condition, or illness. In some embodiments, the neurological inflammatory disease, condition, or illness includes 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 neuropathy (e.g., peripheral neuropathy).

[0223] In some embodiments, the inflammatory disease, condition, or illness includes or is a renal inflammatory disease, condition, or illness. In some embodiments, the renal inflammatory disease, condition, or illness includes or is nephritis, nephritis secondary to Wegener's disease, acute renal failure secondary to acute nephritis, post-obstructive syndrome, renal tubular ischemia, pyelonephritis, glomerulosclerosis, membranous neuropathy, and / or renal artery sclerosis. In some embodiments, nephritis includes or is glomerulonephritis, interstitial nephritis, and / or lupus nephritis.

[0224] In some embodiments, the inflammatory disease, condition, or illness includes, or is an inflammatory disease, condition, or illness of the pancreas. In some embodiments, the inflammatory disease, condition, or illness of the pancreas includes, or is pancreatitis (e.g., acute or chronic pancreatitis).

[0225] In some embodiments, the inflammatory disease, condition, or illness includes or is an arthritic disease, condition, or illness. In some embodiments, the arthritic disease, condition, or illness includes or is rheumatoid arthritis, rheumatoid spondylitis, juvenile rheumatoid arthritis, osteoarthritis, gouty arthritis, psoriatic arthritis, lupus-associated arthritis, ankylosing spondylitis, spondyloarthropathy, degenerative arthritis, and / or synovitis.

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

[0227] In some embodiments, the inflammatory disease, condition, or illness includes, or may include, an endocrine system disorder, condition, or illness. In some embodiments, the inflammatory disease, condition, or illness of the endocrine system includes, or may include, autoimmune thyroiditis (Hashimoto's disease), adrenal cortex inflammation (e.g., acute or chronic adrenal cortex inflammation), and / or type 1 diabetes.

[0228] In some embodiments, the inflammatory disease, condition, or illness includes or is an autoimmune disease, condition, or illness. In some embodiments, the autoimmune disease, condition, or illness includes or is an 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, Sjögren's syndrome, vasculitis, multiple sclerosis, dermatitis (e.g., atopic dermatitis or eczematous dermatitis), myasthenia gravis, IBD, Crohn's disease, colitis, acute inflammatory conditions (e.g., endotoxemia, sepsis, or toxic shock syndrome), transplant rejection, and allergies (e.g., asthma, allergic rhinitis, eczema, allergies). Sexual contact dermatitis or allergic conjunctivitis), Wegener's granulomatosis, vasculitis, polymyalgia rheumatica (PMR), tendinitis, bursitis, immediate-type hypersensitivity reactions (e.g., asthma, hay fever, skin allergies, or acute anaphylactic reactions), acute disseminated encephalomyelitis, Thorgan disease, Addison's disease, heart disease, osteoporosis, generalized alopecia, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, pernicious anemia, autoimmune hepatitis, bullous pemphigoid, endometriosis, Goodpassuia syndrome, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial cystitis, morphine scleroderma, neuromyotonia, temporal arteritis, vasculitis, and / or vitiligo.

[0229] In some embodiments, the inflammatory disease, symptom, or condition includes or is a wound. In one embodiment, a wound includes 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 bleeding.

[0230] In some embodiments, the inflammatory disease, condition, or illness includes or ischemia. In some embodiments, ischemia includes or ischemic heart disease, intestinal disease, cerebral ischemia (e.g., stroke), limb ischemia, mesenteric ischemia, and skin ischemia. In some embodiments, the inflammatory disease, condition, or illness includes or ischemia-reperfusion injury, postperfusion syndrome, and / or transient ischemia of organs (e.g., gastrointestinal tract, bladder, or heart).

[0231] In some embodiments, the inflammatory disease, symptom, or condition includes or is sepsis. In some embodiments, sepsis includes or is sepsis caused by pneumonia, abdominal infection, kidney infection, and / or bloodstream infection. In some embodiments, sepsis includes or is sepsis syndrome, Gram-positive sepsis, Gram-negative sepsis, culture-negative sepsis, fungal sepsis, and / or urinary sepsis.

[0232] In some embodiments, inflammatory diseases, symptoms, or conditions include infectious diseases. In some embodiments, infectious diseases include, or may include, malaria, pneumonia, African trypanosomiasis, tuberculosis, HIV, human cytomegalovirus (HCMV), herpesvirus infection, influenza (e.g., influenza A, influenza B, or influenza C), EBV infection, Chagas disease, bacterial, trichinosis, or fungal myocarditis, meningitis, Legionella, hepatitis (e.g., chronic active hepatitis), pneumonia, Clostridium difficile infection, small intestinal bacterial overgrowth (SIBO), dengue hemorrhagic fever, Lyme disease, meningococcal septicemia, etc. Necrotizing fasciitis, necrotizing enterocolitis, leprosy, streptococcal myositis, infectious colitis, fungal infections (e.g., Candida albicans infection), vancomycin-resistant enterococci (VRE) infection, pneumoglobinitis, peritonitis, hemolytic uremic syndrome, toxic shock syndrome, Pneumocystis carinii, Campylobacter jejuni infection, Helicobacter pylori infection, viral encephalitis, septic arthritis, gas gangrene, pelvic inflammatory disease, intracellular infection of Mycobacterium avium, orchitis, and / or virus-associated hemophagocytic syndrome (VAHS).

[0233] Immune cell modification methods Among other things, this disclosure provides a method for modifying immune cells (e.g., monocytes, macrophages, or dendritic cells), including delivering a nucleic acid construct comprising encoding one or more nucleic acid sequences of: at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent (e.g., a peptide described herein), and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein).

[0234] delivery method Nucleic acid constructs comprising one or more nucleic acid sequences encoding at least one exogenous fibrolytic agent (e.g., a peptide described herein), at least one exogenous anti-inflammatory agent (e.g., a peptide described herein), at least one exogenous regenerative agent, and / or at least one exogenous cytotoxic agent (e.g., a peptide described herein) can be introduced into immune cells (e.g., monocytes, macrophages, or dendritic cells) by physical, chemical, or biological methods. In some embodiments, this disclosure provides methods for modifying immune cells, including in vitro generation of modified immune cells (e.g., monocytes, macrophages, or dendritic cells). In some embodiments, this disclosure provides methods for modifying immune cells, including in vivo generation of modified immune cells (e.g., monocytes, macrophages, or dendritic cells) in a subject. For example, in some embodiments, certain provided methods for modifying immune cells (e.g., macrophages, monocytes, and / or dendritic cells) in a subject include administering / delivering to the subject a composition comprising one or more nucleic acid constructs described herein and a delivery solvent.

[0235] Physical methods for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) may include electroporation, calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, or combinations thereof. The nucleic acid constructs may be introduced into immune cells using commercially available methods, including electroporation (Amaxa Nucleofector-II® (AmaxaBiosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.), GenePulser II® (BioRad, Denver, Colo.), or Multiporator® (Eppendort, Hamburg, Germany)). Nucleic acid constructs can also be introduced into immune cells via mRNA transfection, such as cationic liposome-mediated transfection, lipid transfection, polymer encapsulation, peptide-mediated transfection, or biological projectile particle delivery systems such as “gene guns” (see, for example, Nishikawa et al., Hum Gene Ther., 12(8):861-70 (2001), the whole of which is incorporated herein by reference).

[0236] Biological methods for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) include the use of DNA and RNA vectors. In one embodiment, the vector includes plasmid vectors, viral vectors, transposons, retrotransposons (e.g., piggyback, sleeping beauty), site-directed insertion vectors (e.g., CRISPR, Zn finger nuclease, TALEN), suicide expression vectors, or another vector known in the art. Viral vectors, especially retroviral vectors, have been widely used for inserting genes into mammalian cells (e.g., human cells). Viral vectors may also be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses (e.g., Adf535), or adeno-associated viruses (see, for example, U.S. Patents 5,350,674 and 5,585,362, the entirety of which are incorporated herein by reference). Retroviral vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer, allowing for the long-term stable integration of transgenes and their proliferation in daughter cells.

[0237] The immune cells described herein (e.g., macrophages, monocytes, or dendritic cells) may be resistant to lentiviral transduction due to the expression of the restriction factor SAMHD1, which depletes nucleotide triphosphates available for reverse transcription. For example, SAMHD1 can restrict the replication of human immunodeficiency virus type 1 (HIV-1) by depleting the intracellular library of deoxyribonucleoside triphosphates. Viral protein X (Vpx) is an accessory protein associated with simian immunodeficiency virus (SIV) and HIV-2 that induces the degradation of SAMHD1. In some embodiments, delivery of (a) a viral vector comprising one or more nucleic acid sequences encoding (i) at least one exogenous fibrinolytic agent and / or (ii) at least one exogenous anti-inflammatory agent described herein and (b) at least one Vpx protein can increase the transfection of the immune cells described herein (e.g., macrophages, monocytes, or dendritic cells), for example, relative to the same type of immune cells comprising (i) at least one exogenous fibrinolytic agent and / or (ii) at least one exogenous anti-inflammatory agent but without delivery of 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 cytotoxic agent, thereby achieving long-term, permanent expression of one or more of (i)-(iv). The fact that Vpx-lentivirus-transduced macrophages are phenotypically unaffected by viral transduction enables the production of modified macrophages with phenotypic plasticity as described herein. While other viral vectors (e.g., Ad5f35) induce a pro-inflammatory M1 phenotype, Vpx-lentivirus has no effect on the M1 / M2 phenotype, enabling the production of M0-modified macrophage products that may not possess pro-inflammatory function / toxicity at baseline. Vpx-lentivirus-transduced macrophages retain phenotypic plasticity and can be further polarized to the M1 or M2 phenotype with the aid of cytokines, agonists, peptides, culture media, and other factors. Vpx-lentivirus-transduced macrophages can be exposed to pro-inflammatory signals (such as one or more pro-inflammatory cytokines, such as LPS, IFNa, IFNb, IFNγ, CpG, CD40L, GM-CSF, TNFα, IL-6, or one or more STING ligands (STING-L)) and polarize as M1 macrophages. Vpx-lentivirus-transduced macrophages can be exposed to immunosuppressive signals (such as one or more immunosuppressive cytokines, such as IL-4, IL-10, IL-13, or TGFb) and / or one or more of at least one prostaglandin or at least one or more corticosteroids) and polarize as M2 macrophages.

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

[0240] Chemical approaches for introducing the nucleic acid constructs described herein into immune cells (e.g., monocytes, macrophages, or dendritic cells) include colloidal dispersion systems, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and liposome-nucleic acid complexes).

[0241] Exemplary systems for delivering the nucleic acid constructs described herein are lipid-based systems. The nucleic acid constructs described herein can be encapsulated within an aqueous interior of liposomes, dispersed within a lipid bilayer, linked to liposomes via linker molecules, linked to lipid nanoparticles (LNPs) via linker molecules, embedded in liposomes, embedded in LNPs, complexed with liposomes, complexed with LNPs, dispersed in a solution or suspension comprising lipids, mixed with lipids, complexed with micelles, or otherwise associated with lipids. The lipids used in the methods described herein can be naturally occurring or synthetic lipids. Lipids can also be obtained from commercial sources. For example, myristoylphosphatidylcholine is available from Sigma (St. Louis, MO); diceryl phosphate is available from K & K Laboratories (Plainview, NY); cholesterol is available from Calbiochem-Behring; and myristoylphosphatidylglycerol is available from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. In some embodiments, the lipid-based system may include one or more lipids that promote the targeting of the composition to a desired cell type or cell type (e.g., monocytes, macrophages, or dendritic cells). In some embodiments, the delivery solvent causes the composition to be preferentially taken up (e.g., endocytosis, phagocytosis) by immune cells (e.g., monocytes, macrophages, or dendritic cells) relative to a composition that does not contain the delivery solvent.

[0242] Targeted portion In some embodiments, the delivery solvent may include one or more targeting portions. In some embodiments, the targeting portions may facilitate passive targeting of the composition to a desired target. In some embodiments, the targeting portions may facilitate active targeting of the composition to a desired target.

[0243] In some embodiments, the targeting portion includes or comprises one or more of antibodies (e.g., monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies) or any fragment thereof, such as scFv, aptamers, darpins, centyrin, naturally occurring or synthetic receptors, affibody, or other engineered protein recognition molecules, for example, to bind to one or more of CD14, CD11b, CD163, CD206, CD33, and / or CD209. In some embodiments, the targeting portion may be or comprises a small molecule.

[0244] In some implementations, the targeting portion includes or is a combination of specific lipid or hydrophobic entities, such as those present on or forming the outer surface of liposomes or lipid nanoparticles (e.g., for targeting a specific cell type).

[0245] Nucleic acid molecules In some embodiments of this disclosure, one or more nucleic acid molecules are or include DNA. In some embodiments of this disclosure, one or more nucleic acid molecules are or include messenger RNA (mRNA). In some embodiments, the mRNA according to this disclosure can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance stability. Modification of mRNA may include, for example, modification of RNA nucleotides. Therefore, modified mRNA according to this disclosure may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the steps of modifying mRNA include including modified nucleotides, alterations to the 5' or 3' untranslated region (UTR), cap structures, and / or poly(A) tails.

[0246] In some embodiments, the mRNA of this disclosure (e.g., mRNA 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 cytotoxic agent described herein) may contain RNA backbone modifications. Typically, backbone modifications are modifications that chemically modify the phosphate group of the backbone of the nucleotides contained in the RNA. Exemplary backbone modifications typically include, but are not limited to, modifications selected from: methylphosphonates, methylphosphonamide esters, phosphoramide esters, thiophosphate esters (e.g., cytidine 5'-O-(1-thiophosphate ester)), boron phosphate esters, positively charged guanidine groups, etc., which include replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[0247] In some embodiments, the mRNA of this disclosure (e.g., mRNA 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 cytotoxic agent described herein) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars in the contained nucleotides, including but not limited to sugar modifications selected from: 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O -alkyl oligonucleotides, 2'-deoxy-2'-C-alkyl oligonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligonucleotides and their isomers (2'-cytarabine 5'-triphosphate, 2'-cytarabine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0248] In some embodiments, the mRNA of this disclosure (e.g., mRNA 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 cytotoxic agent described herein) includes modified nucleotides comprising pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine / pseudouridine (5meC PsU), N1-methyl-pseudouridine (N1mPsU), or combinations thereof.

[0249] In some embodiments, the mRNA of this disclosure (e.g., mRNA 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 cytotoxic agent described herein) may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides.

[0250] Typically, mRNA synthesis involves adding a "cap" at the N-terminus (5') and a "tail" at the C-terminus (3'). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the tail is used to protect the mRNA from degradation by exonucleases.

[0251] Therefore, in some embodiments, the mRNA of this disclosure (e.g., mRNA 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 cytotoxic agent described herein) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphates via guanylate transferase, creating a 5' triphosphate bond; and then, 7-N-methylation of guanine is performed via 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, the cap includes a Cap0 structure. The Cap0 structure lacks a 2'-O-methyl residue of the ribose linked to bases 1 and 2. In some embodiments, the cap includes an AGCap1 structure. The AGCap1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap includes a Cap2 structure. The Cap2 structure has a 2'-O-methyl residue attached to both bases 2 and 3. In some embodiments, the cap structure includes AGCap1, m6AGCap1, or an anti-reverse cap analog (ARCA). In some embodiments, the modified mRNA of this disclosure includes m6AGCap1 and a modified nucleotide including pseudouridine (PsU).

[0252] In some embodiments, the mRNA of this disclosure (e.g., mRNA 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 cytotoxic agent described herein) includes a 3' poly(A) tail structure. The 3' poly(A) tail of the 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, the mRNA includes a 3' poly(C) tail structure. A suitable poly(C) tail at the 3' end of mRNA typically comprises approximately 10 to 200 cytidine nucleotides (e.g., approximately 10 to 150 cytidine nucleotides, approximately 10 to 100 cytidine nucleotides, approximately 20 to 70 cytidine nucleotides, approximately 20 to 60 cytidine nucleotides, or approximately 10 to 40 cytidine nucleotides). The poly(C) tail may be added to the poly(A) tail or used as a substitute for it.

[0253] In some embodiments, the mRNA of this disclosure (e.g., mRNA 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 cytotoxic agent described herein) includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the length of the 5' untranslated region may be between about 50 and 500 nucleotides.

[0254] In some embodiments, the 3' untranslated region includes one or more of the following: adenosine monophosphate (APM) signal, a protein binding site that affects the stability or localization of mRNA in the cell, or one or more miRNA binding sites. In some embodiments, the length of the 3' untranslated region may be between 50 and 500 nucleotides or longer.

[0255] Those skilled in the art will recognize that, for a disclosed amino acid sequence, there are several nucleotide sequences capable of generating that amino acid sequence. While certain exemplary nucleotide sequences are disclosed, it is specifically considered that each nucleotide sequence that produces a particular amino acid sequence may be useful in some embodiments.

[0256] Application of additional payload In some embodiments, the method of this disclosure includes one or more steps of treating immune cells (e.g., macrophages, monocytes, or dendritic cells) during the process of modifying immune cells. In some embodiments, the method of this disclosure includes one or more steps of administering to a subject, during the process of modifying immune cells, one or more steps of applying additional payloads to modulate immune cells (e.g., macrophages, monocytes, or dendritic cells). In some embodiments, the composition includes one or more additional payloads. In some embodiments, the composition includes one or more additional payloads in the same delivery medium as one or more nucleic acid molecules. In some embodiments, the composition includes one or more additional payloads in a delivery medium different from the delivery medium used for one or more nucleic acid molecules.

[0257] In some embodiments, the methods of this disclosure include the step of treating immune cells (e.g., macrophages, monocytes, or dendritic cells) with a regulator of a pathway activated by in vitro transcribed mRNA. In some embodiments, the additional payload may be or includes a regulator 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-activating (PKR), retinoic acid-induced gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and 2ʹ-5ʹ-oligoadenylate synthase (OAS)). Signal transduction via these different pathways leads to inflammation associated with interferon type 1 (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), and IL-12, as well as activation of the transcriptional cascade. Overall, these create a pro-inflammatory microenvironment suitable for inducing specific immune responses. Furthermore, downstream effects, such as slowing translation through phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), enhancing RNA degradation through ribonuclease L (RNase L), and overexpression and inhibition of autoamplified mRNA replication, are associated with the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0258] In some embodiments, the regulator of the in vitro transcription mRNA activation pathway includes an RNase inhibitor. In some embodiments, the regulator of the in vitro transcription mRNA activation pathway includes an RNase L, RNase T2, or RNase 1 inhibitor. In some embodiments, the regulator of the in vitro transcription mRNA activation pathway includes an RNase L inhibitor. In some embodiments, the RNase L inhibitor includes sunitinib. In some embodiments, the RNase L inhibitor includes ABCE1.

[0259] In some embodiments, treatment of immune cells (e.g., macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases mRNA stability in the modified immune cells relative to mRNA stability in the same type of modified immune cells not treated with an RNaseL inhibitor. In some embodiments, treatment of immune cells (e.g., macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases the 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 cytotoxic agent in the modified immune cells relative to mRNA stability in the same type of modified immune cells not treated with an RNaseL inhibitor. In some embodiments, treatment of immune cells (e.g., macrophages, monocytes, or dendritic cells) with an RNaseL inhibitor increases effector activity in the modified immune cells relative to effector activity in the same type of modified immune cells not treated with an RNaseL inhibitor.

[0260] In some embodiments, administration of an RNaseL inhibitor to a subject increases the mRNA stability of modified immune cells (e.g., macrophages, monocytes, or dendritic cells) compared to the mRNA stability of the same type of modified immune cells in a subject who has not received an RNaseL inhibitor. In some embodiments, administration of an RNaseL inhibitor to a subject increases the 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 cytotoxic agent in modified immune cells (e.g., macrophages, monocytes, or dendritic cells) compared to the same type of modified immune cells in a subject who has not received an RNaseL inhibitor. In some embodiments, administration of an RNaseL inhibitor to a subject increases the effector activity of modified immune cells (e.g., macrophages, monocytes, or dendritic cells) compared to the effector activity of the same type of modified immune cells in a subject who has not received an RNaseL inhibitor.

[0261] In some embodiments of this disclosure, the step of treating immune cells (e.g., macrophages, monocytes, or dendritic cells) occurs prior to the step of delivering mRNA to the immune cells. In some embodiments of this disclosure, the step of administering an additional payload to the subject occurs prior to the step of administering a composition comprising mRNA to the subject.

[0262] In some embodiments, the method of this disclosure includes the step of culturing immune cells (e.g., macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins. In some embodiments, the method of this disclosure includes the step of administering cytokines or immunostimulatory recombinant proteins to a subject. In some embodiments, cytokines include IFN-α, IFN-β, IFN-γ, TNFα, IL-6, STNGL, LPS, CD40 agonists, 4-1BB ligands, recombinant 4-1BB, CD19 agonists, TLR agonists (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), glucocorticoids, immune complexes, interleukin-1α (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and 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), oncogene M (OSM), TNF-β, CD154, lymphotoxin β (LT-β), 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-associated apoptosis-inducing ligand (TRAIL), TNF-associated weak apoptosis inducer (TWEAK), TNF-associated activation-inducing cytokine (TRANCE), erythropoietin (Epo), thyroid peroxidase precursor (Tpo), FMS-associated tyrosine kinase 3 ligand (FLT-3L), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite surface protein (MSP), nucleotide-binding oligomerization domain-containing protein (NOD) ligands (e.g., NOD1, NOD2, or NOD1 / 2 agonists), RIG-I-like receptor (RLR) ligands (e.g., 5' ppp-dsRNA, 3p-hpRNA, poly(I:C), or poly(dA:dT)), C-type lectin receptor (CLR) ligands (e.g., gel polysaccharides, β-glucan, HKCA, kelp polysaccharide, lecithin, sclerosan, dispersible WGP, soluble WGP, yeast polysaccharide, depleted yeast polysaccharide, furfurman, b-GlcCer, GlcC14C18, HKMT, TDB, TDB-HS15 or TDM), cyclic dinucleotide sensor ligands (e.g., C-Gas agonists or interferon gene stimulating factor (STING) ligands), inflammasome inducers (e.g., alum, ATP, CPPD crystals, Plasmodium pigment, MSU crystals, Nano-SiO2, Nigerian styracin or TDB), aromatic hydrocarbon (AhR) ligands (e.g., FICZ, indirubin, ITE or L-kynurenine), α-protein kinase 1 (ALPK1) ligands, poly-PRR ligands, NFKB / NFAT activators (e.g., concavalin A, ionomycin, PHA-P or PMA) or combinations thereof. In some implementations, the cytokine includes IFN-β.

[0263] In some embodiments of this disclosure, the step of culturing immune cells (e.g., macrophages, monocytes, or dendritic cells) occurs after the step of delivering mRNA to the immune cells. In some embodiments of this disclosure, the step of administering cytokines or immunostimulatory recombinant proteins to the subject occurs after the step of administering a composition comprising mRNA to the subject.

[0264] In some embodiments, culturing modified immune cells (e.g., macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases the viability of the modified immune cells compared to the same type of modified immune cells not cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases protein expression in the modified immune cells compared to the same type of modified immune cells not cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases the persistence of protein expression compared to the same type of modified immune cells not cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases the effector activity of the modified immune cells compared to the same type of modified immune cells not cultured with cytokines or immunostimulatory recombinant proteins. In some embodiments, culturing modified immune cells (e.g., macrophages, monocytes, or dendritic cells) with cytokines or immunostimulatory recombinant proteins increases the pro-inflammatory (M1) polarization of the modified immune cells compared to the same type of modified immune cells not cultured with cytokines or immunostimulatory recombinant proteins.

[0265] In some embodiments, administration of cytokines or immunostimulatory recombinant proteins to a subject increases the activity of modified immune cells (e.g., macrophages, monocytes, or dendritic cells) of the same type as in a subject who has not been administered cytokines or immunostimulatory recombinant proteins. In some embodiments, administration of cytokines or immunostimulatory recombinant proteins to a subject increases the 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 cytotoxic agent in the modified immune cells (e.g., macrophages, monocytes, or dendritic cells) of the subject, relative to the same type of modified immune cells in a subject who has not been administered cytokines or immunostimulatory recombinant proteins. In some embodiments, administration of cytokines or recombinant immunostimulatory proteins to a subject results in a persistent increase in the 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 cytotoxic agent in the modified immune cells (e.g., macrophages, monocytes, or dendritic cells) of the same type of modified immune cells in a subject who have not been administered cytokines or recombinant immunostimulatory proteins. In some embodiments, administration of cytokines or recombinant immunostimulatory proteins to a subject results in an increase in the effector activity of the modified immune cells (e.g., macrophages, monocytes, or dendritic cells) of the modified immune cells in a subject who have not been administered cytokines or recombinant immunostimulatory proteins. In some embodiments, administration of cytokines or recombinant immunostimulatory proteins to a subject results in an increase in the pro-inflammatory (M1) polarization of the modified immune cells (e.g., macrophages, monocytes, or dendritic cells) of the modified immune cells in a subject who have not been administered cytokines or recombinant immunostimulatory proteins.

[0266] Measurement A variety of assays can be performed to confirm the presence of the nucleic acid constructs described herein in immune cells, such as monocytes, macrophages, or dendritic cells. Such assays include, for example, molecular biology assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical assays, such as those using immunological methods (ELISA and Western blotting) to detect the presence or absence of a specific peptide. Other assays disclosed herein include, for example, fluorescence-activated cell sorting (FACS), immunofluorescence microscopy, MSD cytokine analysis, mass spectrometry (MS), RNA-Seq, and functional assays.

[0267] A variety of assays can be performed to determine various characteristics of modified immune cells (e.g., macrophages, monocytes, or dendritic cells), such as, but not limited to, immune cell viability, nucleic acid expression, nucleic acid persistence, protein expression, protein persistence, effector activity, and pro-inflammatory (M1) polarization. Examples of such assays include flow cytometry, quantitative PCR, and in vitro functional assays such as cytokine / chemokine secretion, phagocytosis, cell burial 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 herein in their entirety for reference. Furthermore, materials, methods, and examples are illustrative only and are 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 pertains. While similar or equivalent methods and materials described herein may be used in the practice or testing of this invention, suitable methods and materials are described herein.

[0269] This disclosure is further illustrated by the following examples. These examples are for illustrative purposes only and should not be construed as limiting the scope or content of this disclosure in any way.

[0270] Example The following examples are provided to illustrate to those skilled in the art how to manufacture and use the methods and compositions described herein, and are not intended to limit the scope of this disclosure.

[0271] Example 1: A method for generating and validating viable anti-fibrotic engineered macrophages This embodiment describes the generation and validation of anti-fibrotic engineered mouse macrophages. For example... Figure 1 As shown, primary hematopoietic stem cells (HSCs) were isolated from the bone marrow of wild-type C57BL / 6 (Bl6) mice on day 0. The cells were either untransduced or transduced with troponin retroviruses including exemplary payloads from Table 1.

[0272] Table 1: Exemplary Payload Constructions Transduction efficiency of HSCs was enhanced by culturing them on RetroNectin®-coated containers. HSCs were cultured with 10 ng / mL M-CSF to stimulate differentiation into bone marrow-derived macrophages. On days 2 and 4 of the study, the cell culture medium was replaced with fresh medium containing M-CSF (final concentration 10 ng / mL). On day 5, differentiated BMDM cells were collected and analyzed using an NC200 cell counter. Aliquots of cells from each culture were stored as cryovials in 90% FBS + 10% DMSO to determine freeze-thaw tolerance.

[0273] To assess payload expression over time in vitro, supernatants were collected on days 1, 7, 9, 13, and 14 of the study and analyzed using ELISA. Figure 2 The cell culture medium was changed, and the collected supernatant was replaced with fresh medium containing M-CSF. On day 5, cell resuscitation rate, viability, and expression of transduced payload sequences in BMDM were analyzed using an NC200 analyzer. On day 5, transduced HSCs differentiated into healthy BMDM and exhibited resuscitation rate and viability comparable to standard untransduced macrophages. Figure 3A Furthermore, both untransduced and engineered macrophages were successfully expanded from seeded HSCs (>20-fold expansion) and exhibited high viability (>90%).

[0274] BMDM stored as frozen reserves should be immediately (after thawing) Figure 3B ) and after 48 hours of incubation ( Figure 3C The recovery rate and viability were assessed. All BMDM conditions showed high recovery rates and viability immediately after thawing and maintained viability in vitro for 48 hours.

[0275] In summary, these results indicate that engineered antifibrotic engineered macrophages possess high viability for in vivo and in vitro studies, and that frozen stockpiles are viable immediately upon thawing (similar to their state when administered in in vivo studies) and can maintain viability in vitro for at least 48 hours.

[0276] Example 2: Engineered macrophages can secrete a variety of therapeutic proteins and express therapeutic transmembrane proteins. This embodiment describes the ability of engineered antifibrotic macrophages to secrete therapeutic payloads using ELISA assays. This embodiment also describes the characterization of therapeutic transmembrane proteins expressed by engineered macrophages.

[0277] HSCs were generated and analyzed as described in Example 1. HSCs were either untransduced or transduced with retroviruses encoding exemplary payloads from Table 1, and the production of the immunosuppressive cytokine IL-10 was analyzed. Engineered macrophages expressing the IL-10-P2A-MMP13 payload, the IL-10-P2A-chalasin payload, or the IL-10-P2A-FGF21 payload produced high levels of IL-10 compared to untransduced cells with little or no IL-10 production. Figure 4A ).

[0278] Engineered macrophages expressing IL-10-P2A-FGF21 produced high levels of FGF21, while no expression was detected in untransduced cells or cells expressing IL-10-P2A-MMP13 or IL-10-P2A-chalasin. Figure 4B These results demonstrate the ability to detect two payloads from the same construct (IL-10 and FGF21 from IL-10-P2A-FGF21).

[0279] Engineered macrophages expressing the relaxin construct produce high levels of relaxin ( Figure 4C ), while no expression was detected in untransduced cells.

[0280] Engineered macrophages expressing the IL-10-P2A-core proteoglycan construct produced high levels of core proteoglycan, while no core proteoglycan expression was detected in untransduced cells or cells expressing the anti-HER2 construct. Figure 4D ).

[0281] In summary, these results demonstrate that secreted payloads can be produced at high concentrations. These results also indicate that a single-cell product can co-express multiple proteins.

[0282] The viability of engineered macrophages expressing the transmembrane receptor proteins DN TGFβR2 and IL17AR was also evaluated. Expression of DN TGFβR2 ( Figure 5A ) and IL17AR ( Figure 5B The cells have high vitality. Figure 5C Flow cytometry analysis showed that each receptor had high surface expression compared to untransduced cells. Figure 5D-5E ).

[0283] like Figure 6A As shown, ELISA analysis of cells transduced with IL-10-P2A-GFP revealed high levels of IL-10 in the supernatant of engineered macrophages on day 7, unlike the supernatant of untransduced cells. Furthermore, ELISA results indicated that engineered macrophages maintained IL-10 production in vitro for at least 10 weeks. Figure 6B ).

[0284] In summary, these results demonstrate that secreted payloads can be produced at high concentrations. These results also indicate that a single-cell product can co-express multiple proteins.

[0285] Example 3: Engineered macrophages generated for in vivo use are viable and express the encoded payload in vivo. This embodiment describes a method for evaluating payload expression in vivo. This embodiment also describes the viability characterization and payload expression of engineered macrophages in vivo.

[0286] like Figures 7A-7B As shown, BMDMs were generated and frozen as described in Example 1. HSCs were transduced with a retrovirus encoding either luciferase-P2A-GFP or IL-10-P2A-luciferase. On day 5, the transduced HSCs differentiated into healthy BMDMs. Recovery rate and viability on day 5 were analyzed using an NC200. BMDMs expressing luciferase-P2A-GFP or IL-10-P2A-luciferase were successfully amplified from the inoculated number of HSCs (12 to 20-fold amplification) and exhibited high viability. Figure 8A The same applies to BMDM, which is stored as a frozen reserve and whose viability is assessed immediately after thawing. Figure 8B ).

[0287] To monitor in vivo transport and payload expression, frozen BMDM was thawed and cell counts and viability were assessed. A total of 5 × 10⁶ cells were administered to Balb / c mice via intravenous (“iv”) injection. 6 Cells ( Figure 7B ).

[0288] IVIS was performed on days 1, 7, 10, and 13 after BMDM administration. ® Imaging to monitor luciferase expression ( Figure 7B Engineered macrophages produced detectable luciferase signals in mice at least 13 days after administration. Figures 9A-9B Unbound by theory, these data demonstrate that IL-10 improves the persistence of engineered macrophages. These results confirm that BMDM can generate functional signals in vivo after being transduced as HSCs with a retrovirus, frozen as BMDM on day 5, and then administered intravenously after freeze-thaw.

[0289] Example 4: Method for evaluating the antifibrotic efficacy of engineered macrophages in a mouse model of liver fibrosis This embodiment describes the characterization of engineered antifibrotic macrophages in a carbon tetrachloride (CCl4)-induced liver fibrosis model. Antifibrotic engineered mouse macrophages were generated as described in Example 1. HSCs were either untransduced or transduced using retroviruses encoding the exemplary constructs listed in Table 1.

[0290] like Figure 10 As shown, male Bl6 mice received intraperitoneal injections of freshly prepared CCl4 (1 mL / kg) diluted 1:3 in corn oil 28 days prior to BMDM injection (n=9 per group). On day 28 after the start of CCl4 challenge, BMDM (5 × 10⁻⁶) was administered to the mice via intravenous injection. 6 (cells). On day 42, the mice were sacrificed and the livers were collected for histological analysis.

[0291] To first validate the transduced BMDM expression payload, ELISA and Western blot analysis were used to confirm payload expression. High levels of IL-10 production were detected by ELISA in cells expressing IL-10-P2A-chalasin. Figure 11A Further analysis of the cell culture supernatant was performed using non-denaturing Western blot assays. Figure 11B As shown, relaxin production is specific for engineered macrophages expressing IL-10-P2A-chalasin, while no expression was detected in untransduced cells or BMDM cells expressing other antifibrotic payloads.

[0292] To determine whether engineered macrophages were well tolerated in mice challenged with CCl4, mouse body weight was measured twice weekly throughout the 42-day study. Mice challenged with CCl4 showed weight loss and fluctuation compared to the control group, indicating successful CCl4 administration. Figure 12A Furthermore, CCl4-challenged mice treated with solvent, UTD, or IL-10-P2A-chalasin showed similar body weights on day 43, indicating that the engineered macrophages were well tolerated. Figure 12B ).

[0293] Histological analysis of collagen content was also used to analyze fibrotic liver tissue collected from mice to determine whether the administration of engineered macrophages expressing a therapeutic payload affected CCl4-induced fibrosis. For histological analysis, the mid-segment of the left posterior lobe of the liver was formalin-fixed, paraffin-embedded, and then sectioned. Total collagen content was identified using Masson's trichrome staining. Collagen content was quantified using HALO analysis software.

[0294] Tissue sections from control mice (solvent-treated) showed a significant increase in collagen staining, indicating successful fibrosis induction. Figures 13A-13B Although untransduced macrophages induced a 55% reduction in collagen staining, engineered macrophages expressing IL-10-P2A-chalasin induced a greater than 100% reduction in collagen staining. These results indicate that a single dose of macrophages producing both chalasin and IL-10 significantly reduced CCl4-induced liver fibrosis.

[0295] Example 5: Evaluation of the antifibrotic efficacy of engineered macrophages in a bleomycin-induced pulmonary fibrosis model This example describes the characterization of a mouse model of bleomycin-induced pulmonary fibrosis induced by treatment with engineered macrophages.

[0296] Antifibrotic engineered mouse macrophages were generated as described in Example 1. HSCs were either untransduced or transduced with retroviruses encoding IL-10-P2A-chaserin or IL-10-P2A-DN-TGFβR2 constructs, as listed in Table 1.

[0297] like Figure 14 As shown, to generate a mouse pulmonary fibrosis model, Taconic Bl6 mice (6-7 weeks old) received 1 U / kg of bleomycin (“BLM”) intranasally (n=10-15 per group). On the following day, day 1, engineered macrophages (5 × 10⁶ cells per mouse) were administered intravenously. 6 Mice were treated with either single cell counts or a solvent control. On day 21, mice were sacrificed to assess hydroxyproline content in the lungs.

[0298] BMDM expressing IL-10-P2A-chalasin or IL-10-P2A-DN-TGFβR2 was successfully amplified from the inoculated number of HSCs (12 to 20-fold amplification) and exhibited high activity. Figure 15A To validate the expression payload of transduced BMDM, ELISA and flow cytometry analyses were used to confirm payload expression. High levels of IL-10 production were detected in cells expressing IL-10-P2A-chalasin and IL-10-P2A-TGFβR2 DN by ELISA compared to untransduced cells. Figure 15B ).like Figure 15C As shown, flow cytometry analysis detected dominant and negative TGFβR2 on the surface of cells transduced with IL-10-P2A-TGFβR2 DN, confirming the correct payload expression.

[0299] To determine whether the engineered macrophages were well tolerated by mice challenged with CCl4, mouse body weight was measured twice weekly throughout the 42-day study. Compared to the control group (referred to as "Sham"), mice challenged with BLM showed the expected 10–15% body weight loss followed by a rebound, indicating successful BLM challenge. Figure 16A Furthermore, CCl4-challenged mice treated with solvent, UTD, or IL-10-P2A-chalasin or IL-10-P2A-TGFβR2 DN showed similar body weights on day 21, indicating that the engineered macrophages were well tolerated. Figure 16B ).

[0300] Fibrotic tissue was quantified by measuring the concentration of hydroxyproline (referred to as "HYP" in this paper) from homogenized left lung tissue samples. Compared to the Sham group, BLM-challenged (untreated) mice induced a significant increase in HYP, indicating successful fibrosis induction. Although mice treated with untransduced BMDM showed 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 a decrease in HYP (greater than 90%), such as... Figure 17A As shown. Furthermore, engineered macrophages expressing IL-10-P2A-chalasin induced a 44% reduction in mean HYP, a statistically significant difference compared to untransduced cells. Figure 17B In summary, these results indicate that a single dose of engineered macrophages containing IL-10-P2A-TGFβR2 or IL-10-P2A-chalasin reduces bleomycin-induced pulmonary fibrosis.

[0301] Example 6: Evaluation of antifibrotic engineered macrophages overexpressing TIM4 or TIM4+chalasin Among other things, this embodiment describes the generation of engineered macrophages that overexpress TIM4 or TIM4 and relaxin, and the effects of such engineered macrophages on several indicators of fibrosis in a mouse model and on overall cell burial capacity.

[0302] Primary human macrophages were thawed and transduced either untransduced or with VPX-lentiviral particles encoding full-length human TIM4. Seven days after transduction, engineered and untransduced macrophages were collected, and TIM4 production was analyzed by flow cytometry. Figure 18 Compared to the untransduced (UTD) control, macrophages engineered to express TIM4 produced high levels of TIM4 (…). Figure 19 ).

[0303] Next, a cytotoxicity assay was used to test the cytotoxicity of macrophages engineered to overexpress TIM4. Figure 19 First, primary human macrophages were thawed and preserved as UTD or transduced with VPX-lentiviral particles encoding full-length human TIM4 or a lentiviral control (miRFP670). Jurkat cells were labeled with pHrodo, a pH-sensitive dye whose signal increases in acidic environments, such as during endocytosis. Apoptosis was induced in Jurkat cells using cruciferine. Macrophages were mixed with apoptotic pHrodo-labeled Jurkat cells, and the mixture was monitored using live-cell imaging. Successful phagocytosis of apoptotic target Jurkat cells induced an increase in pHrodo fluorescence signal.

[0304] UTD macrophages did not exhibit strong cell encapsulation. Transduction with lentiviral control did not improve cell encapsulation. However, macrophages engineered to overexpress TIM4 resulted in enhanced cell encapsulation compared to both UTD and lentiviral control transduced macrophages. Figure 21 ).

[0305] In summary, these results demonstrate that the engineered macrophages described in this paper can produce secreted TIM4 at high concentrations. These results also show that macrophages engineered to express TIM4 possess enhanced endocytic capacity.

[0306] Next, as Figure 22 As shown, engineered anti-fibrotic mouse macrophages were generated. Primary myeloid cysts (HSCs) were first isolated from the bone marrow of BI6 mice and then transduced using a troponin-encoding retrovirus encoding TIM4 or TIM4+chalasin (TIM4-P2A-chalasin). Transduction efficiency of HSCs was enhanced by culturing them in RetroNectin-coated containers. HSCs were cultured in fresh medium containing 10 ng / mL M-CSF to mimic differentiation into engineered bone marrow-derived macrophages (BMDMs). Cell cultures were replenished with fresh M-CSF on days 2 and 4. On day 5, differentiated engineered BMDMs were collected and stored in a frozen stock containing 90% FBS and 10% DMSO.

[0307] Engineered macrophages were collected on day 5 and analyzed by flow cytometry to examine viability and CD11b versus F4 / 80 (F4 / 80). Figure 23A ) and TIM4 ( Figure 23B The expression of ). Most macrophages engineered to express TIM4 or TIM4+ relaxin are positive for CD11b and F4 / 80 expression ( Figure 23A Both groups of engineered macrophages also demonstrated TIM4 expression. Macrophages engineered to express TIM4+hyperchalasin showed significantly higher expression than macrophages engineered to express only TIM4.

[0308] Engineered macrophages were also collected on day 7 and analyzed by flow cytometry to assess TIM4 expression. Figure 23C TIM4 expression in engineered macrophages increased from day 5 to day 7.

[0309] Next, the relaxin production of macrophages engineered to express either TMI4 or TIM4+ relaxin was evaluated. Macrophages were generated and engineered using the methods described herein and cultured for 14 days. Supernatants from engineered macrophages were collected on days 5, 7, and 14, and relaxin levels were measured by ELISA. Fresh culture medium was added to the cell culture after collection. On day 14, engineered macrophages expressing TIM4+ relaxin produced high levels of relaxin, while engineered macrophages expressing the TIM4 construct showed no detectable levels of relaxin. Figure 24A ).

[0310] A time-course analysis was also performed on supernatants collected from engineered macrophages expressing TIM4+ relaxin on days 5, 7, and 14 to assess the rate of relaxin production. The rate of relaxin production was calculated as the measured relaxin concentration divided by the time elapsed since the last culture medium change. Although the rate of relaxin production decreased between days 3 and 6, it eventually stabilized from day 6 onwards. Figure 24B ).

[0311] In summary, these results demonstrate that macrophages engineered to express TIM4 produced secreted TIM4, and macrophages engineered to express TIM4+ relaxin produced even higher levels. Engineered macrophages expressing TIM4+ relaxin secreted high levels of relaxin at a stable rate for at least 14 days.

[0312] Example 7: Method for evaluating the antifibrotic efficacy of engineered macrophages overexpressing TIM4 or TIM4+chalasin in a mouse model of liver fibrosis. This example describes the characterization of engineered antifibrotic macrophages in a choline-deficient, L-amino acid-restricted high-fat diet (CDAHFD) liver fibrosis model. Antifibrotic engineered mouse macrophages were generated as described in Example 6. Bone marrow-derived macrophages (BMDM) were transduced with a retrovirus encoding TIM4 or TIM4+ relaxin, or as a control untransduced (UTD) (referred to herein as "control Mɸ").

[0313] like Figure 25 As shown, male Bl6 mice were fed a normal diet and kept untreated (referred to as “healthy liver” in this paper) or a choline-deficient, L-amino acid-restricted high-fat diet (CDAHFD) that induces fibrosis and kept untreated (referred to as “untreated” in this paper) 28 days before BMDM injection, or were treated with the aforementioned engineered macrophages. CDAHFD (also known as “HF-CDAA”) models include, for example, Shi, H. et al., “Loss of TIM4-Dependent Efferocytosis in KupfferCells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis,” bioRxiv The procedure described in 2024.01.30.578023 (2024) is incorporated herein by reference in its entirety. On day 28, after the start of feeding, mice were administered engineered macrophages (5 × 10⁶ cells / mL) via intravenous injection. 6 (cells) or solvent control. On day 42, mice were sacrificed and livers were collected for histological analysis.

[0314] Immunoblotting was performed on TIM4 expression in mouse liver to confirm that CDAHFD-induced liver fibrosis reduced intrahepatic TIM4 levels. Healthy mice (treated with PBS) expressed high levels of TIM4, while CDAHFD-fed mice showed reduced TIM4 protein levels despite treatment with control macrophages (“Ctr Mɸ”). Treatment with engineered TIM4 macrophages rescued TIM4 expression in CDAHFD-fed mice. Figures 26A-26B These results confirm that engineered macrophages overexpressing TIM4 helped restore TIM4 expression as expected.

[0315] Next, in situ analysis of the necrolysis capacity of engineered macrophages was performed using histological sections quantified, as described, for example, in Ampomah et al., “Macrophages use apoptotic cell-derivedmethionine and DNMT3A during efferocytosis to promote tissue resolution,” NatMetab 4, 444-457 (2022), the entire text of which is incorporated herein by reference. Macrophages were identified using the panmaphage marker Mac2. Apoptotic cells were identified using the cleaved caspase 3 (cl-Casp3) marker. Apoptotic cells spatially associated with macrophages were considered to be undergoing necrolysis. The number of apoptotic cells associated with macrophages was normalized relative to the number of apoptotic cells independently located away from macrophages. A larger ratio indicates an increased amount of necrolysis. Figure 27 As shown, treatment with TIM4 or TIM4+chalasin macrophages increased in situ cell burial.

[0316] Next, histological analysis of collagen content was used to analyze fibrotic liver tissue collected from mice to determine whether administration of engineered macrophages expressing TIM4 or TIM4+ relaxin affected CDAHFD-induced fibrosis. For histological analysis, the mid-segment of the left posterior lobe of the liver was formalin-fixed and paraffin-embedded, then sectioned. Staining was performed using picric Sirius red (also referred to herein as "Sirius Red"). Figures 28A-28B ) and type 1 collagen α1 (Col1a1) staining ( Figures 29A-29B The total collagen content was determined using [a specific method / mechanism]. The collagen content was quantified using HALO analysis software.

[0317] Collagen content in tissue sections from solvent-treated (untreated) CDAHFD mice, measured by Sirius red, showed a significant increase in staining, indicating that the fibrosis model successfully activated fibroblasts. Figures 28A-28BMice treated with UTD macrophages (“Control Mɸ”) did not achieve a statistically significant reduction in Sirius red staining, with a reduction in Sirius red area of ​​approximately 16%. Engineered macrophages expressing only TIM4 induced a significant reduction in collagen staining by 49%. In contrast, engineered macrophages co-expressing TIM4 and relaxin induced a significant reduction of 70%.

[0318] Collagen content in tissue sections from untreated CDAHFD mice, measured by Col1A1, showed a significant increase in staining, indicating that the fibrosis model successfully activated fibroblasts. Figures 29A-29B Mice treated with UTD macrophages (“Control Mɸ”) did not achieve statistically significant reduction in Col1A1 staining, with a reduction in Col1A1 area of ​​approximately 5%. Engineered macrophages expressing only TIM4 induced a significant reduction of 30%. In contrast, engineered macrophages co-expressing TIM4 and relaxin induced a significant reduction of 36%.

[0319] Further histological analysis was performed on fibrotic liver tissue collected from mice to assess the cytoskeleton and intracellular components, and to determine whether administration of engineered macrophages expressing TIM4 or TIM4+ relaxin affected CDAHFD-induced fibroblast activation and inflammation. The mid-segment of the left posterior lobe of the liver was formalin-fixed, paraffin-embedded, and then sectioned. Staining with α-smooth muscle actin (“α-SMA”) was performed. Figures 30A-30B ) and osteopontin (“Opn”) staining ( Figures 31A-31B OPN is used as a pro-inflammatory and pro-fibrotic biomarker associated with liver fibrosis. OPN levels were quantified using HALO analysis software.

[0320] Tissue sections from untreated CDAHFD mice showed a significant increase in α-SMA content, indicating that the fibrosis model successfully activated fibroblasts. Figures 30A-30B Mice treated with UTD macrophages (control Mɸ) did not achieve a statistically significant reduction in α-SMA staining, with an α-SMA reduction of approximately 18%. Engineered macrophages expressing only TIM4 induced a significant reduction of 48%. In contrast, engineered macrophages co-expressing TIM4 and relaxin induced a significant reduction of 56%.

[0321] Osteopontin levels in tissue sections from untreated CDAHFD mice showed a significant increase in staining, indicating that the fibrosis model successfully activated fibroblasts. Figures 31A-31BMice treated with UTD macrophages (control Mɸ) did not achieve a statistically significant reduction in Opn staining, with an Opn reduction of approximately 17%. Engineered macrophages expressing only TIM4 induced a significant reduction of 43%. In contrast, engineered macrophages co-expressing TIM4 and relaxin induced a significant reduction of 64%.

[0322] The results presented in this paper demonstrate that engineered macrophages expressing TIM4 or both TIM4 and relaxin achieved a significant reduction in fibrotic area. Even at low transduction levels of 15–20%, TIM4 improved the antifibrotic effect of engineered macrophages compared to UTD macrophages. These results also support increasing macrophage burial capacity as an antifibrotic strategy (e.g., via other burial receptors, synthetic burial receptors, and / or other signaling molecules that upregulate endogenous burial mechanisms). Unlike proteins such as relaxin, TIM4 does not have a readily available systemic counterpart for standalone protein therapy; therefore, the efficacy of TIM4 requires utilization of its function based on cellular solvents. These results also demonstrate that engineered macrophages can indeed achieve an additive antifibrotic effect through the co-expression of TIM4 and relaxin.

[0323] Example 8: Evaluation of anti-fibrotic engineered human macrophages overexpressing TIM4 or TIM4+chalasin in a mouse model of liver fibrosis This embodiment describes the application of antifibrotic engineered human macrophages to NSG. TM Characterization in a mouse model of choline deficiency, L-amino acid-restricted high-fat diet (CDAHFD) liver fibrosis, to enable treatment with human TIM4 or human TIM4+human relaxin without rejection by the mouse's innate immune system.

[0324] On day 0, primary human monocytes were thawed and kept untransduced (UTD; control Mɸ) or transduced with a lentivirus encoding human TIM4 (hu. TIM4 Mɸ). Cells were then allowed to differentiate for five days in the presence of GM-CSF. On day 5, cells were collected and viability and TIM4 expression were analyzed, and formulations for in vivo injection were prepared. Figure 32 ).

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

[0326] The CDAHFD model described in Example 7 was applied to immunodeficient NSG.TM Mice. In nine-week-old female NSG mice. TM Mice were fed a diet or induced with CDAHFD to induce fibrosis. On day 28 after the start of the diet, mice were administered engineered human macrophages (5 × 10⁶ cells) via intravenous injection. 6 (cells) or solvent control. On day 42, mice were sacrificed, and livers were collected for histological analysis (cells) Figure 34 ).

[0327] Next, histological analysis of collagen content was used to analyze fibrotic liver tissue collected from mice to determine whether administration of engineered human macrophages expressing TIM4 affected CDAHFD-induced fibrosis. For histological analysis, the mid-segment of the left posterior lobe of the liver was formalin-fixed and paraffin-embedded, then sectioned. Masson's trichrome staining was used. Figures 35A-35B ), picric acid Sirius red (also referred to as "Sirius red" in this article) staining ( Figures 36A-36B ) and type 1 collagen α1 (Col1a1) staining ( Figures 37A-37B The total collagen content was determined using [a specific method / mechanism]. The collagen content was quantified using HALO analysis software.

[0328] NSG from solvent-treated NSG was measured by Masson's trichrome staining. TM Tissue sections from untreated CDAHFD mice showed a significant increase in collagen staining, indicating successful fibrosis induction. Figures 35A-35B Although untransduced macrophages induced a 29% reduction in collagen staining, engineered human macrophages expressing TIM4 induced a 58% reduction in collagen staining.

[0329] NSG derived from solvent treatment, measured by Sirius red. TM The collagen content in tissue sections of CDAHFD mice showed a significant increase upon staining, indicating that the fibrosis model successfully activated fibroblasts. Figures 36A-36B NSG treated with UTD macrophages (“control Mɸ”) TM Mice did not show a reduction in Sirius red staining. Engineered human macrophages expressing TIM4 induced a significant 47% reduction in collagen staining.

[0330] Measured from untreated NSG by Col1A1 TM The collagen content in tissue sections of CDAHFD mice showed a significant increase upon staining, indicating that the fibrosis model successfully activated fibroblasts. Figures 37A-37B NSG treated with UTD macrophages (“control Mɸ”) TMIn mice, the reduction in Col1A1 staining was not statistically significant, with a decrease of approximately 12% in the area stained. In contrast, engineered human macrophages expressing only TIM4 induced a significant reduction of 44%.

[0331] Further histological analysis was performed on fibrotic liver tissue collected from mice to assess the cytoskeleton and intracellular components and to determine whether administration of TIM4-expressing engineered human macrophages affected CDAHFD-induced fibroblast activation and inflammation. The mid-segment of the left posterior lobe of the liver was formalin-fixed, paraffin-embedded, and then sectioned. Staining with α-smooth muscle actin (“α-SMA”) was performed. Figures 38A-38B ) and osteopontin (“Opn”) staining ( Figures 39A-39B OPN is used as a pro-inflammatory and pro-fibrotic biomarker associated with liver fibrosis. OPN levels were quantified using HALO analysis software.

[0332] From unprocessed NSG TM The α-SMA content in tissue sections of CDAHFD mice showed a significant increase in staining, indicating that the fibrosis model successfully activated fibroblasts. Figures 38A-38B NSG treated with UTD macrophages (control Mɸ) TM In mice, no statistically significant reduction in α-SMA staining was achieved; the reduction was approximately 5%. In contrast, engineered human macrophages expressing TIM4 induced a significant 46% reduction.

[0333] From unprocessed NSG TM The osteopontin content in tissue sections of CDAHFD mice showed a significant increase, indicating that the fibrosis model successfully activated fibroblasts. Figures 39A-39B NSG treated with UTD macrophages (control Mɸ) TM Opn staining was not reduced in mice. Engineered human macrophages expressing TIM4 showed a significant 41% reduction in opn staining.

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

[0335] In summary, these results demonstrate that engineered human macrophages expressing TIM4 achieve a significant reduction in fibrotic area. Compared to UTD macrophages, TIM4 improves the antifibrotic effect of engineered human macrophages. These results also support increasing macrophage burial capacity as an antifibrotic strategy (e.g., via other burial receptors, synthetic burial receptors, and / or other signaling molecules that upregulate endogenous burial mechanisms).

[0336] Example 9: Method for evaluating the antifibrotic efficacy of engineered macrophages overexpressing TIM4 or TIM4+chalasin in a mouse model of advanced liver fibrosis. This example describes the characterization of engineered antifibrotic macrophages in a choline-deficient, L-amino acid-restricted high-fat diet (CDAHFD) model of advanced liver fibrosis, in which the CDAHFD model was induced for an extended duration.

[0337] Antifibrotic engineered mouse macrophages were generated as described in Example 6. The engineered macrophages were kept untransduced (UTD; control Mɸ), transduced to express mouse TIM4 (TIM4 Mɸ; Timd4), or transduced to co-express mouse TIM4 and relaxin. The efficacy of cryopreserved D5 BMDM in a 16-week advanced liver fibrosis model induced by choline deficiency, L-amino acid-limited high-fat diet (CDAHFD) was evaluated.

[0338] For 28 consecutive days, C57BL6 / J mice were fed a normal diet and remained untreated (referred to as "healthy livers" in this paper), or were fed a choline-deficient, L-amino acid-restricted high-fat diet (CDAHFD) that induced fibrosis and remained untreated (referred to as "untreated"), or were treated with the aforementioned engineered macrophages. On day 28, mice began receiving engineered macrophages (5 × 10⁻⁶ cells) every two weeks. 6 (cells) or a solvent control via intravenous injection ( Figure 41 ).

[0339] Sixteen weeks later, the mice were sacrificed, and their livers were collected for histological analysis. Figures 42A-42C As shown, engineered macrophages were well tolerated by mice. Compared to healthy mice, CDAHFD-induced body weight reduction (…) Figure 42A ), increased liver weight ( Figure 42B ) and an increase in the liver-to-body weight ratio ( Figure 42C As expected in this model of advanced liver fibrosis, treatment with macrophages did not exacerbate these changes, indicating that the treatment was well tolerated. Figure 42D As shown, after a 4-hour fast, CDAHFD-fed mice exhibited lower fasting blood glucose levels compared to healthy controls. In contrast, macrophage-treated mice showed blood glucose levels closer to healthy controls than untreated CDAHFD mice, indicating a recovery effect.

[0340] Next, histological analysis of collagen content was used to analyze fibrotic liver tissue collected from mice to determine whether administration of engineered macrophages expressing TIM4 or TIM4+ relaxin affected CDAHFD-induced late fibrosis. For histological analysis, the mid-segment of the left posterior lobe of the liver was formalin-fixed, paraffin-embedded, and then sectioned. Total collagen content was identified using picric Sirius red (also referred to herein as "Sirius Red"). Collagen content was quantified using HALO analysis software.

[0341] Collagen content in tissue sections from solvent-treated (untreated) CDAHFD mice, measured by Sirius red, showed a significant increase in staining, indicating that the fibrosis model successfully activated fibroblasts. Figures 43A-43B Mice treated with UTD macrophages (“control Mɸ”) did not show a significant reduction in Sirius red staining. Engineered macrophages expressing only TIM4 induced a significant 34% reduction in collagen staining. In contrast, engineered macrophages co-expressing TIM4+ relaxin induced a significant 45% reduction.

[0342] In summary, these results demonstrate that engineered macrophages expressing either TIM4 or both TIM4 and relaxin significantly reduced the fibrotic area in a late-stage liver fibrosis model. These results also demonstrate that engineered macrophages can indeed achieve an additive anti-fibrotic effect in a late-stage fibrosis model by utilizing the co-expression of TIM4 and relaxin.

[0343] Example 10: Evaluation of LNP-TIM4 in a mouse model of liver fibrosis Among other things, this embodiment describes the characterization of lipid nanoparticle (“LNP”) delivery of mRNA encoding TIM4 and the effects of such delivery on several indicators of fibrosis in a mouse model and on overall cell burial capacity.

[0344] In short, MC3-lipid nanoparticles were used to encapsulate the mRNA encoding mouse TIM4 (LNP-TIM4). For 28 days, C57BL6 / J mice were fed a normal diet and remained untreated (referred to herein as "healthy liver") or a cholinergic-deficient, L-amino acid-limited high-fat diet (CDAHFD) induced by fibrosis and remained untreated (referred to herein as "untreated"), or treated with the aforementioned LNP-TIM4. On day 28, mice were treated intravenously with LNP-TIM4 or a solvent control. Mice received three single doses, spaced 3–4 days apart. Figure 44 Four days after the last LNP dose, mice were sacrificed for LNP-TIM4 tolerability and efficacy analysis.

[0345] like Figure 45As shown, mouse body weight was measured throughout the study. CDAHFD mice treated with LNP-TIM4 showed similar body weights to mice treated with the solvent, indicating that LNP-TIM4 was well tolerated.

[0346] Next, systemic hepatotoxicity biomarkers were measured to further characterize the safety and tolerability of LNP-TIM4. In mice fed CDAHFD, LNP-TIM4 did not significantly alter the serum toxicity biomarker ALP (Alanine Angiotensin II). Figure 46A ), AST ( Figure 46B ), ALT Figure 46C ), bile acids ( Figure 46D ), triglycerides ( Figure 46E ) or creatine ( Figure 46F In summary, these results indicate that LNP-TIM4 is well tolerated.

[0347] Next, histological analysis of collagen content was used to analyze fibrotic liver tissue collected from mice to determine whether LNP-TIM4 administration affected CDAHFD-induced fibrosis. Histological analysis was performed as described in Example 7. Total collagen content was identified using Masson's trichrome staining. Figure 47A As shown, treatment with LNP-TIM4 resulted in a decrease in collagen levels, but this was not statistically significant.

[0348] Further histological analysis was performed on the collected fibrotic liver tissue, and α-smooth muscle actin (“α-SMA”) staining was performed. Figure 47B ) and osteopontin (“Opn”) staining ( Figure 47C As a pro-inflammatory and pro-fibrotic biomarker associated with liver fibrosis, as described in Example 7. Figure 47C As shown, treatment with LNP-TIM4 resulted in a decrease in α-SMA staining values, but this was not statistically significant. In contrast, LNP-TIM4 treatment significantly reduced Opn.

[0349] In summary, these results demonstrate that LNP-TIM4 appears to be safe and well-tolerated in mice and indicates potential for antifibrotic efficacy. Future experiments will focus on optimizing the dosing regimen and LNP formulation to improve antifibrotic efficacy.

[0350] Example 11: Sequence List The following table provides exemplary amino acid sequences described herein.

[0351] Table 2: Exemplary amino acid sequences Table 3: Exemplary amino acid sequences Table 4: Exemplary amino acid sequences Table 5: Exemplary amino acid sequences Table 6: Exemplary amino acid sequences Equivalent method Those skilled in the art will understand that various changes, modifications, and improvements to this disclosure will be readily apparent to them. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to fall within the spirit and scope of the invention. Therefore, the foregoing description and drawings are by way of example only, and any invention described in this disclosure, if further described in detail, is defined by the following claims.

[0352] Those skilled in the art will understand the typical criteria for attributing deviations or errors to values ​​obtained in the measurements or other processes described herein. Publications, websites, and other references cited herein to describe the background of the invention and to provide additional details for its practice are incorporated herein by reference in their entirety.

Claims

1. A modified immune cell comprising encoding one or more nucleic acid sequences: (i) at least one exogenous fiber pyrolysis agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) At least one exogenous regenerant.

2. The modified immune cells according to claim 1, wherein the at least one exogenous fibrolytic agent comprises relaxin polypeptide, core proteoglycan polypeptide, dominant-negative TGF-β receptor 2 (DN TGFβR2) polypeptide, soluble TGFβ receptor and / or TGFβ switch receptor.

3. The modified immune cells according to claim 1 or 2, wherein the at least one exogenous anti-inflammatory agent comprises one or both of cytokines and cytokine receptors.

4. The modified immune cells according to claim 3, wherein: (i) The cytokines include IL-10, IL-22, IL-27, and / or (ii) The cytokine receptors include or are IL17A switch receptors.

5. The modified immune cells according to any one of claims 1-4, wherein the at least one exogenous fibrolytic agent comprises relaxin polypeptide, and the at least one exogenous anti-inflammatory agent comprises IL-10.

6. The modified immune cells according to any one of claims 1-5, wherein the at least one exogenous regenerative agent is or includes fibroblast growth factor 21 (FGF21), fibroblast growth factor 19 (FGF19), vascular endothelial growth factor α (VEGF-α) and / or hepatocyte growth factor (HGF).

7. The modified immune cell according to 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 retained in or secreted from the immune cell.

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

10. The modified immune cells according to any one of claims 1-9, wherein the modified immune cells comprise macrophages, monocytes, or dendritic cells.

11. The modified immune cells of claim 10, wherein the macrophages are derived from monocytes or precursor immune cells.

12. The modified immune cell according to claim 11, wherein the precursor immune cell includes hematopoietic stem cells, myeloid progenitor cells, myeloid cells, monocytes, premonocytes, or intermediates thereof.

13. The modified immune cell according to 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 according to 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 comprising any one of SEQ ID No: 1-5 or 13-18.

15. A pharmaceutical composition comprising modified immune cells according to any one of claims 1-14.

16. The pharmaceutical composition of claim 15, comprising a pharmaceutically acceptable carrier.

17. A nucleic acid construct comprising encoding one or more nucleic acid sequences: (i) at least one exogenous fiber pyrolysis agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) At least one exogenous regenerant.

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

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

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

21. The pharmaceutical composition of claim 20, wherein the delivery solvent is or comprises lipid nanoparticles (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 a pharmaceutical composition according to any one of claims 15, 16, 18, 19, 20, or 21.

23. The method of claim 22, wherein the fibrosis includes fibrotic diseases, conditions, or illnesses of the liver, lungs, heart, vascular system, kidneys, pancreas, skin, gastrointestinal tract, bone marrow, hematopoietic tissue, nervous system, and / or eyes.

24. The method of claim 23, wherein the liver fibrosis disease, condition or symptom includes fatty liver disease, condition or symptom.

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

26. The method of claim 25, wherein the NAFL includes non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

27. The method of claim 26, wherein the alcoholic liver disease includes alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH).

28. The method according to any one of claims 22-27, wherein the subject has one or more of cirrhosis, liver injury, liver cancer, steatosis, increased risk of liver failure, increased risk of death, and / or hepatitis C infection (HCV).

29. The method according to any one of claims 22-28, wherein the inflammation includes, or is an inflammatory disease, condition, or illness of the liver, gastrointestinal tract, lungs, skin, cardiovascular system, nervous system, kidneys, pancreas, joints, eyes, and / or endocrine system.

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

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

32. The method according to 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 immune cells, comprising delivering a nucleic acid construct to the immune cells, the nucleic acid construct comprising encoding one or more nucleic acid sequences: (i) at least one exogenous fiber pyrolysis agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) At least one exogenous regenerant, This leads to the formation of modified immune cells.

34. The method of claim 33, wherein the delivery comprises electroporation or transfection with mRNA, DNA or chemically modified mRNA.

35. The method of claim 33, wherein the delivery comprises transduction using an adeno-associated virus (AAV) vector, an adenovirus vector, or a retroviral vector.

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

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

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

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

40. The method of claim 39, wherein the Vpx protein is delivered to the immune cells before, simultaneously with, or after the nucleic acid construct.

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

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

43. A method for generating modified immune cells, the method comprising administering a composition to a subject, the composition comprising: (a) One or more nucleic acid constructs comprising encoding one or more nucleic acid sequences of the following: (i) at least one exogenous fibrinolytic agent, (ii) at least one exogenous anti-inflammatory agent, and / or (iii) at least one exogenous regenerative agent, and (b) Delivery of solvent; Upon administration of the composition, the one or more nucleic acid constructs are translated in immune cells to generate the modified immune cells, 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. The modified immune cells mentioned above include macrophages, monocytes, or dendritic cells in the subject.

44. The method of claim 43, wherein the delivery solvent is or includes lipid nanoparticles, liposomes, polymers, adeno-associated virus (AAV) vectors, adenovirus vectors, retroviral vectors, or any combination thereof.

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

46. ​​The modified immune cells of claim 45, wherein the at least one exogenous fibrolytic agent comprises a relaxin polypeptide, and the at least one cytotoxic agent comprises a TIM4 polypeptide.

47. The modified immune cells according to claim 45 or claim 46, wherein the modified immune cells comprise macrophages, monocytes, or dendritic cells.

48. The modified immune cells of claim 47, wherein the macrophages are derived from monocytes or precursor immune cells.

49. The modified immune cell according to claim 48, wherein the precursor immune cell comprises hematopoietic stem cells, myeloid progenitor cells, myeloid cells, monocytes, premonocytes, or intermediates thereof.

50. A pharmaceutical composition comprising the modified immune cells according to any one of claims 45-49.

51. The modified immune cell according to 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 immune cells, comprising delivering a nucleic acid construct to the immune cells, the nucleic acid construct comprising encoding one or more nucleic acid sequences: (i) at least one exogenous fiber pyrolysis agent, and (ii) At least one exogenous necrolysis agent.

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

54. The method of claim 52 or claim 53, wherein the immune cells comprise macrophages, monocytes, or dendritic cells.

55. The method of claim 54, wherein the macrophages are derived from monocytes or precursor immune cells.

56. The method of claim 55, wherein the precursor immune cells include hematopoietic stem cells, myeloid progenitor cells, myeloid cells, monocytes, premonocytes, or intermediates thereof.

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

58. The method according to any one of claims 52-56, wherein the delivery comprises transduction with an adeno-associated virus (AAV) vector, an adenovirus vector, or a retroviral vector.

59. The method according to any one of claims 52-56, wherein the delivery comprises delivering a solvent.

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

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

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

63. The method of claim 62, wherein the Vpx protein is delivered to the immune cells before, simultaneously with, or after the nucleic acid construct.

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

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

66. A nucleic acid construct comprising encoding one or more nucleic acid sequences: (i) at least one exogenous fiber pyrolysis agent, and (ii) At least one exogenous necrolysis agent.

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

68. The nucleic acid construct according to claim 66 or claim 67, further comprising a nucleotide sequence encoding a cleavage peptide, wherein the cleavage peptide comprises one or more of porcine chezinvirus-1 (P2A) peptide, Thoseea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, foot-and-mouth disease virus (F2A) peptide and / or variants thereof.

69. A method for generating modified immune cells, the method comprising administering a composition to a subject, the composition comprising: (a) One or more nucleic acid constructs comprising encoding one or more nucleic acid sequences of the following: (i) at least one exogenous fibrolytic agent and (ii) at least one exogenous cytotoxic agent, and (b) Delivery of solvent; Upon administration of the composition, the one or more nucleic acid constructs are translated in immune cells to produce the modified immune cells, comprising (i) at least one exogenous fibrolytic agent and (ii) at least one exogenous cytotoxic agent. The modified immune cells mentioned above include macrophages, monocytes, or dendritic cells 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 cytotoxic agent comprises a TIM4 polypeptide.

71. The method of claim 69 or claim 70, wherein the delivery solvent is or includes lipid nanoparticles (e.g., MC3-lipid nanoparticles), liposomes, polymers, adeno-associated virus (AAV) vectors, adenovirus vectors, retroviral vectors, or any combination thereof.

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