Engineered Chimeric Fusion Protein Compositions and Methods of Their Use

Abstract

Compositions and methods for generating and using engineered myeloid cells that express chimeric fusion proteins for immunotherapy, and compositions and methods for generating and using engineered T cells that express chimeric antigen receptors for immunotherapy.

Description

[Technical Field]

[0001] Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 192,505, filed May 24, 2021, and U.S. Provisional Patent Application No. 63 / 252,423, filed October 5, 2021, each of which is incorporated by reference in its entirety. [Background technology]

[0002]

[0002] Cellular immunotherapy is a promising new technology that combats the challenges of treating diseases such as cancer and persistent infections, as well as certain diseases refractory to other forms of treatment. A major breakthrough has been the discovery of CAR-T cells and their potential use in immunotherapy. However, new avenues using other cell types for the development of improved therapies are being explored. Myeloid cells are major intracellular compartments of the immune system, including monocytes, dendritic cells, tissue macrophages, and granulocytes. Their significant plasticity and heterogeneity during both homeostasis and disease can be exploited to develop safe and effective therapies. Myeloid cells can be engineered to specifically target and destroy a range of pathogens or pathogenic cell types, initiate robust immune responses, and even induce T cell responses in vivo.

[0003]

[0003] Engineered myeloid cells can be short-lived, phenotypically diverse, sensitive, and plastic in vivo, and they are often difficult to manipulate in vitro. For example, exogenous gene expression in monocytes is more challenging than exogenous gene expression in non-hematopoietic stem cells. There are significant technical challenges associated with transfecting myeloid cells (e.g., monocytes / macrophages). As professional phagocytes, myeloid cells such as monocytes / macrophages contain many potent degradative enzymes that can disrupt the integrity of nucleic acids and inefficiently transfer genes into these cells. This is particularly true for activated macrophages, which undergo dramatic changes in their physiology after exposure to immune or inflammatory stimuli. Viral transduction of these cells is hampered because macrophages are generally terminally differentiated cells; therefore, some vectors that rely on integration into replicative genomes face limited success. Furthermore, macrophages are sensitive to "danger signals," and therefore, some of the original viral vectors used for gene transfer induce strong antiviral responses in these cells, making these vectors unsuitable for gene delivery. Summary of the Invention

[0004] The diverse functionalities of myeloid cells make them ideal candidates for cell therapy, which can be engineered to have numerous therapeutic effects. The present disclosure relates to immunotherapy using myeloid cells of the immune system (e.g., CD14+ cells), particularly phagocytes. Many therapeutic indications could be explored using myeloid cells. For example, myeloid cell immunotherapy could be crucial in cancer, autoimmunity, fibrosis, and infection. The present disclosure relates to immunotherapy using myeloid cells, including phagocytes of the immune system, particularly macrophages. It is an object of the present inventions disclosed herein to inhibit one or more of these functions of myeloid cells for therapeutic use. For example, it is an object of the present inventions disclosed herein to inhibit the phagocytic activity of myeloid cells, including engineered myeloid cells, for therapeutic use. For example, it is an object of the present inventions disclosed herein to inhibit the ability of myeloid cells, including engineered myeloid cells, to promote T cell activation. For example, it is an object of the present inventions disclosed herein to inhibit the ability of myeloid cells, including engineered myeloid cells, to promote the secretion of tumoricidal molecules. For example, inhibiting the ability of myeloid cells, including engineered myeloid cells, to promote the recruitment and trafficking of immune cells and molecules is an objective of the inventions disclosed herein. The present disclosure provides innovative methods and compositions that can induce genetic modifications in myeloid cells with the goal of successfully transfecting or transducing the myeloid cells or otherwise increasing their functional aspects, without further compromising the cells' differentiation potential, maturation potential, and / or their plasticity.

[0005]

[0005] The present disclosure involves the production and use of engineered myeloid cells, such as macrophages or other phagocytes (e.g., CD14+ cells), that can directly or indirectly attack and kill (ATAK) diseased cells, such as cancer cells and infected cells. Engineered myeloid cells, such as macrophages and other phagocytes, can be prepared in vivo by incorporating a nucleic acid sequence (e.g., mRNA, plasmid, viral construct) encoding a chimeric fusion protein (CFP) having an extracellular binding domain specific for a disease-associated antigen (e.g., a cancer antigen) into cells using, for example, recombinant nucleic acid technology, synthetic nucleic acid, gene editing technology (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection, or by using mRNA delivery technologies, including but not limited to, LNP technology. It has been discovered that myeloid cells can be engineered to have a broad and diverse range of activities. For example, it has been discovered that myeloid cells can be engineered to express chimeric fusion proteins (CFPs) containing antigen-binding domains 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, so that when CFP binds to an antigen on a target cell, the cells exhibit increased phagocytosis of the target cell.It has also been found that myeloid cells can be engineered to promote T cell activation, so that when CFP binds to an antigen on a target cell, the cells promote the activation of T cells, for example, T cells in a tumor microenvironment.Engineered myeloid cells can be engineered to promote the secretion of tumoricidal molecules, so that when CFP binds to an antigen on a target cell, the cells promote the secretion of tumoricidal molecules from neighboring cells.Engineered myeloid cells can be engineered to promote the recruitment and transport of immune cells and molecules, so that when CFP binds to an antigen on a target cell, the cells promote the recruitment and transport of immune cells and molecules to the target cell or tumor microenvironment.

[0006]

[0006] The present disclosure is based on the important discovery that engineered myeloid cells overcome at least some of the limitations of CAR-T cells, including being easily mobilized to solid tumors; having a manageable survival time, thus reducing the risk of prolonged persistence resulting in aplasia and immunodeficiency; myeloid cells cannot contaminate T cells; for example, myeloid cells can avoid fratricide 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 bone marrow-derived cells may be a safer immunotherapeutic means of targeting and destroying diseased cells.

[0007]

[0007] Furthermore, myeloid cells such as macrophages are ubiquitously found in the tumor environment (TME), and are particularly the most abundant cells in some tumor types. As part of their role in the immune system, myeloid cells such as macrophages are naturally involved in the elimination of diseased cells. The present invention relates to inhibiting myeloid cell function, particularly targeting, killing, and directly and / or indirectly eliminating diseased cells, as well as delivering payloads such as antigens and cytokines.

[0008]

[0008] The present disclosure is also based on the important discovery that engineered myeloid cells can promote endogenous T cell activity and thus amplify the activity of existing therapies focused on activating T cells. Engineered T cells and myeloid cells can be achieved by in vivo incorporation into cells of nucleic acid sequences (e.g., mRNA, plasmids, viral constructs) encoding chimeric fusion proteins (CFPs) with extracellular binding domains specific for disease-associated antigens (e.g., cancer antigens), e.g., using recombinant nucleic acid technology, synthetic nucleic acids, gene editing technology (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection, or in vivo using mRNA delivery technologies, including but not limited to LNP technology.

[0009]

[0009] In addition to in vivo programming of myeloid cells, the present disclosure includes in vivo programming of T cells. It has been discovered that the combination of T cell and myeloid cell engineering can have potent anti-tumor cell activity. For example, it has been discovered that T cells and myeloid cells can be engineered to express chimeric antigen receptors (CARs) and chimeric fusion proteins (CFPs), respectively, each containing antigen-binding domains with broad and diverse ranges of activity. For example, it has been discovered that T cells can be engineered to have enhanced cytolytic activity, such that upon binding of the CAR to an antigen on a target cell, the cells exhibit increased lysis of the target cell. It has also been discovered that co-engineering myeloid cells with T cells can promote broad immune activity in the tumor microenvironment, resulting in a strong and durable anti-tumor response. Engineered T cells can be achieved by in vivo incorporation into cells of a nucleic acid sequence (e.g., mRNA, plasmid, viral construct) encoding a chimeric antigen receptor (CAR) with an extracellular binding domain specific for a disease-associated antigen (e.g., a cancer antigen), e.g., using recombinant nucleic acid technology, synthetic nucleic acid, gene editing technology (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection, or in vivo using mRNA delivery technologies, including but not limited to LNP technology.

[0010]

[0010] Engineered myeloid cells can be short-lived, phenotypically diverse, sensitive, and plastic in vivo, and they are often difficult to manipulate in vitro. For example, exogenous gene expression in monocytes is more challenging than exogenous gene expression in non-hematopoietic stem cells. There are significant technical challenges associated with transfecting myeloid cells (e.g., monocytes / macrophages). As professional phagocytes, myeloid cells such as monocytes / macrophages contain many potent degradative enzymes that can disrupt the integrity of nucleic acids and inefficiently transfer genes into these cells. This is particularly true for activated macrophages, which undergo dramatic changes in their physiology after exposure to immune or inflammatory stimuli. Viral transduction of these cells is hampered because macrophages are generally terminally differentiated cells; therefore, some vectors that rely on integration into replicative genomes face limited success. Furthermore, macrophages are sensitive to "danger signals," and thus some original viral vectors used for gene transfer induce strong antiviral responses in these cells, making these vectors unsuitable for gene delivery. The present disclosure provides innovative methods and compositions that can induce genetic modifications in myeloid cells with the aim of successfully transfecting or transducing them or otherwise increasing their functional aspects, without further compromising the cells' differentiation potential, maturation potential, and / or their plasticity.

[0011]

[0011] Provided herein is a pharmaceutical composition comprising: (A) a recombinant nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) comprising a phagocytic or tethering receptor (PR) subunit, the chimeric fusion protein comprising (i) a transmembrane domain, and (ii) an intracellular domain comprising an intracellular signaling domain derived from a phagocytic or tethering receptor, wherein the transmembrane domain and the intracellular domain are operably linked; and (B) a pharmaceutically acceptable excipient, wherein the recombinant nucleic acid is a recombinant self-amplifying nucleic acid.

[0012] In some embodiments, the recombinant self-amplifying nucleic acid is a recombinant self-amplifying RNA.

[0013] In some embodiments, the recombinant self-amplifying nucleic acid is a recombinant self-amplifying mRNA.

[0014] In some embodiments, the recombinant self-amplifying nucleic acid comprises a 5' CAP. In some embodiments, the recombinant self-amplifying nucleic acid comprises a 3' polyadenylation (polyA) tail sequence.

[0015] In some embodiments, the recombinant self-amplifying nucleic acid comprises a subgenomic promoter upstream of a sequence encoding a CFP. In some embodiments, the recombinant self-amplifying nucleic acid comprises a sequence encoding one or more replication genes from a virus. In some embodiments, the sequence encoding one or more replication genes from a virus is upstream of the sequence encoding a CFP. In some embodiments, the sequence encoding one or more replication genes from a virus is upstream of a subgenomic promoter. In some embodiments, the recombinant self-amplifying nucleic acid comprises a promoter upstream of a sequence encoding one or more replication genes from a virus. In some embodiments, the one or more replication genes comprise one or more nonstructural proteins (NSPs). In some embodiments, the one or more NSPs comprise one or more of NSP1, NSP2, NSP3, and / or NSP4. In some embodiments, the recombinant self-amplifying nucleic acid lacks one or more viral genes encoding viral structural proteins required for the production of infectious viral particles. In some embodiments, the virus is an RNA virus. In some embodiments, the virus is a single-stranded virus. In some embodiments, the virus is an alphavirus or flavivirus selected from Venezuelan equine encephalitis virus (VEE), Sindbis virus (SINV), vaccinia virus, and Semliki Forest virus (SFV). In some embodiments, the recombinant self-amplifying nucleic acid is encapsulated within a lipid nanoparticle (LNP).

[0016]

[0016] Provided herein is a pharmaceutical composition comprising: (A) a recombinant RNA comprising a sequence encoding a chimeric fusion protein (CFP) comprising a phagocytic or tethering receptor (PR) subunit, the chimeric fusion protein comprising (i) a transmembrane domain, and (ii) an intracellular domain comprising an intracellular signaling domain derived from a phagocytic or tethering receptor, wherein the transmembrane domain and the intracellular domain are operably linked; and (B) a pharmaceutically acceptable excipient, wherein the recombinant RNA is an in vitro glycosylated RNA comprising at least one residue comprising a glycan.

[0017] In some embodiments, the glycan is an N-linked glycan. In some embodiments, at least one residue comprising the glycan is guanosine (G). In some embodiments, the glycan is a sialylated glycan. In some embodiments, the glycan is fucosylated. In some embodiments, the recombinant RNA is a recombinant mRNA. In some embodiments, the recombinant RNA is encapsulated in a lipid nanoparticle (LNP). In some embodiments, the in vitro glycosylated RNA is glycosylated at specific residues. In some embodiments, the recombinant mRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycans. In some embodiments, the recombinant mRNA is a self-amplifying mRNA. Provided herein are pharmaceutical compositions comprising: (A) a recombinant nucleic acid comprising a sequence encoding a chimeric fusion protein (CFP) comprising a phagocytic or tethering receptor (PR) subunit, the chimeric fusion protein comprising (i) a transmembrane domain, and (ii) an intracellular domain comprising an intracellular signaling domain derived from a phagocytic or tethering receptor, wherein the transmembrane domain and the intracellular domain are operably linked; and (B) a pharmaceutically acceptable excipient, wherein the recombinant nucleic acid comprises a sequence complementary to an miRNA, the miRNA being either (i) expressed in myeloid cells, such that binding of the miRNA to the sequence complementary to the miRNA promotes expression of the CFP in the myeloid cells, or (ii) expressed in non-myeloid cells and not substantially expressed in myeloid cells, such that binding of the miRNA to the sequence complementary to the miRNA promotes degradation of the recombinant nucleic acid in the non-myeloid cells.

[0018]

[0018] In some embodiments, the non-myeloid cell is a neuronal cell. In some embodiments, the non-myeloid cell is an epithelial cell. In some embodiments, the non-myeloid cell is a hepatocyte. In some embodiments, the non-myeloid cell is a cardiomyocyte. In some embodiments, the non-myeloid cell is an embryonic stem cell. In some embodiments, the non-myeloid cell is an endothelial cell. In some embodiments, the miRNA is hsa-mir-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302d, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, miR-10a / b, miR-24 / 27, miR-125a, miR-126, or miR-221 / 222.

[0019] In some embodiments, the myeloid cells are CD14+ cells. In some embodiments, the CD14+ cells are CD16- or CD16low. In some embodiments, the composition further comprises one or more lipid and / or carbohydrate or one or more polymer components. In some embodiments, the recombinant mRNA is at least 5 kb in length. In some embodiments, the pharmaceutical composition is formulated for systemic delivery. In some embodiments, the recombinant mRNA is encapsulated in a lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle is about 100 nm in diameter. In some embodiments, the lipid nanoparticle comprises at least a cationic lipid and / or a non-cationic lipid. In some embodiments, the cationic lipid is selected from the group consisting of phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinic acid, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol). In some embodiments, the non-cationic lipid is a neutral lipid. In some embodiments, the non-cationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the molar ratio of mRNA to neutral lipid ranges from about 2:1 to about 8:1. In some embodiments, the lipid composition further comprises a PEGylated lipid. In some embodiments, the PEGylated lipid comprises a linear chain of 50 to 200 carbons.

[0020] In some embodiments, the PEGylated lipid is one or more PEGylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAGs), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(cw-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), or comprises PEGylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate, or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the molar ratio of mRNA to PEGylated lipid ranges from about 100:1 to about 25:1. In some embodiments, the lipid nanoparticle further comprises a steroid or steroid analog. In some embodiments, the steroid or steroid analog is cholesterol. In some embodiments, the molar ratio of mRNA to cholesterol ranges from about 2:1 to 1:1. In some embodiments, the pharmaceutical composition further comprises one or more phospholipids. In some embodiments, the one or more phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. In some embodiments, the CFP further comprises an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell. In some embodiments, the intracellular signaling domain comprises a phagocytic activation domain. In some embodiments, the intracellular signaling domain is derived from a receptor selected from the group consisting of the receptors listed in Table 2.In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain. In some embodiments, the antigen is an antigen or surface molecule expressed by a diseased cell or a pathogenic cell or pathogen. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain, an antibody domain, and the antibody domain is a functional antibody fragment, a single-chain variable fragment (scFv), a Fab, a single-domain antibody (sdAb), a nanobody, a V. H Domain, V L domain, VNAR domain, V HH domains, bispecific antibodies, diabodies, or functional fragments or combinations thereof.

[0021] In some embodiments, the antigen is selected from the group consisting of thymidine kinase (TK1), hypoxanthine-guanine phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-1, mucin-16 (MUC16), MUC1, epidermal growth factor receptor vIII (EGFRvIII), mesothelin, human epidermal growth factor receptor 2 (HER2), mesothelin, EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B-cell maturation antigen (BCMA), glypican 3 (GPC3), follicle-stimulating hormone receptor, fibroblast-activating protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer group 2D (NKG2D) ligand, disialoganglioside 2 (GD2), CD2, CD3, C D4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptors, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, IGLL1, and combinations thereof. In some embodiments, the antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, and CD56.

[0022] In some embodiments, the antigen is human CD5. In some embodiments, the antigen is human HER2.

[0023] In some embodiments, the antigen is human CD19. In some embodiments, the antigen is a viral antigen expressed in mammalian cells. In some embodiments, the antigen is a bacterial antigen expressed in mammalian cells. In some embodiments, binding of CFP to the antigen on a target cell increases the killing activity of cells expressing CFP by at least 20% compared to cells that do not express CFP, and increases the killing or phagocytic activity of cells expressing CFP by at least 20% compared to cells that do not express CFP, wherein the killing or phagocytic activity is measured by flow cytometry. In some embodiments, cells expressing CFP exhibit at least a 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold increase in phagocytosis of target cells expressing an antigen compared to cells not expressing CFP, and phagocytosis is measured by flow cytometry. In some embodiments, the target cells expressing an antigen are diseased cells.

[0024] In some embodiments, cells expressing CFP exhibit increased cytokine production compared to cells that do not express CFP. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferon, and combinations thereof.

[0025] In some embodiments, cells expressing CFP exhibit increased effector activity compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased cross-presentation compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class II proteins compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD80 compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD86 compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class I proteins compared to cells that do not express CFP. In some embodiments, the pharmaceutical composition is aqueous.

[0026]

[0026] In some embodiments, the pharmaceutical composition comprises one or more polynucleic acids, wherein the one or more polynucleic acids comprise: a first sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises a CFP extracellular domain comprising a first antigen-binding domain, and a CFP transmembrane domain operably linked to the CFP extracellular domain, wherein the CFP transmembrane domain is derived from a protein that dimerizes with endogenous FcR-gamma receptors in myeloid cells; and a second sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a CAR extracellular domain comprising a second antigen-binding domain, a CAR transmembrane domain operably linked to the CAR extracellular domain, wherein the CAR transmembrane domain is derived from a protein that functionally interacts with an endogenous T cell receptor (TCR) complex and / or at least one endogenous TCR polypeptide, and a CAR intracellular domain operably linked to the transmembrane domain.

[0027]

[0027] Also provided herein is a pharmaceutical composition comprising one or more polynucleic acids, wherein the one or more polynucleic acids comprise: a first sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises a CFP extracellular domain comprising a first antigen-binding domain, and a CFP transmembrane domain operably linked to the CFP extracellular domain, wherein the CFP transmembrane domain is derived from a protein that dimerizes with endogenous FcR-gamma receptors in myeloid cells; and a second sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a CAR extracellular domain comprising a second antigen-binding domain, a CAR transmembrane domain operably linked to the CAR extracellular domain, wherein the CAR transmembrane domain is derived from a protein that functionally interacts with an endogenous T cell receptor (TCR) complex and / or at least one endogenous TCR polypeptide, and a CAR intracellular domain operably linked to the transmembrane domain.

[0028] In some embodiments, the one or more polynucleic acids comprise a polynucleic acid molecule comprising both a first sequence and a second sequence. In some embodiments, the first sequence and the second sequence are operably linked by a linker sequence. In some embodiments, the linker sequence comprises a protease cleavage site. In some embodiments, the protease cleavage site is a T2A cleavage site or a P2A cleavage site.

[0029] In some embodiments, the one or more polynucleic acids are encapsulated within a nanoparticle delivery vehicle. In some embodiments, the one or more polynucleic acids are encapsulated within the same nanoparticle delivery vehicle.

[0030] In some embodiments, the one or more polynucleic acids comprise a first polynucleic acid molecule comprising a first sequence and a second polynucleic acid molecule comprising a second sequence.

[0031] In some embodiments, the first polynucleic acid molecule is encapsulated in a first nanoparticle delivery vehicle and the second polynucleic acid molecule is encapsulated in a second nanoparticle delivery vehicle, hi some embodiments, the first polynucleic acid molecule and the second polynucleic acid molecule are encapsulated in the same nanoparticle delivery vehicle.

[0032] In some embodiments, after administration of the pharmaceutical composition to the human subject, the CFP is expressed on the surface of myeloid cells in the human subject. In some embodiments, after administration of the pharmaceutical composition to the human subject, the CAR is expressed on the surface of T cells in the human subject.

[0033]

[0033] In some embodiments, the CAR is functionally integrated into the endogenous TCR complex of a T cell of a human subject. In some embodiments, the CAR extracellular domain is a TCR extracellular domain derived from TCR-alpha, TCR-beta, TCR-delta, TCR-gamma, CD3-gamma, CD3-delta, or CD3-epsilon. In some embodiments, the CAR transmembrane domain is a TCR transmembrane domain derived from TCR-alpha, TCR-beta, TCR-delta, TCR-gamma, CD3-zeta, CD3-gamma, CD3-delta, or CD3-epsilon. In some embodiments, the CAR intracellular domain is a TCR intracellular domain derived from CD3-zeta, CD3-gamma, CD3-delta, or CD3-epsilon. In some embodiments, at least two of the TCR extracellular domain, TCR transmembrane domain, and TCR intracellular domain are derived from the same TCR subunit. In some embodiments, each of the TCR extracellular domain, the TCR transmembrane domain and the TCR intracellular domain are from the same TCR subunit.

[0034] In some embodiments, the CAR intracellular domain further comprises a costimulatory domain, which is a functional signaling domain derived from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0035] In some embodiments, the first and / or second antigen-binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain.

[0036] In some embodiments, the CFP extracellular domain is an extracellular domain derived from CD8, CD16a, CD64, CD68, or CD89. In some embodiments, the CFP extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to the CFP transmembrane domain and the first antigen-binding domain.

[0037] In some embodiments, after administration of the pharmaceutical composition to a human subject, the CFP is specifically expressed in myeloid cells, monocytes, or macrophages of the human subject. In some embodiments, after administration of the pharmaceutical composition to a human subject, the CAR is specifically expressed in T cells of the human subject.

[0038]

[0038] In some embodiments, the CFP transmembrane domain is a transmembrane domain derived from CD16a, CD64, CD68, or CD89. In some embodiments, the CFP further comprises a CFP intracellular domain operably linked to the CFP transmembrane domain. In some embodiments, the CFP intracellular domain comprises one or more intracellular signaling domains, wherein the one or more intracellular signaling domains comprise an intracellular signaling domain derived from FcγR, FcαR, FcεR, CD40, or CD3 zeta. In some embodiments, the one or more intracellular signaling domains further comprise a phosphoinositide 3-kinase (PI3K) recruitment domain. In some embodiments, the PI3K recruitment domain comprises a sequence having at least 90% sequence identity to SEQ ID NO:4. In some embodiments, the CFP intracellular domain comprises an intracellular domain derived from CD16a, CD64, CD68, or CD89.

[0039]

[0039] In some embodiments, the one or more polynucleic acids is mRNA.

[0040] In some embodiments, the nanoparticle delivery vehicle comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a polar lipid. In some embodiments, the lipid nanoparticle comprises a non-polar lipid. In some embodiments, the lipid nanoparticle is 100-300 nm in diameter.

[0041] In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to the same antigen. In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to different antigens. In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain bind to an antigen selected from the group consisting of TROP2, GPC3, CD5, HER2, CD137, CD70, Claudin3, Claudin18.2, TMPRSS, CD19, CD22, CD7, PSMA, MSLN, and GP75.

[0042]

[0042] In some embodiments, the pharmaceutical composition inhibits the growth of the cancer when administered to a human subject with cancer.

[0043] Also provided herein are methods of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease. In some embodiments, the subject is a human.

[0044] Incorporation by Reference

[0044] All publications, patents, and patent applications mentioned in this specification are 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.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS 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. [Brief explanation of the drawings]

[0046] [Figure 1A]

[0046] Figure 1 illustrates a diagram showing some of the potentially manipulable functions of myeloid cells. [Figure 1B]

[0047] FIG. 1 illustrates a diagram indicating the presence of various cell types in various types of cancer. Macrophages are the most abundant cells in the cancer types schematized. [Figure 2A]

[0048]

[0014] Figure 1 illustrates a schematic showing an exemplary chimeric receptor fusion protein (CFP) containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain, and a second intracellular signaling domain. The signaling domains can be derived from other receptors and designed to elicit any number of cellular functions. [Figure 2B]

[0049]

[0023] Figure 1 illustrates an exemplary CFP (left) containing an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, as well as a schematic diagram showing a CFP containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain, a second intracellular signaling domain, a third intracellular signaling domain, and one or more additional intracellular signaling domains. The signaling domains can be derived from other receptors and designed to induce any number of cellular functions. [Figure 2C]

[0050] FIG. 1 illustrates a schematic showing an exemplary CFP dimer containing an anti-CD5 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain containing an intracellular domain derived from FcRγ fused to a PI3K recruitment domain. [Figure 2D]

[0051] FIG. 1 illustrates a schematic showing an exemplary CFP dimer containing an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing a phagocytic domain, a PI3K recruitment domain, and a pro-inflammatory domain. [Figure 3]

[0052] Schematic diagram illustrating an exemplary CFP homodimer (left) in which each subunit contains an extracellular domain fused to an scFv that binds a single target, and an exemplary CFP heterodimer (right) in which a first subunit of the heterodimer contains an extracellular domain fused to an scFv that binds a first target and a second subunit of the heterodimer subunit contains an extracellular domain fused to an scFv that binds a second target. [Figure 4A]

[0053] FIG. 1 is a schematic diagram illustrating an exemplary recombinant nucleic acid encoding a CFP containing a signal peptide fused to an antigen-specific scFv fused to the extracellular domain (ECD), transmembrane domain (TM), and intracellular domain of a scavenger receptor. [Figure 4B]

[0054] 4B is a schematic diagram illustrating the CFP of FIG. 4A incorporated into the cell membrane of a myeloid cell. The diagrammed CFP contains an scFv bound to a cancer antigen of a cancer cell. The extracellular, transmembrane, and intracellular domains can be derived from one or more scavenger receptors. [Figure 4C]

[0055] 1 is an exemplary graph illustrating expected results regarding relative phagocytosis in human primary myeloid cells transduced with a vector encoding CFP co-cultured with an empty vector (control) or dye-loaded tumor cells. Phagocytosis is quantified using flow cytometry. [Figure 4D]

[0056] FIG. 1 is an exemplary graph illustrating the expected results regarding the percent specific lysis of tumor cells when incubated in the presence of an empty vector (control) or human primary myeloid cells (effector cells) transduced with a vector encoding CFP co-cultured with luciferase-expressing tumor cells (target cells) at the indicated effector cell:target cell ratio (E:T ratio). [Figure 4E]

[0057] FIG. 1 is an exemplary graph illustrating expected results in terms of percent survival in a mouse xenograft tumor model following treatment with cells transduced with an empty vector (control) or a vector encoding CFP. [Figure 5A]

[0058] FIG. 1 is a schematic diagram illustrating an exemplary recombinant nucleic acid encoding a CFP (M1-CAR) containing a signal peptide fused to an antigen-specific scFv fused to a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular domain containing a phagocytic activation domain and a pro-inflammatory domain. [Figure 5B]

[0059] FIG. 5B is a schematic diagram illustrating the CFP (M1-CAR) of FIG. 5A incorporated into the cell membrane of a myeloid cell. The schematized CFP contains an scFv bound to a cancer antigen on a cancer cell. [Figure 5C]

[0060]

[0023] Figure 1 is an exemplary graph illustrating expected results regarding relative phagocytosis in human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (M1-CAR) co-cultured with dye-loaded tumor cells. Phagocytosis is quantified using flow cytometry. [Figure 5D]

[0061] FIG. 10 is an exemplary graph illustrating expected results in terms of fold increase in production of schematized cytokines in myeloid cells transduced with vector control or vector encoding CFP(M1-CAR). [Figure 5E]

[0062] FIG. 1 is an exemplary graph illustrating expected results in terms of fold increase in production of schematized M1 markers in human primary myeloid cells transduced with vector control or vector encoding CFP(M1-CAR). [Figure 5F]

[0063]

[0023] Figure 1 is an exemplary graph illustrating expected results regarding percent specific lysis of tumor cells when incubated in the presence of an empty vector (control) or human primary myeloid cells (effector cells) transduced with a vector encoding CFP (M1-CAR) co-cultured with luciferase-expressing tumor cells (target cells) at the indicated effector cell:target cell ratio (E:T ratio). Specific lysis is quantified using a luciferase assay. [Figure 5G]

[0064] FIG. 1 is an exemplary graph illustrating expected results in terms of percent survival in a mouse xenograft tumor model following treatment with human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (M1-CAR). [Figure 6A]

[0065] FIG. 1 is a schematic diagram illustrating an exemplary recombinant nucleic acid encoding a CFP (integrin-CAR) containing a signal peptide fused to an antigen-specific scFv fused to a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular phagocytosis and integration activation domain. [Figure 6B]

[0066] 6B is a schematic diagram illustrating the CFP (integrin-CAR) of FIG. 6A integrated into the cell membrane of a myeloid cell. The schematized CFP contains an scFv bound to a cancer antigen on a cancer cell. [Figure 6C]

[0067]

[0023] Figure 1 is an exemplary graph illustrating expected results regarding relative phagocytosis in human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (integrin-CAR) co-cultured with dye-loaded tumor cells. Phagocytosis is quantified using flow cytometry. [Figure 6D]

[0068] FIG. 1 is an exemplary graph illustrating expected results in terms of percent specific lysis of tumor cells when incubated in the presence of an empty vector (control) or human primary myeloid cells (effector cells) transduced with a vector encoding CFP (integrin-CAR) co-cultured with luciferase-expressing tumor cells (target cells) at the indicated effector cell:target cell ratio (E:T ratio). [Figure 6E]

[0069] FIG. 1 is an exemplary graph illustrating expected results regarding the relative infiltration of human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (integrin-CAR). [Figure 6F]

[0070] FIG. 1 is an exemplary graph illustrating expected results in terms of percent survival in a mouse xenograft tumor model following treatment with human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (integrin-CAR). [Figure 7]

[0071] 1 is a schematic diagram illustrating CFP (cross-presentation-CAR) integrated into the cell membrane of myeloid cells. The illustrated cross-presentation-CAR contains an scFv that binds to a cancer antigen of a cancer cell, fused to a CD8 hinge domain, a CD8 transmembrane domain, an intracellular phagocytosis activation domain, and an intracellular cross-presentation activation domain. The cross-presentation-CAR can direct antigens into the cross-presentation pathway. [Figure 8-1]

[0072] FIG. 1 illustrates exemplary flow cytometry data (side scatter (SSC) vs. CD5+) following mock expression or expression of various constructs with an extracellular domain (ECD) comprising an anti-CD5 scFv in myeloid cells. Schematic constructs include: an ECD containing an anti-CD5 scFv fused to an FcRγ intracellular domain, fused to a CD40 intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-CD40-FcR); an ECD containing an anti-CD5 scFv fused to a CD40 intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR-CD40); an ECD containing an anti-CD5 scFv fused to a PI3K recruitment domain, fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR-PI3K); an anti-CD5 fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain ECD containing an scFv (CD5-CD8h-CD8tm-FcR); an ECD containing an anti-CD5 scFv fused to a CD8 transmembrane domain and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-no ICD); an ECD containing an anti-CD5 scFv fused to a PI3K recruitment domain, fused to an FcRγ intracellular domain, fused to a CD28 transmembrane domain, and fused to a CD28 hinge domain (CD5-CD28h-CD28tm-FcR-PI3K); an ECD containing an anti-CD5 scFv fused to a CD8 hinge domain, fused to a CD68 transmembrane domain, fused to an FcRγ intracellular domain, and fused to a PI3K recruitment domain (CD5-CD8h-CD68tm-FcR-PI3K); an anti-CD5 fused to a CD8 transmembrane domain, fused to an FcRγ intracellular domain, fused to a PI3K recruitment domain ECD containing scFv (CD5-CD8tm-FcR-PI3K);An ECD containing an anti-CD5 scFv fused to a CD28 transmembrane domain, fused to an FcRγ intracellular domain, fused to a PI3K recruitment domain (CD5-CD28tm-FcR-PI3K); and an ECD containing an anti-CD5 scFv fused to a CD68 transmembrane domain, fused to an FcRγ intracellular domain, fused to a PI3K recruitment domain (CD5-CD68tm-FcR-PI3K). [Figure 8-2] Continued from Figure 8-1. [Figure 9]

[0073] FIG. 1 illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) following mock expression or expression of various constructs with an extracellular domain (ECD) comprising an anti-CD5 scFv in myeloid cells. Schematic constructs include: an ECD containing an anti-CD5 scFv fused to a PI3K recruitment domain, fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR-PI3K); an ECD containing an anti-CD5 scFv fused to a CD8 transmembrane domain and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR); an ECD containing an anti-CD5 scFv fused to a CD8 transmembrane domain and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-no ICD); an anti-CD5 fused to a CD40 intracellular domain, fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain ECD containing an scFv (CD5-CD8h-CD8tm-FcR-CD40); and ECD containing an anti-CD5 scFv fused to the TNFR2 intracellular domain, fused to the FcRγ intracellular domain, fused to the CD8 transmembrane domain, or fused to the CD8 hinge domain (CD5-CD8h-CD8tm-FcR-TNFR2). [Figure 10A]

[0074] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using coated FITC-labeled beads for antigen targeting FarRed fluorophore-labeled CFP expressed in THP-1 cells. [Figure 10B]

[0075] FIG. 10B illustrates exemplary flow cytometry data (side scatter (SSC) vs. CSFE-FarRed) following mock expression or expression of anti-CD5 CFP using the experimental design of FIG. 10A. [Figure 10C]

[0076] FIG. 10B illustrates an exemplary graph showing relative phagocytosis in human primary myeloid cells transduced with an empty vector (mock) or a vector encoding the schematized CFP co-cultured with FITC-labeled beads coated with BSA or CD5, using the experimental design of FIG. 10A. [Figure 10D-1]

[0077] FIG. 10B depicts an exemplary bar graph of the concentration (pg / mL) of the indicated proteins after mock expression or expression of the indicated anti-CD5 CFPs using the experimental design of FIG. 10A. Each CFP contained an ECD containing an anti-CD5 scFv fused to a CD8 hinge domain fused to a CD8 transmembrane domain fused to the indicated intracellular domain. [Figure 10D-2] Continued from Figure 10D-1. [Figure 10E]

[0078]

[0023] Figure 1 illustrates an exemplary graph measuring the expression of M1-associated markers (CD16 and MHC class I) in primary human monocytic cells expressing anti-CD5 CFP that were incubated for 24 hours in the presence of IL-10, IL-4, and TGFβ, and then incubated with H9 T-cell lymphoma cells. Primary human monocytic cells expressing anti-CD5 CFP demonstrated potent activity in an M2 environment. [Figure 10F]

[0079]

[0023] Figure 1 illustrates an exemplary bar graph of the concentration of TNF-α (pg / mL) after incubating primary human monocytic cells expressing anti-CD5 chimeric antigen receptor (CFP) in the presence of IL-10, IL-4, and TGFβ for 24 hours, followed by overnight incubation in the presence of H9 T-cell lymphoma cells. Primary human monocytic cells expressing anti-CD5 CFP were able to function under tumor microenvironment (TME)-like conditions and produce inflammatory mediators. [Figure 10G]

[0080]

[0023] Figure 1 illustrates an exemplary bar graph of the concentrations (pg / mL) of the indicated chemoattractants (CCL3, CCL4, CXCL10, and CXCL12) after incubating primary human monocytic cells expressing anti-CD5 CFP in the presence of IL-10, IL-4, and TGFβ for 24 hours, followed by overnight incubation in the presence of H9 T cell lymphoma cells. Primary human monocytic cells expressing anti-CD5 CFP were able to function to secrete a wide range of chemokines, including T cell and NK cell chemoattractants, under tumor microenvironment (TME)-like conditions. [Figure 10H]

[0081]

[0023] Figure 1 illustrates an exemplary bar graph of the concentrations (pg / mL) of the indicated chemoattractants (CCL8, CXCL1, eotaxin, and CCL5) after incubating primary human monocytic cells expressing anti-CD5 CFP in the presence of IL-10, IL-4, and TGFβ for 24 hours, followed by overnight incubation in the presence of H9 T-cell lymphoma cells. Primary human monocytic cells expressing anti-CD5 CFP were functional and capable of secreting a wide range of chemokines, including chemokines that activate polymorphonuclear granulocytes (PMNs), eosinophils, and leukocyte chemoattractants. [Figure 11A]

[0082] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using CFSE-labeled target cells targeting FarRed fluorophore-labeled CFP expressed in THP-1 cells. [Figure 11B]

[0083] FIG. 11B illustrates exemplary flow cytometry data (side scatter (SSC) vs. forward scatter (FSC); CSFE vs. FarRed; and cell count CSFE) following mock expression or expression of anti-CD5 CFP in THP-1 cells using the experimental design of FIG. 11A. Myeloid cell lines were electroporated with anti-CD5 CFP and labeled with intracellular FarRed dye. These cells were incubated with CFSE-prelabeled H9 T-cell cancer cells at a 1:3 myeloid cell:tumor cell ratio. After 4 hours, phagocytosis was measured by flow cytometry. [Figure 11C]

[0084] Figure 11B illustrates an exemplary graph showing relative phagocytosis in myeloid cell lines electroporated with an empty vector (mock) or a vector encoding the schematized CFP and labeled with intracellular FarRed dye, using the experimental design of Figure 11A. These cells were incubated with H9 T cell cancer cells prelabeled with CFSE at a myeloid cell:tumor cell ratio of 1:3. After 4 hours, phagocytosis was measured by flow cytometry. [Figure 12A]

[0085] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using pHRod-labeled target cells targeting FarRed fluorophore-labeled CFP expressed in primary human monocytic cells. [Figure 12B]

[0086] Figure 12B illustrates exemplary flow cytometry data (pHRodo vs. FarRed) following mock expression or expression of anti-CD5 CFP in primary human monocytic cells using the experimental design of Figure 12A. Primary human monocytic cells were electroporated with anti-CD5 CFP and labeled with intracellular FarRed dye. These cells were incubated with H9 T cell cancer cells prelabeled with pHRodo. After incubation, phagocytosis was measured by flow cytometry. [Figure 12C]

[0087] FIG. 12B illustrates an exemplary graph quantifying the results of FIG. 12B showing relative phagocytosis after mock expression or expression of a diagrammed anti-CD5 CFP in primary human monocytic cells using the experimental design of FIG. 12A. [Figure 12D-1]

[0088] FIG. 1 illustrates an exemplary bar graph of the concentration (pg / mL) of the indicated proteins after mock expression or expression of the indicated anti-CD5 CFP in monocytic cells after performing a bead-based phagocytosis assay. [Figure 12D-2] Continued from Figure 12D-1. [Figure 13]

[0089]

[0023] Figure 1 illustrates an exemplary graph of relative fluorescence units (RFU) over time following incubation of THP-1 cells expressing anti-CD5 CFP with no cells or CCL2 at the indicated concentrations. The fold increase over control illustrates the ratio of CCL2-induced chemotaxis compared to cells treated with assay buffer alone. Each bar on the graph represents the mean ± SD of duplicate wells. [Figure 14]

[0090]

[0023] Figure 1 illustrates an exemplary graph of relative fluorescence units (RFU) over time following incubation of primary human monocytic cells expressing anti-CD5 CFP with no cells or CCL2 at the indicated concentrations. The fold increase over control illustrates the ratio of CCL2-induced chemotaxis compared to cells treated with assay buffer alone. Each bar on the graph represents the mean ± SD of duplicate wells. [Figure 15A]

[0091]

[0023] Figure 1 illustrates a schematic showing an exemplary experimental flow diagram of a peripheral T-cell lymphoma animal model experiment. Treatment with the indicated amounts of human primary monocytes expressing anti-CD5 CFP began 11 days after tumor inoculation. Tumor burden was measured using IVIS imaging. [Figure 15B]

[0092] FIG. 15B illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) following expression of anti-CD5 CFP in human primary monocytic cells from the experiment shown in FIG. 15A. [Figure 15C]

[0093]

[0033] Figure 15A illustrates exemplary results of a mouse xenograft model treated with vehicle or human primary monocytes expressing anti-CD5 CFP from the experiment shown in Figure 15A. On day 0, NSG mice were injected with luciferase-expressing CD5+ tumor cells. Mice were then either untreated or injected with the indicated regimen of human primary monocytes electroporated with anti-CD5 CFP. [Figure 15D]

[0094] Figure 15B depicts a graph of relative tumor size over time from the results of Figure 15C. Tumor burden was measured using IVIS imaging of luciferase fluorescence. [Figure 16A]

[0095]

[0023] Figure 1 illustrates a schematic showing an exemplary experimental flow diagram of a peripheral T-cell lymphoma animal model experiment. Treatment with the indicated amounts of human primary monocytes expressing anti-CD5 CFP began 11 days after tumor inoculation. [Figure 16B]

[0096] FIG. 16B illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) following expression of anti-CD5 CFP in human primary monocytic cells from the experiment shown in FIG. 16A. The data demonstrate the achievement of 95% transfection efficiency. [Figure 16C]

[0097] Figure 16B depicts a graph of relative tumor size over time from the experiment shown in Figure 16A. Tumor burden was measured using IVIS imaging of luciferase fluorescence. [Figure 16D]

[0098] FIG. 16B illustrates a graph of relative tumor size over time from the experiment shown in FIG. 16A. Tumor burden was measured using caliper measurements. The data demonstrate that treatment was associated with a delay in tumor progression and a statistically significant reduction in tumor burden in a compromised mouse model. Statistical significance was determined using the Bonferroni-Dunn method at alpha=0.5. Each column was analyzed individually, without assuming a constant SD. Number of t-tests: 8 or 4. [Figure 17A]

[0099] FIG. 1 illustrates a schematic showing an exemplary CFP containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from MDA5. [Figure 17B]

[0100] FIG. 1 depicts exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) showing expression in untransfected primary monocytes (top) and primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing an extracellular CD5-binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from MDA5. [Figure 17C]

[0101] FIG. 1 depicts an exemplary bar graph of the concentrations (pg / mL) of the indicated cytokines secreted in untransfected primary monocytes and primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing a CD5 domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from MDA5. [Figure 18A]

[0102] FIG. 1 illustrates a schematic showing an exemplary chimeric receptor fusion protein (CFP) containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR1 or TNFR2. [Figure 18B]

[0103] FIG. 1 illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) showing expression in untransfected primary monocytes (left); primary monocytes transfected with in vitro-transcribed mRNA encoding a CFP containing an extracellular CD5-binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR1 (center); and primary monocytes transfected with in vitro-transcribed mRNA encoding a CFP containing an extracellular CD5-binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR2 (right). [Figure 18C-1]

[0104] FIG. 1 depicts an exemplary bar graph of the concentrations (pg / mL) of the indicated cytokines / chemokines secreted in untransfected primary monocytes; primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing an extracellular CD5 binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR1; and primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing an extracellular CD5 binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR2. [Figure 18C-2] Continued from Figure 18C-1. [Figure 19A]

[0105] FIG. 1 illustrates a schematic showing a CFP containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from CD40, a PI3K recruitment domain, or TNFR2. [Figure 19B]

[0106]

[0023] Figure 1 illustrates a schematic diagram showing an exemplary experimental flow diagram for an M2 stimulation assay. Primary monocytes expressing various CFP constructs were cultured under M2 conditions (IL4, IL10, TGFβ) for 24 hours and then added to culture plates uncoated or coated with recombinant CD5 antigen. Cells were incubated on the plates for 24 hours, and the amounts of various cytokines secreted into the medium were measured. [Figure 19C-1]

[0107]

[0023] Figure 1 depicts an exemplary bar graph of the concentrations (pg / mL) of the indicated cytokines / chemokines (TNFα, IL8, IL1β, IP-10, Gro-alpha / KC, CCL3, CCL4, CCL5, and CXCL12) secreted in non-transfected primary monocytes; primary monocytes transfected with in vitro-transcribed mRNA encoding a CFP containing an extracellular CD5-binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR1; and primary monocytes transfected with in vitro-transcribed mRNA encoding a CFP containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR2. CD5 ligation induced upregulation of several pro-inflammatory cytokines and chemokines, including: [Figure 19C-2] Continuation of Figure 19C-1. [Figure 19C-3] Continued from Figure 19C-2. [Figure 20A]

[0108]

[0023] Figure 1 shows a schematic diagram of an exemplary lentiviral construct encoding a CFP containing an extracellular HER2-binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and either (a) a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain, (b) a first intracellular signaling domain derived from MEGF10 and a second intracellular signaling domain containing a PI3K recruitment domain, or (c) an intracellular signaling domain derived from CD3ζ in THP-1 cells. Also shown is exemplary flow cytometry data (side scatter (SSC) vs. Flag-PE) showing expression in untransduced primary monocytes or primary monocytes transduced with the diagrammed CFP constructs. [Figure 20B]

[0109] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay. [Figure 20C]

[0110]

[0033] Figure 20B illustrates an exemplary bar graph of the percentage of phagocytosis of THP-1 cells transduced with the lentiviral constructs shown in Figure 20A using the phagocytosis assay shown in Figure 20B. Transduced THP-1 cells activated with or without phorbol-12-myristate-13-acetate (PMA) were incubated overnight with FarRed-labeled SKOV3 tumor cells. Exemplary fluorescence microscopy images of cells exhibiting phagocytosis are also shown. [Figure 20D]

[0111] 20B depicts exemplary flow cytometry data (FarRed vs. PE) showing phagocytosis after performing the phagocytosis assay depicted in FIG. 20B. Transduced THP-1 cells, activated with or without PMA, were incubated overnight with FarRed-labeled SKOV3 tumor cells. [Figure 20E]

[0112] FIG. 20B illustrates exemplary flow cytometry data (SSC vs. FSC and FarRed vs. PE) after performing the phagocytosis assay depicted in FIG. 20B. Transduced THP-1 cells, activated with or without PMA, were incubated overnight with FarRed-labeled SKOV3 tumor cells.

[0047]

number

[0048] Also shown is an exemplary bar graph showing percent cell death of target cells calculated by: [Figure 21A]

[0113] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using CD14+ cells isolated from a healthy donor Leukopak and transduced with a lentiviral vector encoding a CFP containing an extracellular HER2-binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain. [Figure 21B]

[0114] 21A illustrates an exemplary bar graph of the percentage of phagocytosis of CD14+ cells transduced with a lentiviral vector encoding a CFP isolated from a healthy donor Leukopak, which contains an extracellular HER2 binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain, using the phagocytosis assay illustrated in FIG. 21A. The transduced cells were incubated overnight with target cells (Jurkat (HER2-) or SKOV3 (HER2+)). Also illustrated is an exemplary fluorescent microscopy image of cells showing the phagocytosis of SKOV3 cells but not Jurkat cells. [Figure 21C-1]

[0115]

[0033] Figure 21B illustrates exemplary flow cytometry data (CSFE vs. PE) showing phagocytosis after performing the phagocytosis assay shown in Figure 21A. Transduced CD14+ cells isolated from a healthy donor, Leukopak, were incubated overnight with CFSE-labeled HER2+ SKOV3 ovarian tumor cells and CFSE-labeled HER2- Jurkat cells. Also shown is an exemplary bar graph showing percent cell death of target cells in the experiment shown in Figure 21A. [Figure 21C-2] Continued from Figure 21C-1. [Figure 22A]

[0116] FIG. 1 illustrates a schematic diagram showing an exemplary experimental flow diagram of an MSTO mesothelioma animal model experiment to investigate the ability of CFP-expressing cells to penetrate tumor sites and to evaluate the activation of CFP-expressing cells after penetration. [Figure 22B]

[0117]

[0023] Figure 1 illustrates fluorescence microscopy images showing bioimaging of tumor samples removed 24 hours after administration of CFSE-labeled CD14+ cells isolated from a healthy donor Leukopak and transduced with a lentiviral vector encoding a CFP containing an extracellular HER2-binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain into MSTO tumor-bearing NSG mice. The transduced cells were observed to migrate into the tumor and accumulate around the tumor cells. [Figure 22C]

[0118] Figure 1 shows fluorescent microscopy images of spleen samples removed 24 hours after administration of CFSE-labeled CD14+ cells isolated from a healthy donor Leukopak and transduced with a lentiviral vector encoding a CFP containing an extracellular HER2-binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain into MSTO tumor-bearing NSG mice. The transduced cells were observed to migrate to the spleen. CFSE-labeled cells isolated from the spleen 24 hours after cell injection were also examined by flow cytometry. The CFSE-labeled cells in the spleen maintained expression of HLA, CD14, and CD303. Interestingly, CCR2 expression was observed to decrease with a concomitant increase in CD370 (CLEC9A), potentially suggesting that the cells migrate to the spleen and develop into professional APCs capable of priming T cell responses. Interestingly, CD206 (mannose) expression was observed to decrease as well as CD45. The decrease in mannose receptor expression can be associated with differentiation towards an M1 phenotype. [Figure 23]

[0119]

[0013] Figure 1 illustrates a schematic showing an exemplary experimental flow diagram of the MSTO mesothelioma animal model experiment. Treatment with the indicated amounts of human primary monocytes expressing anti-HER2 CFP was initiated 21 days after tumor inoculation. Tumor burden was measured using IVIS imaging. [Figure 24]

[0120] Figure 24 illustrates a graph of tumor size over time from the experiment shown in Figure 23. Infusion of human primary monocytes expressing anti-HER2CFP was associated with delayed tumor progression compared to control-treated animals. [Figure 25]

[0121] FIG. 1 illustrates a diagram showing the inhibition of phagocytic receptors by target cell CD47 receptor SIRP-alpha (SIRPα)-mediated signaling. [Figure 26A]

[0122]

[0023] Figure 1 illustrates a diagrammatic representation of the design of a recombinant dominant-negative CFP construct (top panel) and a diagrammatic representation showing the inhibition of endogenous SIRPα by recombinant CFP protein expressed in macrophages. CFP has an extracellular SIRPα domain capable of binding CD47 in target cells, a SIRPα TM domain, but lacks an intracellular signaling domain. [Figure 26B]

[0123] FIG. 1 shows exemplary expected results for relative phagocytosis by control and dominant-negative CFP-transduced cells. [Figure 26C]

[0124] FIG. 1 shows exemplary expected results for target cell lysis (E:T, effector:target) by control and dominant-negative CFP-transduced cells. [Figure 26D]

[0125] FIG. 1 shows an example of expected outcomes of mouse survival in a tumor model after treatment with dominant-negative CFP-transduced macrophages. [Figure 27A]

[0126]

[0023] Figure 1 illustrates a diagrammatic representation of the design of a recombinant CFP, SIRPα-PI3K, (upper panel), which contains a SIRPα extracellular domain capable of binding CD47 in target cells, a SIRPα TM domain, but lacks an intracellular SIRPα signaling domain. At its intracellular end, the CFP is fused to an intracellular signaling domain with a PI3 kinase (PI3K) binding domain. BD: binding domain. The lower panel shows a diagrammatic representation demonstrating the inhibition of endogenous SIRPα by recombinant CFP protein expressed in macrophages. [Figure 27B]

[0127] FIG. 1 shows examples of expected results regarding relative phagocytosis by control and SIRPα-PI3K CFP-transduced cells. [Figure 27C]

[0128] FIG. 1 shows examples of expected results for relative Akt phosphorylation by control and SIRPα-PI3K CFP transduced cells. [Figure 27D]

[0129] FIG. 1 shows the expected results of increased tumor cell lysis by cells expressing CFP (integrin-CAR) compared to control (empty vector-transduced) macrophages. [Figure 27E]

[0130] FIG. 1 shows predicted survival curves in a mouse xenograft model of tumor after treatment with SIRPα-PI3K CFP-transduced macrophages or untreated controls. [Figure 28A]

[0131] The top panel illustrates a diagrammatic representation of the design of a recombinant CFP, (SIRPα-M1) (top panel), that contains a SIRPα extracellular domain capable of binding CD47, a SIRPα TM domain, but lacks an intracellular SIRPα signaling domain. CFP contains an intracellular signaling domain with a pro-inflammatory domain. The bottom panel illustrates a diagrammatic representation showing the inhibition of endogenous SIRPα by recombinant CFP protein when expressed in myeloid cells (e.g., macrophages). The pro-inflammatory domain can induce M1 polarization. [Figure 28B]

[0132] FIG. 1 shows examples of expected results for flow cytometry assays showing increased M1 state marker expression when myeloid cells (e.g., macrophages) are transduced with SIRPα-M1 compared to vector controls. [Figure 28C]

[0133] FIG. 1 shows examples of expected results for flow cytometry assays showing an increase in pro-inflammatory markers when myeloid cells (e.g., macrophages) are transduced with SIRPα-M1 compared to vector controls. [Figure 28D]

[0134] FIG. 1 shows the expected results of increased tumor cell lysis by cells expressing SIRPα-M1 compared to control (empty vector-transduced) myeloid cells (eg, macrophages). [Figure 28E]

[0135] FIG. 1 shows predicted survival curves in a mouse xenograft model of tumor following treatment with SIRPα-M1 transduced myeloid cells (eg, macrophages) or untreated controls. [Figure 29A]

[0136] The top panel illustrates an exemplary schematic diagram of a receptor-based CFP, SIRPαβ, containing an extracellular scFv specific for a cancer antigen fused to the SIRPαβ chain. The extracellular portion of the CD47 receptor SIRPα is fused to the cancer antigen-specific scFv. The ECD of SIRPα is fused to the transmembrane domain of SIRPβ. The intracellular domain of the CFP contains the intracellular domain derived from SIRPβ. Activation of the CFP by binding of the scFv to a target ligand activates the SIRPβ intracellular domain, inducing phagocytosis of the target cell through activation of DAP12. The bottom panel illustrates a diagrammatic representation of recombinant SIRPαβ protein expressed in myeloid cells (e.g., macrophages). [Figure 29B]

[0137] FIG. 1 shows a graphical representation of the phagocytic receptor fusion protein SIRPα□ compared to a vector control. [Figure 29C]

[0138] FIG. 1 shows the expected results of increased lysis of target cells by SIRPα□-transduced macrophages compared to control (empty vector-transduced) macrophages. [Figure 29D]

[0139] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with SIRPαβ-transduced macrophages or untreated controls. [Figure 30A]

[0140] FIG. 1 illustrates an exemplary schematic diagram of a nucleic acid construct comprising a regulatory element sequence, a CFP-encoding sequence, a T2A-encoding sequence, and a sialidase-encoding sequence. The T2A sequence allows for cleavage of the sialidase from the CFP during translation. [Figure 30B]

[0141] FIG. 1 illustrates a diagrammatic representation of enhanced phagocytic engulfment of target cells in the presence of secreted sialidase. [Figure 30C]

[0142] FIG. 1 illustrates predicted results showing enhanced lysis of target cells by engineered myeloid cells expressing CFP in the presence of sialidase. [Figure 30D]

[0143] FIG. 1 illustrates an exemplary schematic diagram of a nucleic acid construct encoding a sialidase with regulatory elements for expression in activated monocytes (eg, macrophages). [Figure 30E]

[0144] 1 illustrates a diagrammatic representation of enhanced phagocytic engulfment of target cells as a result of NF-kappa B (NF-κB) activation in phagocytes. NF-kappa B activation activates expression of a nucleic acid construct encoding a sialidase. [Figure 30F]

[0145]

[0023] Figure 1 illustrates an exemplary schematic diagram of a nucleic acid construct encoding a sialidase with a regulatory element in the 3'UTR. The ARE domain contains a binding sequence motif for an RNA-binding protein, which can be used to target expression of the construct and further extend or shorten the duration of mRNA half-life. [Figure 30G]

[0146] FIG. 6C is a diagrammatic representation of the enhanced phagocytic engulfment of target cells as a result of expressing the sialidase construct shown in FIG. 6F. [Figure 31A]

[0147]

[0023] Figure 1 illustrates an exemplary schematic diagram of an FcRα-based CFP comprising an extracellular scFv specific for a cancer antigen fused to the FcRα chain (top panel). The FcRα chain lacks an intracellular domain. The transmembrane domain trimerizes with the endogenous Fcγ receptor transmembrane domain for expression in macrophages. Activation of the CFP by binding of the scFv to the target antigen activates the FcRα-Fcγ receptor, thereby inducing phagocytosis of the target cell. The bottom panel shows a diagrammatic representation of recombinant FcRα-CFP expressed in myeloid cells (e.g., macrophages). [Figure 31B]

[0148] FIG. 1 illustrates a graphical representation of the relative phagocytic activity of cells expressing CFP(FcRα-CAR) compared to vector control. [Figure 31C]

[0149] FIG. 1 shows the expected results for increased lysis of target cells by CFP (FcRα-CAR)-transduced myeloid cells (e.g., macrophages) compared to control (empty vector-transduced) myeloid cells (e.g., macrophages). [Figure 31D]

[0150] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with CFP (FcRα-CAR)-transduced myeloid cells (e.g., macrophages) or untreated controls. [Figure 32A]

[0151]

[0023] Figure 1 illustrates an exemplary schematic diagram of a CFP (TREM-CAR) containing an extracellular scFv specific for a cancer antigen fused to the ECD of TREM 1 / 2 / 3 (top panel). The CFP contains the TM and ICD of TREM 1 / 2 / 3. The TREM transmembrane domain trimerizes with the endogenous DAP12 transmembrane domain, thereby promoting phagocytosis and regulating inflammation. Activation of the CFP by scFv binding to a target antigen activates TREM-mediated endogenous DAP12 signaling, thereby inducing phagocytosis of the target cell. The bottom panel shows a graphical representation of recombinant CFP (TREM-CAR) expressed in myeloid cells (such as macrophages). [Figure 32B]

[0152] FIG. 1 illustrates a graphical representation of the relative phagocytic activity of cells expressing CFP(TREM-CAR) compared to vector control. [Figure 32C]

[0153] FIG. 1 shows expected results for increased lysis of target cells by CFP (TREM-CAR)-transduced myeloid cells (e.g., macrophages) compared to control (empty vector-transduced) myeloid cells (e.g., macrophages). [Figure 32D]

[0154] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with CFP (TREM-CAR)-transduced myeloid cells (e.g., macrophages) or untreated controls. [Figure 33A]

[0155] Figure 1 shows an exemplary schematic diagram of a caspase-recruiting CFP (caspase-CAR). The construct consists of a signal peptide from the N-terminus to the C-terminus, an antigen-specific scFv, a hinge region (e.g., from CD8), a TM (e.g., from CD8), an ITAM (e.g., FcRγ) containing a phagocytic signaling domain, a T2A sequence for bicistronic expression, an SH2 domain, a caspase cleavage sequence, and a pro-caspase (upper panel). When transduced into macrophages, this construct co-expresses CFP and SH2-procaspase. The pro-caspase is autoinhibited in a quiescent state. Binding of tumor surface antigens to the CAR receptor triggers phosphorylation of the ITAM tyrosine motif, leading to recruitment of the SH2-fused pro-caspase. Clustering of the pro-caspase triggers its autocleavage and activation. The linker between the SH2 and pro-caspase is also cleaved at the recognition site. Activated caspases 1, 4, and 5 mediate potent inflammation (lower panel). [Figure 33B]

[0156]

[0023] Figure 1 shows expected results illustrating increased inflammatory gene expression in cells expressing caspase-recruiting CFP (caspase-CAR) compared to empty vector when human primary myeloid cells (e.g., macrophages) are co-cultured with target tumor cells. Cytokine profiling by ELISA shows increased secretion of pro-inflammatory cytokines and chemokines compared to vector controls. [Figure 33C]

[0157] FIG. 1 shows expected flow cytometry results illustrating increased pro-inflammatory cell surface marker expression in cells expressing caspase-recruiting CFP (caspase-CAR) compared to empty vector when human primary myeloid cells (e.g., macrophages) are co-cultured with target tumor cells. [Figure 33D]

[0158] FIG. 1 shows the expected results for increased lysis of target tumor cells by caspase-recruiting CFP (caspase-CAR)-transduced myeloid cells (e.g., macrophages) compared to control (empty vector-transduced) myeloid cells (e.g., macrophages). [Figure 33E]

[0159] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with caspase-recruiting CFP (caspase-CAR)-transduced macrophages or untreated controls. [Figure 34A]

[0160] FIG. 1 illustrates a diagrammatic illustration of an exemplary modular design of a CFP construct. [Figure 34B]

[0161] FIG. 1 illustrates a diagrammatic illustration of an exemplary modular design of a CFP construct. [Figure 34C]

[0162] FIG. 1 illustrates a diagrammatic illustration of an exemplary modular design of a CFP construct. [Figure 35A]

[0163] Figure 1. Generation of engineered effector (ATAK) monocytes from bone marrow-derived cells. Inflammatory monocytes were isolated using a commercially available kit (EasySep Monocyte Isolation Kit). An average of 3 x 106 cells / donor mouse was obtained with a purity of >90%. The isolation / electroporation process was optimized with a transfection efficiency of 50-80%. A maximum of 12 µg of mRNA was used per transduction for short-term expression. [Figure 35B]

[0164] FIG. 1 shows monocyte enrichment data. [Figure 35C]

[0165] FIG. 1 shows dose-dependent expression of HER2 CFP as determined by binding to HER2. [Figure 36A]

[0166] Figure 36 shows the phenotypic characteristics of effector (ATAK) myeloid cells engineered to express a chimeric fusion protein with a HER2 binder in an in vitro tumor co-culture assay. The assay is summarized graphically in Figure 36A. Figures 36B-36C show data on the % of CD86+ cells and CD40+ cells, respectively, of CD45.1+Ly6C+ cells. Figures 36D and 36E show data demonstrating a Her2-specific increase in MHC-II and Ki67 expression. [Figure 36B] Figure 36 shows the phenotypic characteristics of effector (ATAK) myeloid cells engineered to express a chimeric fusion protein with a HER2 binder in an in vitro tumor co-culture assay. The assay is summarized graphically in Figure 36A. Figures 36B-36C show data on the % of CD86+ cells and CD40+ cells, respectively, of CD45.1+Ly6C+ cells. Figures 36D and 36E show data demonstrating a Her2-specific increase in MHC-II and Ki67 expression. [Figure 36C] Figure 36 shows the phenotypic characteristics of effector (ATAK) myeloid cells engineered to express a chimeric fusion protein with a HER2 binder in an in vitro tumor co-culture assay. The assay is summarized graphically in Figure 36A. Figures 36B-36C show data on the % of CD86+ cells and CD40+ cells, respectively, of CD45.1+Ly6C+ cells. Figures 36D and 36E show data demonstrating a Her2-specific increase in MHC-II and Ki67 expression. [Figure 36D] Figure 36 shows the phenotypic characteristics of effector (ATAK) myeloid cells engineered to express a chimeric fusion protein with a HER2 binder in an in vitro tumor co-culture assay. The assay is summarized graphically in Figure 36A. Figures 36B-36C show data on the % of CD86+ cells and CD40+ cells, respectively, of CD45.1+Ly6C+ cells. Figures 36D and 36E show data demonstrating a Her2-specific increase in MHC-II and Ki67 expression. [Figure 36E]Figure 36 shows the phenotypic characteristics of effector (ATAK) myeloid cells engineered to express a chimeric fusion protein with a HER2 binder in an in vitro tumor co-culture assay. The assay is summarized graphically in Figure 36A. Figures 36B-36C show data for the % of CD86+ cells and CD40+ cells, respectively, of CD45.1+Ly6C+ cells. Figures 36D and 36E show data demonstrating a Her2-specific increase in MHC-II and Ki67 expression. [Figure 37A]

[0167] FIG. 1 shows the ability of effector myeloid cells to kill tumor cells. [Figure 37B] FIG. 1 shows the ability of effector myeloid cells to kill tumor cells. [Fig. 37C-D] FIG. 1 shows the ability of effector myeloid cells to kill tumor cells. [Figure 38A-1]

[0168] FIG. 1 shows cytokines released by effector myeloid cells in the presence of target tumor cells. [Figure 38A-2] Continuation of Figure 38A-1. [Figure 38A-3] Continued from Figure 38A-2. [Figure 38A-4] Continuation of Figure 38A-3. [Figure 38B-1] FIG. 1 shows cytokines released by effector myeloid cells in the presence of target tumor cells. [Figure 38B-2] Continued from Figure 38B-1. [Figure 39]

[0169] Figure 1 shows in vivo data from a syngeneic transgenic human HER2 mouse model. Histological sections (left) compare human Her2 (hHER2) protein expression in the brain and mammary gland, allowing hHer2 tumors to establish without rejection. The analytical list panel shows subsequent studies performed on these mice. [Figure 40]

[0170]

[0023] Figure 1 shows data from an experimental setup testing whether co-administration of effector myeloid cells expressing the HER2 binder CFP with human HER2 tumor cells affected tumor establishment in mice. The experimental setup for using the HER2-expressing tumor cell lines MC38, E0771, and AT3 is illustrated. The microscopic image on the left shows human Her2 protein expression in the brain and mammary gland, allowing hHer2 tumors to establish without rejection. [Figure 41]

[0171] FIG. 1 shows assay setup and preliminary data for effector myeloid cells for administration after tumor progression in a syngeneic model with established AT3 tumors. [Figure 42A]

[0172] FIG. 1 illustrates an exemplary schematic of bicistronic constructs for expression of CFP in myeloid cells and CAR in T cells, respectively, and delivered in vivo using an LNP delivery system. [Figure 42B]

[0173]

[0023] Figure 1 illustrates diagrams of two exemplary recombinant bicistronic mRNA constructs containing a sequence encoding a myeloid cell-specific CFP and a sequence encoding a T cell-specific CAR, with a self-cleaving T2A sequence between the two sequences. The sequences comprising the CAR construct further encode a FLAG or Myc tag. The upper construct, MYL222CD19-FLAG-CD3e-T2A-CD19-Myc-CD89, contains the CD19-CD3e-CAR sequence with a FLAG tag and the CD19-CD89 CFP construct with a Myc tag. The lower construct, MYL223CD19-Myc-CD89-T2A-CD19-FLAG-CD3e, contains the CD19-CD3e-CAR sequence with a FLAG tag and the CD19-CD89 CFP construct with a Myc tag. [Figure 42C]

[0174] FIG. 42B illustrates the expression of Myc-tagged CD19-binding CFP (CD19-Myc-CD89) encoded by the mRNA illustrated in FIG. 42B in THP-1 cells, but not in H9 cells. [Figure 42D]

[0175] Figure 42B illustrates the expression of Myc-tagged CD19-binding CAR (CD19-Myc-CDe) encoded by the mRNA depicted in Figure 42B in H9 cells, but not in THP-1 cells. [Figure 43A]

[0176] FIG. 1 shows FACS data showing activation of primary human T cells using detection of CD25 and CD69 expression. [Figure 43B-1]

[0177] FIG. 1 shows FACS data showing the cell gating strategy (left) and expression of CAR (right) in T cells, encoded by recombinant bicistronic mRNA encoding CFP and CAR. [Figure 43B-2] Continued from Figure 43B-1. [Figure 43B-3] Continued from Figure 43B-2. [Figure 43C-1]

[0178] FIG. 1 shows FACS data demonstrating expression of each construct, MYL222 CD19-FLAG-CD3e-T2A-CD19-Myc-CD89, containing the CD19-CD3e-CAR sequence with a FLAG tag, MYL223 CD19-Myc-CD89-T2A-CD19-FLAG-CD3e, containing the CD19-CD3e-CAR with a FLAG tag, and CD19-CD3e FLAG, but not the myc-tagged CD89 domain containing CFP. [Figure 43C-2] Continued from Figure 43C-1. [Figure 43D]

[0179] FIG. 1 shows the killing of Raji cells in the presence of T cells expressing a construct containing CD19-CD3z. [Figure 44-1]

[0180] Figure 1 illustrates a prophetic example demonstrating that injection of LNPs containing RNA encoding anti-GP75 CFP bearing the CD3ε (CD3 epsilon) chain (alone) in mice does not have any therapeutic activity. Injection of mice with LNPs containing RNA encoding anti-GP75 CFP bearing the CD89 (FcR alpha) chain resulted in a 75% reduction in tumor burden in an established cold tumor model. Administration of a combined construct of anti-GP75 CFP bearing CD3ε (CD3 epsilon) T2A and anti-GP75 CFP bearing CD89 (FcR alpha) resulted in complete tumor clearance and management. In this example, B16 tumors were implanted subcutaneously on day 0. Treatment with either LNP-CART (A), LNP-FcA (B), or LNP-FcA+CART (C) was performed on day 10 (tumors approximately 100 mm3). Four injections of 0.16 mg / mouse (over 12 days) were administered. [Figure 44-2] Continuation of Figure 44-1. [Figure 44-3] Continuation of Figure 44-2. DETAILED DESCRIPTION OF THE INVENTION

[0049]

[0181] All terms are intended to be understood as would be understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0050]

[0182] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0051]

[0183] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may, for clarity, be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0052]

[0184] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily all embodiments.

[0053]

[0185] As used in this specification and claim(s), the terms "comprising" (any form of "comprising," such as "comprise" and "comprises"), "having" (any form of "having," such as "have" and "has"), "including" (any form of "including," such as "includes" and "include"), or "containing" (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 herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0054]

[0186] As used herein, the term "about" or "approximately," when referring to a measurable value, e.g., a parameter, amount, temporal period, etc., is meant to encompass variations of + / -30% or less, + / -20% or less, + / -10% or less, + / -5% or less, or + / -1% or less of the particular value and from the particular value, provided such variations are appropriate to the practice of the present disclosure. It is understood that values ​​to which the modifier "about" or "approximately" is attached are themselves also specifically disclosed.

[0055]

[0187] Provided herein are engineered myeloid cells (including, but not limited to, neutrophils, monocytes, myeloid dendritic cells (mDCs), adipocytes, and macrophages) that are designed to specifically bind to target cells. The engineered myeloid cells can attack and kill target cells directly (e.g., by phagocytosis) and / or indirectly (e.g., by activating T cells). In some embodiments, the target cells are cancer cells.

[0056]

[0188] While cancer is one exemplary embodiment described in detail in this disclosure, the methods and techniques described herein are believed to be useful for targeting infected or otherwise diseased cells within the body. Similarly, therapeutic and vaccine compositions using engineered cells are described herein.

[0057]

[0189] Provided herein are compositions and methods for treating diseases or conditions such as cancer. The compositions and methods provided herein utilize human myeloid cells, including but not limited to neutrophils, monocytes, myeloid dendritic cells (mDCs), adipocytes, and macrophages, to target diseased cells, such as cancer cells. The compositions and methods provided herein eliminate diseased cells, such as cancer cells, and / or diseased tissues through various mechanisms, including T cell activation and recruitment, effector immune cell activation (e.g., CD8 T cell and NK cell activation), antigen cross-presentation, enhanced inflammatory response, reduction of regulatory T cells, and phagocytosis. For example, myeloid cells can be used to sustain an immune response against cancer cells.

[0058]

[0190] Provided herein are compositions comprising recombinant polynucleic acids, wherein one or more polynucleic acids encode one or more chimeric fusion proteins, and wherein the recombinant polynucleic acids, when introduced systemically, are expressed in specific cell types in vivo.

[0059]

[0191] In some aspects, the recombinant polynucleic acid is an mRNA. In some embodiments, the recombinant polynucleic acid is an mRNA comprising a sequence encoding a chimeric fusion protein (CFP) designed for myeloid cell-specific expression.

[0060]

[0192] In some aspects, the recombinant polynucleic acid is mRNA. In some embodiments, the recombinant polynucleic acid is mRNA comprising a sequence encoding a chimeric fusion protein (CFP) designed for non-myeloid cell-specific expression. In some embodiments, the non-myeloid cell is a lymphocyte, e.g., a B cell, a T cell, a NK cell, etc.

[0061]

[0193] In some embodiments, the recombinant polynucleic acid comprises a first sequence comprising a sequence encoding a chimeric fusion protein (CFP) designed for myeloid cell-specific expression and a second sequence encoding a chimeric receptor designed for non-myeloid cell expression. In some embodiments, the non-myeloid cell is a T cell. In some embodiments, a single polynucleotide is designed to comprise a first sequence encoding a CFP predominantly expressed in myeloid cells; and a second sequence encoding a chimeric antigen receptor (CAR) predominantly expressed in T cells. In some embodiments, the first and second sequences are separated by a self-cleaving sequence, e.g., a T2A or P2A sequence, thereby generating two peptides after translation, one containing the CFP and the other containing the CAR. Also provided are recombinant polypeptide translation products of the above recombinant polynucleic acids, including (1) a single polypeptide containing the CFP expressed in myeloid cells, and (2) a single polypeptide containing the CAR expressed in T cells. In some embodiments, a polynucleic acid construct comprising a sequence encoding a CFP arm and a sequence encoding a CAR arm described herein is delivered in vivo in a suitable vehicle, and the CFP is expressed in myeloid cells in vivo and the CAR is expressed in lymphoid cells in vivo.

[0062]

[0194] In some embodiments, the recombinant polynucleic acid is an mRNA comprising: (a) a first sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (i) a CFP extracellular domain comprising a first antigen binding domain, and (ii) a CFP transmembrane domain operably linked to the CFP extracellular domain, wherein the CFP transmembrane domain is derived from a protein that dimerizes with endogenous FcR-gamma receptors in myeloid cells; and (b) a second sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) a CAR extracellular domain comprising a second antigen binding domain, (ii) a CAR transmembrane domain operably linked to the CAR extracellular domain, wherein the CAR transmembrane domain is derived from a protein that functionally interacts with an endogenous T cell receptor (TCR) complex and / or at least one endogenous TCR polypeptide, and (iii) a CAR intracellular domain operably linked to the transmembrane domain. Provided herein is a method for treating a myeloid leukemia comprising: (A) one or more lipid nanoparticles; (B) (a) a first sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (i) a CFP extracellular domain comprising a first antigen-binding domain, and (ii) a CFP transmembrane domain operably linked to a CFP extracellular domain, the CFP transmembrane domain being a transmembrane domain derived from a protein that dimerizes with endogenous FcR-gamma receptors in myeloid cells; and (b) a second sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) a second antigen-binding domain (ii) a CAR extracellular domain comprising a second sequence, the second sequence comprising a CAR extracellular domain operably linked to the CAR extracellular domain, the second sequence comprising a transmembrane domain derived from a protein that functionally interacts with an endogenous T cell receptor (TCR) complex and / or at least one endogenous TCR polypeptide; and (iii) a CAR intracellular domain operably linked to the transmembrane domain, wherein the one or more lipid nanoparticles of (A) encapsulate the one or more polynucleic acids of (B).In some embodiments, the one or more polynucleic acids comprise a polynucleic acid molecule comprising both a first sequence and a second sequence, wherein the first sequence and the second sequence are operably linked by a linker sequence.

[0063]

[0195] In some embodiments, the linker sequence comprises a protease cleavage site, such as a T2A or P2A cleavage site, which can cleave the first and second arms and release the CFP and CAR arms in vivo for independent expression.

[0064]

[0196] In some embodiments, one or more polynucleic acids are encapsulated within a nanoparticle delivery vehicle. In some embodiments, one or more polynucleic acids are encapsulated within the same nanoparticle delivery vehicle.

[0065]

[0197] In some embodiments, the first polynucleic acid molecule is encapsulated in a first nanoparticle delivery vehicle, and the second polynucleic acid molecule is encapsulated in a second nanoparticle delivery vehicle. In some embodiments, the first polynucleic acid molecule and the second polynucleic acid molecule are encapsulated in the same nanoparticle delivery vehicle. In some embodiments, after administration of the pharmaceutical composition to a human subject, the CFP is expressed on the surface of myeloid cells of the human subject. In some embodiments, after administration of the pharmaceutical composition to a human subject, the CAR is expressed on the surface of T cells of the human subject. In some embodiments, the CAR is functionally integrated into the endogenous TCR complex of T cells of the human subject. In some embodiments, the CAR extracellular domain is a TCR extracellular domain derived from TCR-alpha, TCR-beta, TCR-delta, TCR-gamma, CD3-gamma, CD3-delta, or CD3-epsilon. In some embodiments, the CAR transmembrane domain is a TCR transmembrane domain derived from TCR-alpha, TCR-beta, TCR-delta, TCR-gamma, CD3-zeta, CD3-gamma, CD3-delta, or CD3-epsilon. In some embodiments, the CAR intracellular domain is a TCR intracellular domain derived from CD3-zeta, CD3-gamma, CD3-delta, or CD3-epsilon. In some embodiments, at least two of the TCR extracellular domain, TCR transmembrane domain, and TCR intracellular domain are derived from the same TCR subunit. In some embodiments, each of the TCR extracellular domain, TCR transmembrane domain, and TCR intracellular domain is derived from the same TCR subunit. In some embodiments, the CAR intracellular domain further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain derived from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In some embodiments, the first and / or second antigen-binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain. In some embodiments, the CFP extracellular domain is an extracellular domain derived from CD8, CD16a, CD64, CD68, or CD89.In some embodiments, the CFP extracellular domain further comprises a hinge domain derived from CD8, and said hinge domain is operably linked to the CFP transmembrane domain and the first antigen binding domain.In some embodiments, after administering the pharmaceutical composition to human subject, CFP is expressed or specifically expressed in the myeloid cells, monocytes or macrophages of human subject.In some embodiments, after administering the pharmaceutical composition to human subject, CAR is expressed or specifically expressed in the T cells of human subject.

[0066]

[0198] In some embodiments, the CFP transmembrane domain is a transmembrane domain derived from CD16a, CD64, CD68, or CD89. In some embodiments, the CFP further comprises a CFP intracellular domain operably linked to the CFP transmembrane domain. In some embodiments, the CFP intracellular domain comprises one or more intracellular signaling domains, wherein the one or more intracellular signaling domains comprise an intracellular signaling domain derived from FcγR, FcαR, FcεR, CD40, or CD3 zeta. In some embodiments, the one or more intracellular signaling domains further comprise a phosphoinositide 3-kinase (PI3K) recruitment domain. In some embodiments, the PI3K recruitment domain comprises a sequence having at least 90% sequence identity with SEQ ID NO: 4. In some embodiments, the CFP intracellular domain comprises an intracellular domain derived from CD16a, CD64, CD68, or CD89.

[0067]

[0199] In some embodiments, the one or more polynucleic acids are mRNA. In some embodiments, the nanoparticle delivery vehicle comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a polar lipid. In some embodiments, the lipid nanoparticle comprises a non-polar lipid. In some embodiments, the lipid nanoparticle is 100-300 nm in diameter. In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to the same antigen. In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to different antigens.

[0068]

[0200] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain binds to an antigen selected from the group consisting of TROP2, GPC3, CD5, HER2, CD137, CD70, Claudin3, Claudin18.2, TMPRSS, CD19, CD22, CD7, PSMA, MSLN, and GP75. In some embodiments, the T cell is a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a NK T cell, an αβ T cell, or a δγ T cell.

[0069]

[0201] In some embodiments, the pharmaceutical composition inhibits the growth of the cancer when administered to a human subject with cancer.

[0070]

[0202] Also provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described above.

[0071]

[0203] In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease. In some embodiments, the subject is a human.

[0072]

[0204] Provided herein are compositions comprising recombinant nucleic acids encoding chimeric fusion proteins (CFPs), such as phagocytic receptor (PR) fusion proteins (PFPs), scavenger receptor (SR) fusion proteins (SFPs), integrin receptor (IR) fusion proteins (IFPs), or caspase-recruiting receptor (caspase-CAR) fusion proteins. The CFPs encoded by the recombinant nucleic acids may include an extracellular domain (ECD) comprising an antigen-binding domain that binds to an antigen on a target cell. The extracellular domain may be fused to a hinge domain or extracellular domain derived from a receptor, such as CD2, CD8, CD28, CD64, or CD68, a phagocytic receptor, a scavenger receptor, or an integrin receptor. The CFPs encoded by the recombinant nucleic acids may further comprise a transmembrane domain, such as a transmembrane domain derived from CD2, CD8, CD28, CD64, or CD68, a phagocytic receptor, a scavenger receptor, or an integrin receptor. In some embodiments, the CFPs encoded by the recombinant nucleic acids further comprise an intracellular domain comprising an intracellular signaling domain, such as an intracellular signaling domain derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain may include one or more intracellular signaling domains derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain may include one or more intracellular signaling domains that promote phagocytic activity, an inflammatory response, nitric oxide production, integrin activation, enhanced effector cell migration (e.g., via chemokine receptor expression), antigen presentation, and / or enhanced cross-presentation. In some embodiments, the CFP is a phagocytic receptor fusion protein (PFP). In some embodiments, the CFP is a phagocytic scavenger receptor fusion protein (PFP). In some embodiments, the CFP is an integrin receptor fusion protein (IFP). In some embodiments, the CFP is an inflammatory receptor fusion protein. In some embodiments, the CFP encoded by the recombinant nucleic acid further includes an intracellular domain comprising a recruitment domain.For example, the intracellular domain may include one or more PI3K recruitment domains, caspase recruitment domains, or caspase activation and recruitment domains (CARDs).

[0073]

[0205] Provided herein are compositions comprising a recombinant nucleic acid encoding a CFP, wherein the CFP comprises a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)) comprising (i) a transmembrane domain, and (ii) an intracellular domain comprising a phagocytic receptor intracellular signaling domain, and an extracellular antigen-binding domain specific for an antigen, e.g., an antigen of a target cell or an antigen presented on a target cell, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked such that antigen binding to the target by the extracellular antigen-binding domain of the fusion receptor activates the intracellular signaling domain of the phagocytic receptor.

[0074]

[0206] Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP, wherein the CFP comprises a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), which comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and upon binding of the CFP to the antigen of the target cell, the CFP is expressed by a myeloid dendritic cell, such as a neutrophil, monocyte, myeloid dendritic cell (mDC), mast cell, or macrophage. Compositions are provided that increase the killing or phagocytic activity of sexual cells 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 more than 1000% compared to cells that do not express CFP.

[0075]

[0207] Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP, wherein the CFP comprises a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), which comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and wherein upon binding of the CFP to the antigen of the target cell, a neutrophil expressing the CFP is produced. In some embodiments, compositions are provided that increase the killing or phagocytic activity of myeloid cells, such as monocytes, myeloid dendritic cells (mDCs), mast cells, or macrophages, 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, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 75 fold, or 100 fold compared to cells that do not express CFP.

[0076]

[0208] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a myeloid cell, such as a neutrophil, monocyte, myeloid dendritic cell (mDC), mast cell, or macrophage cell, 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 an anti-CD5 binding domain, and (ii) a transmembrane domain operably linked to the extracellular domain; and (b) a pharmaceutically acceptable carrier, wherein the myeloid cell expresses the CFP and exhibits at least a 1.1-fold increase in phagocytosis of CD5-expressing target cells compared to myeloid cells that do not express the CFP. In some embodiments, the CD5-binding domain is a CD5-binding protein comprising an antigen-binding fragment of an antibody, a Fab fragment, an scFv domain, or an sdAb domain. In some embodiments, the CD5-binding domain comprises (i) a variable heavy chain (V) of SEQ ID NO: 1 (see Table 4) or having at least 90% sequence identity to SEQ ID NO: 1. H) sequence of SEQ ID NO:2; and (ii) a variable light chain (V) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2. L ) sequence of SEQ ID NO: 33 or an scFv having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the HER2 binding domain comprises (i) a variable heavy chain (V) of SEQ ID NO: 8 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. H ) sequence of SEQ ID NO:9, and (ii) a variable light chain (V) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9. L ) sequence. In some embodiments, the CD5 binding domain comprises an scFv comprising SEQ ID NO: 32 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32. In some embodiments, the CFP further comprises an intracellular domain, wherein the intracellular domain comprises one or more intracellular signaling domains, and wherein a wild-type protein comprising the intracellular domain does not comprise an extracellular domain.

[0077]

[0209] In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, the hinge domain operably linked to the transmembrane domain and the anti-CD5 binding domain. In some embodiments, the extracellular hinge domain comprises SEQ ID NO:7 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7.

[0078]

[0210] In some embodiments, the CFP comprises an extracellular domain fused to a transmembrane domain of SEQ ID NO: 30 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30. In some embodiments, the CFP comprises an extracellular domain fused to a transmembrane domain of SEQ ID NO: 31 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31.

[0079]

[0211] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises SEQ ID NO: 6 or 29 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6 or 29. In some embodiments, the transmembrane domain comprises SEQ ID NO: 18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises SEQ ID NO: 34 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises SEQ ID NO: 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19.

[0080]

[0212] In some embodiments, the CFP comprises one or more intracellular signaling domains including a phagocytosis signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, MerTk, FcRα, and Bai1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, MerTk, FcR, and Bai1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than CD3ζ. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from FcRγ, FcRα, and FcRε. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from CD3ζ. In some embodiments, the CFP comprises an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 20, 27, and 28. In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3-kinase (PI3K) recruitment domain. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO: 4 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the pro-inflammatory signaling domain is derived from the intracellular signaling domain of CD40. In some embodiments, the proinflammatory signaling domain comprises SEQ ID NO:5 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5.In some embodiments, the CFP comprises an intracellular signaling domain of SEQ ID NO: 21 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises an intracellular signaling domain of SEQ ID NO: 23 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23.

[0081]

[0213] In some embodiments, the CFP comprises SEQ ID NO: 14 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the CFP comprises SEQ ID NO: 15 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the CFP comprises SEQ ID NO: 16 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the CFP comprises SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises SEQ ID NO: 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25.

[0082]

[0214] In some embodiments, the CFP comprises (a) (i) an scFv that specifically binds to CD5, and (ii) an extracellular domain comprising a hinge domain derived from CD8; at least a portion of a hinge domain derived from CD28 or an extracellular domain derived 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 at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise (i) a first intracellular signaling domain derived from FcRα, FcRγ, or FcRε, and (ii) a second intracellular signaling domain: (A) comprising a PI3K recruitment domain, or (B) derived from CD40. In some embodiments, as an alternative to (c) above, the CFP comprises an intracellular domain comprising at least two intracellular signaling domains, wherein the intracellular domain comprising at least two intracellular signaling domains comprises (i) a first intracellular signaling domain derived from a phagocytic receptor intracellular domain, and (ii) a second intracellular signaling domain derived from a scavenger receptor phagocytic receptor intracellular domain comprising a PI3K recruitment domain (A) or derived from CD40 (B). Exemplary scavenger receptors from which the intracellular signaling domain may be derived can be found in Table 2. In some embodiments, the CFP comprises an intracellular signaling domain derived from the intracellular signaling domain of an innate immune receptor.

[0083]

[0215] In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the recombinant polynucleic acid is circRNA. In some embodiments, the recombinant polynucleic acid is a viral vector. In some embodiments, the recombinant polynucleic acid is delivered via a viral vector.

[0084]

[0216] In some embodiments, the myeloid cells are CD14+ cells, CD14+ / CD16- cells, CD14+ / CD16+ cells, CD14- / CD16+ cells, CD14- / CD16- cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages, or mosaic myeloid cells / macrophages / dendritic cells.

[0085]

[0217] In some embodiments, the T cells are T cell precursors. In some embodiments, the T cells are undifferentiated and / or non-polarized. In some embodiments, the T cells are CD4 T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the T cells are gamma delta T cells. In some embodiments, the cells are NK T cells.

[0086]

[0218] In one aspect, the description provides a method of treating cancer in a human subject in need thereof, comprising administering to the human subject a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) myeloid cells comprising a recombinant polynucleic acid sequence, wherein the polynucleic acid sequence comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (i) an extracellular domain comprising an anti-CD5 binding domain, and (ii) a transmembrane domain operably linked to the extracellular domain; and (b) a pharmaceutically acceptable carrier, wherein the myeloid cells express the CFP.

[0087]

[0219] In some embodiments, when CFP binds to CD5 expressed by the target cancer cells of a subject, the killing or phagocytic activity of myeloid cells increases by more than 20% compared to myeloid cells that do not express CFP. In some embodiments, tumor growth is inhibited in a human subject.

[0088]

[0220] In some embodiments, the cancer is a CD5+ cancer. In some embodiments, the cancer is a leukemia, T-cell lymphoma, or B-cell lymphoma.

[0089]

[0221] In some embodiments, the anti-CD5 binding domain is a CD5 binding protein comprising an antigen-binding fragment of an antibody, an scFv domain, a Fab fragment, or an sdAb domain. In some embodiments, the anti-CD5 binding domain is a protein or fragment thereof that binds to CD5, such as a ligand of CD5 (e.g., a natural ligand of CD5).

[0090]

[0222] In some embodiments, the CFP further comprises an intracellular domain, wherein the intracellular domain comprises one or more intracellular signaling domains, wherein the one or more intracellular signaling domains comprise a phagocytic signaling domain, and wherein the wild-type protein comprising the intracellular domain does not comprise an extracellular domain.

[0091]

[0223] In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, MerTk, FcRα, and Bai 1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from FcRγ, FcRα, or FcRε.

[0092]

[0224] In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3 kinase (PI3K) recruitment domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the extracellular domain comprises at least a portion of a hinge domain derived from CD8, a hinge domain derived from CD28, or an extracellular domain derived from CD68.

[0093]

[0225] In some embodiments, the CFP comprises: (a) (i) an scFv that specifically binds to CD5, and (ii) an extracellular domain comprising at least a portion of a hinge domain derived from CD8, a hinge domain derived from CD28, or an extracellular domain derived 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 at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from FcRγ or FcRε, and (ii) a second intracellular signaling domain, wherein (A) comprises a PI3K recruitment domain or (B) is derived from CD40. In some embodiments, the recombinant nucleic acid is an mRNA or a circRNA. In some embodiments, the myeloid cells are CD14+ cells, CD14+ / CD16- cells, CD14+ / CD16+ cells, CD14- / CD16+ cells, CD14- / CD16- cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages, or mosaic myeloid cells / macrophages / dendritic cells.

[0094]

[0226] In some embodiments, the method further comprises administering an additional therapeutic agent selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits a GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to PFPs, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4,5-trisphosphate production, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, an agent that promotes lymphocyte segregation in primary and / or secondary lymphoid organs, an agent that increases the concentration of naive T cells and central memory T cells in secondary lymphoid organs, and any combination thereof.

[0095]

[0227] In some embodiments, the myeloid cells further comprise (a) an endogenous peptide or protein that dimerizes with CFP, (b) a non-endogenous peptide or protein that dimerizes with CFP, and / or (c) a second recombinant polynucleic acid sequence, wherein the second recombinant polynucleic acid sequence comprises a sequence encoding a peptide or protein that interacts with CFP; and the dimerization or interaction enhances phagocytosis by the myeloid cells that express CFP compared to myeloid cells that do not express CFP.

[0096]

[0228] In some embodiments, the myeloid cells exhibit (i) increased effector activity, cross-presentation, respiratory burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4,5-trisphosphate production, trogocytosis by antigen-expressing target cells, resistance to CD47-mediated inhibition of phagocytosis, resistance to LILRB1-mediated inhibition of phagocytosis, or any combination thereof; and / or (ii) increased expression of IL-1, IL3, IL-6, IL-10, IL-12, IL-13, IL-23, TNFα, the TNF family of cytokines, CCL2, CXCL9, CXCL10, CXCL11, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL18, CXCL19 ... and / or any combination thereof.

[0097]

[0229] In some embodiments, the intracellular signaling domain is derived from a phagocytic or tethering receptor, or the intracellular signaling domain comprises a phagocytic activation domain. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR-alpha, or Bai1. In some embodiments, the intracellular signaling domain is selected from TNFR1, MDA5, CD40, lectin, Dectin-1, CD206, Scavenger receptor A1 (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, Fcα receptor I, CR1, CD35, CD3ζ, complement receptor, CR3, CR4, Tim-1, Tim-4, and CD169 (e.g., a phagocytic receptor). In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0098]

[0230] In some embodiments, the intracellular signaling domain is derived from an ITAM domain-containing receptor.

[0099]

[0231] Provided herein are compositions comprising a recombinant nucleic acid encoding a CFP, such as a phagocytic or tethered receptor (PR) fusion protein (PFP), wherein the CFP comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is derived from a phagocytic receptor other than Megf10, MerTk, FcRα, or Bai1.

[0100]

[0232] In some embodiments, binding of the CFP to an antigen on a target cell increases the killing activity of cells expressing the CFP 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 more than 1000% compared to cells that do not express the CFP. In some embodiments, when CFP is expressed in cells, CFP is functionally integrated into the cell membrane of cells. In some embodiments, when CFP binds to the antigen of target cells, the killing activity of the cells that express CFP increases at least 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 50 times, 75 times or 100 times compared with the cells that do not express CFP.

[0101]

[0233] In some embodiments, the intracellular signaling domain is selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCR The intracellular signaling domain is derived from a receptor such as a phagocytic receptor selected from the group consisting of B4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fcα receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD 169. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain.

[0102]

[0234] Provided herein are compositions comprising a recombinant nucleic acid encoding a CFP, such as a phagocytic or tethering receptor (PR) fusion protein (PFP), wherein the CFP comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is selected from the group consisting of TNFR1, MDA5, CD40, lectin, Dectin-1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, S Compositions are provided that are derived from receptors such as phagocytic receptors selected from the group consisting of CARA3, 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, Fcα receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD169.

[0103]

[0235] In some embodiments, binding of the CFP to an antigen on a target cell increases the killing activity of cells expressing the CFP 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 more than 1000% compared to cells that do not express the CFP. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, and Bai1. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain, such as a PI3K recruitment domain derived from CD19. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0104]

[0236] Provided herein are compositions comprising a recombinant nucleic acid encoding a CFP, such as a phagocytic or tethered receptor (PR) fusion protein (PFP), wherein the CFP comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0105]

[0237] Provided herein are compositions of engineered CFPs, such as phagocytic receptor fusion proteins, that can be expressed in cells, such as myeloid cells, e.g., to generate engineered myeloid cells capable of targeting target cells, such as diseased cells.

[0106]

[0238] The target cell may be, for example, a cancer cell. In some embodiments, the engineered myeloid cells can present cancer antigens on their cell surface after phagocytosis of the cancer cell, activating T cells. An "antigen" is a molecule capable of stimulating an immune response. Antigens recognized by T cells, helper T lymphocytes (helper T (TH) cells), or cytotoxic T lymphocytes (CTLs) are not recognized as intact proteins, but as small peptides associated with MHC proteins (e.g., class I or class II MHC proteins) on the cell surface. During a natural immune response, antigens recognized in association with class II MHC molecules on antigen-presenting cells (APCs) are acquired from the outside of the cell, internalized, and processed into small peptides associated with class II MHC molecules.

[0107]

[0239] In some embodiments, binding of the CFP to an antigen on a target cell increases the killing activity of cells expressing the CFP 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 more than 1000% compared to cells that do not express the CFP. In some embodiments, when CFP is expressed in cells, CFP is functionally integrated into the cell membrane of cells. In some embodiments, when CFP binds to the antigen of target cells, the killing activity of the cells that express CFP increases at least 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 50 times, 75 times or 100 times compared with the cells that do not express CFP.

[0108]

[0240] In some embodiments, the antigen-expressing target cells are cancer cells. In some embodiments, the antigen-expressing target cells are at least 0.8 microns in diameter.

[0109]

[0241] In some embodiments, cells expressing CFP exhibit increased phagocytosis of antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit at least a 1.1-fold increase in phagocytosis of antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, or 50-fold increase in phagocytosis of antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased cytokine production compared to cells that do not express CFP. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferon, and combinations thereof. In some embodiments, cells expressing CFP exhibit increased effector activity compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased cross-presentation compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class II proteins compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD80 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD86 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class I protein compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TRAIL / TNF family death receptor compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of B7-H2 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of LIGHT compared to cells not expressing CFP.In some embodiments, cells expressing CFP exhibit increased expression of HVEM compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD40 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TL1A compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of 41BBL compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of OX40L compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of GITRL death receptor compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD30L compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TIM4 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TIM1 ligand compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of SLAM compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD48 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD58 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD155 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD112 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of PDL1 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of B7-DC compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased respiratory burst compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased ROS production compared to cells not expressing CFP.In some embodiments, cells expressing CFP exhibit increased iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased extracellular vesicle production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased trogocytosis by target cells that express an antigen compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased resistance to CD47-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased resistance to LILRB1-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased phosphatidylinositol 3,4,5-trisphosphate production.

[0110]

[0242] In some embodiments, the extracellular domain of the CFP comprises an Ig-binding domain. In some embodiments, the extracellular domain comprises an IgA, IgD, IgE, IgG, IgM, FcRγI, FcRγIIA, FcRγIIB, FcRγIIC, FcRγIIIA, FcRγIIIB, FcRn, TRIM21, or FcRL5-binding domain. In some embodiments, the extracellular domain of the CFP comprises an FcR extracellular domain. In some embodiments, the extracellular domain of the CFP comprises an FcRα, FcRβ, FcRε, or FcRγ extracellular domain. In some embodiments, the extracellular domain comprises an FcRα (FCAR) extracellular domain. In some embodiments, the extracellular domain comprises an FcRβ extracellular domain. In some embodiments, the extracellular domain comprises an FCERI extracellular domain. In some embodiments, the extracellular domain comprises an FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, or FCGR3B extracellular domain. In some embodiments, the extracellular domain comprises an integrin domain or an integrin receptor domain. In some embodiments, the extracellular domain comprises one or more integrin α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, or β8 domains.

[0111]

[0243] In some embodiments, the CFP further comprises an extracellular domain and an extracellular antigen-binding domain operably linked to the transmembrane domain. In some embodiments, the extracellular domain further comprises the extracellular domain of a receptor, a hinge, a spacer, and / or a linker. In some embodiments, the extracellular domain comprises the extracellular portion of a phagocytic receptor. In some embodiments, the extracellular portion of the CFP is derived from the same receptor from which the intracellular signaling domain is derived. In some embodiments, the extracellular domain comprises the extracellular domain of a scavenger receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises the extracellular domain of an immunoglobulin or an immunoglobulin hinge region. In some embodiments, the extracellular domain comprises a phagocytic domain. In some embodiments, the extracellular domain comprises a structure capable of multimer assembly. In some embodiments, the extracellular domain comprises a multimerization scaffold. In some embodiments, the extracellular domain is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. In some embodiments, the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids in length. In some embodiments, the extracellular antigen-binding domain specifically binds to an antigen on a target cell. In some embodiments, the extracellular antigen-binding domain comprises an antibody domain. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain, an antibody domain, and the antibody domain is a functional antibody fragment, a single-chain variable fragment (scFv), a Fab, a single-domain antibody (sdAb), a nanobody, a V H Domain, V L domain, VNAR domain, V HHIn some embodiments, the extracellular antigen-binding domain comprises an extracellular domain of a ligand, a receptor, or an adaptor. In some embodiments, the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain specific for a single antigen. In some embodiments, the extracellular antigen-binding domain comprises at least two extracellular antigen-binding domains, each of which is specific for a different antigen.

[0112]

[0244] In some embodiments, the antigen is a cancer-associated antigen, a lineage-associated antigen, a pathogenic antigen, or an autoimmune antigen. In some embodiments, the antigen comprises a viral antigen. In some embodiments, the antigen is a T lymphocyte antigen. In some embodiments, the antigen is an extracellular antigen. In some embodiments, the antigen is an intracellular antigen. In some embodiments, the antigen is thymidine kinase (TK1), hypoxanthine-guanine Phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-1, mucin-16 (MUC16), MUC1, epidermal growth factor receptor vIII (EGFRvIII), mesothelin, human epidermal growth factor receptor 2 (HER2), EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B-cell maturation antigen (BCMA), glypican 3 (GPC3), follicle-stimulating hormone receptor, fibroblast activation protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer group 2D (NKG2D) ligand, disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, The antigen is selected from the group consisting of CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptors, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, protease, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is a protein antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, and CD56. In some embodiments, the antigen is an ovarian cancer antigen or a T lymphoma antigen. In some embodiments, the antigen is an integrin receptor antigen.In some embodiments, the antigen is an antigen of an integrin receptor or integrin selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, and β8. In some embodiments, the antigen is an antigen of an integrin receptor ligand. In some embodiments, the antigen is an antigen of fibronectin, vitronectin, collagen, or laminin. In some embodiments, the antigen-binding domain can bind to two or more different antigens.

[0113]

[0245] In some embodiments, the antigen-binding domain comprises an autoantigen, such as Dsg1 or Dsg3, or a fragment thereof. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain or an antibody domain, and the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.

[0114]

[0246] In some embodiments, the transmembrane domain and the extracellular antigen-binding domain are operably linked through a linker, such as the hinge region of CD8α, IgG1, or IgG4.

[0115]

[0247] In some embodiments, the extracellular domain comprises a multimerization scaffold.

[0116]

[0248] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises an FcR transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRγ transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRα transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRβ transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRε transmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane domain from a syntaxin, such as syntaxin 3 or syntaxin 4 or syntaxin 5. In some embodiments, when the CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when the CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an exogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the intracellular signaling domain is derived. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the extracellular domain is derived. In some embodiments, the transmembrane domain comprises the transmembrane domain of a phagocytic receptor. In some embodiments, the transmembrane domain and the extracellular domain are derived from the same protein. In some embodiments, the transmembrane domain is derived from the same protein as the intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes a DAP12 recruitment domain. In some embodiments, the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12.

[0117]

[0249] In some embodiments, the transmembrane domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length, hi some embodiments, the transmembrane domain is at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length.

[0118]

[0250] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, and Bai1. In some embodiments, the intracellular signaling domain is selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin-1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor selected from the group consisting of .5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a scavenger receptor. In some embodiments, the intracellular domain comprises a CD47 inhibitory domain. In some embodiments, the intracellular domain comprises a Rac inhibitory domain, a Cdc42 inhibitory domain, or a GTPase inhibitory domain. In some embodiments, the Rac inhibitory domain, Cdc42 inhibitory domain, or GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase in the phagocytic cup of PFP-expressing cells. In some embodiments, the intracellular domain comprises an F-actin dissociation activation domain, an ARHGAP12 activation domain, an ARHGAP25 activation domain, or an SH3BP1 activation domain. In some embodiments, the intracellular domain comprises a phosphatase inhibitory domain. In some embodiments, the intracellular domain comprises an ARP2 / 3 inhibitory domain. In some embodiments, the intracellular domain comprises at least one ITAM domain.In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ITAM domains. In some embodiments, the intracellular domain comprises at least one ITAM domain selected from the group consisting of CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and ITAM domains of these amino acid sequences having at least one to no more than 20 modifications thereto. In some embodiments, at least one ITAM domain comprises a Src family kinase phosphorylation site. In some embodiments, at least one ITAM domain comprises a Syk recruitment domain. In some embodiments, the intracellular domain comprises an F-actin depolymerization activation domain. In some embodiments, the intracellular domain lacks enzymatic activity.

[0119]

[0251] In some embodiments, the intracellular domain does not include a domain derived from the CD3 zeta intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the MerTK intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the TLR4 intracellular domain. In some embodiments, the intracellular domain includes a CD47 inhibitory domain. In some embodiments, the intracellular signaling domain includes a domain that activates an integrin, such as the intracellular region of PSGL-1. In some embodiments, the intracellular signaling domain includes a domain that activates Rap1 GTPase, such as from EPAC and C3G. In some embodiments, the intracellular signaling domain is derived from paxillin. In some embodiments, the intracellular signaling domain activates focal adhesion kinase. In some embodiments, the intracellular signaling domain is derived from a single phagocytic receptor. In some embodiments, the intracellular signaling domain is derived from a single scavenger receptor. In some embodiments, the intracellular domain includes a phagocytosis-enhancing domain.

[0120]

[0252] In some embodiments, the intracellular domain comprises a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain. In some embodiments, the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain. In some embodiments, the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, an NLRP family member, NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), a caspase domain, a pro-caspase binding domain, or any combination thereof.

[0121]

[0253] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a connexin (Cx) protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Cx43, Cx46, Cx37, Cx40, Cx33, Cx50, Cx59, Cx62, Cx32, Cx26, Cx31, Cx30.3, Cx31.1, Cx30, Cx25, Cx45, Cx47, Cx31.3, Cx36, Cx31.9, Cx39, Cx40.1, or Cx23. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Cx43.

[0122]

[0254] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a SIGLEC protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-3 (CD33), Siglec-4 (MAG), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-13, Siglec-14, Siglec-15, Siglec-16, or Siglec-17.

[0123]

[0255] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a C-type lectin protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a mannose receptor protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from an asialoglycoprotein receptor protein. For example, the intracellular domain is a protein encoding macrophage galactose-type lectin (MGL), DC-SIGN (CLEC4L), Langerin (CLEC4K), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), DC-associated C-type lectin 1 (Dectin-1) subfamily protein, Dectin-1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily protein, DCIR / CLEC4A, Dectin-2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), Mincle (macrophage-attracting The signaling domain may include an intracellular signaling domain such as an intracellular signaling domain derived from an NLR protein, such as C-type lectin 4E (CLEC4E), a NOD-like receptor protein, a NOD-like receptor MHC class II transactivator (CIITA), IPAF, BIRC1, a RIG-I-like receptor (RLR) protein, RIG-I, MDA5, LGP2, NAIP5 / Bircle, an NLRP protein, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP8, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP14, an NLR protein, NOD1 or NOD2, or any combination thereof.

[0124]

[0256] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a cell adhesion molecule. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from an IgCAM, cadherin, integrin, C-type lectin-like domain protein (CTLD), and / or proteoglycan molecule. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a selectin, such as E-selectin, L-selectin, or P-selectin.

[0125]

[0257] In some embodiments, the GFP does not comprise a full-length intracellular signaling domain. In some embodiments, the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. In some embodiments, the intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length.

[0126]

[0258] In some embodiments, the recombinant nucleic acid encodes an FcR α chain extracellular domain, an FcR α chain transmembrane domain, and / or an FcR α chain intracellular domain. In some embodiments, the recombinant nucleic acid encodes an FcR β chain extracellular domain, an FcR β chain transmembrane domain, and / or an FcR β chain intracellular domain. In some embodiments, the FcR α chain or FcR β chain forms a complex with FcRγ when expressed in a cell. In some embodiments, the FcR α chain or FcR β chain forms a complex with endogenous FcRγ when expressed in a cell. In some embodiments, the FcR α chain or FcR β chain is not incorporated into the cell membrane of a cell that does not express FcRγ. In some embodiments, the CFP does not comprise an FcR α chain intracellular signaling domain. In some embodiments, the CFP does not comprise an FcR β chain intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes a TREM extracellular domain, a TREM transmembrane domain, and / or a TREM intracellular domain. In some embodiments, the TREM is TREM1, TREM2, or TREM3.

[0127]

[0259] In some embodiments, the recombinant nucleic acid comprises a sequence encoding a pro-inflammatory polypeptide. In some embodiments, the composition further comprises a pro-inflammatory nucleotide or nucleotides in the recombinant nucleic acid, such as ATP, ADP, UTP, UDP, and / or UDP-glucose.

[0128]

[0260] In some embodiments, the composition further comprises a pro-inflammatory polypeptide. In some embodiments, the pro-inflammatory polypeptide is a chemokine or cytokine. In some embodiments, the chemokine is selected from the group consisting of IL-1, IL3, IL5, IL-6, IL8, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon.

[0129]

[0261] In some embodiments, myeloid cells are specifically targeted for delivery. Myeloid cells can be targeted using specific biodegradable polymers, such as PLGA (polylactic-co-glycolic acid) and / or polyvinyl alcohol (PVA). In some embodiments, one or more compounds can be selectively incorporated into such polymeric structures to affect myeloid cell function. In some embodiments, the targeting structure is multilayered, e.g., consisting of one or more PLGA and one or more PVA layers. In some embodiments, the targeting structure is assembled in a layer-by-layer sequence. In some embodiments, the targeting polymeric structure is organized into specifically shaped components, such as labile structures, that can adhere to the myeloid cell surface and deliver one or more components, such as growth factors and cytokines, to maintain myeloid cells in a microenvironment that confers a specific polarization. In some embodiments, the polymeric structures can remain attached to the surface without being phagocytosed by myeloid cells. In some embodiments, the one or more growth factors can be M1-polarizing factors, such as cytokines. In some embodiments, the one or more growth factors can be M2-polarizing factors, such as cytokines. In some embodiments, the one or more growth factors can be macrophage-activating cytokines, such as INFγ. In some embodiments, the polymeric structure is capable of sustained release of the one or more growth factors in an in vivo environment, such as a solid tumor.

[0130]

[0262] In some embodiments, the recombinant nucleic acid comprises a sequence encoding a homeostatic regulator of inflammation. In some embodiments, the homeostatic regulator of inflammation is a sequence in an untranslated region (UTR) of an mRNA. In some embodiments, the sequence in the UTR is a sequence that binds to an RNA-binding protein. In some embodiments, binding of the RNA-binding protein to the sequence in the untranslated region (UTR) inhibits or prevents translation. In some embodiments, the sequence in the UTR comprises the consensus sequence WWWU(AUUUA)UUUW, where W is A or U. In some embodiments, the recombinant nucleic acid is expressed in a bicistronic vector.

[0131]

[0263] In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments, the target cell comprises a cell infected by a pathogen. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a cancer cell that is a lymphocyte. In some embodiments, the target cell is a cancer cell that is an ovarian cancer cell. In some embodiments, the target cell is a cancer cell that is a breast cell. In some embodiments, the target cell is a cancer cell that is a pancreatic cell. In some embodiments, the target cell is a cancer cell that is a glioblastoma cell.

[0132]

[0264] In some embodiments, the recombinant nucleic acid is DNA. In some embodiments, the recombinant nucleic acid is RNA. In some embodiments, the recombinant nucleic acid is mRNA. In some embodiments, the recombinant nucleic acid is unmodified mRNA. In some embodiments, the recombinant nucleic acid is modified mRNA. In some embodiments, the recombinant nucleic acid is circRNA. In some embodiments, the recombinant nucleic acid is tRNA. In some embodiments, the recombinant nucleic acid is microRNA.

[0133]

[0265] Also provided herein is a vector comprising a recombinant nucleic acid sequence encoding a CFP described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the vector further comprises a promoter operably linked to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector is polycistronic. In some embodiments, each of the at least one nucleic acid sequence is operably linked to a separate promoter. In some embodiments, the vector further comprises one or more internal ribosome entry sites (IRES). In some embodiments, the vector further comprises a 5'UTR and / or 3'UTR adjacent to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.

[0134]

[0266] Also provided herein are polypeptides encoded by the recombinant nucleic acids of the compositions described herein.

[0135]

[0267] Also provided herein are cells comprising the compositions described herein, vectors described herein, or polypeptides described herein. In some embodiments, the cells are phagocytes. In some embodiments, the cells are stem cell-derived cells, myeloid cells, macrophages, dendritic cells, lymphocytes, mast cells, monocytes, neutrophils, microglia, or astrocytes. In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are M1 cells. In some embodiments, the cells are M2 cells. In some embodiments, the cells are M1 macrophage cells. In some embodiments, the cells are M2 macrophage cells. In some embodiments, the cells are M1 myeloid cells. In some embodiments, the cells are M2 myeloid cells.

[0136]

[0268] Also provided herein is a pharmaceutical composition comprising a composition described herein, such as a recombinant nucleic acid described herein, a vector described herein, a polypeptide described herein, or a cell described herein; and a pharmaceutically acceptable excipient.

[0137]

[0269] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes the production of phosphatidylinositol 3,4,5-triphosphate, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, and any combination thereof. In some embodiments, the pharmaceutically acceptable excipient comprises a serum-free medium, a lipid, or a nanoparticle.

[0138]

[0270] Also provided herein are methods for treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is selected from the group consisting of suitable cancers including ovarian cancer, ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is leukemia or lymphoma. In some embodiments, the liquid cancer is T-cell lymphoma. In some embodiments, the disease is a T-cell malignancy.

[0139]

[0271] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent, wherein the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4,5-trisphosphate production, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, and any combination thereof.

[0140]

[0272] In some embodiments, the administering step comprises infusing or injecting. In some embodiments, the administering step comprises directly administering to the solid tumor. In some embodiments, the administering step comprises a circRNA-based delivery procedure, an anon-particle-encapsulated mRNA-based delivery procedure, an mRNA-based delivery procedure, a virus-based delivery procedure, a particle-based delivery procedure, a liposome-based delivery procedure, or an exosome-based delivery procedure. In some embodiments, a CD4+ T cell response or a CD8+ T cell response is elicited in the subject.

[0141]

[0273] Also provided herein are methods of preparing a cell, the methods comprising contacting the cell with a composition described herein, a vector described herein, or a polypeptide described herein. In some embodiments, the contacting comprises transducing. In some embodiments, the contacting comprises chemical transfection, electroporation, nucleofection, or viral infection or transduction.

[0142]

[0274] Also provided herein are methods of preparing a pharmaceutical composition, comprising contacting a lipid with a composition described herein or a vector described herein, in some embodiments, the contacting comprises forming lipid nanoparticles.

[0143]

[0275] Also provided herein are methods for preparing a pharmaceutical composition, comprising contacting an antibody with a composition described herein or a vector described herein, in some embodiments, the contacting comprises forming lipid nanoparticles.

[0144] definition

[0276] An "agent" can refer to any cell, small molecule chemical compound, antibody or fragment thereof, nucleic acid molecule, or polypeptide.

[0145]

[0277] An "alteration" or "change" can refer to an increase or decrease. For example, an alteration can be a 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or up to 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90, or 100% increase or decrease. For example, an alteration can be a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, or up to 40-fold, 50-fold, 60-fold, or even up to 70-fold, 75-fold, 80-fold, 90-fold, or 100-fold increase or decrease.

[0146]

[0278] As used herein, "antigen-presenting cells" or "APCs" include 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). APCs can express major histocompatibility complex (MHC) molecules and present antigens in complexes with MHC on their surface that can be recognized by T cells, inducing T cell activation and immune responses. Professional antigen-presenting cells, particularly dendritic cells, play an important role in stimulating naive T cells. Non-professional antigen-presenting cells, such as fibroblasts, can also contribute to this process. APCs can also process exogenous antigens and cross-present peptide antigens by displaying the processed antigens on class I MHC molecules. Antigens that give rise to proteins recognized in association with class I MHC molecules are generally proteins produced intracellularly, which are processed and associated with class I MHC molecules.

[0147]

[0279] A "biological sample" can refer to any tissue, cell, body fluid, or other substance of biological origin.

[0148]

[0280] The term "epitope" can refer to any protein determinant, such as a sequence, structure, or amino acid residue, capable of binding to an antibody or binding fragment thereof, a T cell receptor, and / or an antibody-like molecule. Epitope 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.

[0149]

[0281] Chimeric fusion protein (CFP) can be used to refer to the chimeric receptor produced by recombinant DNA technology and described herein. In some cases, CFP is referred to as phagocytic receptor fusion protein (PFP), and the terms can be used interchangeably. For the purpose of describing a construct, such as a bicistronic construct, having one arm of a recombinant polynucleic acid containing a sequence encoding a chimeric receptor for expression in myeloid cells, and another arm containing a sequence encoding a chimeric receptor for expression in T cells, the chimeric receptor designed for expression in T cells is referred to as CAR, to distinguish it from the chimeric protein designed for expression in myeloid cells, which is referred to as CFP. As described herein, for the purpose of discussing these double constructs from bicistronic recombinant nucleic acids, the CAR construct can contain a CD3z domain, and the CFP construct can contain a CD89 transmembrane domain.

[0150]

[0282] Engineered cells, such as engineered myeloid cells, can refer to cells that have at least one exogenous nucleic acid sequence in the cell, even if the expression is transient. The step of expressing the exogenous nucleic acid can be performed by various methods described elsewhere, including methods known in the art. The present disclosure relates to the preparation and use of engineered cells, such as engineered myeloid cells, for example, engineered phagocytes. The present disclosure particularly relates to engineered cells that contain an exogenous nucleic acid encoding, for example, a chimeric fusion protein (CFP).

[0151]

[0283] 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 can be influenced by modulation of T cell costimulation and NK cell costimulation. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxic activity. Furthermore, immune responses include immune responses indirectly influenced by NK cell activation, B cell activation, and / or T cell activation, such as antibody production (humoral response), and activation of cytokine-responsive cells, such as macrophages. Immune responses include adaptive immune responses. The adaptive immune system can react to foreign molecular structures, such as antigens of invading organisms. Unlike the innate immune system, the adaptive immune system is highly pathogen-specific. Adaptive immunity can also provide long-lasting protection. Adaptive immune responses include humoral and cell-mediated immune responses. In humoral immune responses, antibodies secreted into body fluids by B cells bind to antigens from pathogens and result in their elimination through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells are activated, which are capable of destroying other cells. For example, when disease-associated proteins are presented by cells, they can be proteolytically fragmented into peptides within the cell. Specific cellular proteins can then attach themselves to antigens or peptides formed in this way, transport them to the cell's surface, and present them to molecular defense mechanisms such as T cells. Cytotoxic T cells can recognize these antigens and kill cells bearing these antigens.

[0152]

[0284] A "ligand" can refer to a molecule capable of binding to or forming a complex with another molecule, such as a receptor. Ligands can include, but are not limited to, proteins, glycoproteins, carbohydrates, lipoproteins, hormones, fatty acids, phospholipids, or any component that binds to a receptor. In some embodiments, a receptor has a specific ligand. In some embodiments, a receptor can exhibit promiscuous binding to ligands, in which case the receptor can bind to several ligands that share at least similarities in conformational configuration, charge distribution, or any other physicochemical characteristics. A ligand can be a biomolecule. A ligand can also be a non-biological substance. For example, a ligand can be a negatively charged particle that is a ligand for the scavenger receptor MARCO. For example, a ligand can be TiO2, which is a ligand for the scavenger receptor SRA1.

[0153]

[0285] The terms "major histocompatibility complex (MHC)," "MHC molecule," or "MHC protein" refer to proteins capable of binding to antigenic peptides and presenting them to T lymphocytes. Such antigenic peptides can represent T cell epitopes. Human MHC is also called the HLA complex. Therefore, 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. Although the proteins of the two HLA classes are very similar in structure, they have very different functions. Class I HLA proteins are presented on the surface of almost all cells in the body, including most tumor cells. Class I HLA proteins are loaded with antigens, usually from endogenous proteins or intracellular pathogens, and then presented to naive or cytotoxic T lymphocytes (CTLs). HLA class II proteins are presented by antigen-presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages, which primarily present peptides processed from external antigen sources, e.g., the outside of cells, to helper T cells.

[0154]

[0286] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can endocytose material into phagosomes—although B cells more commonly endocytose into endosomes—which fuse with lysosomes, whose acidic enzymes cleave the endocytosed proteins 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.

[0155]

[0287] Presentation of peptides to CD4+ helper T cells by HLA class II molecules can result in an immune response 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 the differentiation of other immune cells. HLA class II molecules are typically heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than the class I peptide-binding groove.

[0156]

[0288] HLA alleles are typically expressed in a codominant manner. For example, each person has two alleles of each of the three class I genes (HLA-A, HLA-B, and HLA-C), allowing them to express six different types of class II HLA. At the class II HLA locus, each person inherits a pair of HLA-DP genes (DPAl and DPB1, which encode the α and β chains), HLA-DQ (DQA1 and DQB1, which encode the α and β chains), one gene, HLA-DRα (DRA1), and one or more genes, HLA-DRβ (DRB1 and DRB3, -4, or -5). For example, HLA-DRB1 has nearly 400 known alleles. This means that a heterozygous individual can inherit six or eight functional class II HLA alleles: three or more from each parent. Therefore, HLA genes are highly polymorphic; many different alleles exist in different individuals within a population. Genes that encode HLA proteins have many possible variations, allowing each person's immune system to respond to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is given a specific number. In some embodiments, 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, the class II HLA allele is HLA-DRB* 01:01, HLA-DRB * 01:02, HLA-DRB * 11:01, HLA-DRB * 15:01, and HLA-DRB * It is 07:01.

[0157]

[0289] "Myeloid cells" can broadly refer to cells of the myeloid lineage of hematopoietic stem cells, excluding, for example, lymphoid lineages. Myeloid cells include, for example, cells of the granulocyte and monocytic lineages. Myeloid cells differentiate from a common ancestor derived from hematopoietic stem cells in the bone marrow. Commitment to the myeloid lineage can be governed by the activation of different transcription factors; therefore, myeloid cells can be characterized as cells with a level of plasticity and the ability to further differentiate into terminal cell types based on extracellular and intracellular stimuli. Myeloid cells can be rapidly recruited to local tissues via various chemokine receptors on their surface. Myeloid cells respond to various cytokines and chemokines.

[0158]

[0290] Myeloid cells may be cells that arise in the bone marrow from hematopoietic stem cells 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, myeloid cells are progenitor cells. In some embodiments, myeloid cells may be cells that have characteristics of a common myeloid precursor, or a granulocyte precursor, a myeloblast, or a monocyte-dendritic cell precursor, or a combination thereof. Myeloid cells may include granulocytes or monocytes or their precursors. Myeloid cells may include immature granulocytes, immature monocytes, immature macrophages, immature neutrophils, and immature dendritic cells. Myeloid cells may include monocytes or promonocytic cells or monocyte precursors. In some cases, as used herein, myeloid cells may refer to monocytes with an M0 phenotype, an M1 phenotype, or an M2 phenotype. Myeloid cells may include dendritic cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, pre-dendritic cells, or DC precursors. Myeloid cells may include neutrophils, which may be mature neutrophils, neutrophil precursors, or polymorphonuclear cells (PMNs). Myeloid cells may include macrophages, monocyte-derived macrophages, tissue macrophages, and macrophages of M0, M1, or M2 phenotypes. Myeloid cells may include tumor-infiltrating monocytes (TIMs). Myeloid cells may include tumor-associated monocytes (TAMs). Myeloid cells may include myeloid-derived suppressor cells (MDSCs). Myeloid cells may include tissue-resident macrophages. Myeloid cells may include tumor-associated DCs (TADCs). Thus, myeloid cells may express one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD38, CCR5, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, SIGLEC family proteins and CLEC family proteins.In some cases, myeloid cells may be characterized by high or low expression of one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, or a combination thereof.

[0159]

[0291] "Phagocytosis" is used interchangeably with "engulfment" and can refer to the process by which cells engulf particles, such as cancer cells or infected cells. This process can result in an internal compartment (phagosome) containing the particle. This process can be used to ingest and / or remove particles, such as cancer cells or infected cells, from the body. Phagocytic receptors can be involved in the process of phagocytosis. The process of phagocytosis can be closely related to immune responses and antigen presentation. Processing of exogenous antigens is followed by uptake into professional antigen-presenting cells by some type of endocytic event. Phagocytosis can also promote antigen presentation. For example, antigens from phagocytosed cells or pathogens, including cancer antigens, can be processed and presented on the cell surface of APCs.

[0160]

[0292] A "polypeptide" can refer to a molecule containing amino acids linked together via peptide bonds, such as a glycoprotein, lipoprotein, intracellular protein, or 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, a polypeptide may comprise more than one peptide sequence in a single amino acid chain, which may be separated by a spacer, linker, or peptide cleavage sequence. A polypeptide may be a fusion polypeptide. A polypeptide may comprise one or more domains, modules, or moieties.

[0161]

[0293] A "receptor" can refer to a chemical structure composed of a signal-transducing polypeptide, such as a polypeptide that transduces an extracellular signal into a cell. A receptor can contribute to transmitting information in a cell, a cell formation, or an organism. A receptor includes at least one receptor unit and can contain two or more receptor units, each receptor unit including a protein molecule, e.g., a glycoprotein molecule. A receptor can include 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 after binding to the ligand on the surface of the cell.

[0162]

[0294] The term "antibody" refers to the class of proteins commonly known as immunoglobulins, including, but not limited to, IgG1, IgG2, IgG3, and IgG4, IgA (including IgA1 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 (sdAbs), and antigen-binding fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab', and F(ab'), Fd(V). H and C H 1), single chain variable fragments (scFv), single chain antibodies, disulfide-linked variable fragments (dsFv) and V L and / or V H Antibodies may be derived from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, may contain the variable region(s) alone or in combination with one or more of the hinge region, CH1 domain, CH2 domain, and CH3 domain. Also included are any combinations of the variable region and the hinge region, CH1, CH2, and CH3 domain. Antibodies may be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind, for example, an HLA-associated polypeptide or HLA-peptide complex.

[0163]

[0295] The term "recombinant nucleic acid" refers to a nucleic acid prepared, expressed, created, or isolated by recombinant means. Recombinant nucleic acids may contain nucleotide sequences that are not naturally occurring. Recombinant nucleic acids may be synthesized in a laboratory. Recombinant nucleic acids may be prepared by using recombinant DNA techniques, for example, enzymatic modification of DNA, such as restriction enzyme digestion, ligation, and DNA cloning. Recombinant nucleic acids may be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA may be transcribed ex vivo or in vitro to produce messenger RNA (mRNA). Recombinant mRNA may be isolated, purified, and used to transfect cells. Recombinant nucleic acids may encode proteins or polypeptides.

[0164]

[0296] The process of introducing or incorporating nucleic acids into cells can be via transformation, transfection, or transduction. Transformation is the process of uptake of foreign nucleic acids by bacterial cells. This process is applied to the propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that can take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially introduced in a laboratory setting. Transfection is the introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. Transfection can also refer to the introduction of bacteriophage into bacterial cells. "Transduction" is often used to describe the introduction of recombinant viral vector particles into target cells, while "infection" refers to the natural infection of humans or animals with wild-type viruses.

[0165]

[0297] The term "vector" can refer to a nucleic acid molecule capable of autonomous replication in a host cell, which allows for the cloning of a nucleic acid molecule. As known to those skilled in the art, vectors include, but are not limited to, plasmids, cosmids, phagemids, viral vectors, phage vectors, yeast vectors, mammalian vectors, etc. 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 the replication and / or maintenance of a nucleic acid sequence in a host cell. In some embodiments, the vectors or plasmids provided herein are expression vectors. Expression vectors are capable of directing the expression of genes and / or nucleic acid sequences to which they are operably linked. In some embodiments, expression vectors or plasmids are 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, the nucleic acid sequence of a plasmid is not integrated into the genome or chromosome of the host cell after introduction. For example, a plasmid may contain elements for transient or stable expression of a nucleic acid sequence in a host cell, such as a gene or open reading frame carried by the plasmid. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that replicates autonomously in host cells. In some embodiments, the nucleic acid sequence of the plasmid is integrated into the genome or chromosome of the host cell when introduced into the host cell. Expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors. The vector can be a DNA or RNA vector. In some embodiments, the vector provided herein is an RNA vector, such as a retroviral vector or a lentiviral vector, that can be integrated into the genome of the host cell (e.g., via reverse transcription) when introduced into the host cell.Other forms of expression vectors known to those of skill in the art which serve equivalent functions, e.g., autonomously replicating extrachromosomal vectors or vectors capable of integrating into a host genome, can also be used. Exemplary vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked.

[0166]

[0298] The term "spacer" or "linker," when used in reference to a fusion protein, refers to a peptide sequence that connects two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than to link or maintain some minimum distance or other spatial relationship between protein or RNA sequences. In some embodiments, the constituent amino acids of the spacer can be selected to affect certain properties of the molecule, such as the folding, flexibility, net charge, or hydrophobicity of the molecule. Suitable linkers for use in embodiments of the present disclosure are well known to those of skill in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, a linker is used to separate two or more polypeptides, such as two antigenic peptides, by a distance sufficient to ensure that each antigenic peptide folds correctly. Exemplary peptide linker sequences adopt a flexible, extended conformation and do not exhibit the properties required for forming a regular secondary structure. The amino acids of the flexible linker protein region may include Gly, Asn, and Ser, or any permutation of an amino acid sequence containing Gly, Asn, and Ser. Other nearby natural amino acids, such as Thr and Ala, may also be used in the linker sequence.

[0167]

[0299] The terms "treat," "treated," "treating," "treatment," and the like are meant to refer to reducing, preventing, or ameliorating the associated disorder and / or symptoms (e.g., neoplasm or tumor or infectious agent or autoimmune disease). "Treating" can refer to the administration of a therapy to a subject after the onset or suspected onset of a disease (e.g., cancer or infection by an infectious agent or autoimmune disease). "Treating" includes the concept of "alleviating" and can refer to reducing the frequency or severity of the occurrence or recurrence of any symptoms or other adverse effects associated with a disease and / or side effects associated with a treatment. The term "treating" also encompasses the concept of "managing" and refers to reducing the severity of a patient's disease or disorder, e.g., extending lifespan or the survival rate of a patient with a disease, or delaying its recurrence, e.g., extending the period of remission for a patient suffering from a disease. Although not excluded, treating a disorder or condition may not require the complete elimination of the associated disorder, condition, or symptoms. As used herein, the terms "prevent," "preventing," "prevention," and their grammatical equivalents may refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who does not experience such symptoms at the time of initiation of administration of a drug or compound. In certain embodiments, treating a subject or patient, as described herein, includes administering a therapeutic composition, such as a drug, metabolite, prophylactic component, nucleic acid, peptide, or protein encoding or otherwise forming a drug, metabolite, or prophylactic component. In some embodiments, the treating step comprises administering the cell or population of cells to a subject in need thereof.In some embodiments, the treating step includes administering to the subject one or more engineered cells described herein, e.g., one or more engineered myeloid cells, such as phagocytes. Treating includes treating a disease, condition, or 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 include administering a therapeutic vaccine. In some embodiments, engineered phagocytes are administered to a patient or subject. In some embodiments, the cells administered to a human subject result in reduced immunogenicity. For example, engineered phagocytes may not result in or reduce graft-versus-host disease (GVHD) or fratricide. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., have a compatible HLA subtype naturally expressed in the subject). Subject-specific HLA alleles or the subject's HLA genotype may be determined by any method known in the art. In exemplary embodiments, the method includes determining a polymorphism genotype, which may include generating an alignment of reads extracted from sequencing data set to a genetic reference set that includes allelic variants of the polymorphic gene; determining a first posterior probability or posterior probability-derived score for each allelic variant in the alignment; identifying the allelic variant with the highest first posterior probability or posterior probability-derived score as the first allelic variant; identifying one or more overlapping reads aligned with the first allelic variant and one or more other allelic variants; determining a second posterior probability or posterior probability-derived score for the one or more other allelic variants using a weighting factor; identifying the second allelic variant by selecting the allelic variant with the highest second posterior probability or posterior probability-derived score, the first and second allelic variants defining a genotype of the polymorphic gene; and providing an output of the first and second allelic variants.

[0168]

[0300] 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 the reference protein or nucleic acid.

[0169]

[0301] The terms "isolated," "purified," "biologically pure," and their grammatical equivalents refer to a material that is free to varying degrees from the components that normally accompany it as found in its natural state. "Isolate" indicates a degree of separation from its original source or surrounding materials. "Purify" indicates a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that any impurities do not significantly 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 when it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or from chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be separately purified.

[0170]

[0302] The term "neoplasia" or "cancer" refers to any disease caused by or resulting from 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 term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells with typical characteristics of cancer-causing cells, such as uncontrolled growth, immortality, metastatic potential, rapid proliferation and growth rate, and certain characteristic morphological properties. Cancer cells are often in the form of tumors, but such cells can exist alone in an animal or can be non-tumorigenic cancer cells, such as leukemia cells.

[0171]

[0303] The term "vaccine" is understood to mean a composition for generating immunity for the prevention and / or treatment of disease (e.g., neoplasm / tumor / infectious agent / autoimmune disease). Thus, as used herein, a vaccine is a pharmaceutical comprising a recombinant nucleic acid or cells containing and expressing a recombinant nucleic acid, and is intended for use in humans or animals to generate specific defenses and protectants by vaccination. A "vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent. Aspects of the present disclosure relate to the use of technology in the preparation of phagocyte-based vaccines.

[0172]

[0304] The term "pharmaceutically acceptable" refers to approved or approvable by a federal or state government regulatory agency, or listed in the United States Pharmacopoeia 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 a drug, does not destroy the pharmacological activity thereof, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0173]

[0305] Nucleic acid molecules useful in the methods of the present disclosure include, but are not limited to, any nucleic acid molecule that has an activity or encodes a polypeptide. A polynucleotide having substantial identity to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to a pair of nucleic acid molecules forming a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507.) For example, stringent salt concentrations may typically 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 achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergents, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved 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 may occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA).In another exemplary embodiment, hybridization may occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, post-hybridization wash steps may also vary in stringency. Wash stringency conditions may be defined by salt concentration and temperature. As noted above, wash stringency may be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for wash steps may 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 wash steps may include temperatures of at least about 25°C, at least about 42°C, or at least about 68°C. In an exemplary embodiment, the wash step may occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash step may occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash step may occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be readily apparent to one of ordinary skill 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.

[0174]

[0306] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Such sequences may be at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). 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. An exemplary method for determining the degree of identity is to use the BLAST program, with a probability score between e-3 and em° indicating similar sequences. The "reference" is the basis for comparison. It will be understood that the numbering of specific positions or residues in each sequence depends on the specific protein and the numbering scheme used. Numbering may differ, for example, between the precursor of a mature protein and the mature protein itself, and sequence differences between species may affect numbering. Those skilled in the art will be able to identify any homologous protein and each residue in its encoding nucleic acid by methods well known in the art, such as by sequence alignment with a reference sequence and determining homologous residues.

[0175]

[0307] The term "subject" or "patient" refers to an organism, such as an animal (e.g., a human), who 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 non-human primate, a mouse, a cow, a horse, a dog, a sheep, or a cat, or other non-human mammal.

[0176]

[0308] The term "therapeutic effect" refers to some alleviation of one or more symptoms of a disorder (e.g., a neoplasm, a tumor, or an infection by an infectious agent or an autoimmune disease) or its associated pathology. As used herein, a "therapeutically effective amount" refers to an amount of an agent that, upon administration to a cell or subject in single or multiple doses, is effective in extending the survival chances of a patient with such a disorder, reducing, preventing, or delaying one or more signs or symptoms of the disorder beyond that expected in the absence of such treatment, etc. A "therapeutically effective amount" is intended to quantify the amount needed to achieve a therapeutic effect. A physician or veterinarian having ordinary skill can readily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the required pharmaceutical composition.

[0177] Engineered myeloid cells are "targeted" to attack diseased cells

[0309] The present disclosure includes compositions and methods for preparing targeted killer myeloid cells by leveraging their innate functional roles in immune defense, ranging from detection of foreign bodies, particles, diseased cells, cellular debris, inflammatory signals, chemoattractants; activation of endogenous DAMP and PAMP signaling pathways; induction of myelopoiesis; extravasation; chemotaxis; phagocytosis; pinocytosis; recruitment; phagocytosis; scavenging; activation of intracellular oxidative burst and lysis or killing of pathogens; detection, engulfment and killing of diseased or damaged cells; removal of unwanted cells, tissues or cellular debris in vivo; roles in antigen presentation and activation of innate immunity; activation and regulation of immune response cascades; activation of the T cell repertoire; autophagy; inflammatory and non-inflammatory apoptosis; pyroptosis, immune editing in response to stress and properties related to restoration of tissue homeostasis. In one aspect, the present disclosure provides methods and compositions for enhancing one or more functions of myeloid cells for use in therapeutic applications, where the one or more functions may be one or more of: detection of foreign bodies, particles, diseased cells, cell debris, inflammatory signals, and chemoattractants; activation of endogenous DAMP and PAMP signaling pathways; induction of myelopoiesis; extravasation; chemotaxis; phagocytosis; pinocytosis; recruitment; trogocytosis; phagocytosis; scavenging; activation of intracellular oxidative burst and intracellular lysis or killing of pathogens; detection, phagocytosis, and killing of diseased or damaged cells; removal of unwanted cells, tissues, or cell debris in vivo; a role in antigen presentation and activation of innate immunity; activation and regulation of immune response cascades; activation of the T cell repertoire; autophagy; inflammatory and non-inflammatory apoptosis; pyroptosis; immunoediting in response to stress; and restoration of tissue homeostasis. In one embodiment, the compositions and methods are also directed to enhancing the targeting and killing functions of specific myeloid cells through genetic modification of these cells.The compositions and methods described herein may also be directed to the production of engineered myeloid cells that contain at least one genetic modification to recognize and induce effector functions against pathogens, diseased cells, such as tumor or cancer cells, such that the engineered myeloid cells are capable of recognizing, targeting, phagocytosis, killing and / or eliminating pathogens or diseased or cancer cells, and further may activate specific immune response cascades following phagocytosis, killing and / or elimination of the pathogen or diseased cells.

[0178]

[0310] Myeloid cells appear to be the most abundant cells in tumors (Figure 1B). Myeloid cells can recognize tumor cells over healthy normal cells and amplify the body's immune response to tumor cells. As sentinels of the innate immune response, myeloid cells can identify non-self or abnormal cell types and eliminate them through a process called phagocytosis. This can be therapeutically beneficial in driving myeloid cell-mediated phagocytosis and tumor cell lysis. However, these naturally occurring tumor-infiltrating myeloid cells (TIM) can be influenced by the tumor microenvironment (TME). TIM comprise a heterogeneous population of cells. Many TIM arise from circulating monocytes and granulocytes, which in turn derive from bone marrow-derived hematopoietic stem cells. However, in the presence of persistent stimulation by tumor-derived factors, monocyte and granulocyte precursors can transform their intrinsic pathway of terminal differentiation into mature macrophages, DCs, or granulocytes, becoming tumor-promoting myeloid cell types. Differentiation into pathological or activated immature myeloid cells is favored. These immature myeloid cells include tumor-associated DCs (TADCs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). Instead of this acute myelopoiesis, TAMs may arise from tissue-resident macrophages, which may then be of embryonic or monocytic origin. These tissue-resident macrophages undergo phenotypic and functional changes during carcinogenesis, and proliferation may help maintain TAMs derived from tissue-resident macrophages. The tumor microenvironment may drive tumor-infiltrating myeloid cells to become myeloid-derived suppressor cells and acquire the ability to suppress T cells. Consequently, innovative methods are needed to generate therapeutically effective TAMs that can infiltrate tumors and target tumor cells for phagocytic uptake and killing.

[0179]

[0311] In one aspect, the present disclosure provides engineered myeloid / phagocytic cells capable of targeting specific target cells, such as tumor or pathogenic cells. In some embodiments, the engineered myeloid cells provided herein are effective in infiltrating, targeting, and killing tumor cells. The engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids encoding one or more proteins that assist in targeting phagocytic cells to target cells, such as tumor or cancer cells. In one embodiment, the engineered myeloid cells are capable of readily infiltrating tumors. In one embodiment, the engineered myeloid cells have high specificity for target cells while circulating, with no or negligible cross-reactivity to non-tumor, non-disease cells of interest. In one embodiment, the engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids that will help the cells overcome / evade the effects of the TME and mount a potent anti-tumor response. In one embodiment, the engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids that increase phagocytosis of target cells. In another embodiment, the engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids to increase the reduction or elimination of trogocytosis and / or enhance phagocytic lysis of target cells.

[0180]

[0312] Thus, in some embodiments, the compositions described herein include myeloid cells comprising a recombinant nucleic acid encoding a chimeric receptor fusion protein (CFP), e.g., a phagocytic receptor (PR) fusion protein (PFP). The recombinant nucleic acid can include a sequence encoding a PR subunit comprising (i) a transmembrane domain and (ii) an intracellular domain comprising a PR intracellular signaling domain, and an extracellular antigen-binding domain specific for an antigen on a target cell, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked; the PR intracellular signaling domain is derived from a receptor having a signaling domain. The recombinant nucleic acid further encodes one or more polypeptides comprising one or more cell membrane receptors that help phagocytes bind to target cells and enhance their phagocytic activity.

[0181]

[0313] In some embodiments, the myeloid cells described herein comprise one or more recombinant proteins comprising a chimeric receptor, wherein the chimeric receptor is capable of responding to a first phagocytic signal directed toward a target cell, which may be a diseased cell, tumor cell, or pathogen, and a second signal, which is an inflammatory signal that enhances the phagocytic and killing response toward the target initiated by the first signal.

[0182] phagocytes

[0314] Provided herein are methods and compositions for immunotherapy that include "improving" or "modifying" or "engineering" phagocytes and targeting them toward specific targets, which may be specific cell types or classes of cells in a patient or subject. In some embodiments, the subject is a patient with a disease. The terms subject and patient are often used interchangeably in this section. In some embodiments, the phagocytes are derived from a subject with a disease, the disease being, for example, cancer. Autologous cells from the subject may be modified in vitro or administered to the subject, and the modified phagocytes are redesigned to specifically attack and kill the subject's cancer cells.

[0183]

[0315] In some embodiments, the subject has a disease that is not cancer.

[0184]

[0316] In some embodiments, the subject has a disease that is an infection. In some embodiments, the immunotherapeutic methods and compositions provided herein are for "improving" or "modifying" or "engineering" phagocytes and targeting them to the infection, e.g., infected cells, in the subject.

[0185]

[0317] In some embodiments, the subject has a disease that is a viral, bacterial, fungal, or protozoan infection. In some embodiments, the immunotherapy methods and compositions provided herein are for "improving," "modifying," or "engineering" phagocytes and targeting them to viral, bacterial, fungal, or protozoan-infected cells in an infected subject. In some embodiments, the immunotherapy methods and compositions provided herein are for "improving," "modifying," or "engineering" phagocytes and targeting them to a virus, bacteria, fungus, or any pathogen in the subject, so that the virus, bacteria, fungus, or pathogen in the subject is phagocytosed and / or killed. In some embodiments, the immunotherapy methods and compositions provided herein are for "improving," "modifying," or "engineering" phagocytes and targeting them to a viral antigen, bacterial antigen, fungal antigen, or pathogen antigen in the subject, so that there is at least one improved immune response in the subject against the virus, bacteria, fungus, or pathogen in the subject.

[0186]

[0318] In some embodiments, the myeloid cells, such as phagocytes, are allogeneic. In some embodiments, the methods and compositions for immunotherapy provided herein include obtaining myeloid cells, such as phagocytes, from an allogeneic source. The myeloid cells, such as phagocytes, can then be modified or engineered and introduced into a diseased subject so that the modified or engineered cells from the allogeneic source can attack, phagocytose, and / or directly or indirectly kill diseased cells in the subject, or improve at least one immune response of the subject to the disease. In some embodiments, the allogeneic source is a human. In some embodiments, the allogeneic source is a healthy human.

[0187]

[0319] Phagocytes are natural sentinels of the immune system, forming the body's first line of defense. They engulf pathogens, pathogen-infected cells, foreign bodies, or cancer cells, eliminating them from the body. Most potential pathogens are quickly neutralized by this system before they can cause serious infections, for example. This can involve receptor-mediated uptake via the clathrin-coated pit system, pinocytosis, and especially macropinocytosis, as a result of membrane ruffling and phagocytosis. Thus, phagocytes can be activated by a variety of non-self (and self) elements and exhibit a level of plasticity in their "target" recognition.

[0188]

[0320] The mononuclear phagocyte system (MPS), composed of monocytes, macrophages, and dendritic cells, is essential for maintaining tissue homeostasis and determining the balance of immune responses through its role in antigen presentation. The MPS is a cell lineage that arises from bone marrow progenitor cells and gives rise to blood monocytes, tissue macrophages, and dendritic cells. Thus, the process of generating macrophages from the MPS begins with BM promonocytes that undergo a process of differentiation into monocytes poised to enter the systemic circulation. After a short period of circulation (<48 hours), these newly formed monocytes rapidly infiltrate peripheral tissues, where most of them differentiate into macrophages or dendritic cells (DCs). Antimicrobial phagocytosis eliminates and degrades disease-causing microorganisms, induces proinflammatory signaling through cytokine and chemokine secretion, and recruits immune cells to initiate an effective inflammatory response. This type of phagocytosis is often referred to as "inflammatory phagocytosis" (or "immunogenic phagocytosis"). However, in some cases, such as certain persistent infections, an anti-inflammatory response follows microbial uptake. Antimicrobial phagocytosis is generally carried out by professional phagocytes of the myeloid lineage, such as immature dendritic cells (DCs) and macrophages, as well as tissue-resident immune cells. Phagocytosis of damaged, apoptotic cells or cells is typically a non-inflammatory (also called "non-immunogenic") process. Transformed or malignant cells (autologous cells) and cells undergo phagocytosis, and apoptotic cells are rapidly removed without causing damage to surrounding tissues or inducing a pro-inflammatory immune response. This type of apoptotic cell clearance is non-inflammatory and involves the release of a "find me" signal from the apoptotic cell to recruit phagocytes to its location; an "eat me" signal exposed on the apoptotic cell's surface that is bound by phagocytes via specific receptors; cytoskeletal rearrangements that engulf the apoptotic cell; and subsequent digestion of the ingested apoptotic cell, which attracts specific phagocytic receptors (e.g., secretion of anti-inflammatory cytokines).

[0189]

[0321] Phagocytosis, the cellular uptake of microparticles, e.g., particles >0.5 μm within the plasma membrane envelope, is closely related to and partially overlaps with fluid-phase macropinocytosis and receptor-mediated endocytosis of soluble ligands. Variants associated with the uptake of apoptotic cells, also known as efferocytosis, and those of necrotic cells result from infection and inflammation (necroptosis and pyroptosis). The uptake of exogenous particles (heterophagy) shares characteristics with autophagy, an endogenous process for the segregation and lysosomal disposal of damaged organelles. There is a spectrum of uptake mechanisms, depending on particle size, diversity of receptor-ligand interactions, and cytoskeletal involvement. Once internalized, phagosomes can selectively fuse with transient lysosomes or products of the endoplasmic reticulum (ER) and Golgi complex to form secondary phagolysosomes (Russell, DG (2011). Immunol. Rev. 240, 252-268). This pathway is dynamic, undergoing fusion and division with intracellular and secretory vesicles, macrophages, DCs, osteoclasts, and eosinophils. Antimicrobial phagocytosis eliminates and degrades disease-causing microorganisms, induces proinflammatory signaling through cytokine and chemokine secretion, and recruits immune cells to initiate an effective inflammatory response. This type of phagocytosis is often referred to as "inflammatory phagocytosis" (or "immunogenic phagocytosis"). However, in some cases, such as certain persistent infections, an anti-inflammatory response follows microbial uptake. Antimicrobial phagocytosis is generally carried out by professional phagocytes of the myeloid lineage, such as immature dendritic cells (DCs) and macrophages, as well as tissue-resident immune cells. In contrast, phagocytosis (e.g., efferocytosis) of damaged, autologous, apoptotic cells or cell debris is typically a non-inflammatory (also called "non-immunogenic") process. Billions of damaged, dying, and unwanted cells undergo apoptosis every day. Unwanted cells include, for example, excess cells generated during development, senescent cells, infected cells (intracellular bacteria or viruses), transformed or malignant cells, and cells irreversibly damaged by cytotoxic agents.

[0190]

[0322] The bone marrow is a source of circulating neutrophils and monocytes that replace selected tissue-resident macrophages during inflammation and infection, expanding the tissue myeloid population. After phagocytosis, newly recruited monocytes and tissue macrophages secrete their products by generating them from pre-existing phospholipids and arachidonic acid in the cell membrane and by releasing radicals generated by activation of the respiratory burst or induction of inducible nitric oxide synthesis. Apart from the synthesis of low-molecular-weight products (arachidonic acid metabolites, superoxide anion, and nitric oxide) generated as described above, macrophage phagocytosis-induced secretion is primarily achieved by de novo synthesis of RNA and a change in pH, resulting in gradual acidification.

[0191]

[0323] In some embodiments, the phagocytes provided herein are monocytes or cells of the monocyte lineage.

[0192]

[0324] In some embodiments, myeloid cells are phagocytic macrophages that are MARCO+SignR1+ and are found in the outer marginal zone, where they rapidly eliminate encapsulated bacteria. Similar CD169+ F4 / 80 macrophage strains are present in the subcapsular sinus of lymph nodes and have been implicated in viral infections. Endothelial macrophages, including Kupffer cells in the liver, are known to eliminate microorganisms and antigenic ligands from the blood and lymph nodes, providing sinusoidal immune functions comparable to but distinct from mucosal immunity. Despite expressing typical macrophage markers, not all tissue macrophages are constitutively phagocytic. In the marginal zone of rodent spleens, metallophilic macrophages lacking F4 / 80 strongly express CD169 and sialic acid-binding immunoglobulin (Ig)-like lectin 1 (SIGLEC1 [sialoadhesin]) but are weakly phagocytic. Non-professional phagocytes include epithelial cells and fibroblasts. Fibroblasts are "working-class phagocytes" that eliminate apoptotic debris by using integrins other than CD11b-CD18 through adhesion molecules ICAM and vitronectin receptors. Astrocytes have also been reported to phagocytose apoptotic corpses, although they do not efficiently degrade them. Cell membrane receptors associated with phagocytosis can be opsonins, primarily FcRs (activating or inhibiting), a conserved domain of IgG antibodies, and complement receptors, such as CR3 of iC3b accumulated by the classical pathway of complement activation (IgM or IgG) or the alternative lectin pathway. CR3 can also mediate recognition in the absence of opsonins, possibly by accumulating macrophage-derived complement. Antimicrobial phagocytosis is generally carried out by professional phagocytes of the myeloid lineage, such as immature dendritic cells (DCs) and macrophages, and by tissue-resident immune cells.

[0193]

[0325] In some embodiments, due to the instant cell engineering programs disclosed herein, the cells used to engineer for immunotherapeutic use are strongly phagocytic.

[0194]

[0326] In some embodiments, for the instant cell engineering programs disclosed herein, the cells used to engineer for use in immunotherapy are obtained from whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, or spleen tissue.

[0195]

[0327] In some embodiments, the cells used to engineer for use in immunotherapy are obtained from peripheral blood.

[0196]

[0328] Within liver MPS, various structural and functional distinctions have been characterized as both stimulatory and inhibitory with a view to generating cells for cancer immunotherapy.

[0197] [Table 1]

[0198]

[0329] In some embodiments, the myeloid cells engineered for use in immunotherapy of the immediate application comprise myeloid cells selected from the group consisting of macrophages, dendritic cells, adipocytes, monocytes, neutrophils, microglia, and astrocytes.

[0199]

[0330] In some embodiments, the myeloid cells engineered for use in immunotherapy are phagocytes, hi some embodiments, the phagocytes are monocytes.

[0200]

[0331] In some embodiments, the myeloid cells engineered for use in immunotherapy of the immediate application are monocytes, monocyte-derived macrophages, and / or dendritic cells.

[0201]

[0332] In some embodiments, the myeloid cells engineered for use in immunotherapy of the immediate application are monocytes or macrophages.

[0202]

[0333] In some embodiments, the myeloid cells are obtained from peripheral blood.

[0203]

[0334] In some embodiments, the myeloid cells are labeled with the selectable marker CD14 + CD16 low In some embodiments, the myeloid cells are selected via elutriation.

[0204]

[0335] In some embodiments, the myeloid cells are isolated from a leukapheresis column in a subject, hi some embodiments, the subject is the same subject to which the pharmaceutical composition comprising the engineered cells is administered.

[0205]

[0336] In some embodiments, the subject is not the same subject that is administered the pharmaceutical composition comprising the engineered cells.

[0206]

[0337] In some embodiments, leukapheresis is performed on the same subject once a week to recover more myeloid cells. In some embodiments, leukapheresis is performed on the same subject more than once over an 8-10 day period to recover more myeloid cells. In some embodiments, leukapheresis is performed on the same subject more than twice over a one month period to recover more myeloid cells.

[0207]

[0338] In some embodiments, myeloid cells are isolated from a leukapheresis sample or a peripheral blood sample. In some embodiments, the myeloid cells are progenitor cells. In some embodiments, the myeloid cells are monocyte progenitor cells. In some embodiments, the myeloid cells described herein do not differentiate into end-stage cells or exhibit end-stage cell phenotypes, such as tissue macrophages. In some embodiments, the myeloid cells comprise CD14+ cells. In some embodiments, the myeloid cells do not express CD16. In some embodiments, the myeloid cells express low amounts of CD16. In some embodiments, myeloid cells are preselected for manipulation from a biological sample, such as peripheral blood or an apheresis sample, by selection of CD14+ cells. In some embodiments, selection is performed without contacting or binding with the selected myeloid cells. In some embodiments, myeloid cells are selected prior to manipulation from a biological sample by sorting, e.g., flow cytometry-based cell sorter (FACS). In some embodiments, myeloid cells expressing CD16 are captured with an antibody, and the remaining myeloid cells are collected and used for manipulation. In some embodiments, to obtain myeloid cells, one or more other cell surface molecules, such as CD3, CD8, CD11c, CD40, or CD206, were targeted in addition to CD16 for capture in a negative selection process.

[0208]

[0339] In one aspect, the present disclosure provides a myeloid cell comprising at least one exogenous recombinant nucleic acid encoding a fusion protein. The fusion protein may be a chimeric protein comprising at least one transmembrane domain and an extracellular domain comprising a region capable of binding to a target cell. For example, the chimeric protein may bind to a target, such as a target antigen, antigenic peptide, or ligand of the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the target is a cancer antigen. In some embodiments, the chimeric protein is expressed in a myeloid cell and activates the myeloid cell, overcomes TME-induced inhibitory signals, and acts as an activated pro-inflammatory myeloid cell. In one embodiment, the chimeric protein expressed in the myeloid cell is capable of responding to a second signal other than the target (first signal), where the second signal is a pro-inflammatory signal and an activating signal. In some embodiments, the chimeric protein expressed in the myeloid cell is capable of responding to multiple signals other than the target or the first signal. The chimeric protein may be capable of responding to one, two, three, four, five, or more signals other than the target or the first signal.

[0209]

[0340] In another embodiment, the chimeric protein expressed in myeloid cells specifically binds to a target. In some embodiments, the target is a cancer antigen. Expression of the chimeric protein contributes to targeting specificity to myeloid cells.

[0210]

[0341] In one embodiment, chimeric proteins expressed in myeloid cells can be multiplexed, e.g., with multiple domains for activation and processing of more than one signal or signal type. In some embodiments, activation of multiple domains simultaneously results in an increased effector response to myeloid cells. Myeloid cell effector responses include, for example, enhanced phagocytosis, pro-inflammatory activation, and target cell killing. In some embodiments, chimeric proteins expressed in myeloid cells and capable of multiplexing can bind to more than one ligand, such as a target antigen and a helper molecule. In some embodiments, chimeric proteins can bind to multiple target antigens on cancer cells. In some embodiments, chimeric proteins capable of multiplexing can bind to multiple target antigens on multiple cells. In some embodiments, chimeric proteins can bind to macrophage-monocyte inhibitory targets on cancer cells and generate a stimulatory signal upon contact using a pro-inflammatory domain fused to the intracellular tail, a process referred to as "signal switching." For example, the extracellular domain of the chimeric protein may contain a CD47-binding domain, whereas the chimeric fusion protein lacks the transmembrane and / or intracellular domain of the native CD47 receptor but contains a PI3K recruitment domain in the intracellular region, thereby converting a macrophage-monocyte inhibitory signal from contact with tumor cells into a pro-inflammatory phagocytosis-enhancing signal.

[0211]

[0342] In some embodiments, the chimeric protein is capable of binding multiple units of the expressed chimeric protein, e.g., multimerizing. Multimerization includes the formation of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers. In some embodiments, multimerization can occur through the association of transmembrane regions, extracellular regions, or intracellular regions, or a combination thereof. For example, a chimeric protein containing the collagen domain of the phagocytic receptor MARCO may form a trimer for its effective function. In some embodiments, the chimeric protein is capable of associating with other molecules, e.g., another receptor. For example, the chimeric protein may contain an Fc-alpha transmembrane domain that dimerizes with an Fcγ TM domain, where Fcγ may be an endogenous receptor.

[0212]

[0343] In some embodiments, the chimeric protein that can be multiplexed comprises multiple intracellular domains that can be activated by more than one signal and then activate multiple intracellular signaling molecules.For example, the chimeric protein can comprise a phagocytic receptor domain and a pro-inflammatory domain.For example, the chimeric protein can comprise an FcR signaling domain and an additional phosphorylation domain that recruits procaspase.

[0213] Phagocytosis receptor (PR) subunit of the PFP fusion protein

[0344] Provided herein is a recombinant nucleic acid encoding a phagocytic receptor (PR) fusion protein (PFP). A PFP may comprise a PR subunit comprising a transmembrane (TM) domain and an intracellular domain (IDC) comprising a PR intracellular signaling domain. In some embodiments, when a recombinant nucleic acid encoding a PFP is expressed in a cell, the PFP is functionally integrated into the cell membrane of the cell. In some embodiments, the recombinant nucleic acid encodes a transmembrane domain that is specifically integrated into the membrane of a phagocyte, for example, a myeloid cell such as a macrophage.

[0214]

[0345] In some embodiments, suitable PR is selected after screening a library of transmembrane proteins. The PR subunit is fused with a cancer cell-binding antibody at the extracellular domain. In some embodiments, the PR may be fused with one or more additional domains at the intracellular end.

[0215] Intracellular domain of CFP fusion protein

[0346] In some embodiments, the CFP subunit comprises the TM domain of a phagocytic receptor.

[0216]

[0347] In some embodiments, the CFP subunit comprises the ICD domain of a phagocytic receptor.

[0217]

[0348] In some embodiments, the phagocytic receptor is a scavenger receptor. While many scavenger receptors cooperate in the detection and uptake of materials, not all receptors exclusively engage in phagocytosis-induced engulfment. The association of specific phagocytic and scavenger receptors can have dramatic effects on downstream immune responses. For example, induction of the A-type scavenger receptor MARCO by 500 nm negatively charged nanoparticles is associated with an anti-inflammatory and tolerogenic immune response. On the other hand, positively charged particles are engulfed by a subset of phagocytic receptors that activate pro-inflammatory pathways such as NLRP3 and / or fibrotic responses. Furthermore, certain scavenger receptor pathways, such as the scavenger receptor expressed by endothelial cells (SREC-1), have been shown to play a role in antigen cross-presentation. Therefore, identifying and understanding potential receptors that can be utilized to enhance macrophage activity and clinical efficacy is a critical step in the CFP development platform.

[0218]

[0349] Non-opsonic receptors naturally and variably expressed by professional phagocytes include lectin-like recognition molecules such as CD169 and CD33, as well as related receptors for sialylated residues. Additionally, phagocytes express Dectin-1 (a receptor for fungal β-glucans with well-defined signaling capabilities), related C-type lectins (e.g., MICL, Dectin-2, Mincle, and DNGR-1), and a group of scavenger receptors. SR-A, MARCO, and CD36 differ in domain structure and have overlapping yet distinct recognition of apoptotic and microbial ligands. CD36-related family members reveal that apoprotein ligands bind to the receptor helix bundle, while their extracellular surface domains form channels that translocate lipids, such as cholesterol, across the membrane bilayer.

[0219] [Table 2-1]

[0220] [Table 2-2]

[0221] [Table 3-1]

[0222] [Table 3-2]

[0223]

[0350] In some embodiments, the recombinant nucleic acid encodes a chimeric antigen receptor for phagocytosis (CAR-P). In some embodiments, the recombinant nucleic acid encodes a phagocytic receptor (PR) fusion protein.

[0224]

[0351] In some embodiments, the ICD of the CFP encoded by the recombinant nucleic acid comprises a domain from a protein selected from the group consisting of TNFR1, CD40, MDA5, lectin, Dectin 1, mannose receptor (CD206), scavenger receptor A1 (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), and CD169 receptor.

[0225]

[0352] In some embodiments, the ICD is selected from the group consisting of lectin, dectin-1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO (collagenous macrophage receptor, also known as SRA6, SCARA2), CD36 (thrombosponge receptor, also known as scavenger receptor class B, member 3), CD163 (scavenger receptor, cysteine-rich type 1), MSR1, SCARA3, COLEC12 (also known as C-type lectin, SCARA4) , or scavenger receptor with collectin 12), SCARA5, SCARB1, SCARB2, CD68 (SCARD, microsialin), OLR1 (oxidized low-density lipoprotein receptor 1, Lox-1, or C-type lectin domain family 8 member A), SCARF1, SCARF2, SRCRB4D, SSC5D, and CD169 (also known as sialoadhesin receptor, SIGLEC1).

[0226]

[0353] In some embodiments, the recombinant nucleic acid encodes, for example, the intracellular domain of human MARCO. The PR subunit may include an intracellular domain having the 44 amino acid ICD of human MARCO, which has the amino acid sequence: MRNKKILKEDELLSETQQAAFHQIAMEPFEINVPKPKRRNGVNF. In some embodiments, the PR subunit includes a variant that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of MARCO. In some embodiments, the PR includes the transmembrane region of human MARCO.

[0227]

[0354] In some embodiments, the recombinant nucleic acid encodes the intracellular domain of human SRA1. The CFP comprises an intracellular domain having the 50 amino acid ICD of human SRA1, which has the amino acid sequence: MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFK. In some embodiments, the PR subunit comprises a variant that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of human SRA1. The intracellular region of SRA contains phosphorylation sites.

[0228]

[0355] In some embodiments, the CFP comprises the transmembrane region of human SRA1.

[0229]

[0356] In some embodiments, the recombinant nucleic acid comprises a sequence encoding the intracellular domain of CD36. In some embodiments, the recombinant nucleic acid comprises a sequence encoding the TM domain of CD36. Naturally occurring full-length CD36 has two TM domains and two short intracellular domains, and the extracellular domain of CD36 binds to oxidized LDL. Both intracellular domains contain acylated fatty acid cysteine ​​pairs. It lacks known signaling domains (e.g., kinase, phosphatase, G-protein binding, or scaffolding domains). The N-terminal cytoplasmic domain is extremely short (5-7 amino acid residues) and closely associates with the inner leaflet of the plasma membrane. The carboxy-terminal domain contains 13 amino acids containing a CXCX5K motif, which is homologous to regions of the intracellular domains of CD4 and CD8 known to interact with signaling molecules. The intracellular domain of CD36 is capable of assembling signaling complexes that activate lyn kinase, MAP kinase, and focal adhesion kinase (FAK) and inactivating Src homology 2-containing phosphotyrosine phosphatase (SHP-2). Members of the guanine nucleotide exchange factors (GEFs) have been identified as potential key signaling intermediates.

[0230]

[0357] In some embodiments, the recombinant nucleic acid encodes, for example, the intracellular domain of human SCARA3. The CFP may comprise the intracellular domain of human SCARA3 having the 56 amino acid ICD, which has the amino acid sequence: MKVRSAGGDGDALCVTEEDLAGDDEDMPTFPCTQKGRPGPRCSRCQKNLSLHTSVR. In some embodiments, the CFP comprises a variant that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of human SCARA3. In some embodiments, the CFP comprises the TM domain of SCARA3.

[0231]

[0358] In some embodiments, the TM domain of a PR is about 20-30 amino acids in length. In some embodiments, the TM domain comprises multiple transmembrane spans. In some embodiments, the TM domain comprises about 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or more amino acids in length. In some embodiments, the TM domain of an SR is about 20-30 amino acids in length.

[0232]

[0359] Scavenger receptors can occur as homo- or heterodimers. For example, MARCO occurs as a homotrimer. In some embodiments, the scavenger receptor is a monomer. In some embodiments, the scavenger receptor is a homodimer. In some embodiments, the scavenger receptor is a heterodimer. In some embodiments, the scavenger receptor is a homotrimer. In some embodiments, the scavenger receptor is a heterotrimer. In some embodiments, the scavenger receptor is a homotetramer. In some embodiments, the scavenger receptor is a heterotetramer. In some embodiments, the scavenger receptor is a multimer comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more subunits, which may be the same or different.

[0233]

[0360] In some embodiments, the TM domain or ICD domain of the PSP is not derived from FcR, Megf10, Bai1 or MerTK. In some embodiments, the ICD of the RP does not comprise the CD3 zeta intracellular domain.

[0234]

[0361] In some embodiments, the intracellular and transmembrane domains are derived from FcRβ.

[0235]

[0362] In one embodiment, the recombinant nucleic acid encodes a chimeric antigen receptor with enhanced phagocytosis (CAR-P), which is a phagocytic scavenger receptor (PSR) fusion protein (PFP) comprising: (a) an extracellular domain comprising an extracellular antigen-binding domain specific for an antigen of a target cell; (b) a transmembrane domain; and (c) a recombinant PSR intracellular signaling domain, wherein the recombinant PSR intracellular signaling domain comprises a first portion derived from a phagocytic receptor and a second portion derived from a non-phagocytic receptor.

[0236]

[0363] In some embodiments, the second moiety is not a PI3K recruitment domain.

[0237]

[0364] The second portion derived from a non-phagocytic receptor may include an intracellular signaling domain that enhances phagocytosis and / or the inflammatory potential of engineered myeloid cells, such as phagocytes, that express the recombinant nucleic acid. In some embodiments, the second portion derived from a non-phagocytic receptor includes more than one intracellular domain (ICD). In some embodiments, the second portion derived from a non-phagocytic receptor includes a second ICD. In some embodiments, the second portion derived from a non-phagocytic receptor includes a second and a third ICD. In some embodiments, the second portion derived from a non-phagocytic receptor includes a second, a third, and a fourth ICD, and the second portion is encoded by a recombinant nucleic acid. In some embodiments, the intracellular portion includes two, three, four, five, six, seven, or more ICDs. Respective second portions including a second, a third, or a fourth ICD from a non-phagocytic receptor are described below.

[0238] Chimeric antigen receptors enhance intracellular signaling and inflammatory activation

[0365] In one embodiment, the recombinant nucleic acid encodes a second intracellular domain in addition to the phagocytic ICD, conferring potent pro-inflammatory immune activation, such as in myeloid cells such as macrophages involved in infection defense. The second intracellular domain (second ICD) is fused to the cytoplasmic end of the first phagocytic ICD. The second intracellular domain provides the second signal required to trigger inflammasomes and pro-inflammatory signals. Nod-like receptors (NLRs) are a subset of receptors activated in the innate immune response. They oligomerize to form multiprotein complexes that serve as platforms for recruiting pro-inflammatory caspases and inducing their cleavage and activation. This often leads to the direct activation of ROS, resulting in severe cell death known as pyroptosis. There are four inflammasome complexes: NLRP1m, NLRP3, IPAF, and AIM2.

[0239]

[0366] The tumor microenvironment (TME) constitutes an immunosuppressive environment. The effects of IL-10, glucocorticoid hormones, apoptotic cells, and immune complexes can interfere with innate immune cell function. Immune cells, including phagocytes, adopt a tolerogenic phenotype. In myeloid cells, such as macrophages, this phenotype, commonly known as the M2 phenotype, differs from the M1 phenotype in that the cells are capable of killing pathogens. Myeloid cells, such as macrophages, exposed to LPS or IFNγ, for example, can polarize to the M1 phenotype, whereas myeloid cells, such as macrophages, exposed to IL-4 or IL-13, can polarize to the M2 phenotype. LPS or IFNγ interacts with Toll-like receptor 4 (TLR4) on the surface of myeloid cells such as macrophages, inducing the Trif and MyD88 pathways and inducing activation of transcription factors IRF3, AP-1, and NFKB, thereby activating genes such as TNF, interferon, CXCL10, NOS2, and IL-12. Similarly, IL-4 and IL-13 bind to IL-4R, activate the Jak / Stat6 pathway, and regulate the expression of genes associated with anti-inflammatory responses (M2 responses), such as CCL17, ARG1, IRF4, IL-10, and SOCS3. The expression of CD14, CD80, and D206, but low expression of CD163, is indicative of the polarization of myeloid cells such as macrophages toward an M1 phenotype.

[0240]

[0367] In some embodiments, the recombinant nucleic acid encodes one or more additional intracellular domains comprising a cytoplasmic domain for an inflammatory response. In some embodiments, expression of a recombinant nucleic acid encoding a phagocytic receptor (PR) fusion protein (PFP) comprising a cytoplasmic domain for an inflammatory response in engineered myeloid cells, such as macrophages, confers a strong pro-inflammatory response resembling an M1 phenotype.

[0241]

[0368] In some embodiments, the cytoplasmic domain for an inflammatory response comprises the intracellular signaling domain of TLR3, TLR4, TLR9, MYD88, TRIF, RIG-1, MDA5, CD40, an IFN receptor, NLRP-1, NLRP-2, NLRP-3, NLRP-4, NLRP-5, NLRP-6, NLRP-7, NLRP-8, NLRP-9, NLRP-10, NLRP-11, NLRP-12, NLRP-13, NLRP-14, NOD1, NOD2, Pyrin, AIM2, NLRC4 and / or CD40.

[0242]

[0369] In some embodiments, the phagocytic scavenger receptor (PR) fusion protein (PFP) comprises a pro-inflammatory cytoplasmic domain for activation of the IL-1 signaling cascade.

[0243]

[0370] In some embodiments, the cytoplasmic portion of the chimeric receptor (e.g., phagocytic receptor (PR) fusion protein (PFP)) comprises a cytoplasmic domain from a Toll-like receptor, such as the intracellular signaling domain of Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TLR8), or Toll-like receptor 9 (TLR9).

[0244]

[0371] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from interleukin-1 receptor-associated kinase (IRAK1).

[0245]

[0372] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from the differentiation primary response protein (MYD88).

[0246]

[0373] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from myelin and lymphocyte protein (MAL).

[0247]

[0374] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from the retinoic acid-inducible gene (RIG-1).

[0248]

[0375] In some embodiments, the cytoplasmic portion of the CFP comprises the cytoplasmic domain of any one of MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, or IRAK1.

[0249]

[0376] In some embodiments, the recombinant CFP intracellular signaling domain comprises a first portion derived from a phagocyte receptor and a second portion derived from a non-phagocytic receptor, wherein the second portion derived from the non-phagocytic receptor comprises a phosphorylation site. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for autophosphorylation. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for phosphorylation by a Src family kinase. In some embodiments, the phosphorylation site comprises an amino acid sequence capable of binding to the SH2 domain of a kinase upon phosphorylation. In some embodiments, a receptor tyrosine kinase domain is fused to the cytoplasmic end of PFP in addition to the first cytoplasmic portion.

[0250]

[0377] In some embodiments, the phosphorylation is tyrosine phosphorylation.

[0251]

[0378] In some embodiments, the second intracellular domain is an immunoreceptor tyrosine-based activation motif (ITAM), which is present in mammalian α and β immunoglobulin proteins, TCRγ receptors, FCRγ receptor subunits, CD3 chain receptors, and NFAT activating molecules.

[0252]

[0379] In some embodiments, the PFP intracellular domain comprises one ITAM motif. In some embodiments, the PFP intracellular domain comprises more than one ITAM motif. In some embodiments, the PFP intracellular domain comprises two or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises three or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises four or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises five or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises six or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises seven or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises eight or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises nine or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises ten or more ITAM motifs.

[0253]

[0380] In some embodiments, one or more domains of the first phagocytic ICD comprise a mutation.

[0254]

[0381] In some embodiments, one or more domains of the second ICD comprise mutations that enhance a kinase binding domain, create a phosphorylation site, create an SH2 docking site, or a combination thereof.

[0255] Co-expression of inflammatory genes

[0382] In one aspect, the recombinant nucleic acid comprises a coding sequence for a pro-inflammatory gene that is co-expressed with PFP in the engineered cell. In some embodiments, the pro-inflammatory gene is a cytokine. Examples include, but are not limited to, TNF-α, IL-1α, IL-1, IL-6, CSF, GMCSF, or IL-12 or an interferon.

[0256]

[0383] The recombinant nucleic acid encoding the pro-inflammatory gene can be monocistronic, wherein the two coding sequences, (a) the PSP and (b) the pro-inflammatory gene, are cleaved post-transcriptionally or post-translationally for independent expression.

[0257]

[0384] In some embodiments, the two coding sequences include a self-cleaving domain, for example, encoding a P2A sequence.

[0258]

[0385] In some embodiments, the two coding regions are separated by an IRES site.

[0259]

[0386] In some embodiments, the two coding sequences are encoded by a bicistronic genetic element. The coding regions for (a) PSP and (b) the pro-inflammatory gene can be unidirectional, each under separate regulatory control. In some embodiments, both coding regions are bidirectional, driving in opposite directions. Each coding sequence is under separate regulatory control.

[0260]

[0387] Co-expression of pro-inflammatory genes is designed to provide a strong inflammatory stimulus for myeloid cells such as macrophages, activating the surrounding tissues in inflammation.

[0261] Integrin Activation Domain

[0388] Cell-cell and cell-substratum adhesion are mediated by the binding of integrin extracellular domains to diverse protein ligands, but cellular regulation of these adhesive interactions and their translation into dynamic cellular responses, such as cell spreading or migration, requires the integrin cytoplasmic tails. These short tails bind to intracellular ligands that connect the receptor to signaling pathways and cytoskeletal networks (Calderwood DA, 2004, Integrin Activation, Journal of Cell Science 117, 657-666, incorporated herein in its entirety). Integrins are heterodimeric adhesion receptors formed by the noncovalent association of α and β subunits. Each subunit is a type I transmembrane glycoprotein with a relatively large extracellular domain and, except for the β4 subunit, a short cytoplasmic tail. Individual integrin family members have the ability to recognize multiple ligands. Integrins can bind to many extracellular matrix proteins (bone matrix proteins, collagen, fibronectin, fibrinogen, laminin, thrombospondin, vitronectin, and von Willebrand factor), representing their primary function in cell adhesion to the extracellular matrix. Many "counter-receptors" are ligands, representing the role of integrins in mediating cell-cell interactions. Integrins undergo conformational changes that increase ligand affinity.

[0262]

[0389] The integrin β2 subfamily consists of four distinct receptors: α M β2 (CD11b / CD18, Mac-1, CR3, Mo-1), α L β2 (CD11a / CD18, LFA-1), α X β2 (CD11c / CD18), and α D β2 (CD11d / CD18). These leukocyte integrins are involved in virtually all aspects of leukocyte function, including immune responses, adhesion to and transmigration from the endothelium, phagocytosis of pathogens, and leukocyte activation.

[0263]

[0390] All β2 integrin α subunits contain an inserted region of approximately 200 amino acids, called the I or A domain. Highly conserved I domains are found in several other integrin α subunits and in other proteins, such as certain coagulation and complement proteins. I domains mediate protein-protein interactions, and in integrins, they are integrally responsible for binding protein ligands. While the I domain governs the ligand-binding function of these integrins, other regions of the α subunit influence ligand recognition. For example, α M In β2, mAb (OKM1) recognizes an epitope outside the I domain, whereas in α M The α subunit inhibits ligand binding; L The EF-hand regions of β2 and α2β1, as well as I-domain integrins in their α subunits, contribute to ligand recognition. M The α subunit, and possibly other α subunits, contain lectin-like domains involved in binding non-protein ligands, the occupancy of which may regulate the function of the I domain.

[0264]

[0391] When integrins lack enzymatic activity, signaling is instead induced by the assembly of signaling complexes on the cytoplasmic surface of the cell membrane. The formation of these complexes is achieved in two ways: first, by receptor clustering, which increases the avidity of molecular interactions and thereby increases the on-rate for effector molecule binding; and second, by inducing conformational changes in the receptor that create or expose effector-binding sites. Within the ECM, integrins have the ability to bind fibronectin, laminin, collagen, tenascin, vitronectin, and thrombospondin. Clusters of integrin / ECM interactions form focal adhesions, concentrating cytoskeletal components and intracellular signaling molecules. The cytoplasmic tails of integrins serve as binding sites for α-actinin and talin, which then recruit vinculin, a protein involved in binding F-actin to membranes. Talin is activated by kinases such as protein kinase C (PKCα).

[0265]

[0392] Integrins are activated by selectins. Leukocytes express L-selectin, activated platelets express P-selectin, and activated endothelial cells express E- and P-selectin. P-selectin-mediated adhesion allows chemokine- or platelet-activating factor-induced activation of β2 integrins and stabilizes adhesion. It also promotes the release of chemokines from adherent leukocytes. The cytoplasmic domain of P-selectin glycoprotein ligand 1 forms a constitutive complex with Nef-related factor 1. After P-selectin binding, Src kinase phosphorylates Nef-related factor 1, recruiting the phosphoinositide-3-OH kinase p85-p110δ heterodimer, leading to leukocyte integrin activation. E-selectin ligand transduces signals that also affect β2 integrin function. Selectins induce activation of Src family kinases. SFKs activated by selectin binding phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of DAP12 and FcRγ. In part, CD44 is sufficient to transduce signals from E-selectin. CD44 induces inside-out signaling of integrins. The final common step in integrin activation is the binding of talin to the cytoplasmic tail of the β subunit. Kindlin, another group of cytoplasmic adaptors, binds to a distinct region of the integrin β tail. Kindlin increases the clustering of talin-activated integrins. Kindlin is responsive to selectin signaling, but is found predominantly in hematopoietic stem cells such as neutrophils. Selectin signaling and signaling upon integrin activation by chemokine components share common components, including SFKs, Syk, and SLP-76.

[0266]

[0393] In some embodiments, the intracellular domain of the recombinant CFP comprises an integrin activation domain. The integrin activation domain comprises the intracellular domain of a selectin, such as P-selectin, L-selectin, or E-selectin.

[0267]

[0394] In some embodiments, the intracellular domain of the recombinant CFP comprises the integrin activation domain of laminin.

[0268]

[0395] In some embodiments, the intracellular domain of the recombinant CFP comprises an integrin activation domain for activation of talin.

[0269]

[0396] In some embodiments, the intracellular domain of the recombinant CFP comprises an integrin activation domain fused to the cytoplasmic end of a phagocytic receptor ICD domain.

[0270] Chimeric receptors for enhancing antigen cross-presentation

[0397] In some embodiments, the recombinant nucleic acid encodes a domain capable of enabling antigen cross-presentation. Generally, MHC class I molecules present self- or pathogen-derived antigens synthesized within cells, whereas exogenous antigens derived from endocytic uptake are loaded onto MHC class II molecules for presentation to CD4+ T cells. MHC I is limited to presenting endogenous antigens, and peptides are generated by proteosomes. However, in some cases, DCs can process exogenous antigens into the MHC-I pathway for presentation to CD8+ T cells. This is called antigen cross-presentation. Soluble or exogenous antigen components may be degraded by lysosomal proteases in the vacuole and cross-presented by DCs instead of through the endocytic pathway. In some cases, chaperones such as heat shock protein 90 (Hsp90) have been shown to facilitate antigen cross-presentation by specific APCs. HSP-peptide complexes are known to be internalized by a different set of receptors compared to free polypeptides. These receptors are from the scavenger receptor family and include LOX-1, SREC-I / SCARF-I, and FEEL1 / stabilin-1. Both SREC-I and LOX-1 have been shown to mediate cross-presentation of molecular chaperone-bound antigens, leading to activation of CD8+ T lymphocytes.

[0271]

[0398] SREC-1 (scavenger receptor expressed by endothelial cells) does not show significant homology to other types of scavenger receptors but has a unique domain structure. It contains 10 repeats of an EGF-like cysteine-rich motif in its extracellular domain. Recently, the structure of SREC-1 has been shown to be similar to that of a transmembrane protein with 16 EGF-like repeats encoded by the Caenorhabditis elegans gene ced-I, which functions as a cell surface phagocytic receptor that recognizes apoptotic cells.

[0272]

[0399] Cross-presentation of cancer antigens through class I MHC pathway leads to enhanced CD8+ T cell response, which is associated with cytotoxicity and therefore beneficial to tumor regression. In some embodiments, the intracellular domain of PFP comprises SREC1 intracellular domain. In some embodiments, the intracellular domain of PFP comprises SRECII intracellular domain.

[0273]

[0400] In some embodiments, the CFP comprises an intracellular domain that includes a PSR intracellular signaling domain from SREC1 or SRECII.

[0274]

[0401] In some embodiments, the CFP comprises (i) a transmembrane domain and (ii) an intracellular domain comprising a CFP intracellular signaling domain derived from SREC1 or SRECII.

[0275]

[0402] In some embodiments, the CFP comprises (i) a transmembrane domain, (ii) an intracellular domain comprising an intracellular signaling domain, and (iii) an extracellular domain derived from SREC1 or SRECII.

[0276] Transmembrane domain of PFP fusion protein

[0403] In some embodiments, the TM encoded by the recombinant nucleic acid comprises a sequence encoding a domain of a scavenger receptor (SR). In some embodiments, the TM can be a TM domain of, or derived from, any one or more of: lectin, dectin-1, mannose receptor (CD206), SRA1, MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, SRCRB4D, SSC5D, and CD169.

[0277]

[0404] In some embodiments, the TM domain is about 20-30 amino acids in length. The TM domain of an SR is about 20-30 amino acids in length.

[0278]

[0405] In some embodiments, the TM domain or ICD domain of the CFP is not derived from Megf10, Bai1, or MerTK. In some embodiments, the ICD of the CFP does not include the CD3ζ intracellular domain.

[0279]

[0406] In some embodiments, the TM is derived from the same phagocytic receptor as the ICD.

[0280]

[0407] In some embodiments, the TM region is derived from a cell membrane protein. The TM may be selected from an Fc receptor (FcR). In some embodiments, nucleic acid sequences encoding domains from specific FcRs are used for cell-specific expression of recombinant constructs. An FCR-alpha region containing the TM domain may be used for specific expression of the construct in myeloid cells such as macrophages. FcRα recombinant proteins may be expressed in adipocytes.

[0281]

[0408] In some embodiments, the PFP comprises a TM of FcRβ.

[0282]

[0409] In some embodiments, PFP comprises both an FcRβ and an ICD domain, hi some embodiments, PFP comprises both an FcRα and an ICD domain.

[0283]

[0410] In some embodiments, the TM domain is derived from CD8.

[0284]

[0411] In some embodiments, the TM is derived from CD2.

[0285]

[0412] In some embodiments, the TM is derived from FcRα.

[0286] The extracellular domain of the PFP protein

[0413] In some embodiments, the extracellular domain of the PFP fusion protein provided herein comprises an antigen-binding domain that binds to one or more targets. The binding target can be an antigen or a ligand. For example, the binding target can be an antigen on a target cell. In some embodiments, the target-binding domain is specific to the target. In some embodiments, the extracellular domain can comprise an antibody, or an antigen-binding domain selected from an intrabody, a peptibody, a nanobody, a single-domain antibody, a SMIP, and a multispecific antibody.

[0287]

[0414] In some embodiments, the antibody fragment comprises a portion of an intact antibody, e.g., the antigen-binding region or variable region of the intact antibody. In a further aspect of the invention, the anti-HIV antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, diabodies, linear antibodies, multispecific antibodies formed from antibody fragments and scFv fragments, as well as other fragments described below. In another embodiment, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, or other antibody class or isotype described herein. (See, e.g., Hudson et al., Nat. Med. 9:129-134 (2003); Pluckthin, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); WO 93 / 01161; and U.S. Pat. Nos. 5,571,894, 5,869,046, 6,248,516, and 5,587,458.) A full-length, intact, or whole antibody is an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0288]

[0415] Fv is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment comprises a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops from each of the H and L chains) that contribute antigen-binding amino acid residues and confer antigen-binding specificity to the antibody. However, even a single variable region (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0289]

[0416] Single-chain Fvs (sFvs or scFvs) are VFs linked in a single polypeptide chain. H Antibody domains and V L sFv polypeptides are antibody fragments containing the V H Domains and V L A polypeptide linker may further be included between the domains, which enables the sFv to form the desired structure for antigen binding. (See, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.) sFvs can be used in chimeric antigen receptors (CARs).

[0290]

[0417] Diabodies are V domains that are engineered to achieve interchain, rather than intrachain, pairing of V domains, resulting in bivalent fragments. H Domains and V L A bispecific diabody is a small antibody fragment prepared by constructing an sFv fragment with a short linker (approximately 5-10 residues) between the V domains of two antibodies. H Domains and V LIt is a heterodimer of two crossover sFv fragments in which the domains are present on different polypeptide chains (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0291]

[0418] Domain antibodies (dAbs), which can be produced in fully human form, are the smallest known antigen-binding fragments of antibodies, ranging from about 11 kDa to about 15 kDa. dAbs are composed of the heavy and light chains (V and V, respectively) of immunoglobulins. H and V L ) are robust variable regions. They are highly expressed in microbial cell culture and exhibit favorable biophysical properties, including, for example, but not limited to, solubility and temperature stability, making them well suited for selection and affinity maturation by in vitro selection systems such as phage display. dAbs are biologically active as monomers, and their small size and inherent stability allow them to be formatted into larger molecules to generate drugs with extended serum half-lives or other pharmacological activities. (See, e.g., WO9425591 and US20030130496.)

[0419] Fv and sFv are the only species with intact binding sites devoid of constant regions. Therefore, they are suitable for reducing nonspecific binding when used in vivo. sFv fusion proteins can be constructed to fuse an effector protein to either the amino or carboxy terminus of an sFv. Antibody fragments can also be "linear antibodies." (See, e.g., U.S. Pat. No. 5,641,870.) Such linear antibody fragments can be monospecific or bispecific.

[0292]

[0420] In some embodiments, the extracellular domain comprises a Fab binding domain, hi yet other such embodiments, the extracellular domain comprises an scFv.

[0293]

[0421] In some embodiments, the chimeric antigen receptor is an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a V H Domain, V L Domains, Single Domain Antibodies (sdAbs), VNAR Domains, and V HH In some embodiments, the antibody comprises an extracellular antigen-binding domain derived from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a V domain, a bispecific antibody, a diabody, or a functional fragment of any of these. H Domain, V L Domains, Single Domain Antibodies (sdAbs), VNAR Domains, and V HH The domain, bispecific antibody, diabody, or functional fragment of any of these specifically binds to one or more antigens.

[0294]

[0422] In some embodiments, the antigen is a cancer antigen and the target cell is a target cancer cell. In some embodiments, the antigen on the target cell is CD3, CD4, CD5, CD7, CD19, CCR2, CCR4, CD30, CD37, TCRB1 / 2, TCRαβ, TCRαδ, CD22, HER2 (ERBB2 / neu), mesothelin, PSCA, CD123, CD30, CD171, CD138, CS-1, CLECL1, CD33, CD79b, EGFRvIII, GD2, GD3, BCMA, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3 (CD276), KIT (CD117), CD213A2, IL-1 Selected from the group consisting of IRa, PRSS21, VEGFR2, CD24, MUC-16, PDGFR-β, SSEA-4, CD20, MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, FAP, EphA2, GM3, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CD97, CD179a, ALK, and IGLL1.

[0295]

[0423] In some embodiments, the target antigen is an autoimmune antigen. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a B cell that produces autoantibodies. In some embodiments, the target antigen is Dsg1 or Dsg3.

[0296]

[0424] Various cancer antigen targets can be selected from cancer antigens known to those skilled in the art.Depending on the cancer and cell type involved, cancer antigens are mutated natural proteins.Antigen binding domains are screened for specificity to mutated / cancer antigens, not natural antigens.

[0297]

[0425] In some embodiments, for example, the cancer antigen for the target cancer cells is a mutant / cancer antigen: MUC16, CCAT2, CTAG1A, CTAG1B, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA6, PRAME, PCA3, MAGEC1, MAGEC2, MAGED2, AFP, MAGEA8, MAGE9, MAGEA11, MAGEA12, IL13RA2, PLAC1, S It may be one or more of DCCAG8, LSP1, CT45A1, CT45A2, CT45A3, CT45A5, CT45A6, CT45A8, CT45A10, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A8, CT47A9, CT47A10, CT47A11, CT47A12, CT47B1, SAGE1, and CT55.

[0298]

[0426] In some embodiments, for example, the cancer antigen of the target cancer cells may be one or more of the following mutant / cancer antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30, CXCR4, CD45, CD56, and the cancer is a T-cell lymphoma.

[0299]

[0427] In some embodiments, for example, the cancer antigen of the target cancer cells may be one or more of the following mutated / cancer antigens: IDH1, ATRX, PRL3, or ETBR, and the cancer is glioblastoma.

[0300]

[0428] In some embodiments, for example, the cancer antigen of the target cancer cells may be one or more of the following mutant / cancer antigens: CA125, β-hCG, urinary gonadotropin fragment, AFP, CEA, SCC, inhibin, or extradiol, and the cancer is ovarian cancer.

[0301]

[0429] In some embodiments, the cancer antigen of the target cancer cell can be CD5.

[0302]

[0430] In some embodiments, the cancer antigen of the target cancer cells can be HER2.

[0303]

[0431] In some embodiments, the cancer antigen of the target cancer cells can be EGFR variant III.

[0304]

[0432] In some embodiments, the cancer antigen of the target cancer cell can be CD19.

[0305]

[0433] In some embodiments, the SR subunit region comprises the extracellular domain (ECD) of a scavenger receptor. In some embodiments, the ECD of the scavenger receptor comprises an ECD domain of an SR, which includes an ICD domain and a TM domain. In some embodiments, the target antigen is an SR ligand on a cancer cell, for example, any one of the ligand components in Table 2 or Table 3. In some embodiments, the SR-ECD contributes to the binding of phagocytes to target cells, and is then activated to activate phagocytosis of the target cells.

[0306]

[0434] In some embodiments, the CFP comprises the ECD of a scavenger receptor or a portion thereof. In some embodiments, the CFP comprises the ICD of a scavenger receptor or a portion thereof. In some embodiments, the CFP comprises the TM domain of a scavenger receptor. In some embodiments, the ECD encoded by the recombinant nucleic acid comprises a domain selected from the group consisting of lectin, Dectin 1, mannose receptor (CD206), scavenger receptor A1 (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), and CD169. The extracellular domains of most scavenger receptors comprise scavenger receptors with broad binding specificities that can be used to distinguish between self and non-self in nonspecific, antibody-independent recognition of foreign substances. The type I and type II class A scavenger receptors (SR-AI1 and SR-AII) are trimeric membrane glycoproteins with a small NH2-terminal intracellular domain and an extracellular portion containing a short spacer domain, an α-helical coiled-coil domain, and a triple-helical collagen domain. Type I receptors additionally contain a cysteine-rich COOH-terminal (SRCR) domain. These receptors are present on myeloid cells, such as macrophages, in various tissues throughout the body and exhibit very broad ligand binding specificity. They bind a wide variety of polyanions, including chemically modified proteins such as modified LDL, and are involved in cholesterol deposition during atherogenesis. They may also play a role in macrophage-associated host defense and cell adhesion processes in inflammatory conditions.

[0307]

[0435] In some embodiments, the SR ECD is designed to bind to pro-apoptotic cells. In some embodiments, the scavenger receptor ECD comprises a binding domain for a cell surface molecule of a cancer cell or an infected cell.

[0308]

[0436] In some embodiments, the extracellular domain of the PR subunit is linked by a linker to a target cell binding domain, e.g., an antibody or portion thereof, specific for a cancer antigen.

[0309]

[0437] In some embodiments, the extracellular antigen-binding domain comprises one antigen-binding domain. In some embodiments, the extracellular antigen-binding domain comprises more than one binding domain. In some embodiments, the binding domain is an scFv. Figure 2 shows a schematic diagram of an embodiment in which PFP targets a single target (left) or multiple targets (right) on cancer cells. One or more scFvs are fused to a recombinant PR at the extracellular domain. In some embodiments, the scFv fraction and the extracellular domain of the PR are linked via a linker.

[0310]

[0438] In some embodiments, the ECD antigen binding domain is capable of binding to an intracellular antigen. In some embodiments, the intracellular antigen is a cancer antigen.

[0311]

[0439] In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 1000 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 500 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 450 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 400 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 350 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 250 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 200 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 100 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity in the range of 200 nM to 1000 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity in the range of 300 nM to 1.5 nM. In some embodiments, the antigen binding domain binds to the target ligand with an affinity of >200 nM, >300 nM, or >500 nM.

[0312]

[0440] In some embodiments, the extracellular antigen-binding domain binds to a target ligand, where the target ligand is a T cell, and its binding characteristics are such that the target T cell is not triggered to activate T cell-mediated lysis of the engineered cell. In some embodiments, binding of the TCR to the ligand on the engineered cell is avoided, bypassed, or inhibited.

[0313] Linker

[0441] Linkers can be used to link any of the polypeptide or peptide domains of the present disclosure. The PFP fusion proteins described herein can include one or more linkers. For example, one or more domains and subunits of a PFP fusion protein can be directly fused to another domain or subunit, or can be linked to another domain or subunit via a linker. In some embodiments, an extracellular antigen-binding domain comprising an antibody specific for an antigen on a target cell, a portion of an antibody capable of specifically binding to an antigen on a target cell, or an scFv specific for an antigen on a target cell is linked to a TM domain or other extracellular domain by a linker. In some embodiments in which more than one scFv is present in the extracellular antigen-binding domain, the more than one scFv are linked to each other by a linker.

[0314]

[0442] In some embodiments, the linker is a short peptide sequence.

[0315]

[0443] The linker can be a single, covalent bond, or a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or amino acid-based. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.).

[0316]

[0444] In some embodiments, the linker is a single amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, a group, a polymer, or a chemical moiety. In some embodiments, the linker is about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length. In some embodiments, the linker is a stretch of glycine residues and one or more serine residues. Other preferred amino acids for short peptide linkers include, but are not limited to, threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), alanine (Ala), arginine (Arg), phenylalanine (Phe), and glutamic acid (Glu). Among these, Pro, Thr, and Gln are frequently used amino acids in natural linkers. Pro is a unique amino acid with a cyclic side chain that induces highly restricted conformations. Pro-rich sequences have been used as interdomain linkers, including the linker between the lipoyl and E3-binding domains of pyruvate dehydrogenase (GA2PA3PAKQEA3PAPA2KAEAPA3PA2KA). For the purposes of this disclosure, empirical linkers can be flexible linkers, rigid linkers, and cleavable linkers. Sequences such as (G4S)x (where x is multiple copies of the moiety, designated 1, 2, 3, 4, etc.) comprise flexible linker sequences. Other flexible sequences used herein include several repeats of glycine, such as (Gly)6 or (Gly)8. On the other hand, rigid linkers can be used, for example, the linker (EAAAK)x (where x is an integer, 1, 2, 3, 4, etc.) produces a rigid linker.A variety of linker lengths and interdomain or intersubunit flexibility can be used in the fusion proteins provided herein to achieve optimal length, ranging from highly flexible linkers of the form (GGGS)n, (GGGGS)n, and (G)n, to more rigid linkers of the form (EAAAK)n, (SGGS)n, SGSETPGTSESATPES (see, e.g., Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014;32(6):577-82; the entire contents of which are incorporated herein by reference), and (XP)n. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, where n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGGGGSG. In some embodiments, the linker comprises the amino acid sequence GSGS.

[0317]

[0445] In some embodiments, the linker is flexible. In some embodiments, the linker comprises a hinge region. When included, such a spacer or linker domain can position the binding domain away from the host cell surface, further enabling proper cell-to-cell contact, binding, and activation. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, the selected binding epitope, and the size and affinity of the binding domain. In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In some embodiments, the linker or spacer used herein comprises an IgG4 hinge region having the sequence: ESKYGPPCPPCP. In some embodiments, the hinge region comprises a hinge or spacer comprising a sequence present in the extracellular region of a type 1 membrane protein, such as CD8a, CD4, CD28, and CD7, which may be wild-type or a variant thereof. In some embodiments, the extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof. In some embodiments, the spacer or linker may be further modified by post-translational modification, such as glycosylation.

[0318]

[0446] In some embodiments, the extracellular spacer domain may comprise the stalk region of a type II C lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D. In yet further embodiments, the extracellular spacer domain may be derived from the scavenger receptor MERTK.

[0319]

[0447] In some embodiments, the linker comprises at least 2 or at least 3 amino acids. In some embodiments, the linker comprises 4 amino acids. In some embodiments, the linker comprises 5 amino acids. In some embodiments, the linker comprises 6 amino acids. In some embodiments, the linker comprises 7 amino acids. In some embodiments, the linker comprises 8 amino acids. In some embodiments, the linker comprises 9 amino acids. In some embodiments, the linker comprises 8 amino acids. In some embodiments, the linker comprises 10 amino acids. In some embodiments, the linker comprises more than 10 amino acids. In some embodiments, the linker comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, there are 12 or more amino acids in the linker. In some embodiments, there are 14 or more amino acids in the linker. In some embodiments, there are 15 or more amino acids in the linker.

[0320] Other fusion proteins for enhancing phagocytosis

[0448] In one embodiment of the present disclosure, recombinant nucleic acids are prepared that encode one or more chimeric receptors that enhance phagocytosis in myeloid cells, such as macrophages, primarily by blocking inhibitory signals. Particularly in tumor environments, myeloid cells, such as macrophages, face phagocytosis-suppressing or inhibitory signals, such as CD47-mediated antiphagocytic activity, against target cells, such as cancer cells. Chimeric receptors are generated that block CD47 signaling when expressed in phagocytes.

[0321]

[0449] In some embodiments, other CAR fusion proteins can be designed for expression in phagocytes, which can enhance phagocytosis. In one embodiment, provided herein is a composition comprising a recombinant nucleic acid encoding a chimeric antigen receptor (CAR) fusion protein (CFP), wherein the CFP comprises (a) a subunit comprising (i) an extracellular domain and (ii) a transmembrane domain, and (b) an extracellular antigen-binding domain specific to CD47 on a target cell, wherein the extracellular domain and the extracellular antigen-binding domain of the subunit are operably linked; and the subunit does not comprise a functional intracellular domain of an endogenous receptor that binds to CD47 or an intracellular domain that activates phosphatase. In some embodiments, the extracellular antigen-binding domain is derived from signal-regulatory protein alpha (SIRPα). In some embodiments, the extracellular antigen-binding domain is derived from signal-regulatory protein alpha (SIRPβ). In some embodiments, the transmembrane domain is derived from SIRPα. In some embodiments, the transmembrane domain is derived from SIRPβ.

[0322]

[0450] In some embodiments, additional CAR fusion protein (CFP) can be co-transfected with the above-mentioned recombinant PFP. In some embodiments, the scavenger receptor intracellular domain comprises a second intracellular domain that comprises a signal transduction domain that activates phagocytosis; or a pro-inflammatory domain at the cytoplasmic end, and these are operably linked. The signal transduction domain that activates phagocytosis is derived from a receptor selected from the group consisting of the receptors listed in Table 2.

[0323]

[0451] In some embodiments, the intracellular domain having the phagocytosis signaling domain comprises a domain having one or more immunoreceptor tyrosine-based activation motif (ITAM) motifs. ITAMs are conserved sequences present in the cytoplasmic tails of several receptors of the immune system, such as T cell receptors, immunoglobulins (Ig), and FcRs. They are located at defined intervals (YXXL / IX 6-8ITAMs share a conserved amino acid sequence motif consisting of paired YXXL / I motifs (Y = tyrosine, L = lysine, and I = isoleucine) separated by a -YXXL / I. Furthermore, most ITAMs contain a negatively charged amino acid (D / E) at the +2 position relative to the first ITAM tyrosine. Phosphorylation of residues within the ITAM recruits several signaling molecules that activate phagocytosis. The ITAM motif is also present in the intracellular adaptor protein, DNAX activator protein 12 (DAP12).

[0324]

[0452] In some embodiments, the phagocytic signaling domain of the intracellular region comprises a PI3 kinase (PI3K) recruitment domain (also referred to as a PI3K binding domain). The PI3K binding domain used herein may be the PI3K binding domain of CD19, CD28, CSFR, or PDGFR. Recruitment of PI3 kinase to the binding domain leads to the activation of the Akt-mediated signaling cascade and phagocytosis. The PI3K-Akt signaling pathway is important in phagocytosis, inflammatory response control, and other activities, including vesicle transport and cytoskeletal reorganization. The PI3 kinase recruitment domain is an intracellular domain of a plasma membrane protein that contains a tyrosine residue that can be phosphorylated and then recognized by the Src homology domain (SH2) domain of PI3Kp85. The SH2 domain of p85 recognizes the phosphorylated tyrosine on the cytosolic domain of the receptor. This results in the allosteric activation of p110 and the production of phosphatidylinositol-3,4,5-triphosphate (PIP3), which is recognized by the enzyme Akt and the constitutively active 3'-phosphoinositide-dependent kinase 1 (PDK1) through their pleckstrin homology domains. The interaction of Akt with PIP3 leads to a conformational change in Akt and its phosphorylation at Thr308 and Ser473 by PDK1 and the rictor-mTOR complex, respectively. Phosphorylation of these two residues results in the activation of Akt, which then phosphorylates, among other substrates, the enzyme glycogen synthase kinase-3 (GSK-3). GSK-3 has two isoforms, GSK-3α and GSK-3β, both of which are constitutively active. These isoforms are structurally related but functionally non-redundant. Inactivation of GSK-3 is observed when the Ser21 residue of GSK-3α or the Ser9 residue of GSK-3β, both located in their regulatory N-terminal domains, are phosphorylated by Akt and other kinases. Inhibition of GSK-3 by phosphorylation is important in the modulation of inflammation and the phagocytic process.

[0325]

[0453] In some embodiments, the recombinant PFP comprises (a) an extracellular CD47-binding domain SIRPα, (b) a SIRPβ transmembrane domain, and (c) an intracellular domain of SIRPβ. SIRPβ signaling can activate pro-phagocytic signaling by participating in the activation of DAP12.

[0326]

[0454] Various members of this family transduce checkpoint signals upon contact with sialylated glycans on membrane proteins. In some members, the intracellular domain of Siglec proteins contains multiple immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The ITIMs share a consensus amino acid sequence in their cytoplasmic tails: (I / V / L / S)-XYXX-(L / V), where X represents any amino acid, I = isoleucine, V = valine, L = lysine, S = serine, and Y = tyrosine. Phosphorylation of the tyrosine residues in the ITIM motifs recruits two SH2 domain-containing negative regulators: the inositol phosphatase SHIP (Src homology 2-containing inositol polyphosphate 5-phosphatase) or the tyrosine phosphatase SHP-1 (Src homology 2-containing protein tyrosine phosphatase-1). Leucine at the (Y+2) position favors binding to SHIP, while isoleucine at the (Y-2) position favors binding to SHP-1. ITIMs can also bind to another tyrosine phosphatase, SHP-2, although evidence for a functional role for SHP-2 in ITIM-mediated inhibition is less clear than for other mediators. Thus, activation of Siglec membrane proteins at their extracellular ligand-binding domains by binding to sialic acid residues (e.g., by sialylated membrane glycan proteins) causes ITIMs to receive intracellular signals, become phosphorylated, and initiate SHP-mediated signaling for immune regulation, including reduced phagocytic activity.

[0327]

[0455] In some embodiments, the compositions described herein include a recombinant nucleic acid construct encoding a chimeric Siglec receptor (SgR) fusion protein (SgFP), wherein the SgFP comprises: (a) an SgR subunit comprising (i) a transmembrane domain and (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising an antigen-binding domain specific for sialylated glycans on a cell surface protein of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and (i) the SgFP does not comprise a functional intracellular domain of an endogenous receptor that binds to sialylated glycans, or (ii) the SgFP comprises an intracellular signaling domain that activates phagocytosis or an inflammatory pathway. In some embodiments, the chimeric receptor lacks the intracellular domain and thus acts as a blocker of Siglec-induced immunoregulatory intracellular signaling. This is achieved by deleting the nucleic acid region encoding the intracellular domain and cloning the remaining coding sequence of the Siglec receptor. This construct may be designated a siglec intracellular domain deletion construct [Siglec ICD]. In some embodiments, the recombinant nucleic acid construct encodes a recombinant chimeric antigen receptor comprising a cancer antigen-specific scFv fused to the extracellular domain (ECD) of a siglec receptor, allowing the construct to target cancer cells. The chimeric receptor comprises the TM and ICD of a siglec receptor, which may be the endogenous ICD or an ICD fused to an additional phagocytosis-promoting domain, such as one or more PI3K-binding domains. In some embodiments, the chimeric receptor comprising the extracellular siglec domain is coexpressed with a sialidase. A nucleic acid encoding the sialidase can be incorporated into an expression vector expressing the chimeric domain with a secretion signal sequence. Because the sialidase is expressed by the same cells in which the CAR-siglec receptor is expressed, expression of the sialidase prevents the ECD of the siglec from binding to its natural ligand, but activates it through binding of the scFv to the receptor, thereby ensuring specificity of the chimeric receptor's action on cells expressing the cancer antigen.

[0328]

[0456] In some embodiments, the chimeric receptor comprises one or more domains from a TREM protein fused at the extracellular region to an antigen-binding domain capable of specifically binding to a cancer antigen, e.g., a cancer antigen-specific antibody or a portion or fragment thereof. In some embodiments, the recombinant nucleic acid encoding the TREM chimeric antigen receptor encodes a fusion protein comprising (a) at least a TREM transmembrane domain (TM) and a TREM intracellular domain (ICD), and (b) an extracellular domain (ECD) comprising an antigen-binding domain capable of specifically binding to a cancer antigen. The fusion protein is designed to target cancer cells and bind to the target cancer cells via the ECD comprising the antigen-binding domain, and this binding induces and enhances phagocytosis by signaling through the TREM TM and / or intracellular domain. The TREM transmembrane domain trimerizes with the DAP12 transmembrane domain, triggering an intracellular pro-phagocytic signaling cascade. In some embodiments, the TREM domain is provided by a TREM1, TREM2, or TREM3 member. The extracellular antigen-binding domain is fused to the extracellular end of the TREM domain via a short spacer or linker.

[0329]

[0457] In some embodiments, the extracellular antigen-binding domain comprises an antibody specific for a cancer antigen, hi some embodiments, the extracellular antigen-binding domain comprises an antibody or antigen-binding portion thereof that specifically binds to an antigen on the surface of a cancer cell.

[0330]

[0458] In some embodiments, the extracellular antigen-binding domain is an antibody specific for a cancer antigen. In some embodiments, the extracellular antigen-binding domain is a fragment of an antibody, and the fragment can specifically bind to a cancer antigen on cancer cells. In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv) specific for the cancer antigen-binding domain.

[0331]

[0459] In some embodiments, the chimeric fusion protein (CFP) comprises a heavy chain variable region (V) having the amino acid sequence set forth in, for example, SEQ ID NO: 1. H In some embodiments, the chimeric CFP comprises a CD5-binding heavy chain variable domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1. In some embodiments, the extracellular domain (ECD) targeted to bind to CD5 (CD5-binding domain) comprises a light chain variable domain (V) having the amino acid sequence set forth in SEQ ID NO: 2. L In some embodiments, the chimeric CFP comprises a CD5-binding light chain variable domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2.

[0332]

[0460] In some embodiments, the CFP comprises an extracellular domain targeted to bind to HER2 (a HER2-binding domain), e.g., having a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 8 and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the CFP comprises a HER2-binding heavy chain variable domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 8. In some embodiments, the CFP comprises a HER2-binding light chain variable domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 9.

[0333]

[0461] In some embodiments, the CFP comprises a hinge that connects the ECD to the transmembrane (TM). In some embodiments, the hinge comprises the amino acid sequence of the hinge region of the CD8 receptor. In some embodiments, the CFP may comprise a hinge having the amino acid sequence set forth in SEQ ID NO: 7 (CD8 alpha chain hinge domain). In some embodiments, the PFP hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identical to SEQ ID NO: 7.

[0334]

[0462] In some embodiments, the CFP comprises a CD8 transmembrane region having, for example, the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the CFP TM region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 6.

[0335]

[0463] In some embodiments, the CFP comprises an intracellular domain having an FcR domain, hi some embodiments, the CFP comprises an intracellular domain having an FcR domain comprising the amino acid sequence set forth in SEQ ID NO:3, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.

[0336]

[0464] In some embodiments, the CFP comprises an intracellular domain having a PI3K recruitment domain. In some embodiments, the PI3K recruitment domain comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the PI3K recruitment domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[0337]

[0465] In some embodiments, the CFP comprises an intracellular domain that has a CD40 intracellular domain. In some embodiments, the CD40 ICD comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CD40 ICD comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5.

[0338]

[0466]

[0339] [Table 4-1]

[0340] [Table 4-2]

[0341] [Table 4-3]

[0342] [Table 4-4]

[0343] [Table 4-5]

[0344] [Table 4-6]

[0345] [Table 4-7]

[0346] [Table 4-8]

[0347] [Table 4-9]

[0348] [Table 5]

[0349]

[0467] The present disclosure thus provides all of the necessary components for construct design contemplated using any one domain in combination with another to construct chimeric proteins bearing the information provided herein. Any such domain can be used to generate a phagocytic receptor fusion protein, i.e., a CFP, or a CAR or dual CFP-CAR construct.

[0350] Characteristics of PFP

[0468] PFP is structurally integrated into the cell membrane of the cell in which it is expressed. A specific leader sequence, such as a signal peptide, of the nucleic acid construct directs the expression of the encoded protein at the cell membrane. The transmembrane domain encoded by the construct integrates the expressed protein into the cell membrane.

[0351]

[0469] In some embodiments, the transmembrane domain comprises the TM domain of the FcR-alpha receptor, which dimerizes with the endogenous FcRgamma receptor on myeloid cells, e.g., macrophages, ensuring myeloid cell-specific expression.

[0352]

[0470] In some embodiments, PFP provides cells expressing it with potent phagocytosis. When a recombinant nucleic acid encoding PFP is expressed in a cell, the cell exhibits increased phagocytosis of target cells bearing the target cell's antigen compared to cells that do not express the recombinant nucleic acid. When a recombinant nucleic acid is expressed in a cell, the cell exhibits increased phagocytosis of target cells bearing the target cell's antigen compared to cells that do not express the recombinant nucleic acid. In some embodiments, when a recombinant nucleic acid is expressed in a cell, the cell exhibits at least a two-fold increase in phagocytosis of target cells bearing the target cell's antigen compared to cells that do not express the recombinant nucleic acid. In some embodiments, when a recombinant nucleic acid is expressed in a cell, the cell exhibits at least a three-fold, four-fold, five-fold, six-fold, seven-...

Claims

1. A composition comprising an RNA comprising a sequence encoding a chimeric fusion protein (CFP) comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the transmembrane domain and the intracellular domain are operably linked, and the extracellular domain and the transmembrane domain intracellular domain are operably linked, wherein the RNA comprises a sequence complementary to an miRNA, and the miRNA is (i) expressed in myeloid cells, and binding of the miRNA to the sequence complementary to the miRNA promotes the expression of CFP in myeloid cells, or (ii) expressed in non-myeloid cells, not substantially expressed in myeloid cells, and binding of the miRNA to the sequence complementary to the miRNA promotes the degradation of the RNA in non-myeloid cells, the composition.

2. The composition according to claim 1, wherein the non-myeloid cells are neuronal cells, epithelial cells, hepatocytes, cardiomyocytes, embryonic stem cells, or endothelial cells.

3. The composition according to claim 1, wherein the RNA is mRNA.

4. The composition according to claim 3, wherein the mRNA comprises a 3'-untranslated region (3'UTR), and the sequence complementary to the miRNA is incorporated into the 3'UTR.

5. The composition according to claim 1, wherein the miRNA is miR-24 / 27, hsa-miR-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302d, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, miR-10a / b, miR-125a, miR-126, or miR-221 / 222.

6. The composition according to claim 1, wherein the transmembrane domain comprises a transmembrane domain derived from CD16a, CD64, CD68, or CD89, or a fragment thereof.

7. The composition according to claim 6, wherein the transmembrane domain comprises a transmembrane domain derived from CD89.

8. The composition according to claim 1, wherein the intracellular domain comprises an intracellular domain derived from CD16a, CD64, CD68, or CD89, or a fragment thereof.

9. The composition according to claim 8, wherein the intracellular domain comprises an intracellular domain derived from CD89.

10. The composition according to claim 1, formulated for systemic delivery.

11. The composition according to claim 1, wherein the RNA is encapsulated within lipid nanoparticles (LNP).

12. The composition according to claim 1, wherein the myeloid cells are CD14+ cells.

13. The composition according to claim 12, wherein the CD14+ cells are CD16- or CD16 ｌｏｗ cells.

14. The composition according to claim 1, wherein the extracellular domain comprises an antigen-binding domain specific for an antigen of a target cell.

15. The composition according to claim 14, wherein the target cell is a cancer cell.

16. The extracellular antigen-binding domain comprises a receptor domain or an antibody domain, and the antibody domain comprises a functional antibody fragment, a single-chain variable fragment (scFv), Fab, a single-domain antibody (sdAb), a nanobody, a V Ｈ domain, a V Ｌ domain, a VNAR domain, a V ＨＨ domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof of these, the composition according to claim 1.

17. The composition according to claim 14, wherein the antigen is selected from the group consisting of TROP2, glypican 3 (GPC3), human epidermal growth factor receptor 2 (HER2), and CD5.

18. The composition according to claim 17, wherein the antigen is TROP2.

19. The composition according to claim 17, wherein the antigen is GPC3.

20. The composition according to claim 17, wherein the antigen is HER2.

21. The composition according to claim 1, wherein the RNA comprises at least two copies of a sequence complementary to the miRNA.

22. The composition according to claim 21, wherein the RNA comprises two, three, four, or five copies of a sequence complementary to the miRNA.

23. A pharmaceutical composition comprising the composition according to any one of claims 1 to 22 and a pharmaceutically acceptable excipient.

24. The composition according to any one of claims 1 to 22 for use in treating a disease or condition in a subject in need thereof.

25. The composition according to claim 24, wherein the disease or condition comprises cancer.

26. The composition according to claim 25, wherein the cancer is selected from the group consisting of ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, lung cancer, and T cell lymphoma.

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