Engineered chimeric fusion protein compositions and methods of use thereof

Abstract

Provided herein are methods and compositions for treating autoimmune diseases and conditions using engineered myeloid cells. In one aspect of the present disclosure, a myeloid cell can be engineered to express a chimeric fusion protein (CFP) comprising (i) an extracellular domain comprising a target binding domain, (ii) a transmembrane domain operably linked to the extracellular domain; and (iii) an intracellular domain comprising an intracellular signaling domain from an engulfment and cell motility protein domain (ELMO1) or a functional variant thereof.

Description

ENGINEERED CHIMERIC FUSION PROTEIN COMPOSITIONSAND METHODS OF USE THEREOFCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 584,218, filed on September 21, 2023, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Chimeric receptors have been the focus of interest in the research community as well as in the development of targeted cell therapy and immunotherapy. Immune cells are both the activators and targets of various human ailments. Ability to manipulate immune cells to exercise a particular therapeutic function in the body at relevant tissue or organ sites within the living system and at a particular period of time is both an exciting opportunity and a challenge for modem medicine.

[0003] Myeloid cell lineage, that encompasses cells such as the monocytes, macrophages, dendritic cells, among others, harbors the gatekeepers of the immune defense. Innovative concepts are outlined and pursued such as described here, towards rendering myeloid cells as drivers of a successful immune response. A successful immune response can be directed against a foreign pathological agent, such as bacteria, fungi and viral organism, or an internal etiological agent such as a tumor, or to regulate inflammatory and autoimmune actions by an overactive or uncontrolled immune system thereby curing or preventing diseases resulting from autoinflammatory sequelae or autoimmune conditions.SUMMARY

[0004] Provided herein is a composition comprising a polynucleic acid, wherein the polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising an target binding domain, wherein the target binding domain is an antibody or antigen-binding domain thereof, (ii) a transmembrane domain operably linked to the extracellular domain; and (iii) an intracellular domain comprising an intracellular signaling domain from an engulfment and cell motility protein, ELMO1. In some embodiments, the CFP further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1.

[0005] In some embodiments, the extracellular domain comprising the target binding domain comprises an antibody or binding domains thereof, an Fab, a nanobody, a bispecific antibody, a diabody, a single chain variable fragment (scFv), single domain antibody (SdAb), variable heavychain (VHH) domain, a VH domain, a VL domain, a VNAR domain, a ligand or a receptor or a functional fragment thereof or a combination thereof.

[0006] In some embodiments, the extracellular domain comprising the target binding domain is an scFv or a sdAb.

[0007] In some embodiments, the target binding domain binds to an inflammatory cell, an autoimmune cell, or an apoptotic cell.

[0008] In some embodiments, the target binding domain binds to a cell surface molecule of a target cell, wherein the target cell is an inflammatory cell, an autoimmune cell, or an apoptotic cell.

[0009] In some embodiments, the target binding domain binds to cell surface molecule selected from a group consisting of Keratinocyte adhesion protein, Dsg3, Myelin oligodendrocyte glycoprotein, Fibroblast activation protein, an AAV Capsid protein, and Citrullinated vimentin. In some embodiments, the CFP further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1.

[0010] In some embodiments, the extracellular domain further comprises a hinge domain between the target binding domain and the transmembrane domain.

[0011] In some embodiments, the hinge domain comprises an extracellular hinge region from a protein selected from CD8oc, CD2, CD28, CD16, CD64, CD89, BAI1, TIM4, MerTK, and phagocytosis receptor. In some embodiments, the CFP further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1.

[0012] In some embodiments, the transmembrane domain is a transmembrane domain from a protein selected from CD16, CD89, CD64, CD3 zeta, BAI1, TIM4, MerTK.

[0013] In some embodiments, the hinge is from the same protein as the transmembrane protein.

[0014] In some embodiments, the intracellular domain further comprises one or more intracellular domains from one or more proteins.

[0015] In some embodiments, the one or more intracellular domains comprise an intracellular signaling domain.

[0016] In some embodiments, the one or more intracellular domain is a phagocytic receptor intracellular domain.

[0017] In some embodiments, the one or more intracellular domains is an intracellular domain from a pattern recognition molecule.

[0018] In some embodiments, the one or more intracellular domains is an intracellular domain from a cytokine or a chemokine.

[0019] In some embodiments, the CFP further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the CFP may further comprise one or more intracellular domains that is selected from a group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor Al (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF 1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, CR1, CD35, CD3^, a complement receptor, CR3, CR4, Tim-1, Tim-4 and CD169, CD40, a cytokine receptor, a chemokine receptor, Fea receptor I, Fc gamma receptor, and Fc epsilon receptor Ig (FCER1G).

[0020] In some embodiments, the intracellular signaling domain promotes activation of phagocytosis.

[0021] In some embodiments, the target binding domain binds to an apoptotic cell surface marker.

[0022] In some embodiments, the apoptotic cell surface marker is phosphatidyl inositol or phosphatidyl serine.

[0023] In some embodiments, activation of the CFP upon contacting the target does not result in sustained inflammatory cytokine or chemokine production by a cell expressing the CFP.

[0024] In some embodiments, the inflammatory cytokine is TNF alpha, interferon gamma, interleukin 2, or interleukin 1.

[0025] In some embodiments, the chemokine is a C-C chemokine, or a CXC- chemokine.

[0026] In some embodiments, the polynucleic acid is an RNA. In some embodiments, the polynucleic acid is an mRNA. In some embodiments, the RNA is 2kb - 20,000kb long. In some embodiments, the RNA is an in vitro transcribed (IVT) mRNA. In some embodiments, the mRNA comprises a 5’ UTR and a 3’UTR. In some embodiments, the mRNA comprises a 5’ guanosyl CAP. In some embodiments, the 5’ guanosyl CAP is modified from a native 5’ CAP. In some embodiments, the 5’ guanosyl CAP comprises a 2’-O-methyl modification. In some embodiments, the 5’ guanosyl CAP comprises an N6 methyl modification. In some embodiments, the mRNA comprises a 3’-polyA tail, wherein the poly A tail is 50-250 nucleotides long. In some embodiments, the 5’ guanosyl CAP is added post-transcriptionally to the IVT mRNA.

[0027] In some embodiments, the composition further comprises a delivery vehicle. In some embodiments, the delivery vehicle comprises one or more lipids. In some embodiments, the lipid is a cationic lipid. In some embodiments, the lipid further comprises a neutral lipid and a PEGylated lipid. In some embodiments, the delivery vehicle is a liposome. In some embodiments, the polynucleic acid is encapsulated in a liposome. In some embodiments, the liposome is a lipid nanoparticle.

[0028] Provided herein is a cell comprising the composition of any of the embodiments described herein. Also provided herein is a pharmaceutical composition comprising the composition or cell of any one of embodiments described herein. Provided herein is a method of treating an inflammatory or autoimmune disease is a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a polynucleic acid, wherein the polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising a target binding domain, (ii) a transmembrane domain operatively linked to the extracellular domain; and (iii) an intracellular domain comprising an intracellular signaling domain from ELMO1. In some embodiments, the CFP further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the target binding domain is an antibody or binding domain thereof. In some embodiments, the subject is a human. In some embodiments, the administering is performed systemically. In some embodiments, the pharmaceutical composition is formulated for intravenous or intramuscular or subcutaneous injections. In some embodiments, the pharmaceutical composition is formulated for oral, or parenteral administration.

[0029] In some embodiments, the autoimmune disease is Pemphigus vulgaris, Multiple sclerosis, Cardiac fibrosis, Immune response to AAV-based gene therapies, or Rheumatoid Arthritis.INCORPORATION BY REFERENCE

[0030] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0032] FIG. 1 depicts a schematic showing an exemplary construct design for anti-autoimmune or anti-inflammatory chimeric fusion proteins that may be expressed in myeloid cells for producing engineered myeloid cells for therapeutic purpose. The polypeptide construct is shown from N terminal to C-terminal, that comprises an exemplary scFv that comprises a binding domain that can bind to any one of the following antigen targets that are expressed on the surface of a cell and can trigger an inflammatory reaction (or flare): Keratinocyte adhesion protein Dsg3; Myelin oligodendrocyte glycoprotein; Fibroblast activation protein; AAV Capsid or Citrullinated vimentin. Each of these proteins are normal endogenous proteins that are either overexpressed inautoimmune or inflammatory conditions, or can otherwise trigger an inflammatory response. The CFP is designed such that when myeloid cells are engineered to express the CFP, cells that express the target antigen are readily phagocytosed and removed by the engineered myeloid cells, and the inflammatory or autoimmune reaction is reduced. The construct additionally comprises an ELM01 intracellular domain, that is designed for activation of phagocytosis upon receptor activation and helps reduce inflammation. The construct also comprises a transmembrane domain as shown and described herein. Some of the transmembrane domain render the CFP to be specifically expressed in myeloid cells.

[0033] FIG. 2 depicts an exemplary CFP construct comprising, from N-terminus to C-terminus, an anti -FAP scFv domain, a CD89 or a TIM4 domain, and an ELMO1 intracellular signaling domain.

[0034] FIG. 3 depicts flow cytometry results measuring expression of FLAG to indicate CFP expression in cells.

[0035] FIG. 4A is a quantification of frequency of cell expressing the CFP construct.

[0036] FIG. 4B is a quantification of mean fluorescence intensity of CFP expression.

[0037] FIG. 5A depicts flow cytometry results measuring FAP expression on phagocytosis target beads.

[0038] FIG. 5B depicts a histogram of FAP expression on phagocytosis target beads.

[0039] FIGs. 6A-6B depict results of a phagocytosis assay. FIG. 6A depicts results organized within each construct design. FIG. 6B depicts results organized by target bead utilized.

[0040] FIGs. 7A-7B depict exemplary flow cytometry plots measuring results of a phagocytosis assay. FIG. 7A depicts results obtained using a mock electroporation (left panels) or a FAP-CD89 construct (right panels). FIG. 7B depicts results obtained using a FAP-CD89-ELMO (left panels) construct or a FAP-TIM4-ELMO construct (right panels).

[0041] FIGs. 8A-8B depict flow cytometry results measuring expression of FLAG to indicate CFP expression in cells. FIG. 8A depicts CFP expression at 24 hours (upper panels) and 48 hours (lower panels) post electroporation. FIG. 8B depicts CFP expression at 72 hours post electroporation.

[0042] FIGs. 9A-9B depict results measuring cytokine concentration in the supernatant of CFP- expression cell cultures. FIG. 9A depicts results measuring TNF, IL-6, MCP-1 / CCL2, and MIP- l-alpha / CCL3. FIG. 9B depicts results measuring IL-10, IL-lbeta, MIP-lbeta / CCL4, and RANTES / CCL5.DETAILED DESCRIPTION

[0043] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0044] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0045] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the disclosure can also be implemented in a single embodiment.

[0046] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.

[0047] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0048] The term “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -30% or less, + / -20% or less, + / - 10% or less, + / -5% or less, or + / -1% or less of and from the specified value, insofar such variations are appropriate to perform in the present disclosure. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically disclosed.

[0049] An “agent” can refer to any cell, small molecule chemical compound, antibody or fragment thereof, nucleic acid molecule, or polypeptide.

[0050] An “alteration” or “change” can refer to an increase or decrease. For example, an alteration can be an increase or decrease of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%. For example, an alteration can be an increase or decrease of 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, or by 40-fold, 50-fold, 60-fold, or even by as much as 70-fold, 75-fold, 80-fold, 90-fold, or 100-fold.

[0051] An “antigen presenting cell” or “APC” as used herein includes professional antigen presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, Langerhans cells),as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells). An APC can express Major Histocompatibility complex (MHC) molecules and can display antigens complexed with MHC on its surface which can be recognized by T cells and trigger T cell activation and an immune response. Professional antigen-presenting cells, notably dendritic cells, play a key role in stimulating naive T cells. Nonprofessional antigen-presenting cells, such as fibroblasts, may also contribute to this process. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens on class I MHC molecules. Antigens that give rise to proteins that are recognized in association with class I MHC molecules are generally proteins that are produced within the cells, and these antigens are processed and associate with class I MHC molecules.

[0052] An apoptotic cell is a cell that is undergoing programmed cell death. Programmed cell death is a normal process of cellular turnover, a process that is essential for morphogenesis, growth, repair and regeneration. This is often contrasted with necrosis where a cell that is not yet programmed for death undergoes an accidental death process, e.g., in response to unfavorable circumstances, and it is held that apoptosis and necrosis are physiologically and molecular process-wise distinct in several respects. For example, cell membrane blebbing and DNA laddering may be features of an apoptotic cell that are not noted in necrotic cell. A cell prior to undergoing apoptosis may show transient signs of alterations that may be variously considered pre-apoptotic and pro-apoptotic. For example, Caspase 8 activation may promote apoptosis and may be detected in a pre-apoptotic cell. While there is no clear consensus between various literatures on the subject of distinction between pre-apoptotic and pro-apoptotic cells, a pro- apoptotic cell may be generally thought of as a cell that is committed to pass into an apoptotic stage, and a pre-apoptotic cell may be a cell that can linger before committing to apoptotic phase.

[0053] A “biological sample” can refer to any tissue, cell, fluid, or other material derived from an organism.

[0054] The term “epitope” can refer to any protein determinant, such as a sequence or structure or amino acid residues, capable of binding to an antibody or binding fragment thereof, a T cell receptor, and / or an antibody-like molecule. Epitopic determinants typically consist of chemically active surface groups of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. A “T cell epitope” can refer to a peptide or peptide-MHC complex recognized by a T cell receptor.

[0055] An engineered cell, such as an engineered myeloid cell, can refer to a cell that has at least one exogenous nucleic acid sequence in the cell, even if transiently expressed. Expressingan exogenous nucleic acid may be performed by various methods described elsewhere, and encompasses methods known in the art. The present disclosure relates to preparing and using engineered cells, for example, engineered myeloid cells, such as engineered phagocytic cells. The present disclosure relates to, inter alia, an engineered cell comprising an exogenous nucleic acid encoding, for example, a chimeric fusion protein (CFP).

[0056] The term “immune response” includes, but is not limited to, T cell mediated, NK cell mediated and / or B cell mediated immune responses. These responses may be influenced by modulation of T cell costimulation and NK cell costimulation. Exemplary immune responses include T cell responses, e.g., cytokine production, and cellular cytotoxicity. In addition, immune responses include immune responses that are indirectly affected by NK cell activation, B cell activation and / or T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. Immune responses include adaptive immune responses. The adaptive immune system can react to foreign molecular structures, such as antigens of an intruding organism. Unlike the innate immune system, the adaptive immune system is highly specific to a pathogen. Adaptive immunity can also provide long-lasting protection. Adaptive immune reactions include humoral immune reactions and cell-mediated immune reactions. In humoral immune reactions, antibodies secreted by B cells into bodily fluids bind to pathogen- derived antigens leading to elimination of the pathogen through a variety of mechanisms, e.g., complement-mediated lysis. In cell-mediated immune reactions, T cells capable of destroying other cells are activated. For example, if proteins associated with a disease are present in a cell, they can be fragmented proteolytically to peptides within the cell. Specific cell proteins can then attach themselves to the antigen or a peptide formed in this manner, and transport them to the surface of the cell, where they can be presented to molecular defense mechanisms, such as T cells. Cytotoxic T cells can recognize these antigens and kill cells that harbor these antigens.

[0057] A “ligand” can refer to a molecule which is capable of binding or forming a complex with another molecule, such as a receptor. A ligand can include, but is not limited to, a protein, a glycoprotein, a carbohydrate, a lipoprotein, a hormone, a fatty acid, a phospholipid, or any component that binds to a receptor. In some embodiments, a receptor has a specific ligand. In some embodiments, a receptor may have promiscuous binding to a ligand, in which case it can bind to several ligands that share at least a similarity in structural configuration, charge distribution or any other physicochemical characteristic. A ligand may be a biomolecule. A ligand may be an abiotic material. For example, a ligand may be a negative charged particle that is a ligand for scavenger receptor MARCO. For example, a ligand may be TiO?, which is a ligand for the scavenger receptor SRA1.

[0058] The term “major histocompatibility complex (MHC)”, “MHC molecule”, or “MHC protein” refers to a protein capable of binding an antigenic peptide and present the antigenic peptide to T lymphocytes. Such antigenic peptides can represent T cell epitopes. The human MHC is also called the HLA complex. Thus, the terms “human leukocyte antigen (HLA)”, “HLA molecule” or “HLA protein” are used interchangeably with the terms “major histocompatibility complex (MHC)”, “MHC molecule”, and “MHC protein”. HLA proteins can be classified as HLA class I or HLA class II. The structures of the proteins of the two HLA classes are very similar; however, they have very different functions. Class I HLA proteins are present on the surface of almost all cells of the body, including most tumor cells. Class I HLA proteins are loaded with antigens that usually originate from endogenous proteins or from pathogens present inside cells, and are then presented to naive or cytotoxic T-lymphocytes (CTLs). HLA class II proteins are present on antigen presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages. They mainly present peptides which are processed from external antigen sources, e.g., outside of cells, to helper T cells.

[0059] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can take up entities by phagocytosis into phagosomes - though B cells exhibit the more general endocytosis into endosomes - which fuse with lysosomes whose acidic enzymes cleave the uptaken protein into many different peptides. Autophagy is another source of HLA class II peptides. The most studied subclass II HLA genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0060] Presentation of peptides by HLA class II molecules to CD4+ helper T cells can lead to immune responses to foreign antigens. Once activated, CD4+ T cells can promote B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells can also secrete cytokines and chemokines that activate and induce differentiation of other immune cells. HLA class II molecules are typically heterodimers of a-and P-chains that interact to form a peptide-binding groove that is more open than class I peptide-binding grooves.

[0061] HLA alleles are typically expressed in codominant fashion. For example, each person carries 2 alleles of each of the 3 class I genes, (HLA-A, HLA-B and HLA-C) and so can express six different types of class II HLA. In the class II HLA locus, each person inherits a pair of HLA- DP genes (DPA1 and DPB1, which encode a and P chains), HLA-DQ (DQA1 and DQB1, for a and P chains), one gene HLA-DRa (DRA1), and one or more genes HLA-DRP (DRB 1 and DRB3, -4 or-5). HLA-DRB1, for example, has more than nearly 400 known alleles. That means that one heterozygous individual can inherit six or eight functioning class II HLA alleles: three or more from each parent. Thus, the HLA genes are highly polymorphic; many different alleles exist in the different individuals inside a population. Genes encoding HLA proteins have many possiblevariations, allowing each person’s immune system to react to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is given a particular number. In some embodiments, the class I HLA alleles are HLA-A*02:01, HLA-B* 14:02, HLA- A*23:01, HLA-E*01 :01 (non-classical). In some embodiments, class II HLA alleles are HLA- DRB*01:01, HLA-DRB*01:02, HLA-DRB* 11:01, HLA-DRB*15:01, and HL A-DRB *07:01.

[0062] A “myeloid cell” can refer broadly to cells of the myeloid lineage of the hematopoietic cell system, and can exclude, for example, the lymphocytic lineage. Myeloid cells comprise, for example, cells of the granulocyte lineage and monocyte lineages. Myeloid cells are differentiated from common progenitors derived from the hematopoietic stem cells in the bone marrow. Commitment to myeloid cell lineages may be governed by activation of distinct transcription factors, and accordingly myeloid cells may be characterized as cells having a level of plasticity, which may be described as the ability to further differentiate into terminal cell types based on extracellular and intracellular stimuli. Myeloid cells can be rapidly recruited into local tissues via various chemokine receptors on their surface. Myeloid cells are responsive to various cytokines and chemokines.

[0063] A myeloid cell, for example, may be a cell that originates in the bone marrow from a hematopoietic stem cell under the influence of one or more cytokines and chemokines, such as G- CSF, GM-CSF, Flt3L, CCL2, VEGF and S100A8 / 9. In some embodiments, the myeloid cell is a precursor cell. In some embodiments, the myeloid cell may be a cell having characteristics of a common myeloid progenitor, or a granulocyte progenitor, a myeloblast cell, or a monocyte- dendritic cell progenitor or a combination thereof. A myeloid cell can include a granulocyte or a monocyte or a precursor cell thereof. A myeloid cell can include an immature granulocyte, an immature monocyte, an immature macrophage, an immature neutrophil, and an immature dendritic cell. A myeloid cell can include a monocyte or a pre-m onocytic cell or a monocyte precursor. In some cases, a myeloid cell as used herein may refer to a monocyte having an M0 phenotype, an Ml phenotype or an M2 phenotype. A myeloid cell can include a dendritic cell (DC), a mature DC, a monocyte derived DC, a plasmacytoid DC, a pre-dendritic cell, or a precursor of a DC. A myeloid cell can include a neutrophil, which may be a mature neutrophil, a neutrophil precursor, or a polymorphonucleocyte (PMN). A myeloid cell can include a macrophage, a monocyte-derived macrophage, a tissue macrophage, a macrophage of an M0, an Ml or an M2 phenotype. A myeloid cell can include a tumor infiltrating monocyte (TIM). A myeloid cell can include a tumor associated monocyte (TAM). A myeloid cell can include a myeloid derived suppressor cell (MDSC). A myeloid cell can include a tissue resident macrophage. A myeloid cell can include a tumor associated DC (TADC). Accordingly, a myeloid cell may express one or more cell surface markers, for example, CDl lb, CD14, CD15, CD16,CD38, CCR5, CD66, Lox-1, CDl lc, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CDlc, CD83, CD141, CD209, CD205, P selectin, integrins, ICAMS, VCAMS, MHC-II, CD123, CD303, CD304, a SIGLEC family protein and a CLEC family protein. In some cases, a myeloid cell may be characterized by a high or a low expression of one or more of cell surface markers, for example, CDl lb, CD14, CD15, CD16, CD66, Lox-1, CDl lc, CD64, CD68, CD163, CCR2, CCR5, HLA- DR, CDlc, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304 or a combination thereof

[0064] “Phagocytosis” is used interchangeably with “engulfmenf ’ and can refer to a process by which a cell engulfs a particle, such as a cancer cell or an infected cell. This process can give rise to an internal compartment (phagosome) containing the particle. This process can be used to ingest and or remove a particle, such as a cancer cell or an infected cell from the body. A phagocytic receptor may be involved in the process of phagocytosis. The process of phagocytosis can be closely coupled with an immune response and antigen presentation. The processing of exogenous antigens follows their uptake into professional antigen presenting cells by some type of endocytic event. Phagocytosis can also facilitate antigen presentation. For example, antigens from phagocytosed cells or pathogens, including cancer antigens, can be processed and presented on the cell surface of APCs.

[0065] A “polypeptide” can refer to a molecule containing amino acids linked together via a peptide bond, such as a glycoprotein, a lipoprotein, a cellular protein or a membrane protein. A polypeptide may comprise one or more subunits of a protein. A polypeptide may be encoded by a recombinant nucleic acid. In some embodiments, polypeptide may comprise more than one peptide sequence in a single amino acid chain, which may be separated by a spacer, a linker or peptide cleavage sequence. A polypeptide may be a fused polypeptide. A polypeptide may comprise one or more domains, modules or moieties.

[0066] A “receptor” can refer to a chemical structure composed of a polypeptide, which transduces a signal, such as a polypeptide that transduces an extracellular signal to a cell. A receptor can serve to transmit information in a cell, a cell formation or an organism. A receptor comprises at least one receptor unit and can contain two or more receptor units, where each receptor unit comprises a protein molecule, e.g., a glycoprotein molecule. A receptor can contain a structure that binds to a ligand and can form a complex with the ligand. Signaling information can be transmitted by a conformational change of the receptor following binding with the ligand on the surface of a cell.

[0067] The term “antibody” refers to a class of proteins that are generally known as immunoglobulins, including, but not limited to IgGl, IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, IgM, and IgY. The term “antibody” includes, but is not limited to, full length antibodies, single-chain antibodies, single domain antibodies (sdAb) and antigen-bindingfragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab’ and F(ab’)2, Fd (consisting of VH and CHI), single-chain variable fragment (scFv), single-chain antibodies, disulfide-linked variable fragment (dsFv) and fragments comprising a VL and / or a VH domain. Antibodies can be from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, can comprise variable region(s) alone or in combination with tone or more of a hinge region, a CHI domain, a CH2 domain, and a CH3 domain. Also included are any combinations of variable region(s) and hinge region, CHI, CH2, and CH3 domains. Antibodies can be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies which, e.g., specifically bind an HLA-associated polypeptide or an HLA- peptide complex. As used herein, a “binder” represents a polypeptide that comprises a binding domain that can bind to a target, wherein target for the binder can be a protein e.g., a cancer antigen, a glycoprotein etc. A binder is often used to represent a CFP, e.g., a CAR, and designated by the target it binds, e.g., CD5-binder that comprises a binding domain for CD5 antigen. In some cases, the same is alternatively or interchangeably termed, anti-CD5 binder, or anti-TROP2 binder. As generally used, the term binder refers to any molecule that has a binding domain. In some cases, when described in context, a binder may be a BiME or a TRiME.

[0068] The term “polynucleic acid” or its grammatical equivalents refers to a polymer of nucleotides (single stranded or double stranded). In some embodiments, a polynucleic acid is a recombinant polynucleic acid that is prepared, expressed, created or isolated by recombinant means. A polynucleic acid can contain a nucleotide sequence that is not naturally occurring. A polynucleic acid may be synthesized in the laboratory. A polynucleic acid may be prepared by using recombinant DNA technology, for example, enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. A polynucleic acid can be DNA, RNA, analogues thereof, or a combination thereof. A DNA may be transcribed ex vivo or in vitro, such as to generate a messenger RNA (mRNA). An mRNA may be isolated, purified and used to transfect a cell. A polynucleic acid may encode a protein or a polypeptide.

[0069] The process of introducing or incorporating a polynucleic acid into a cell can be via transformation, transfection or transduction. Transformation is the process of uptake of foreign nucleic acid by a bacterial cell. This process is adapted for propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in a laboratory setting. Transfection is the introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. Transfection may also refer to the introduction of bacteriophage into bacterial cells. “Transduction” is mostly used to describe the introductionof recombinant viral vector particles into target cells, while ‘infection’ refers to natural infections of humans or animals with wild-type viruses.

[0070] The term “vector” can refer to a nucleic acid molecule capable of autonomous replication in a host cell, and which allow for cloning of nucleic acid molecules. As known to those skilled in the art, a vector includes, but is not limited to, a plasmid, cosmid, phagemid, viral vectors, phage vectors, yeast vectors, mammalian vectors and the like. For example, a vector for exogenous gene transformation may be a plasmid. In certain embodiments, a vector comprises a nucleic acid sequence containing an origin of replication and other elements necessary for replication and / or maintenance of the nucleic acid sequence in a host cell. In some embodiments, a vector or a plasmid provided herein is an expression vector. Expression vectors are capable of directing the expression of genes and / or nucleic acid sequence to which they are operatively linked. In some embodiments, an expression vector or plasmid is in the form of circular double stranded DNA molecules. A vector or plasmid may or may not be integrated into the genome of a host cell. In some embodiments, nucleic acid sequences of a plasmid are not integrated in a genome or chromosome of the host cell after introduction. For example, the plasmid may comprise elements for transient expression or stable expression of the nucleic acid sequences, e.g., genes or open reading frames harbored by the plasmid, in a host cell. In some embodiments, a vector is a transient expression vector. In some embodiments, a vector is a stably expressed vector that replicates autonomously in a host cell. In some embodiments, nucleic acid sequences of a plasmid are integrated into a genome or chromosome of a host cell upon introduction into the host cell. Expression vectors that can be used in the methods as disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages or viral vectors. A vector can be a DNA or RNA vector. In some embodiments, a vector provide herein is an RNA vector that is capable of integrating into a host cell’s genome upon introduction into the host cell (e.g., via reverse transcription), for example, a retroviral vector or a lentiviral vector. Other forms of expression vectors known by those skilled in the art which serve the equivalent functions can also be used, for example, self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome. Exemplary vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked.

[0071] The terms “spacer” or “linker” as used in reference to a fusion protein refers to a peptide sequence that joins two other peptide sequences of the fusion protein. In some embodiments, a linker or spacer has no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the proteins or RNA sequences. In some embodiments, the constituent amino acids of a spacer can be selected to influence some property of the molecule such as the folding, flexibility, net charge, or hydrophobicity of the molecule.Suitable linkers for use in an embodiment of the present disclosure are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, a linker is used to separate two or more polypeptides, e.g., two antigenic peptides by a distance sufficient to ensure that each antigenic peptide properly folds. Exemplary peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure. Amino acids in flexible linker protein region may include Gly, Asn and Ser, or any permutation of amino acid sequences containing Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, also can be used in the linker sequence.

[0072] The terms “treat,” “treated,” “treating,” “treatment,” and the like are meant to refer to reducing, preventing, or ameliorating a disorder and / or symptoms associated therewith (e.g., a neoplasia or tumor or infectious agent or an autoimmune disease). “Treating” can refer to administration of the therapy to a subject after the onset, or suspected onset, of a disease (e.g., cancer or infection by an infectious agent or an autoimmune disease). “Treating” includes the concepts of “alleviating”, which can refer to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to the disease and / or the side effects associated with therapy. The term “treating” also encompasses the concept of “managing” which refers to reducing the severity of a disease or disorder in a patient, e.g., extending the life or prolonging the survivability of a patient with the disease, or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. The term “prevent”, “preventing”, “prevention” and their grammatical equivalents as used herein, can refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. In certain embodiments, treating a subject or a patient as described herein comprises administering a therapeutic composition, such as a drug, a metabolite, a preventive component, a nucleic acid, a peptide, or a protein that encodes or otherwise forms a drug, a metabolite or a preventive component. In some embodiments, treating comprises administering a cell or a population of cells to a subject in need thereof. In some embodiments, treating comprises administering to the subject one or more of engineered cells described herein, e.g., one or more engineered myeloid cells, such as phagocytic cells. In some embodiments, treating comprises treating a disease or a condition or a syndrome, which may be a pathological disease, condition or syndrome, or a latent disease, condition or syndrome. In some cases, treating, as used herein may comprise administering a therapeutic vaccine. In some embodiments, the engineered myeloid cell such as the phagocyticcell is administered to a patient or a subject. In some embodiments, the engineered myeloid cell administered to a human subject results in reduced immunogenicity as compared to a naturally occurring myeloid cell counterpart and / or an engineered T cell counterpart. For example, an engineered myeloid cell such as the phagocytic cell may lead to no or reduced graft versus host disease (GVHD) or fratricide effect as compared to a naturally occurring myeloid cell counterpart and / or an engineered T cell counterpart. In some embodiments, an engineered cell administered to a human subject is immunocompatible to the subject (i.e., having a matching HLA subtype that is naturally expressed in the subject). Subject specific HLA alleles or HLA genotype of a subject can be determined by any method known in the art. In exemplary embodiments, the methods include determining polymorphic gene types that can comprise generating an alignment of reads extracted from a sequencing data set to a gene reference set comprising allele variants of the polymorphic gene, determining a first posterior probability or a posterior probability derived score for each allele variant in the alignment, identifying the allele variant with a maximum first posterior probability or posterior probability derived score as a first allele variant, identifying one or more overlapping reads that aligned with the first allele variant and one or more other allele variants, determining a second posterior probability or posterior probability derived score for the one or more other allele variants using a weighting factor, identifying a second allele variant by selecting the allele variant with a maximum second posterior probability or posterior probability derived score, the first and second allele variant defining the gene type for the polymorphic gene, and providing an output of the first and second allele variant.

[0073] A “fragment” can refer to a portion of a protein or nucleic acid. In some embodiments, a fragment retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of a reference protein or nucleic acid.

[0074] The terms “isolated,” “purified”, “biologically pure” and their grammatical equivalents refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the present disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a proteinthat can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications can give rise to different isolated proteins, which can be separately purified.

[0075] The terms “neoplasia” or “cancer” refers to any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is one non-limiting example of a neoplasia or cancer. The terms “cancer” or “tumor” or “hyperproliferative disorder” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non- tumorigenic cancer cell, such as a leukemia cell.

[0076] The term “vaccine” is to be understood as meaning a composition for generating immunity for the prophylaxis and / or treatment of diseases (e.g., neoplasia / tumor / infectious agents / autoimmune diseases). Accordingly, vaccines as used herein are medicaments which comprise recombinant nucleic acids, or cells comprising and expressing a recombinant nucleic acid and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination. A “vaccine composition” can include a pharmaceutically acceptable excipient, carrier or diluent. Aspects of the present disclosure relate to use of the technology in preparing a myeloid cell such as phagocytic cell-based vaccine.

[0077] The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. A “pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0078] Nucleic acid molecules useful in the methods of the disclosure include, but are not limited to, any nucleic acid molecule with activity or that encodes a polypeptide. Polynucleotides having substantial identity to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridize” refers to when nucleic acid molecules pair to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentration can ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridizationcan be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions can ordinarily include temperatures of at least about 30° C, at least about 37°C, or at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In an exemplary embodiment, hybridization can occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization can occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In another exemplary embodiment, hybridization can occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps can include a temperature of at least about 25°C, of at least about 42°C, or at least about 68°C. In exemplary embodiments, wash steps can occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In other exemplary embodiments, wash steps can occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, wash steps can occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0079] “Substantially identical” refers to a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Such a sequence can be at least 60%, 80% or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705. Other programs such as BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs can also be used). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program can be used, with a probability score between e-3 and e-m° indicating a closely related sequence. A “reference” is a standard of comparison. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment to a reference sequence and determination of homologous residues.

[0080] The term “subject” or “patient” refers to an organism, such as an animal (e.g., a human) which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0081] The term “therapeutic effect” refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia, tumor, or infection by an infectious agent or an autoimmune disease) or its associated pathology. “Therapeutically effective amount” as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. “Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required.

[0082] Provided herein are specific compositions, methods and uses of polynucleic acids encoding a chimeric fusion protein (CFP) and specific compositions, methods and uses of myeloid cells, e.g., engineered myeloid cells comprising the polynucleic acids or the CFP that can reduce, suppress, or otherwise eliminate inflammation and autoimmune response. In some embodiments, myeloid cells are engineered ex vivo and administered to a subject in need thereof. In someembodiments, compositions and methods are described herein that specifically target myeloid cells in vivo, that are activated or engineered by specific uptake and / or expression of recombinant nucleic acid compositions. In some embodiments, the CFP comprises an intracellular domain comprising an intracellular signaling domain from an engulfment and cell motility protein domain (ELM01) or a functional variant thereof.

[0083] The diverse functionality of myeloid cells makes them an ideal cell therapy candidate that can be engineered to have numerous therapeutic effects. The present disclosure is related to immunotherapy using myeloid cells (e.g., CD14+ cells) of the immune system, particularly phagocytic cells. A number of therapeutic indications could be contemplated using myeloid cells. For example, myeloid cell immunotherapy could be exceedingly important in cancer, autoimmunity, fibrotic diseases and infections. The present disclosure is related to immunotherapy using myeloid cells, including phagocytic cells of the immune system, particularly macrophages.

[0084] It is an object of the invention disclosed herein to harness one or more of these functions of myeloid cells for therapeutic uses. For example, it is an object of the invention disclosed herein to harness the phagocytic activity of myeloid cells, including engineered myeloid cells, for therapeutic uses. For example, it is an object of the invention disclosed herein to harness the ability of myeloid cells, including engineered myeloid cells, to promote T cell activation. For example, it is an object of the invention disclosed herein to harness the ability of myeloid cells, including engineered myeloid cells, to promote secretion of tumoricidal molecules. For example, it is an object of the invention disclosed herein to harness the ability of myeloid cells, including engineered myeloid cells, to promote recruitment and trafficking of immune cells and molecules.

[0085] The present disclosure provides innovative methods and compositions that can successfully transfect or transduce a myeloid cell, or otherwise induce a genetic modification in a myeloid cell, with the purpose of augmenting a functional aspect of a myeloid cell, additionally, without compromising the cell’s differentiation capability, maturation potential, and / or its plasticity. The myeloid cell may reside in vivo inside the body or be engineered ex vivo. In some cases, the functional aspect of a myeloid cell comprises promoting engulfment, antigen clearance, or immune tolerance.

[0086] The present disclosure involves programming myeloid cells in vivo or ex vivo, making and using engineered myeloid cells (e.g., CD14+ cells, such as macrophages or other phagocytic cells, which can attack and kill (ATAK) diseased cells directly and / or indirectly, such as cancer cells and infected cells. Engineered myeloid cells, such as macrophages and other phagocytic cells, can be directed to a pathogenic agent / cell or site in the body by incorporating nucleic acid sequences (e.g., mRNA, plasmids, viral constructs) encoding a chimeric fusion protein (CFP), that has an extracellular binding domain specific to a disease associated antigens (e.g., relevant to theinstant case, fungal, bacterial and / or viral antigens), using nucleic acid technology such as recombinant nucleic acid technology, synthetic nucleic acids, gene editing techniques (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection, or in vivo using mRNA delivery technology including but not limited to LNP technology. It has been found that myeloid cells can be engineered to have a broad and diverse range of activities. For example, it has been found that myeloid cells can be engineered to express a chimeric fusion protein (CFP) containing an antigen binding domain to have a broad and diverse range of activities. For example, it has been found that myeloid cells can be engineered to have enhanced phagocytic activity such that upon binding of the CFP to an antigen on a target cell, the cell exhibits increased phagocytosis of the target cell, thereby limiting the prevalence or prolongation of a disease-causing agent in the first place. Myeloid cells also exhibit a variety of scavenger receptors, which do not require a very high level of specificity in the cognition of the foreign matter in order to mount a response, e.g., phagocytosis. Instead, upon scavenging and / or phagocytosis, monocytes, macrophages or dendritic cells in turn activate T cells for further antigen specific immune response. This flexibility of myeloid cells makes them excellent candidates for use in cell therapy over a variety of immune conditions.

[0087] It has also been found that activated myeloid cells promote activation of T cells which are then reactive to the target cell or organism expressing the antigen. The present disclosure is also based on the important finding that engineered myeloid cells can promote endogenous T cell activity. CD8+ T cells exert cytotoxic effect, which kill target cells and limit the spread of the foreign agent. CD4+T helper and memory cells prepare the immune system for sustained or future assaults by the foreign organism. Myeloid cells can be engineered to promote recruitment and trafficking of immune cells and molecules such that upon binding of the CFP to an antigen on a target cell, the cell promotes recruitment and trafficking of immune cells and molecules to the target cell, a site of infection, inflammation, autoimmune response or a tumor microenvironment.

[0088] Engineered myeloid cells can also be short-lived in vivo, phenotypically diverse, sensitive, and plastic. The present disclosure is based on the important finding that engineered myeloid cells overcome at least some of the limitations of CAR-T cells, including being readily recruited to sites of infection or inflammation; having short, as well as engineerable duration of survival, therefore lowering the risk of prolonged persistence resulting in aplasia and immunodeficiency; myeloid cells cannot be contaminated with T cells; myeloid cells can avoid fratricide, at least because they do not express the same antigens as malignant T cells; and myeloid cells have a plethora of anti-tumor functions that can be deployed. In some respects, engineered myeloid-derived cells can be safer immunotherapy tools to target and destroy diseased cells.

[0089] The present disclosure involves the programming of myeloid cells. Engineered myeloid cells can be achieved through the in vivo incorporation of nucleic acid sequences (e.g., mRNA, plasmids, viral constructs) encoding a chimeric fusion protein (CFP), that has an extracellular binding domain specific to disease associated antigens (e.g., cancer antigens), into the cells using, for example, ex vivo using nucleic acid technology such as recombinant nucleic acid technology, synthetic nucleic acids, gene editing techniques (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection, or in vivo using mRNA delivery technology including but not limited to LNP technology. However, myeloid cells are often found to be difficult to manipulate in vitro. For example, exogenous gene expression in monocytes has been difficult compared to exogenous gene expression in non-hematopoietic cells. There are significant technical difficulties associated with transfecting myeloid cells (e.g., monocytes / macrophages), while concurrently still seeking to maintain their flexibility, pliability, chemotaxis efficiency and maturing ability. As professional phagocytes, myeloid cells, such as monocytes / macrophages comprise many potent degradative enzymes that can disrupt nucleic acid integrity and make gene transfer into these cells an inefficient process. This is especially true of activated macrophages which undergo a dramatic change in their physiology following exposure to immune or inflammatory stimuli. Viral transduction of these cells has been hampered because macrophages are end-stage cells that generally do not divide; therefore, some of the vectors that depend on integration into a replicative genome have met with limited success. Furthermore, macrophages are quite responsive to “danger signals,” and therefore several of the original viral vectors that were used for gene transfer induced potent anti-viral responses in these cells making these vectors inappropriate for gene delivery. The present disclosure provides innovative methods and compositions that can successfully transfect or transduce a myeloid cell, or otherwise induce a genetic modification in a myeloid cell, with the purpose of augmenting a functional aspect of a myeloid cell, additionally, without compromising the cell’s differentiation capability, maturation potential, and / or its plasticity.

[0090] Provided herein are therapeutic agents directed to bind to certain antigens that are expressed on a diseased cell, for example, a cancer cell, and binding of the therapeutic agents (e.g., targeted “binders”) to a target antigen on target diseased cells initiates the process of destruction of the target cell.Chimeric Fusion Proteins

[0091] Provided herein are chimeric fusion proteins (CFP) or recombinant polynucleic acids encoding the CFPs, which when expressed in cells such as myeloid cells, e.g., engineered myeloid cells, said cells are useful in performing at least one biochemical action that is anti-inflammatory.

[0092] In one aspect, a CFP described herein comprises (I) an extracellular domain, (II) a transmembrane domain and (III) an intracellular domain. In some embodiments, a chimeric fusion protein described herein comprises an intracellular domain that comprises a sequence from the intracellular domain of ELMO1. The CFP when activated as a result of binding to a target via the extracellular domain, the intracellular domain is activated and ELMO1 triggers intracellular signaling to the cell which promotes engulfment, antigen clearance and / or immune tolerance. In one embodiment, the CFP comprises a sequence from human ELMO1 (NCBI Reference Sequence: NP 055615.8). In one embodiment, the CFP comprises a fragment of the human E1MO1. In one embodiment, the CFP comprises amino acids 532-727 of human ELMO1 as an intracellular domain. In some embodiments, the intracellular domain comprises a sequence that has at least 80% sequence identity to the sequence comprising amino acids 532-727 of human ELMO1 :

[0093] PILELKEKIQPEILELIKQQRLNRLVEGTCFRKLNARRRQDKFWYCRLSPNHKVL HYGDLEESPQGEVPHDSLQDKLPVADIKAVVTGKDCPHMKEKGALKQNKEVLELAFSI LYDSNCQLNFIAPDKHEYCIWTDGLNALLGKDMMSDLTRNDLDTLLSMEIKLRLLDLE NIQIPDAPPPIPKEPSNYDFVYDCN (SEQ ID NO: 1).

[0094] In some embodiments, the intracellular domain comprises a sequence that has at least 81% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 82% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 83% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 84% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 86% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 87% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 88% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 89% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 91% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 92% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 93% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 94% sequence identity to SEQ ID NO: 1. In someembodiments, the intracellular domain comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the intracellular domain comprises a sequence that has 100% sequence identity to SEQ ID NO: 1.

[0095] In some embodiments, the extracellular domain is a target binding domain. A target binding domain may be an antigen binding domain, e.g., an antibody or binding domains thereof, a single chain variable fragment (scFv), single domain antibody (SdAb), variable heavy chain (VHH) compositions, or ligands or receptors or parts thereof. In some embodiments, any one of binding domains of the therapeutic agent comprises the binding domain of an antibody, a functional fragment of an antibody, a variable domain thereof, a VH domain, a VL domain, a VNAR domain, a VHH domain, a single chain variable fragment (scFv), an Fab, a single-domain antibody (sdAb), a nanobody, a bispecific antibody, a diabody, or a functional fragment or a combination thereof.

[0096] In some embodiments, a target may be an antigen on a target cell, a protein, lipid, carbohydrate, lipoprotein, glycoprotein, a ligand, a receptor, a membrane bound protein, etc. In some embodiments, a target cell may be an inflammatory cell, an autoimmune cell, or a cell that can lead to an autoimmune or inflammatory reaction. In some embodiments, a target cell is an immune cell. The extracellular domain can help creating or establishing contact between the target cell and the effector cell, where, in one embodiment, the effector cell is the cell that is expressing the CFP - and is capable of destroying the target cell. In some embodiments, the extracellular domain upon contacting the target cell surface molecule on the target cell activates the CFP intracellular domain to undergo a biochemical change, such as a phosphorylation reaction or initiates intracellular signaling to activate the effector cell to activate killing the target cell. In some embodiments, killing of a target cell by an effector cell wherein the effector cell is a myeloid cell may encompass phagocytosis of the target cell. Phagocytosis can be complete, where the phagocytic cell, e.g., the macrophage, monocyte or dendritic cell, e.g., the myeloid cell, engulfs the target cell completely. Phagocytosis may also be incomplete or partial. In such cases, the phagocytic cell may partially engulf the target cell, and the target cell is thereafter lysed or programmed for cell death, and may be subject to further phagocytosis and removal. Where the target cell is a culprit for an inflammatory flare up, an autoimmune reaction flare up or an allergic reaction flare up, phagocytosis and destruction of such cells for reducing the resultinginflammatory cycle is desired, and the therapeutic strategy may be programmed thus to engage engineered myeloid cells that have a CFP designed for killing and destruction of the source inflammatory cell or immune cell. In some cases, an undesired prolongation of an inflammatory phase may ensue following an infection or injury. For example, certain viral infections can result in an inflammatory sequelae, which can be even more harmful for the subject than the infection alone. For example, respiratory infections such as influenza, post viral bacterial infection, such as post -influenza streptococcal infection, can result in a cycle of inflammatory reactions that are sustained, and do not resolve easily. Inflammatory signaling molecules, such as cytokines and chemokines feed and multiply the inflammatory reaction, and maintain steady influx of inflammatory cells into the tissue and organ of the inflammation which damage the tissue further, exposing more of the surrounding tissue and damage of blood vessels. A “sustained” inflammatory reaction or outbreak, for example, such as described above, may be one that is characterized by repeated cyclic inflammatory outbreak in a site of infection or injury, e.g., organ, tissue etc., that lasts longer than 3 days, 4 days, 5 days, 6 days, 7 days, e.g., 1 week, 2 weeks, 3 weeks beyond the expected period of recovery as determined by a clinically trained individual. In some embodiments, an inflammatory flare up, autoimmune reaction flare up, or allergic reaction flare up is detectable or measurable by inflammatory cytokine or chemokine release (e.g., by the target cell), rate of immune cell influx in the surrounding, release of histamines, and other means. Clinically, inflammatory, autoimmune or allergic reactions can be detected by a medically trained individual and diagnosis results in assessment of an etiological cause (e.g., cells that cause the harmful effect). It is contemplated, therefore that engineered myeloid cells such as described herein, that can target and eliminate the sources of multiplying inflammatory and autoimmune signals (e.g., the targets for the engineered myeloid cells), can be deployed at the site to subdue the inflammatory or autoimmune reaction. The engineered myeloid cells, as described herein may comprise a recombinant polynucleic acid encoding a chimeric fusion protein (CFP) that is expressed on the surface of the myeloid cell, the CFP comprising an extracellular target binding domain, that can bind to a target antigen on a target cell, a transmembrane domain and an intracellular signaling domain that can facilitate activation of the myeloid cell to efficiently recognize an apoptotic, damaged, or inflammatory cell target, phagocytose and eliminate such target from the system, and subside the inflammation. Several target cell surface antigens have thus far been identified as associated with an inflammatory or autoimmune disease. For examples, the target cell surface antigen comprises keratinocyte adhesion protein, Dsg3, myelin oligodendrocyte glycoprotein, fibroblast activation protein, an AAV Capsid protein, citrullinated vimentin. In some cases, the target cell surface antigen comprises FAP. In some embodiments, the target antigen is not expressed on a cell surface. In some embodiments, the target antigen is presentin an extracellular space. In some embodiments, the target antigen is extracellular DNA or chromatin. In some embodiments, the target antigen is derived from neutrophil extracellular traps.

[0097] In another embodiment, the target cell is an apoptotic cell. Apoptotic turnover is a major function of multicellular organisms for development and homeostasis. Disruption of any part of this normal apoptotic turnover in the organism’s system may result in apoptotic or necrotic cells being the source of inflammation and autoimmune diseases and conditions. It has been observed that lack of or delayed clearing of apoptotic cells can lead to a large variety of autoinflammatory and autoimmune diseases. Thus, a need remains for a therapy that can clear the apoptotic cells and prevent / treat autoinflammatory, autoimmune, or other inflammatory diseases.

[0098] Myeloid cells are considered professional phagocytes that scavenge and remove pre- apoptotic, pro-apoptotic, apoptotic cells and post-apoptotic debris. Engagement of myeloid cells engineered to express a CFP that comprises for example binding domains from a scavenger receptor, or binding domains that can bind apoptotic cell surface markers; and the CFP comprises, for example, specialized intracellular domains that can activate the myeloid cell to perform rapid phagocytosis and clearance of the apoptotic cell and debris, without activating the inflammatory network can be useful in such circumstances.

[0099] In some embodiments, the compositions provided herein comprise recombinant polynucleic acid encapsulated in, for example, a lipid nanoparticle, that can be administered to a subject in need thereof, and further, that can be taken up and expressed preferentially or specifically in myeloid cells in the organismic system. Such compositions can therefore provide a significant therapeutic advantage.

[0100] In some embodiments, a chimeric antigen receptor is designed for expressing in vitro or in vivo in myeloid cells, in order to generate engineered myeloid cells that efficiently phagocytose and remove apoptotic, inflammatory and autoimmune cells, and thereby reduce, suppress or eliminate an inflammatory and autoimmune response.

[0101] In some embodiments, the recombinant polynucleic acid encoding a chimeric antigen receptor (e.g., the CFP described above) is designed for the CFP to bind to a specific target antigen on an inflammatory cell or an autoimmune cell. The CFP may be designed to comprise an scFv within an extracellular antigen binding domain, wherein the extracellular antigen binding domain specifically binds to an antigen on a target autoimmune or inflammatory cell.

[0102] In some embodiments, provided herein is a recombinant polynucleic acid encoding a CFP, comprising an extracellular antigen binding domain that is an scFv, wherein the scFv can specifically bind to an epithelial cell antigen as target Keratinocyte adhesion protein such as Desmoglein-3 (Dsg3). In some embodiments, the recombinant polynucleic acid encoding a CFP comprises an extracellular antigen binding domain that binds to a target keratinocyte adhesionprotein Dsg3, and further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. Without being bound by any particular theory, keratinocyte adhesion protein Dsg3 targeting autoantibodies are responsible for an autoimmune reaction and inflammatory flare in the disease Pemphigus vulgaris (PV), which causes skin blistering. In one embodiment, a CFP directed to Dsg3 expressing cell may eliminate Dsg3 overexpressing keratinocytes and reduce the autoimmune flare. In another embodiment, myeloid cells expressing the CFP that can bind to Dsg3 may be additionally engineered to render the inflamed tissue cold, by inducing desensitization and anti-inflammation. For example, the myeloid cell may be engineered to secrete anti-inflammatory cytokines, such as IL10. For example, the CFP may comprise one or more additional intracellular signaling domains that can induce anti-inflammatory domain. Exemplary signaling domains may comprise certain TAM domains but are not limited to it. TAM receptors may have both inflammatory and anti-inflammatory roles depending on the cell and the microenvironment. However, activation of tyrosine kinase activity of TAM receptor domains may be involved in the process of phagocytosis to go forward, and may prevent inflammatory cytokine or chemokine generation in the process. In one embodiment, the myeloid cells are engineered ex vivo, e.g., electroporated with recombinant polynucleic acid encoding a CFP comprising an extracellular domain comprising an scFv that can specifically bind to keratinocyte adhesion protein Dsg3, wherein the myeloid cells are autologous cells isolated from the subject’s peripheral blood. In one embodiment, the subject is administered a composition comprising the recombinant polynucleic acid encoding the CFP in a suitable delivery vehicle, such as a liposome or a composition comprising one or more lipids or a polymeric composition, such that the recombinant polynucleic acid expressed preferentially or substantially in the myeloid cells in the subject.

[0103] In some embodiments, provided herein is a polynucleic acid encoding a CFP, comprising an extracellular antigen binding domain that is an scFv, wherein the scFv can specifically bind to myelin oligodendrocyte glycoprotein (MOG), which can be associated with an autoimmune disease such as myelin oligodendrocyte glycoprotein (MOG) antibody disease (MOGAD) or multiple sclerosis. In some embodiments, the recombinant polynucleic acid encoding a CFP comprises an extracellular antigen binding domain that binds to a target myelin oligodendrocyte glycoprotein, and further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In one embodiment, the myeloid cells are engineered ex vivo, e.g., electroporated with polynucleic acid encoding a CFP comprising an extracellular domain comprising an scFv that canspecifically bind to myelin oligodendrocyte glycoprotein, wherein the myeloid cells are autologous cells isolated from the subject’s peripheral blood. In some embodiments, the ex vivo engineered myeloid cells are then administered to the subject. In some embodiments, the administration of the engineered cells is directly at the site of autoimmune injury or inflammation. In some embodiments, the administration is systemic. In one embodiment, the subject is administered a composition comprising the polynucleic acid encoding the CFP in a suitable delivery vehicle, such as a liposome or a composition comprising one or more lipids or a polymeric composition, such that the polynucleic acid expressed preferentially or substantially in myeloid cells in the subject.

[0104] In some embodiments, provided herein is a polynucleic acid encoding a CFP, comprising an extracellular antigen binding domain that is an scFv, wherein the scFv can specifically bind to extracellular DNA or chromatin that is generated by neutrophil extracellular traps (NETs), which can be associated with an autoimmune disease. In some embodiments, the recombinant polynucleic acid encoding a CFP comprises an extracellular antigen binding domain that binds to a target myelin oligodendrocyte glycoprotein, and further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In one embodiment, the myeloid cells are engineered ex vivo, e.g., electroporated with polynucleic acid encoding a CFP comprising an extracellular domain comprising an scFv that can specifically bind to myelin oligodendrocyte glycoprotein, wherein the myeloid cells are autologous cells isolated from the subject’s peripheral blood. In some embodiments, the ex vivo engineered myeloid cells are then administered to the subject. In some embodiments, the administration of the engineered cells is directly at the site of autoimmune injury or inflammation. In some embodiments, the administration is systemic. In one embodiment, the subject is administered a composition comprising the polynucleic acid encoding the CFP in a suitable delivery vehicle, such as a liposome or a composition comprising one or more lipids or a polymeric composition, such that the polynucleic acid expressed preferentially or substantially in myeloid cells in the subject.

[0105] In some embodiments, provided herein is a polynucleic acid encoding a CFP, comprising an extracellular antigen binding domain that is an scFv, wherein the scFv can bind to citrullinated protein such as vimentin. In some embodiments, the polynucleic acid encoding a CFP comprises an extracellular antigen binding domain that binds to a target citrullinated vimentin, and further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. Citrullinatedvimentin can be associated with an autoimmune disorder, such as rheumatoid arthritis (RA). RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients. Myeloid cells that are engineered to express a CFP having a binding domain to citrullinated vimentin can help reduce or eliminate the cells that express, or overexpress citrullinated vimentin, thereby reducing the autoimmune flare.

[0106] In some embodiments, provided herein is a polynucleic acid encoding a CFP, comprising an extracellular antigen binding domain that is an scFv, wherein the scFv can bind to fibroblast activation protein (FAP). In some embodiments, the polynucleic acid encoding a CFP comprises an extracellular antigen binding domain that binds to a target FAP, and further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. Fibroblast activation protein can be associated with rheumatoid arthritis (RA). Fibroblast activation protein can be a therapeutic target for joint pain and inflammation in the autoimmune disease, rheumatoid arthritis, as it is overexpressed in the diseased joints. FAP can also promote cardiac fibrosis. In some cases, the myeloid cells are engineered to target and remove cells overexpressing FAP by phagocytosis without inducing any side effects. In one embodiment, the myeloid cells are engineered ex vivo, e.g., electroporated with polynucleic acid encoding a CFP, wherein the myeloid cells are autologous cells isolated from the subject’s peripheral blood, and wherein the ex vivo engineered myeloid cells are administered to the subject. In some embodiments, the administration of the engineered cells is directly at the site of autoimmune injury or inflammation. In some embodiments, the administration is systemic. In one embodiment, the subject is administered a composition comprising the polynucleic acid encoding a CFP in a suitable delivery vehicle, such as a liposome or a composition comprising one or more lipids or a polymeric composition, such that the polynucleic acid expressed preferentially or substantially in myeloid cells in the subject.

[0107] In some embodiments, the scFv comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18. In some embodiments, the scFv comprises a heavy chain comprising a sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8. In embodiments, the heavy chain comprises a CDR1 sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 9-11. In embodiments, the heavy chain comprises a CDR2 sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 12-14. In embodiments, the heavy chain comprises a CDR3 sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 15 or 16. In some embodiments, the scFv comprises a light chain comprising a sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, the light chain comprises a CDR1 sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3 or 4. In some embodiments, the light chain comprises a CDR2 sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5 or 6. In some embodiments, the light chain comprises a CDR3 sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7. In some embodiments, the scFv comprises a linker sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17.

[0108] In some embodiments, provided herein is a polynucleic acid encoding a CFP, comprising an extracellular antigen binding domain that is an scFv, wherein the scFv can bind to an AAV Capsid protein. In some embodiments, the CFP further comprises a transmembrane domain and an intracellular domain comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. Adeno-associated virus (AAV) can be a source of autoimmune response in subjects that undergo AAV based gene therapies (Mingozzi F, High KA. Immune Responses to AAV Vectors: Overcoming Barriers to Successful Gene Therapy, Blood. (2013) 122(1 ):23--36. doi: 10.1182 / blood-2013-01-306647; Flotte TR. Revisiting the “New” Inflammatoiy Toxicities of Adeno- Associated Virus Vectors. Hum Gene li r (2020) 31(7- 8):398 -9. doi: 10.1089 / hum .2020.29117.trf). It is noted that the formation of anti-rAAV antibody restricts administration of gene therapy to a mere single dose. Therefore in such situation, it is necessary to have alternative and safe methods for treating the autoimmune response, e.g., by administering engineered myeloid cells as described above that express CFP that can bind to AAV protein antigens and can thereafter phagocytose and eliminate cells expressing the AAV proteins on the cell surface such as following a gene therapy course, once the latter is completed, in order to subdue the autoimmune response to the AAV proteins. In one embodiment, the myeloid cells are engineered ex vivo, e.g., electroporated with polynucleic acid encoding a CFP, wherein themyeloid cells are autologous cells isolated from the subject’s peripheral blood, and wherein the ex vivo engineered myeloid cells are administered to the subject. In some embodiments, the administration of the engineered cells is directly at the site of autoimmune injury or inflammation. The AAV capsid protein can comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, or any variants thereof. In some embodiments, the target antigen and / or disease indication is one or more of those listed in Table 1.

[0109] Table 1. Exemplary target antigens and associated diseases:

[0110] In some embodiments, the administration is systemic. In one embodiment, the subject is administered a composition comprising the polynucleic acid encoding a CFP in a suitable delivery vehicle, such as a liposome or a composition comprising one or more lipids or a polymeric composition, such that the polynucleic acid expressed preferentially or substantially in myeloid cells in the subject.[OHl] In some embodiments, the polynucleic acid encoding a CFP is designed for the CFP to bind to a specific target antigen on an inflammatory cell when expressed on a myeloid cell and activating the CFP intracellular domains, in turn activating phagocytosis by the myeloid cell on which the CFP is expressed and removal of the target inflammatory cell. In some embodiments, upon binding of the CFP to the antigen of the target cell, the phagocytosis or killing activity of a cell expressing the CFP is increased by at least 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%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to a cell not expressing theCFP. In some embodiments, the CFP functionally incorporates into a cell membrane of a cell when the CFP is expressed in the cell. In some embodiments, upon binding of the CFP to the antigen of the target cell, the killing activity of a cell expressing the CFP is increased by at least 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5- fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, -fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75- fold, or 100-fold compared to a cell not expressing the CFP.

[0112] In some embodiments, the polynucleic acid encoding the CFP is designed for the CFP to bind to a specific target antigen on an inflammatory cell. In some embodiments, the level of proinflammatory cytokine or chemokine expression and / or secretion by the cell is reduced by at least 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%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to a cell not expressing the CFP. In some embodiments, the level of anti-inflammatory cytokine expression and / or secretion by the cell is increased by at least 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%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to a cell not expressing the CFP.

[0113] In some embodiments, the CFP may comprise an intracellular signaling domain that is derived from a myeloid cell receptor, such as a phagocytic receptor, selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor Al (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fea receptor I, CR1, CD35, CD3< CR3, CR4, Tim-1, Tim-4 and CD169.

[0114] In some embodiments, the polynucleic acid encoding the CFP is designed for expressing preferentially or substantially in a myeloid cell and not substantially expressed in a non-myeloid cell, e.g., a T cell; when a composition comprising a polynucleic acid encoding a CFP as described herein is contacted to a heterogenous population of cells comprising myeloid cells and non- myeloid cells. For example, the recombinant polynucleic acid encoding a chimeric antigen receptor as described herein is contacted to a heterogenous population of cells comprising myeloid cells and non-myeloid cells and the contact occurs in vitro. In some cases, the polynucleic acid encoding a CFP as described herein is contacted to a heterogenous population of cells comprising myeloid cells and non-myeloid cells and the contact occurs in vivo, e.g., when a compositioncomprising the polynucleic acid encoding a CFP is administered to a subject in need thereof, systemically in vivo.

[0115] In one embodiment, a target cell is an apoptotic cell, a pro-apoptotic cell, or a pre- apoptotic cell. A cell undergoing apoptosis exhibits a variety of structural and biochemical changes, transforming from pre-apoptotic through pro-apoptotic and apoptotic stages. .Apoptosis may be characterized by a series of typical morphological features, such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and ultimately rapid phagocytosis byneighboring cells. Activation of cysteine proteases called caspases may play a major role in the execution of apoptosis. These proteases may selectively cleave vital cellular substrates, which results in apoptotic morphology and inter-nucleosomal fragmentation of DNA by selectively- activated DNases. In response to several pro-apoptotic signals, mitochondria may release caspase activating factors, that initiate an escalating caspase cascade and commit the cell to die. Members of the Bcl-2 oncoprotein family control mitochondrial events and can prevent, or induce, both apoptotic and non-apoptotic types of cell death. This suggests that different types of cell death can share common mechanisms in the early phases, whereas activation of caspases determines the phenotype of cell death. Detection of apoptotic cells in tissue samples currently relies on the TUNEL assay. TUNEL-positive cardiomyocytes show morphological features of apoptosis and the typical ladder pattern in DNA electrophoresis. Under normal course, apoptosis and engulfment of apoptotic cells by circulating phagocytes can be a natural and non-inflammatory process. However, under certain disease conditions phagocytic clearance may be ineffective and delayed, and can lead to a variety of inflammatory and autoimmune diseases. For example, a potential source of antigenic components in systemic lupus erythematosus is the abundant generation of nucleosomes in cells undergoing apoptosis, as inter-nucleosomal cleavage of DNA is a well- defined biochemical feature of the apoptotic mode of cell death. The morphologic sequence of apoptosis has been well-defined, and follows an orderly process of nuclear condensation, surface blebbing, cytoplasmic contraction, and packaging of cellular components within membranes before their budding from the cell as apoptotic bodies. This reorganization is striking, as membrane, cytoplasmic, and nuclear components can appear at the surface of the dying cell. Apoptosis occurs in different cell types in a wide variety of organisms. It may occur in response to many different stimuli, including DNA damage, growth factor deficiency, ligation of a family of cell surface receptors, heat shock, and bacterial or viral infection. Many of these stimuli can generate reactive oxygen species, with consequent oxidative damage to multiple cellular targets. Furthermore, exposure to H2O2 (which generates -OH radicals) can also result in cell death with an apoptotic morphology, suggesting that oxidant stresses might function in a final commonpathway leading to cell death. Bcl-2, a member of an interacting family of homologous proteins that modulate apoptosis, can function in an antioxidant pathway that prevents apoptosis.

[0116] Certain autoantigens resulting from an apoptotic cell compartment can be found to be clustered in two discrete populations of blebs at the surface of apoptotic cells. In one population, small, abundant RNA containing surface blebs can be observed which stainintensely for Ro, and contain fragmented endoplasmic reticulum (ER) and ribosomes. The other population of larger blebs may be less numerous and may contain DNA (often nicked), surrounded by a rim of ribonucleoprotein antigens including Ko, La, and the snKNPs. Earlier studies (e.g., Casciola- Rosen, L., et al., J. Exp. Med., Volume 179 April 1994 1317-1330), indicate that the clusters of autoantigens in lupus may be confined to a cellular domain characterized by its proximity to ER and nuclear membranes.

[0117] A proapoptotic or an apoptotic cell may express cell surface markers such as phosphatidyl inositol or phosphatidyl serine (PtdSer). PtdSer may be sequestered on the inner leaflet of the plasma membrane, and during apoptosis PtdSer may be exposed on the outer leaflet of the plasma membrane, where it can interact with PtdSer receptors on phagocytes. Ligation of PtdSer receptors on phagocytes can trigger phagocytic cup formation, which then can facilitate the engulfment of the dying cell. Clearance by such cells by myeloid cells that are engineered to bind to and phagocytose apoptotic or proapoptotic cells can help reduce inflammation. Provided herein are one or more recombinant polynucleic acids encoding a CFP, comprising an extracellular antigen binding domain from natural macrophage receptors that can recognize apoptotic cell markers. In one embodiment, an exemplary scavenger receptor with an extracellular domain that can bind to PtdSer is Scavenger Receptor Type F family member 1 (SCARF 1). A recombinant polynucleic acid encoding a CFP as described herein may comprise a binding domain from the extracellular region of SCARF 1 or a functional variant thereof. In one embodiment, an exemplary scavenger receptor with an extracellular domain that can bind to PtdSer is MEGF10. A recombinant polynucleic acid encoding a CFP as described herein may comprise a binding domain from the extracellular region of MEGF10 or a functional variant thereof.

[0118] Exemplary receptors for apoptotic cell recognition in myeloid cells may include: (i) Brain angiogenesis inhibitor 1 (BAI1). BAI1 is an adhesion G-protein coupled receptor 1 that can directly bind to PtdSer via thrombospondin repeats. This receptor activates intracellular signaling via its engulfment and cell motility protein domain, ELM01. Signaling takes place by interaction with Dock 180, Rael module; (ii) Brain angiogenesis inhibitor 3 (BAB), which is also called adhesion G-protein coupled receptor 3. This receptor activates intracellular signaling via its engulfment and cell motility protein domain, ELMO1. Signaling takes place by interaction with Dock 180, Rael module; (iii) T cell immunoglobulin and mucin receptor 1 (TIM 1). TIM 1 can interactwith PtdSer via direct binding of the immunoglobulin domain. The receptor possesses tyrosine phosphorylation sites in intracellular domain and may signal via Fyn kinase. Similar to TIM1, TIM4 also belongs to the same class and exhibits similar characteristics; and / or (iv) TAM domain receptors Axl, Tyro3 and MerTK, which can bind to PtdSer via indirect binding, for example, via Gas6 or Protein S. These proteins can phosphorylate ELM01 and may elicit Rac 1 activation.

[0119] Exemplary constructs of CFPs are shown in FIG. 1. Exemplary anti -FAP binding domains are summarized in Table 4. Full length construct designs are summarized in Table 5.

[0120] Table 4. Anti-FAP Binding Domains

[0121] Table 5. CFP Construct Designs

[0122] Provided herein are one or more polynucleic acids encoding a CFP, comprising an extracellular antigen binding domain from natural macrophage receptors that can recognize apoptotic cell markers, such as extracellular binding domains from SCARF 1, MEG10, or BAI3, TIM3 and additional receptor proteins described above, wherein the CFP additionally comprises an intracellular domain ELMO1. The ELMO1 intracellular domain can potentiate phagocytosis. Provided herein are myeloid cells that are engineered to express the CFP comprising (i) an extracellular antigen binding domain from natural macrophage receptors that can recognize apoptotic cell markers, such as extracellular binding domains from SCARF 1, MEG10, BAB, TIM3, or additional receptor proteins described above, or a functional variant thereof, and (ii) an intracellular domain ELMO1 or a functional variant thereof.

[0123] In some embodiments, any one of the CFPs described herein further comprises a transmembrane domain derived from a CD2, CD8, CD28, CD64 or CD68 protein TM domain.

[0124] In some embodiments, the CFP comprises a transmembrane domain from a protein that dimerizes with endogenous FcR-gamma receptors in myeloid cells, monocytes or macrophages; wherein after administration of a pharmaceutical composition comprising the nucleic acid encoding the CFP to a human subject, the CFP is specifically expressed in myeloid cells, monocytes or macrophages of the human subject. In some embodiments, the transmembrane domain is a transmembrane domain from CD 16a, CD64, CD68, TIM4, or CD89. In some embodiments, the CFP comprising a transmembrane domain from CD89, TIM4, CD 16, CD64, or CD68 protein, may be substantially expressed in a myeloid cell, and not substantially expressed in a non-myeloid cell. A myeloid cell may fall under a diverse cell category such as a CD 14+ cell, a CD14+ and CD16- cell (i.e., a CD14+ / CD16- cell), a CD14+and CD16+ cell (i.e., a CD14+ / CD16+ cell), a CD14- and CD16+ cell (i.e., a CD14- / CD16+ cell), CD14- and CD16- cell (i.e., a CD14- / CD16- cell), a CD14+ and CD161ow cell, a dendritic cell, an MO macrophage, an M2 macrophage, an Ml macrophage or a mosaic myeloid cell / macrophage / dendritic cell. A non- myeloid cell may be a T cell, a B cell, a neuron, a fibroblast or an epithelial cell, for example. In some embodiments, the CFP comprising a transmembrane domain from CD89, CD 16, CD64, or CD68 protein, may be substantially expressed in CD14+ cells in a population of CD14+ cells, such as, for example, greater than 50% CD14+ cells express the CFP, wherein the CD14+ cells represent the exemplary myeloid cells.

[0125] In some embodiments, the CFP comprising a transmembrane domain from CD89, TIM4, CD 16, CD64, or CD68 protein and is not substantially expressed on a T cell as exemplary non- myeloid cell, such as, for example, less than 5% of cells in a population of the T cells express the CFP. In some embodiments, less than 4%, less than 3%, less than 2%, or less than 1% cells of the population of T cells express the CFP.

[0126] In some embodiments, the transmembrane domain is associated with a short extracellular domain from CD 16a, CD64, CD68, TIM4, or CD89 respectively. In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular domain or portion thereof from CD 16a, CD64, CD68, TIM4, or CD89 respectively.

[0127] In some embodiments, the CFP comprises an extracellular antigen binding domain as described previously, and a transmembrane domain from any one of the CD 16a, CD64, CD68, TIM4, or CD89 protein. In some embodiments, the CFP comprises an extracellular antigen binding domain as described in the above section, an extracellular hinge domain, for example a CD8a or CD68 hinge domain, and a transmembrane domain from any one of the CD 16a, CD64, CD68, TIM4, or CD89 protein. In some embodiments, the CFP comprises an extracellular antigen binding domain as described in the above section, an extracellular hinge domain, an extracellular domain from the same protein as the transmembrane domain, such as a transmembrane domain from any one of the CD 16a, CD64, CD68, TIM4, or CD89 protein. In some embodiments, the CFP comprises an extracellular antigen binding domain as described previously, an extracellular hinge domain, an extracellular domain from the same protein as the transmembrane domain, such as a transmembrane domain from any one of the CD 16a, CD64, CD68, TIM4, or CD89 proteins and an intracellular domain from the same protein as the transmembrane domain, e.g., from any one of the CD 16a, CD64, CD68, TIM4, or CD89 respectively. In some embodiments, the dimerization of the CFP with an endogenous FcR receptor activates and signals to the cell via FcR mediated intracellular signaling.

[0128] In some embodiments, the CFP further comprises one or more intracellular signaling domains, and wherein the one or more intracellular signaling domains comprises an intracellular signaling domain from FcyR, FcaR, FcsR, CD40 or CD3zeta. In some embodiments, the one or more intracellular signaling domains further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain.

[0129] In some embodiments, a CFP described herein may comprise at least 2 intracellular signaling domains. For example, a CFP described herein may comprise a phosphoinositide 3- kinase (PI3K) recruitment domain and a CD40 intracellular signaling domain. In some embodiments, a CFP described herein may comprise at least the ELMO1 intracellular signaling domain or a functional variant thereof. In some embodiments, the CFP comprises an ELMO1 domain or a functional variant thereof and at least one additional intracellular signaling domain, selected from phosphoinositide 3-kinase (PI3K) recruitment domain and a CD40 intracellular signaling domain.

[0130] In some embodiments, provided herein is a therapeutic composition comprising a chimeric fusion protein, such as a chimeric fusion receptor protein (CFP), the CFP comprises: (a) an extracellular domain comprising: (i) a scFv that specifically binds any one of the targets disclosed herein, and (ii) a hinge domain derived from CD8, a hinge domain derived from CD28 or at least a portion of an extracellular domain from CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain or a CD68 transmembrane domain; and (c) an intracellular domain comprising one or more intracellular signaling domains, wherein the one or more intracellular signaling domain comprises an ELMO1 domain or a functional variant thereof. In some embodiments, the CFP further comprises an intracellular signaling domain comprising a phosphoinositide 3-kinase (PI3K) recruitment domain.

[0131] In some embodiments, provided herein is a therapeutic composition comprising a chimeric fusion protein, such as a chimeric fusion receptor protein (CFP), the CFP comprises: (a) an extracellular domain comprising: (i) a scFv that specifically binds any one of the targets disclosed herein, and (ii) a hinge domain derived from CD8, a hinge domain derived from CD28 or at least a portion of an extracellular domain from CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain or a CD68 transmembrane domain; or a transmembrane domain from CD89; and (c) an intracellular domain comprising one or more intracellular signaling domains, wherein the one or more intracellular signaling domain comprises a phosphoinositide 3-kinase (PI3K) recruitment domain, a CD40 intracellular signaling domain, a cytokine intracellular signaling domain, a TAM intracellular signaling domain; and an additional signaling domain comprising the ELMO1 domain or a functional variant thereof.

[0132] In some embodiments, provided herein is a composition comprising a CFP, comprising (a) an extracellular domain comprising: (a) an extracellular domain comprising a scFv that specifically binds any one of the targets disclosed herein, (b) a transmembrane domain from CD89, (c) an intracellular domain comprising a sequence of amino acid residues 523-727 of human ELMO1 (SEQ ID NO: 1).

[0133] In some embodiments, provided herein is a composition comprising a CFP, comprising (a) an extracellular domain comprising: (a) an extracellular domain comprising a scFv that specifically binds any one of the targets disclosed herein, (b) a transmembrane domain from TIM4, (c) an intracellular domain comprising a sequence of amino acid residues 523-727 of human ELMO1 (SEQ ID NO: 1).

[0134] In some embodiments, the extracellular domain of the CFP further comprises a hinge domain derived from CD8, a hinge domain derived from CD28 or at least a portion of an extracellular domain from CD68.

[0135] In some embodiments, the CFP comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 20 or 21.

[0136] In some embodiments, the CFP comprises a sequence that has at least 81% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 82% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 83% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 84% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 85% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 86% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 87% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 88% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 89% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 91% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 92% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 93% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 94% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 96% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 97% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 98% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, the CFP comprises a sequence that has 100% sequence identity to SEQ ID NO: 20.

[0137] In some embodiments, the CFP comprises a sequence that has at least 81% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 82% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 83% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 84% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 85% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 86% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 87% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequencethat has at least 88% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 89% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 91% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 92% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 93% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 94% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 96% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 97% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 98% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises a sequence that has 100% sequence identity to SEQ ID NO: 21.

[0138] In some embodiments, the CFP comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 23 or 24.

[0139] In some embodiments, the CFP comprises a sequence that has at least 81% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 82% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 83% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 84% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 85% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 86% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 87% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 88% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 89% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 91% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 92% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 93% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 94% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 95% sequence identity to SEQ IDNO: 23. In some embodiments, the CFP comprises a sequence that has at least 96% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 97% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 98% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 23. In some embodiments, the CFP comprises a sequence that has 100% sequence identity to SEQ ID NO: 23.

[0140] In some embodiments, the CFP comprises a sequence that has at least 81% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 82% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 83% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 84% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 85% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 86% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 87% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 88% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 89% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 91% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 92% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 93% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 94% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 96% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 97% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 98% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence that has 100% sequence identity to SEQ ID NO: 24.CFP designed for myeloid cell specific expression and in vivo delivery

[0141] One aspect of developing nucleic acid encoding chimeric antigen receptor for delivery in vivo is rendering macrophage specific uptake, expression and function of the CFP in monocytic cells, and reduce expression in non-myeloid cells which do not have phagocytic capability. Insome embodiments, the nucleic acid encoding the CFP is delivered to a subject in need thereof, wherein the nucleic acid is encapsulated in a nanoparticle, e.g., a lipid nanoparticle, and the delivery is achieved by a systemic route, e.g., intravenous delivery. Accordingly, in one aspect, the CFP is designed such that the expression of CFP is dependent on the presence or expression of another myeloid cell specific protein. As for example, contemplated herein is the concept of applying a myeloid specific promoter for expression of the CFP. The myeloid specific promoter can be encoded in a vector or otherwise in the recombinant nucleic acid, at a position upstream of and operatively linked to the sequence encoding the CFP. Certain myeloid cell-specific promoters are disclosed in the art. For example, CD68S promoter is a myeloid specific promoter (Scharenberg et al., Nat Cotrnmi i 11, 3327 (2020).)

[0142] In one aspect, the nucleic acid is mRNA. Contemplated herein are methods for myeloid specific expression that can be utilized irrespective of whether the nucleic acid is delivered as mRNA or DNA. Accordingly, in one aspect, the CFP is designed such that it leverages the presence of a myeloid endogenous protein for its own expression and functionality. In one aspect, the myeloid specific expression of the CFP is achieved through a design in which the CFP is expressed in the membrane only when it dimerizes (or multimerizes) with one monocyte specific protein. In some embodiments, the myeloid specific functionality of the CFP is achieved through a design in which the CFP is expressed on the membrane only when it dimerizes (or multimerizes) with a myeloid specific protein. FcR alpha receptors oligomerize with FcR gamma receptors for membrane expression on myeloid cells and / or function. Several Fc receptors (FcRs) are endogenously expressed in monocytes and myeloid cells. Following their crosslinking by immune complexes, FcRs play various roles such as modulation of the immune response by released cytokines or of phagocytosis. FcR alpha receptors and FcRgamma receptors oligomerize via transmembrane domains.

[0143] In some embodiments, the CFP comprises a transmembrane domain and / or an intracellular domain from a Fc receptor, which oligomerize with an endogenous FcR. Exemplary FcRs and their cellular expression patterns are listed in Table 2. Sequences of FcR domains are provided in Table 3.

[0144] Table 2 FcRs and their cellular expression patterns

[0145] Table 3 Exemplary FcR domain sequences.

[0146] In some embodiments, the CFP comprises a transmembrane domain and / or an intracellular domain from TIM4. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has least 80% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 81% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 82% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 83% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 84% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 85% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 86% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 87% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 88% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 89% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 90% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 91% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 92% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 93% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 94% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 95% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 96% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 97% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequencethat has at least 98% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has at least 99% sequence identity to SEQ ID NO: 22. In some embodiments, the CFP comprises a domain from TIM4 comprising a sequence that has 100% sequence identity to SEQ ID NO: 22.

[0147] In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of protein selected from FcyRl (CD64). In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of protein selected from FcyRIIIA (CD16). In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of protein selected from FcyRIIA (CD32a). In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of protein selected from FcaRl (CD89). Any of the domains in the CFP described herein can be from a suitable mammalian origin, either depending on or irrespective of the origin of any other adjoining domains. Non-human mammalian transmembrane domains are contemplated within the scope of the instant disclosure.Therapeutic compositionsCell Therapy Compositions

[0148] Provided herein, in one aspect, is a myeloid cell, such as a CD14+ cell, a CD14+ / CD16- cell, a CD14+ / CD16+ cell, a CD14- / CD16+ cell, CD14- / CD16- cell, a dendritic cell, a macrophage, an M0 macrophage, an M2 macrophage, an Ml macrophage or a mosaic myeloid cell / macrophage / dendritic cell that is engineered ex vivo to comprise a recombinant polynucleic acid encoding a chimeric fusion protein (CFP) as described in the disclosure. In some embodiments, provided herein is a therapeutic composition comprising a cell population comprising at least 20%, at least 30%, at least 40% or at least 50% CD14+ cells. In some embodiments, the therapeutic composition comprises a cell population comprising at least 20%, at least 30%, at least 40% or at least 50% CD14+ / CD16- cells. In some embodiments, provided herein is a therapeutic composition comprising a cell population comprising less than 20%, less than 15%, less than 10% or less than 5% dendritic cells.

[0149] In some embodiments depending on its use for specific purposes, a therapeutic composition may comprise a cell population comprising 50% or more dendritic cells. In some embodiments, for example, where desensitization of an immunoreactive antigen is needed, the therapeutic composition described herein may comprise a cell population comprising at least about 50% dendritic cells. In some embodiments, the therapeutic composition may comprise a cell population comprising about or greater than 50% CD14+ cells. In some embodiments, thetherapeutic composition may comprise a cell population comprising about or greater than 50% CD14+ / CD16+ cells.

[0150] The myeloid cell for the therapeutic composition as described herein, comprises a recombinant nucleic acid that encodes a chimeric fusion protein encoding a CFP receptor protein or an engager protein as described herein. The myeloid cell for the therapeutic composition as described herein, expresses the CFP encoded by the recombinant nucleic acid or expresses an engager protein encoded by the recombinant nucleic acid as described herein. The therapeutic composition comprises a therapeutically effective number of cells that are described above, and a pharmaceutically acceptable excipient. In some embodiments, the therapeutically effective number of cells may range from 10A5 cells to 10A10 cells. In some embodiments, the therapeutically effective number of cells comprises at least about lxl0A5 CD14+ cells, 2xlOA5 CD14+ cells, 3xl0A5 CD14+ cells, 4xlOA5 CD14+ cells, 5xl0A5 CD14+ cells, 6xlOA5 CD14+ cells, 7xlOA5 CD14+ cells, 8xl0A5 CD14+ cells, 9xlOA5 CD14+ cells, 10A6 CD14+ cells, or about 5X10A6 CD14+ cells, about 10A7 CD14+cells, about 5X10A7 CD14+cells, about 10A8 cells, about 5X10A8 CD14+cells, about 10A9 CD14+cells, about 5X10A9 CD14+cells, or about 10A10 CD14+cells.Nucleic acid compositions for in vivo expression

[0151] Provided herein, in one aspect, is a composition, e.g., a therapeutic composition comprising one or more polynucleic acid(s) encoding one or more recombinant proteins that can be a chimeric fusion protein such as a receptor, e.g., chimeric fusion protein (CFP) as described in the disclosure. In some embodiments, the polynucleic acid(s) is an mRNA. In some embodiments, the mRNA is purified. In some embodiments, the mRNA is in vitro transcribed. In some embodiments, the mRNA comprises at least one modification. In some embodiments, the polynucleic acid comprises a circRNA. In some embodiments, the polynucleic acid is encompassed in a viral vector. In some embodiments, the polynucleic acid is delivered via a viral vector.

[0152] In some embodiments, the CFP, designed for in vivo delivery via a delivery vehicle, such as an LNP, is designed to be suitable for expression preferentially by a myeloid cell in vivo. In some embodiments, as described above, the CFP comprises a transmembrane domain that can dimerize with an endogenous membrane protein in myeloid cells, thereby ensuring myeloid cell specific expression of the CFP. In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of protein selected from FcyRl (CD64, FcyRIIIA (CD 16, FcyRIIA (CD32a) and FcaRl (CD89). In some embodiments, the CFP is designed to comprise a transmembrane domain derived from the transmembrane domain of CD89 or CD 16.

[0153] In some embodiments, the therapeutic composition comprises the one or more polynucleic acid(s) encoding one or more recombinant proteins and further comprises a delivery vehicle. In some embodiments, the delivery vehicle comprises a lipid. In some embodiments, the lipid is a cationic lipid. In some embodiments the lipid is a combination of lipids, comprising at least a cationic lipid. In some embodiments, the delivery vehicle comprises a cationic lipid, a noncationic lipid, a neutral lipid, and / or a PEGylated lipid. In some embodiments, the lipid is a liposome.

[0154] In some embodiments the one or more polynucleic acid(s) encoding one or more proteins is encapsulated in a liposome, wherein the liposome is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a non-cationic lipid, a neutral lipid, and / or a PEGylated lipid.

[0155] In some embodiments, the one or more polynucleic acid(s) encoding one or more proteins is encapsulated in polymeric nanoparticle. In some embodiments, the polymeric nanoparticle comprises synthetic polymers.In vitro and in vivo delivery of recombinant nucleic acid

[0156] In one aspect, the nucleic acid encoding the CFP is encapsulated in a suitable lipid nanoparticle that in a therapeutic composition for delivery in vivo. The nucleic acid may be DNA, circRNA or mRNA. In some embodiments, naked DNA or messenger RNA (mRNA) may be used to introduce the nucleic acid inside a cell. In some embodiments, the DNA or mRNA encoding the chimeric fusion protein is introduced into the phagocytic cell by lipid nanoparticle (LNP) encapsulation. The mRNA can be codon optimized. In some embodiments the mRNA may comprise one or more modified or unnatural bases such as 5 ’-methylcytosine, or pseudouridine. The mRNA may be 50-10,000 bases long. In one aspect, the transgene is delivered as an mRNA. The mRNA may comprise greater than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 bases. In some embodiments, the mRNA may be more than 10,000 bases long. In some embodiments, the mRNA may be about 11,000 bases long. In some embodiments, the mRNA may be about 12,000 bases long.

[0157] In some embodiments, a pharmaceutical composition delivered locally or systemically in a subject comprises a messenger RNA encoding a chimeric fusion protein described herein, associated with one or more lipid components. In some embodiments, the one or more lipid components can comprise a cationic lipid. In some embodiments, the one or more lipid components may comprise a non-cationic lipid. In some embodiments, the one or more lipid components may comprise a polar lipid, and / or a non-polar lipid. In some embodiments, the one or more lipid components may comprise a neutral lipid. In some embodiments, the one or more lipid components may comprise a conjugated lipid. In some embodiments, the one or more lipid components may comprise a liposome around themRNA. In some embodiments, the one or more lipid components may comprise a lipid nanoparticle encapsulating the mRNA.

[0158] In some embodiments, an LNP encapsulated DNA or RNA can be used for transfecting myeloid cells, such as monocytes or macrophages, or can be administered to a subject. The LNP may be designed for targeted delivery for uptake by a myeloid cell when delivered locally or systemically to a subject. In some embodiments, the LNP may comprise one or more targeting moieties, such as an antibody or a ligand, or a biomolecule or part thereof that binds to a surface element of a myeloid cell and facilitates uptake of the DNA or RNA encapsulated LNP by myeloid cells. In some embodiments, the LNP can be targeted to a tissue by one or more antibodies, ligands, binders, aptamers that me be associated or incorporated into the LNP.

[0159] In one embodiment, the LNP comprises one or more polymers.

[0160] In one embodiment, the LNP comprises a synthetic polymer.

[0161] In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP comprises a non-cationic lipid. In some embodiments the LNP comprises a neutral lipid. In some embodiments, the LNP comprises one or more PEGylated lipids. In some embodiments, the LNP is about 100 nm to about 200 nm in diameter. In some embodiments, the LNP is about 150 nm in diameter, such as 80-120 nm, 100-140 nm, 100-130 nm, 70-140 nm, 80-150 nm, or 90-180 nm in diameter. In some embodiments, the LNP is less than 100 nm, such as less than 90 nm, or less than 80 nm in diameter.

[0162] In some embodiments, the delivery vehicle is any one of the lipid vehicles as described above, e.g., freely associated lipid components with the recombinant nucleic acids, encapsulating liposomes or LNPs, and wherein the mRNA is designed for preferential expression in myeloid cells, as described elsewhere within the specification.

[0163] In some embodiments, the mRNA with the delivery vehicle described anywhere in the specification is used for formulating a composition, for in vitro delivery of the mRNA encoding a CFP in a myeloid cell, such as a monocyte in a population of cells comprising a monocyte, wherein the myeloid cell comprising the mRNA encoding the CFP is formulated into a pharmaceutical composition for delivery into a mammalian subject, such as a human subject. In some embodiments, the mRNA comprises a CFP that comprises an extracellular antigen binding domain that binds to a cell surface antigen on an immune cell, an antigen on an inflammatory cell, an antigen on an autoimmune cell, or an antigen on an apoptotic cell, as described in the specification. In some embodiments, the mRNA with the delivery vehicle described anywhere in the specification is used for formulating a pharmaceutical composition for delivery into a mammalian subject, such as a human subject. In some embodiments, the mRNA comprising a sequence that encodes an antigen binding CFP as designed and described in the sections above for preferential or substantial expression in myeloid cells, and the delivery vehicle is taken up specifically by a myeloid cell in a heterogenouspopulation of cells. In some respects, for example, in a population of myeloid and non-myeloid cells, at least 50% of the myeloid cells will take up and express the CFP, whereas less than 10% non-myeloid cells take up and / or express the CFP. In some embodiments, the mRNA comprising a sequence that encodes an antigen binding CFP, is taken up specifically by myeloid and non-myeloid cells in a heterogenous population of cells, but is only expressed and / or functional in a myeloid cell within the heterogenous population of cells.

[0164] In some embodiments, the mRNA described above is electroporated into a cell. In some embodiments, the composition comprising the mRNA is electroporated into a cell in vitro. In some embodiments, the mRNA is associated with one or more lipid components, as described above, e.g., in an LNP. In some embodiments, the composition comprising the mRNA (comprising the one or more lipid components, for example) is electroporated into myeloid cell within a heterogenous population of cells in vitro; and the composition is designed for delivery into a myeloid cell in vitro.Methods for generation of novel chimeric receptors fusion proteins (CFP) constructs

[0165] In one aspect, provided herein is a method for generating novel chimeric receptor proteins, including, for example, identification of novel domains that can be useful in augmenting a myeloid cell function such that when the fusion receptor is expressed in a myeloid cell, it functions as an effector myeloid cell of the specifications described herein. Generation of fusion proteins as described herein can be performed using well known molecular cloning techniques, and the sequences can be verified after generating of the recombinant nucleic acid.Preparation of Recombinant Nucleic Acid Encoding CAR:

[0166] Recombinant nucleic acid constructs are prepared that encode chimeric antigen receptor (CAR) and are incorporated in plasmid vectors for amplification and / or testing expression in a eukaryotic cell. The recombinant CARs can be constructed using molecular cloning techniques known in the art. A recombinant CAR protein can comprise an intracellular domain, a transmembrane domain and an extracellular domain. Each domain or subsection of a domain can be encoded by a nucleic acid sequence that is generated by PCR from heterologous source sequences, and pieced together by cloning individually into the vector, or ligated into a longer nucleic acid that is then inserted into the multi-cloning sites of a suitable plasmid or vector with appropriate promoter and 3 ’-regulatory elements for amplification. Briefly, an exemplary CAR is prepared by incorporating a nucleic sequence encoding one or more signaling domains, (e.g., a PI3 Kinase recruiting domain or an ELMO1 domain), a nucleic acid sequence encoding a hinge and transmembrane domain, a nucleic acid sequence encoding an extracellular domain, having a sequence encoding a target antigen binding scFv at the extracellular end. In some embodiments, a standard CFP polypeptide structure comprises an N terminal signal peptide. In someembodiments, the N terminal signal peptide is a GMCSF signal peptide having a sequence that is at least 90% sequence identity of MWLQSLLLLGTVACSIS (SEQ ID NO: 19).

[0167] Certain constructs can include a FLAG peptide sequence at the extracellular end designed such that it does not pose hindrance to the scFv binding to its target antigen. These components can be ligated together into a sequence that encode a fully functional transmembrane CAR. The nucleic acid subunits encoding individual domains of the recombinant protein can be designed to include intervening short flexible linker sequences between two domains. The construct can be ligated in a plasmid having a promoter and 3’ stabilizing structural units. In one variation, the construct is placed within an Alu retrotransposon element that encodes ORF2p and has the respective 5’- and 3’-UTR sequences, a CMV promoter. The plasmid is amplified in E. coli, validated by sequencing or stored in (-) 80°C. mRNA Preparation:

[0168] mRNA can be prepared by in vitro transcription using the digested plasmid as template and purified to remove contaminant DNA and polyadenylated. The RNA product is purified, resuspended to Img / ml in RNase free water and stored in cryovials.

[0169] Identification of useful CFP extracellular domains, transmembrane domains, intracellular domains, and antigen binding domains for the generation of novel CFPs can be done using the method described herein. Briefly, a large number of potential candidate proteins can be screened for enhanced phagocytic properties and their respective phagocytosis related intracellular signaling. The useful domains can be then used for generation of novel CFPs. The screen can be divided in two parts: A. Screening for the phagocytic receptor (PR) domains,' B. Screening for the extracellular antigen binding domains.Phagocytosis assay:

[0170] Antigen-linked silica or polysterene beads ranging in diameters 1 nm, 5 nm or 10 nm can be used for a screen of macrophages. Inert beads can be coated in a supported lipid bilayer and the antigens can be ligated to the lipid bilayer. J774 macrophage cell lines can be prepared, each cell line expressing a cloned recombinant plasma membrane protein. The recombinant plasma membrane protein may also express a fluorescent tag. The cell lines can be maintained and propagated in complete RPMI media with heat inactivated serum and antibiotics (Penicillin / Streptomycin). On the day of the assay, cells can be plated at a density of lxlOA6 cells / ml per well in 6 well plates or in a relative proportion in 12 or 24 well plates, and incubated for 2-6 hours. The cells can be then washed once in Phosphate Buffer Saline, and the beads can be added in serum depleted or complement depleted nutrient media. Cells can be visualized by light microscopy at 30 minutes and 2 hours after addition of the beads. Immunofluorescence reaction may be performed using tagged antibody, and fluorescent confocal microscopy is used todetect the interaction and co-localization of cellular proteins at engulfment. Confidence levels can be determined by Kruskal -Wallis test with Dunn’s multiple comparison correction.

[0171] In some examples, dye loaded tumor cells can be fed to macrophage cell lines and phagocytosis is assessed by microscopy.Cytokine production:

[0172] Macrophage cell lines can be cultured as described above. In one assay, each J774 cell line expressing a plasma membrane protein is plated in multi-wells and challenged with antigen- linked beads and cytokine production was assayed by collecting the supernatants at 4 hours and 24 hours. Cytokines can be assayed from the supernatant by ELISA. In another fraction, cells can be collected at 4 and 24 hours after incubation with the beads and flow cytometry is performed for detection of cytokines. In each case, multiple cytokines can be assayed in a multiplex format, which can be selected from: IL-la, IL-ip, IL-6, IL-12, IL-23, TNF-a, GMCSF, CXCL1, CXCL3, CXCL9, CXCL-10, MIPl-a and MIP-2. A macrophage inflammatory cytokine array kit (R&D Systems) can be used used.

[0173] Intracellular signaling pathway for inflammatory gene and cytokine activation can be identified by western blot analysis for phosphorylation of MAP kinases, JNK, Akt signaling pathway, interferon activation pathway including phosphorylation and activation of STAT-1. Screening for high affinity antigen binding domains:

[0174] Cancer ligands can be subjected to screening for antibody light chain and heavy chain variable domains to generate extracellular binding domains for the CFPs. Human full length antibodies or scFv libraries can be screened. Also potential ligands can be used for immunizing llamas for development of novel immunoglobulin binding domains in llamas, and preparation of single domain antibodies.

[0175] Specific useful domains identified from the screens can be then reverse transcribed, and cloned into lentiviral expression vectors to generate the CFP constructs. A recombinant nucleic acid encoding a CFP can generated using one or more domains from the extracellular, TM and cytoplasmic regions of the highly phagocytic receptors generated from the screen. Briefly plasma membrane receptors showing high activators of pro-inflammatory cytokine production and inflammasome activation can be identified. Bioinformatics studies can be performed to identify functional domains including extracellular activation domains, transmembrane domains and intracellular signaling domains, for example, specific kinase activation sites, SH2 recruitment sites. These screened functional domains can be then cloned in modular constructions for generating novel CFPs. These can be candidate CFPs, and each of these chimeric constructs is tested for phagocytic enhancement, production of cytokines and chemokines, and / or tumor cell killing in vitro and / or in vivo. A microparticle based phagocytosis assay can be used to examinechanges in phagocytosis. Briefly, streptavidin coupled fluorescent polystyrene microparticles (6 pm diameter) can be conjugated with biotinylated recombinantly expressed and purified cancer ligand. Myeloid cells expressing the novel CFP can be incubated with the ligand coated microparticles for 1-4 h and the amount of phagocytosis was analyzed and quantified using flow cytometry. Plasmid or lentiviral constructions of the designer CFPs can be then prepared and tested in macrophage cells for cancer cell lysis.Method of manufacturing myeloid cells from a subjectMyeloid cell isolation from PBMCs:

[0176] Peripheral blood mononuclear cells can be separated from normal donor buffy coats by density centrifugation using Histopaque 1077 (Sigma). After washing, CD14+ monocytes can be isolated from the mononuclear cell fraction using CliniMACS GMP grade CD14 microbeads and LS separation magnetic columns (Miltenyi Biotec). Briefly, cells can be resuspended to appropriate concentration in PEA buffer (phosphate-buffered saline [PBS] plus 2.5 mmol / L ethylenediaminetetraacetic acid [EDTA] and human serum albumin [0.5% final volume of Alburex 20%, Octopharma]), incubated with CliniMACS CD14 beads per manufacturer’s instructions, then washed and passed through a magnetized LS column. After washing, the purified monocytes can be eluted from the demagnetized column, washed and re-suspended in relevant medium for culture. Isolation of CD14+ cells from leukapheresis: PBMCs can be collected by leukapheresis from cirrhotic donors who gave informed consent to participate in the study. Leukapheresis of peripheral blood for mononuclear cells (MNCs) is carried out using an Optia apheresis system by sterile collection. A standard collection program for MNC is used, processing 2.5 blood volumes. Isolation of CD14 cells is carried out using a GMP-compliant functionally closed system (CliniMACS Prodigy system, Miltenyi Biotec). Briefly, the leukapheresis product is sampled for cell count and an aliquot taken for pre- separation flow cytometry. The percentage of monocytes (CD14+) and absolute cell number can be determined, and, if required, the volume is adjusted to meet the required criteria for selection (< 20 x 109total white blood cells; < 400 x 106white blood cells / mL; < 3.5 x 109CD14 cells, volume 50-300 mL). CD14 cell isolation and separation is carried out using the CliniMACS Prodigy with CliniMACS CD14 microbeads (medical device class III), TS510 tubing set and LP-14 program. At the end of the process, the selected CD14+ positive monocytes can be washed in PBS / EDTA buffer (CliniMACS buffer, Miltenyi) containing pharmaceutical grade 0.5% human albumin (Alburex), then re-suspended in TexMACS (or comparator) medium for culture.Cell count and purity:

[0177] Cell counts of total MNCs and isolated monocyte fractions can be performed using a Sysmex XP-300 automated analyzer (Sysmex). Assessment of macrophage numbers is carried outby flow cytometry with TruCount tubes (Becton Dickinson) to determine absolute cell number, as the Sysmex consistently underestimated the number of monocytes. The purity of the separation is assessed using flow cytometry (FACSCanto II, BD Biosciences) with a panel of antibodies against human leukocytes (CD45-VioBlue, CD15-FITC, CD14-PE, CD16-APC), and product quality is assessed by determining the amount of neutrophil contamination (CD45int, CD15pos). Cell culture — development of cultures with healthy donor samples:

[0178] Optimal culture medium for macrophage differentiation is investigated, and three candidates can be tested using for the cell product. In addition, the effect of monocyte cryopreservation on deriving myeloid cells and macrophages for therapeutic use is examined. Functional assays can be conducted to quantify the phagocytic capacity of myeloid cells and macrophages and their capacity for further polarization, and phagocytic potential as described elsewhere in the disclosure.Full-scale process validation with subject samples:

[0179] Monocytes cultured from leukapheresis from Prodigy isolation can be cultured at 2 x 106monocytes per cm2and per mL in culture bags (MACS GMP differentiation bags, Miltenyi) with GMP-grade TexMACS (Miltenyi) and 100 ng / mL M-CSF. Monocytes can be cultured with 100 ng / mL GMP-compliant recombinant human M-CSF (R&D Systems). Cells can be cultured in a humidified atmosphere at 37°C, with 5% CO2 for 7 days. A 50% volume media replenishment is carried out twice during culture (days 2 and 4) with 50% of the culture medium removed, then fed with fresh medium supplemented with 200 ng / mL M-CSF (to restore a final concentration of 100 ng / mL).Cell harvesting:

[0180] For normal donor-derived macrophages, cells can be removed from the wells at day 7 using Cell Dissociation Buffer (Gibco, Thermo Fisher) and a pastette. Cells can be resuspended in PEA buffer and counted, then approximately IxlO6cells per test can be stained for flow cytometry. Leukapheresis-derived macrophages can be removed from the culture bags at day 7 using PBSZEDTA buffer (CliniMACS buffer, Miltenyi) containing pharmaceutical grade 0.5% human albumin from serum (HAS; Alburex). Harvested cells can be resuspended in excipient composed of two licensed products: 0.9% saline for infusion (Baxter) with 0.5% human albumin (Alburex).Flow cytometry characterization:

[0181] Monocyte and macrophage cell surface marker expression can be analyzed using either a FACSCanto II (BD Biosciences) or MACSQuant 10 (Miltenyi) flow cytometer. Typically, approximately 20,000 events can be acquired for each sample. Cell surface expression of leukocyte markers in freshly isolated and day 7 matured cells is carried out by incubating cellswith specific antibodies (final dilution 1 : 100). Cells are incubated for 5 min with FcR block (Miltenyi) then incubated at 4°C for 20 min with antibody cocktails. Cells can be washed in PEA, and dead cell exclusion dye DRAQ7 (BioLegend) is added at 1 : 100. Cells can be stained for a range of surface markers as follows: CD45-VioBlue, CD14-PE or CD14-PerCP-Vio700, CD163- FITC, CD169-PE and CD16-APC (all Miltenyi), CCR2-BV421, CD206-FITC, CXCR4-PE and CD115-APC (all BioLegend), and 25F9-APC and CD115-APC (eBioscience). Both monocytes and macrophages can be gated to exclude debris, doublets and dead cells using forward and side scatter and DRAQ7 dead cell discriminator (BioLegend) and analyzed using FlowJo software (Tree Star). From the initial detailed phenotyping, a panel is developed as Release Criteria (CD45- VB / CD206-FITC / CD14-PE / 25F9 APC / DRAQ7) that defined the development of a functional macrophage from monocytes. Macrophages can be determined as having mean fluorescence intensity (MFI) five times higher than the level on day 0 monocytes for both 25F9 and CD206. A second panel is developed which assessed other markers as part of an Extended Panel, composed of CCR2-BV421 / CD163-FITC / CD169-PE / CD14-PerCP-Vio700 / CD16-APC / DRAQ7), but is not used as part of the Release Criteria for the cell product.

[0182] Monocytes and macrophages can be isolated from withdrawing a buffy coat layer formed in a sucrose gradient centrifugation sample of isolated peripheral blood cells. CD14 cells can be tested for phagocytic uptake using pHRodo beads, which fluoresce only when taken into acidic endosomes. Briefly, monocytes or macrophages can be cultured with 1-2 uL of pHRodo Escherichia coli bioparticles (Life Technologies, Thermo Fisher) for 1 h, then the medium is taken off and cells are washed to remove non-phagocytosed particles. Phagocytosis is assessed using an EVOS microscope (Thermo Fisher), images captured and cellular uptake of beads quantified using ImageJ software (NIH). The capacity to polarize toward defined differentiated macrophages is examined by treating day 7 macrophages with ZFNy (50 ng / mL) or IL-4 (20 ng / mL) for 48 h to induce polarization to Ml or M2 phenotype (or M[IFNy] versus M[IL-4], respectively). After 48 h, the cells can be visualized by EVOS bright-field microscopy, then harvested and phenotyped as before. Further analysis is performed on the cytokine and growth factor secretion profile of macrophages after generation and in response to inflammatory stimuli. Macrophages can be generated from healthy donor buffy coats as before, and either left untreated or stimulated with TNFa (50 ng / mL, Peprotech) and polyinosinic:polycytidylic acid (poly I:C, a viral homolog which binds TLR3, 1 g / mL, Sigma) to mimic the conditions present in the inflamed liver, or lipopolysaccharide (LPS, 100 ng / mL, Sigma) plus fFNy (50 lU / mL, Peprotech) to produce a maximal macrophage activation. Day 7 macrophages can be incubated overnight and supernatants collected and spun down to remove debris, then stored at -80°C until testing. Secretome analysisis performed using a 27-plex human cytokine kit and a 9-plex matrix metalloprotease kit run on a Magpix multiplex enzyme linked immunoassay plate reader (BioRad).Product stability:

[0183] Various excipients can be tested during process development including PBS / EDTA buffer; PBS / EDTA buffer with 0.5% HAS (Alburex), 0.9% saline alone or saline with 0.5% HAS. The 0.9% saline (Baxter) with 0.5% HAS excipient is found to maintain optimal cell viability and phenotype (data not shown). The stability of the macrophages from cirrhotic donors after harvest is investigated in three process optimization runs, and a more limited range of time points assessed in the process validation runs (n = 3). After harvest and re-suspension in excipient (0.9% saline for infusion, 0.5% human serum albumin), the bags can be stored at ambient temperature (21-22°C) and samples taken at 0, 2, 4, 6, 8, 12, 24, 30 and 48 h postharvest. The release criteria antibody panel is run on each sample, and viability and mean fold change from day 0 is measured from geometric MFI of 25F9 and CD206.Statistical analysis:

[0184] Results can be expressed as mean ± SD. The statistical significance of differences is assessed where possible with the unpaired two-tailed / -test using GraphPad Prism 6. Results can be considered statistically significant when the P value is < 0.05.

[0185] Provided herein is a method for administering a therapeutic comprising any one of the compositions described above. In some embodiments, the therapeutic is administered via a parenteral administration route.

[0186] In some embodiments, the therapeutic is administered via intramuscular administration route. In some embodiments, the therapeutic is administered via intravenous administration route. In some embodiments, the therapeutic is administered via subcutaneous administration route.

[0187] Also provided herein is a method of preparing a pharmaceutical composition comprising the one or more recombinant nucleic acids described herein and a lipid in an aqueous composition described herein. In some embodiments, the composition comprises a vector described herein. In some embodiments, the lipid comprises forming a lipid nanoparticle.Methods of Treatment

[0188] The pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing anyundesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; com oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non - toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0189] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 - butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0190] The injectable formulations can be sterilized, for example, by filtration through a bacterial - retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0191] Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0192] The modified particles can be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the modified particles. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens,Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

[0193] It will also be appreciated that the modified particles and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another anti - inflammatory agent), or they may achieve different effects (e.g., control of any adverse effects).

[0194] In certain embodiments, the pharmaceutical compositions containing the engineered myeloid cells or the pharmaceutical compositions comprising the polynucleotide acid encoding the CFP of the present disclosure further comprise one or more additional therapeutically active ingredients (e.g., anti-inflammatory and / or palliative). For purposes of the invention, the term “palliative” refers to treatment that is focused on the relief of symptoms of a disease and / or side effects of a therapeutic regimen, but is not curative. The invention provides methods of regulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to the individual the pharmaceutical composition described herein. Methods of immunoregulation provided by the invention include those that suppress and / or inhibit an innate immune response or an adaptive immune response, including, but not limited to, an immune response stimulated by immunostimulatory polypeptides or viral or bacterial components. The pharmaceutical compositions are administered in an amount sufficient to regulate an immune response. As described herein, regulation of an immune response may be humoral and / or cellular, and is measured using standard techniques in the art and as described herein.

[0195] In certain embodiments, the individual suffers from a disorder associated with unwanted immune activation, such as allergic disease or condition, allergy and asthma. An individual having an allergic disease or asthma is an individual with a recognizable symptom of an existing allergic disease or asthma. In certain embodiments, the individual suffers from a disorder associated with unwanted immune activation, such as atherosclerosis, ischemic reperfusion injury, and myocardial infarction.

[0196] In certain embodiments, the individual suffers from a disorder associated with unwanted immune activation, such as autoimmune disease and inflammatory disease. An individual having an autoimmune disease or inflammatory disease is an individual with a recognizable symptom of an existing autoimmune disease or inflammatory disease.

[0197] In some embodiments, the human autoimmune antigen is associated with an autoimmune disease, such as an autoimmune disease. In some embodiments, the human autoantigen is associated with any one of Achalasia, Addison’s disease, Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM / Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan’s syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn’s disease, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’s syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Lyme disease chronic, Meniere’s disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myelin Oligodendrocyte Glycoprotein Antibody Disorder, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonage-Tumer syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary Bihary Cholangitis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRC A), Pyoderma gangrenosum, Raynaud’s phenomenon, Reactive Arthritis, Reflexsympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren’s syndrome, Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac’s syndrome, Sympathetic ophthalmia (SO), Takayasu’s arteritis, Temporal arteritis / Giant cell arteritis, Thrombocytopenic purpura (TTP), Thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi- Harada Disease.

[0198] In some embodiments, the human autoimmune antigen is associated with a disease or condition that is selected from the group consisting of multiple sclerosis (MS), rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, Addison’s disease, Sjogren’s syndrome, pernicious anemia, celiac disease and vasculitis.

[0199] In some embodiments, the human autoimmune antigen is associated with Celiac Disease. In some embodiments, the human autoimmune antigen is associated with Multiple Sclerosis. In some embodiments, the human autoimmune antigen is associated with Myasthenia gravis. In some embodiments, the human autoimmune antigen is associated with Type 1 Diabetes. In some embodiments, the human autoimmune antigen is associated with rheumatoid arthritis. In some embodiments, the human autoimmune antigen is associated with systemic lupus erythematosus(lupus). In some embodiments, the human autoimmune antigen is associated with inflammatory bowel disease (IBD). In some embodiments, the human autoimmune antigen is associated with ulcerative colitis. In some embodiments, the human autoimmune antigen is associated with Crohn's disease, type 1 diabetes mellitus. In some embodiments, the human autoimmune antigen is associated with chronic inflammatory demyelinating polyneuropathy. In some embodiments, the human autoimmune antigen is associated with psoriasis. In some embodiments, the human autoimmune antigen is associated with Graves' disease. In some embodiments, the human autoimmune antigen is associated with Hashimoto's thyroiditis. In some embodiments, the human autoimmune antigen is associated with Sjogren’s syndrome. In some embodiments, the human autoimmune antigen is associated with pernicious anemia. In some embodiments, the human autoimmune antigen is associated with vasculitis.

[0200] In some embodiments, the human autoimmune antigen may be selected from the group consisting of MBP13-32, MBP83-99, MBP111-129, MBP146-170, MOG1-20, MOG35-55, and PLP139-15 polypeptide and fragments thereof.EXAMPLESExample 1. Phagocytic Activity of Cells Expressing CFPs Targeting FAP

[0201] In order to determine the effect of CFPs comprising an anti-FAP extracellular domain, CFP constructs were designed as illustrated in FIG. 2. Tested constructs included an extracellular scFv domain targeting fibroblast activation protein (FAP) and an ELMO intracellular domain. One construct included an additional CD89 domain (“aFAP-CD89-ELMO” in FIG. 3) and another construct included an additional TIM4 domain (“aFAP-TIM4-ELMO” in FIG. 3). A construct without an ELMO domain (“aFAP-CD89” in FIG. 3) was also utilized. Constructs also included a FLAG domain to measure CFP expression.

[0202] THP-1 monocytes were electroporated using lOOug / mL mRNA encoding the CFP. Cells were incubated for 22hr at 37°C. At 24hr post-electroporation, expression of the CFP was measured via flow cytometry using a fluorescent (BV421) anti -FLAG antibody. As shown in the flow cytometry plots in FIG. 3 and summarized in FIGs. 4A-4B, CFP expression was detected in over 90% of the cells across groups.

[0203] To test the phagocytic activity of cells expressing CFPs targeting FAP, a phagocytosis assay was designed. Streptavidin YG beads were conjugated with either biotin conjugated FAP or Biotin conjugated-bovine serum albumin (BSA) as a negative control. Effective conjugation was verified by flow cytometry as shown in FIGs. 5A and 5B. THP-1 monocytes were electroporated using lOOug / mL mRNA and incubated for 22hr at 37°C. Then, cells were incubated with either of the conjugated beads at a 1 : 1 effector to target (E:T) ratio. After 2 hours, cells were analyzed via flow cytometry to measure phagocytosis. Prior to the flow cytometry analysis, cells were stained with a DRAQ5 viability dye, which stains live cells. To determine phagocytic activity, cells were assayed for YG (beads) and DRAQ5 (live THP-1 cells). Percent phagocytic activity among live cells was determined using the formula (Quadrant 2) / (Quadrant 2+Quadrant 3). As summarized in FIGs. 6A and 6B, effective phagocytosis was observed in each of the constructs tested, with FAP-TIM4-ELMO demonstrating the highest levels of phagocytic activity. Individual flow cytometry graphs are depicted in FIGs. 7A and 7B. Taken together, these data demonstrate that CFPs targeting FAP can effectively induce phagocytosis of FAP-expressing targets by myeloid cells expressing the CFP.Example 2. Anti-Inflammatory Activity of ELMO domains

[0204] In order to test the anti-inflammatory activity of cells expressing CFPs comprising an ELMO domain, constructs were designed as in Example 1. Primary monocytes from human donors were electroporated using 200ug / mL of mRNA. Following a 2 hour incubation at 37°C, expression of the CFP was measured via flow cytometry using a fluorescent (BV421) anti -FLAG antibody at 24-, 48-, and 72 hours post electroporation. As shown in FIGs. 8A and 8B, cells maintained expression of the CFP even at 72 hours post electroporation.

[0205] In order to determine the anti-inflammatory effects of cells expressing the CFP, electroporated monocytes were plated on wells coated with lOug / mL of recombinant human FAP for 24 hours. Following stimulation, supernatant was harvested and analyzed for cytokine expression via a multiplex cytokine assay. As shown in FIGs. 9A and 9B, CFP constructs containing ELMO were effective in reducing expression of cytokines such as TNF, IL-6, IL-lbeta as well as chemokines such as CCL4 and CCL5 compared to constructs not containing the ELMO domain.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a polynucleic acid, wherein the polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising a target binding domain, wherein the target binding domain is an antibody or antigen-binding domain thereof, (ii) a transmembrane domain operably linked to the extracellular domain; and (iii) an intracellular domain comprising an intracellular signaling domain from an engulfment and cell motility protein domain (ELM01) or a functional variant thereof.

2. The composition of claim 1, wherein the intracellular domain comprises a sequence having at least 80% identity to SEQ ID NO: 1.

3. The composition of claim 1 or 2, wherein the extracellular domain comprising the target binding domain comprises an antibody or binding domains thereof, an Fab, a nanobody, a bispecific antibody, a diabody, a single chain variable fragment (scFv), single domain antibody (SdAb), variable heavy chain (VHH) domain, a VH domain, a VL domain, a VNAR domain, a ligand or a receptor or a functional fragment thereof or a combination thereof.

4. The composition of any one of claims 1-3, wherein the extracellular domain comprising the target binding domain is an scFv or a sdAb.

5. The composition of any one of claims 1-4, wherein the target binding domain binds to an inflammatory cell, an autoimmune cell, or an apoptotic cell.

6. The composition of any one of claims 1-5, wherein the target binding domain binds to a cell surface molecule of a target cell, wherein the target cell is an inflammatory cell, an autoimmune cell, or an apoptotic cell.

7. The composition of any one of claims 1-6, wherein the target binding domain binds to a cell surface molecule selected from the group consisting of keratinocyte adhesion protein, Dsg3, myelin oligodendrocyte glycoprotein, fibroblast activation protein, an AAV Capsid protein, and citrullinated vimentin.

8. The composition of any one of claim 1-6, wherein the target binding domain binds to extracellular DNA and / or chromatin.

9. The composition of any one of claims 1-6, wherein the target binding domain binds to fibroblast activation protein.

10. The composition of claim 9, wherein the target binding domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 18.

11. The composition of claim 9, wherein the target binding domain comprises a heavy chain comprising a sequence with at least 80% sequence identity to SEQ ID NO: 8.

12. The composition of claim 11, wherein the heavy chain comprises a CDR1 sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-11.

13. The composition of claim 11 or 12, wherein the heavy chain comprises a CDR2 sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-14.

14. The composition of any one of claims 11-13, wherein the heavy chain comprises a CDR3 sequence having at least 80% sequence identity to SEQ ID NO: 15 or 16.

15. The composition of claim 9, wherein the target binding domain comprises a light chain comprising a sequence with at least 80% sequence identity to SEQ ID NO: 2.

16. The composition of claim 11, wherein the light chain comprises a CDR1 sequence having at least 80% sequence identity to SEQ ID NO: 3 or 4.

17. The composition of claim 11 or 12, wherein the light chain comprises a CDR2 sequence having at least 80% sequence identity to SEQ ID NO: 5 or 6.

18. The composition of any one of claims 11-13, wherein the light chain comprises a CDR3 sequence having at least 80% sequence identity to SEQ ID NO: 7.

19. The composition of any one of claims 1-18, wherein the extracellular domain further comprises a hinge domain between the target binding domain and the transmembrane domain.

20. The composition of claim 19, wherein the hinge domain comprises an extracellular hinge region from a protein selected from the group consisting of CD8oc, CD2, CD28, CD16, CD64, CD89, BAI1, TIM4, MerTK, and a phagocytosis receptor.

21. The composition of any one of claims 1-20, wherein the transmembrane domain is a transmembrane domain from a protein selected from the group consisting of CD16, CD89, CD64, CD3 zeta, BAI1, TIM4, and MerTK.

22. The composition of any one of claims 19 or 20, wherein the hinge domain is from the same protein as the transmembrane domain.

23. The composition of any one of claims 1-21, wherein the intracellular domain further comprises one or more additional intracellular domains from one or more proteins.

24. The composition of claim 23, wherein the one or more additional intracellular domains comprise an intracellular signaling domain.

25. The composition of any one of claims 23 or 24, wherein the one or more additional intracellular domains is a phagocytic receptor intracellular domain.

26. The composition of any one of claims 23 or 24, wherein the one or more additional intracellular domains is an intracellular domain from a pattern recognition molecule.

27. The composition of any one of claims 23 or 24, wherein the one or more additional intracellular domains is an intracellular domain from a cytokine or a chemokine receptor.

28. The composition of any one of claims 23-26, wherein the one or more additional intracellular domains is derived from a protein selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor Al (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, 0LR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, CR1, CD35, CD3^, a complement receptor, CR3, CR4, Tim-1, Tim-4 and CD169, CD40, a cytokine receptor, a chemokine receptor, Fea receptor I, Fc gamma receptor, and Fc epsilon receptor Ig (FCER1G).

29. The composition of any one of claims 23-26, wherein the one or more additional intracellular domains is derived from CD89 or Tim-4.

30. The composition of claim 29, wherein the one or more additional intracellular domain is derived from Tim-4.

31. The composition of claim 30, wherein the one or more additional intracellular domain comprises a sequence having at least 80% identity to SEQ ID NO: 22.

32. The composition of claim 29, wherein the one or more additional intracellular domain is derived from CD89.

33. The composition of claim 32, wherein the one or more additional intracellular domain comprises a sequence having at least 80% identity to SEQ ID NO: 19.

34. The composition of any one of claims 24-33, wherein the intracellular signaling domain promotes activation of phagocytosis.

35. The composition of any one of claims 1-34, wherein the target binding domain binds to an apoptotic cell surface marker.

36. The composition of claim 35, wherein the apoptotic cell surface marker is phosphatidyl inositol or phosphatidyl serine.

37. The composition of any one of claims 1-36, wherein activation of the CFP upon contacting the target does not result in sustained inflammatory cytokine or chemokine production by a cell expressing the CFP.

38. The composition of claim 37, wherein the inflammatory cytokine is TNF alpha, interferon gamma, interleukin 2, or interleukin 1.

39. The composition of claim 37, wherein the chemokine is a C-C chemokine, or a CXC- chemokine.

40. The composition of any one of claims 1-39, wherein the polynucleic acid is an RNA.

41. The composition of any one of claims 1-40, wherein the polynucleic acid is an mRNA.

42. The composition of claim 40 or 41, wherein the polynucleic acid is 2kb - 20,000kb long.

43. The composition of claim 41 or 42, wherein the polynucleic acid is an in vitro transcribed (IVT) mRNA.

44. The composition of any one of claims 41-43, wherein the mRNA comprises a 5’ UTR and a 3’UTR.

45. The composition of any one of claims 41-43, wherein the mRNA comprises a 5’ guanosyl CAP.

46. The composition of claim 45, wherein the 5’ guanosyl CAP is modified from a native 5’ CAP.

47. The composition of claim 46, wherein the 5’ guanosyl CAP comprises a 2’-O-methyl modification.

48. The composition of claim 46, wherein the 5’ guanosyl CAP comprises an N6 methyl modification.

49. The composition of any one of claims 41-48, wherein the mRNA comprises a 3’-polyA tail, wherein the poly A tail is 50-250 nucleotides long.

50. The composition of any one of claims 45-49, wherein the 5’ guanosyl CAP is added post- transcriptionally to the IVT mRNA.

51. The composition of any one of claims 1-50, further comprising a delivery vehicle.

52. The composition of any one of claims 1-51, further comprising one or more lipids.

53. The composition of claim 52, wherein the one or more lipids comprise a cationic lipid.

54. The composition of claim 52, wherein the one or more lipids comprise a neutral lipid and a PEGylated lipid.

55. The composition of any one of claims 51-54, wherein the delivery vehicle is a liposome.

56. The composition of any one of claims 1-55, wherein the polynucleic acid is encapsulated in a liposome.

57. The composition of claim 55 or 56, wherein the liposome is a lipid nanoparticle.

58. The composition of any one of claims 1-57, wherein the CFP comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 20, 21, 23, and 24.

59. A cell comprising the composition of any one of claims 1-58.

60. A pharmaceutical composition comprising the composition of any one of claims 1-58 or the cell of claim 59, and a pharmaceutically acceptable excipient.

61. A method of treating an inflammatory or autoimmune disease in a subject in need thereof comprising administering to the subject the pharmaceutical composition of claim 60.

62. A method of treating an inflammatory or autoimmune disease in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising a target binding domain, (ii) a transmembrane domain operatively linkedto the extracellular domain; and (iii) an intracellular domain comprising an intracellular signaling domain from ELM01.

63. The method of claim 62, wherein the target binding domain is an antibody or binding domain thereof.

64. The method of claim 62, wherein the target binding domain binds to a cell surface molecule selected from the group consisting of keratinocyte adhesion protein, Dsg3, myelin oligodendrocyte glycoprotein, fibroblast activation protein, an AAV Capsid protein, extracellular DNA / chromatin, and citrullinated vimentin.

65. The method of claim 62, wherein the target binding domain binds to extracellular DNA and / or chromatin.

66. The method of claim 62, wherein the target binding domain binds to fibroblast activation protein.

67. The method of claim 66, wherein the target binding domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 18.

68. The method of claim 66, wherein the target binding domain comprises a heavy chain comprising a sequence with at least 80% sequence identity to SEQ ID NO: 8.

69. The method of claim 68, wherein the heavy chain comprises a CDR1 sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-11.

70. The method of claim 68 or 69, wherein the heavy chain comprises a CDR2 sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-14.

71. The method of any one of claims 68-70, wherein the heavy chain comprises a CDR3 sequence having at least 80% sequence identity to SEQ ID NO: 15 or 16.

72. The method of claim 66, wherein the target binding domain comprises a light chain comprising a sequence with at least 80% sequence identity to SEQ ID NO: 2.

73. The method of claim 72, wherein the light chain comprises a CDR1 sequence having at least 80% sequence identity to SEQ ID NO: 3 or 4.

74. The method of claim 72 or 73, wherein the light chain comprises a CDR2 sequence having at least 80% sequence identity to SEQ ID NO: 5 or 6.

75. The method of any one of claims 72-74, wherein the light chain comprises a CDR3 sequence having at least 80% sequence identity to SEQ ID NO: 7.

76. The method of any one of claims 62-75, wherein the intracellular domain further comprises one or more additional intracellular domains from one or more proteins.

77. The method of claim 76, wherein the one or more additional intracellular domains is from CD89 or Tim-4.

78. The method of claim 77, wherein the one or more additional intracellular domain is derived from Tim-4.

79. The method of claim 78, wherein the one or more additional intracellular domain comprises a sequence having at least 80% identity to SEQ ID NO: 22.

80. The method of claim 77, wherein the one or more additional intracellular domain is derived from CD89.

81. The method of claim 80, wherein the one or more additional intracellular domain comprises a sequence having at least 80% identity to SEQ ID NO: 19.

82. The method of any one of claims 62-81 , wherein the CFP comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 20, 21, 23, and 24.

83. The method of any one of claims 61-82, wherein the subject is a human.

84. The method of any one of claims 61-82, wherein the administering is performed systemically.

85. The method of any one of claims 61-82, wherein the pharmaceutical composition is formulated for intravenous or intramuscular or subcutaneous injections.

86. The method of claim 61 or 62, wherein the pharmaceutical composition is formulated for oral, or parenteral administration.

87. The method of any one of claims 61-86, wherein the inflammatory or autoimmune disease is Pemphigus vulgaris, Multiple sclerosis, Cardiac fibrosis, Immune response to AAV-based gene therapies, autoimmune-associated neutrophil extracellular traps, or Rheumatoid Arthritis.

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