Engineered Chimeric Fusion Protein Compositions and Methods of Use Thereof

Abstract

Compositions and methods for making and using engineered cells, such as engineered bone marrow cells, that express a chimeric fusion protein having a binding domain capable of binding to a surface molecule on a target cell, such as a diseased cell.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 162,352, filed March 17, 2021, U.S. Provisional Patent Application No. 63 / 172,922, filed April 9, 2021, U.S. Provisional Patent Application No. 63 / 243,947, filed September 14, 2021, and U.S. Provisional Patent Application No. 63 / 255,540, filed October 14, 2021, each of which is incorporated by reference in its entirety into this specification. [Background technology]

[0002]

[0002] Cellular immunotherapy is a promising new technology for combating difficult-to-treat diseases, such as cancer and persistent infections, as well as certain diseases that are refractory to other forms of treatment. The discovery of CAR-T cells and their potential use in immunotherapy has resulted in a major breakthrough. CAR-T cells are T lymphocytes that express chimeric antigen receptors (CARs), which help target the T cells to specific diseased cells, such as cancer cells, and, depending on the intracellular domain employed and the immunosuppressive cytokines coexpressed, can either induce a cytotoxic response designed to kill the targeted cancer cells or induce immunosuppression and / or immune tolerance. While CAR T cells remain a promising tool for cancer treatment, several limitations along the way have slowed progress in CAR-T cells and diminished their promise in clinical trials.

[0003] Understanding the limitations of CAR-T cells is key to harnessing the technology and sustaining innovation toward successful immunotherapy models. Specifically, CAR-T cells face significant challenges in T-cell malignancies. Because CAR-T cells and malignant T cells share surface antigens in most T-cell lymphomas (TCLs), CAR-T cells, like cancer cells, are subject to cytotoxic effects. In some cases, CAR-T products may be contaminated by malignant T cells. In addition, T-cell aplasia is a potential problem for prolonged CAR-T cell survival. Other limitations include the poor ability of CAR-T cells to penetrate solid tumors and the potent tumor microenvironment, which acts to downregulate their antitumor potential. CAR-T cell function is also negatively affected by the immunosuppressive tumor microenvironment (TME), which leads to the inactivation and exhaustion of endogenous T cells.

[0004]

[0004] Myeloid cells, including macrophages, are derived from the myeloid lineage and belong to the innate immune system. They originate from bone marrow stem cells, which escape into the blood and migrate to tissues. Some of their primary functions include phagocytosis, activation of T cell responses, and removal of cellular debris and extracellular matrix. Myeloid cells also play important roles in maintaining homeostasis and in the initiation and resolution of inflammation. Furthermore, myeloid cells can differentiate into numerous downstream cells, including macrophages, which can display distinct responses ranging from pro-inflammatory to anti-inflammatory, depending on the type of stimuli they receive from the surrounding microenvironment. Furthermore, tissue macrophages have been shown to play a broad regulatory and activating role on other immune cell types, including CD8+ and CD4+ T effector cells, NK cells, and regulatory T cells. Macrophages have been shown to be the primary immune infiltrate within malignant tumors and to exert a broad immunosuppressive effect on effector immune infiltration and immune function. Summary of the Invention

[0005] The diverse functionality of bone marrow cells makes them ideal candidates for cellular therapy, which can be engineered to exert multiple therapeutic effects. The present disclosure relates to immunotherapy using bone marrow cells of the immune system (e.g., CD14+ cells), particularly phagocytes. Numerous therapeutic applications using bone marrow cells may be envisioned. For example, immunotherapy with bone marrow cells may be crucial in cancer, autoimmune, fibrotic diseases, and infectious diseases. The present disclosure relates to immunotherapy using phagocytes of the immune system, particularly bone marrow cells, including monocytes. It is an object of the present invention to utilize one or more of these functions of bone marrow cells for therapeutic use. For example, it is an object of the present invention to utilize the phagocytic activity of bone marrow cells, including engineered bone marrow cells, for therapeutic use. For example, it is an object of the present invention to utilize the ability of bone marrow cells, including engineered bone marrow cells, to promote T cell activation. For example, it is an object of the present invention to utilize the ability of bone marrow cells, including engineered bone marrow cells, to promote the secretion of tumor-killing molecules. For example, it is an object of the invention disclosed herein to harness the ability of bone marrow cells, including engineered bone marrow cells, to promote the recruitment and transport of immune cells and immune molecules. In one aspect, the present disclosure provides new and useful chimeric constructs that, when expressed in bone marrow cells, drive the bone marrow cells to target and phagocytose a molecule, molecular assembly, object, or target, e.g., a cell containing a target antigen on its surface. One of the many aspects of the present disclosure is (i) to enhance the phagocytic ability of bone marrow cells (e.g., engineered bone marrow cells expressing new and improved chimeric constructs); helping to elicit an organized and sustained immune response against a target (e.g., a target antigen). The present disclosure presents novel methods and compositions that can successfully transfect or transduce bone marrow cells or otherwise induce genetic modifications in bone marrow cells, with the aim of enhancing the functional aspects of the bone marrow cells, while not impairing the cells' differentiation potential, their maturation potential, and / or plasticity. The resulting cells may be referred to as therapeutically effective, engineered or effector bone marrow cells.One strategy for the improvements described herein is to induce the inflammatory phenotype of bone marrow cells to develop into effector bone marrow cells. One strategy is to generate effector bone marrow cells that can assume an inflammatory phenotype upon engagement with a target. In one embodiment, the effector bone marrow cells can induce or activate a type I interferon response in the bone marrow cells upon engagement with their target. Another strategy is to enhance the type I interferon response in the bone marrow cells to cause these cells to develop into effector bone marrow cells. One strategy for inducing an inflammatory phenotype is to induce or activate an NF-kB response in the effector bone marrow cells upon engagement with their target. One strategy for developing bone marrow cells isolated from a biological sample into effector bone marrow cells for the immunotherapy described herein is to induce and / or enhance a type I interferon response, an NF-kappaB response, or both in the bone marrow cells.

[0006]

[0006] The present disclosure involves the production and use of engineered bone marrow cells (e.g., CD14+ cells), such as macrophages or other phagocytes, that can directly and / or indirectly attack and kill diseased cells, such as cancer cells and infected cells (ATAK). Engineered bone marrow cells, such as macrophages and other phagocytes, can be prepared by incorporating into cells, using recombinant nucleic acid technology, synthetic nucleic acids, gene editing methods (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection, nucleic acid sequences (e.g., mRNA, DNA, plasmids, viral constructs) encoding chimeric fusion proteins (CFPs) with extracellular binding domains specific for disease-associated antigens (e.g., cancer antigens). It has been discovered that bone marrow cells can be engineered to exert a broad and diverse range of activities. For example, it has been found that bone marrow cells can be engineered to express chimeric fusion proteins (CFPs) containing antigen-binding domains to exert a wide and diverse range of activities. For example, it has been found that bone marrow cells can be engineered to have enhanced phagocytic activity, such that upon binding of the CFP to an antigen on a target cell, the cells exhibit increased phagocytosis of the target cell. It has also been found that bone marrow cells can be engineered to promote T cell activation, such that upon binding of the CFP to an antigen on a target cell, the cells promote the activation of T cells, such as T cells, within the tumor microenvironment. Engineered bone marrow cells can be engineered to promote the secretion of tumor-killing molecules from nearby cells, such that upon binding of the CFP to an antigen on the target cell, the cells promote the recruitment and transport of immune cells and immune molecules to the target cell or tumor microenvironment, such that upon binding of the CFP to an antigen on the target cell, the cells promote the recruitment and transport of immune cells and immune molecules to the target cell or tumor microenvironment.

[0007]

[0007] The present disclosure is based on the important discovery that engineered bone marrow cells overcome at least some of the limitations of CAR-T cells, including that they are readily recruited to solid tumors; their survival is manipulable, thus reducing the risk of prolonged survival resulting in aplasia and immunodeficiency; bone marrow cells cannot be contaminated with T cells; bone marrow cells may avoid fratricide because, for example, they do not express the same antigens as malignant T cells; and bone marrow cells have a wealth of anti-tumor functions that can be deployed. In some respects, engineered bone marrow-derived cells may be a safer immunotherapy tool for targeting and destroying diseased cells.

[0008]

[0008] Furthermore, myeloid cells such as macrophages are found ubiquitously within 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 the utilization of myeloid cell function, particularly the targeting, killing, and direct and / or indirect elimination of diseased cells, as well as the delivery of payloads such as antigens and cytokines.

[0009] Engineered bone marrow cells are also short-lived, phenotypically diverse, highly sensitive, and plastic in vivo, often proving difficult to manipulate in vitro. For example, expressing exogenous genes in monocytes is more challenging than expressing exogenous genes in nonhematopoietic cells. There are significant technical challenges associated with transfecting bone marrow cells (e.g., monocytes / macrophages). As professional phagocytes, bone marrow cells such as monocytes / macrophages contain many potent degradative enzymes that can disrupt the integrity of nucleic acids, making gene transfer into these cells an inefficient process. This is particularly true for activated macrophages, which undergo dramatic changes in their physiology after exposure to immune or inflammatory stimuli. Because macrophages are generally non-dividing, end-stage cells, viral transduction of these cells is not ideal; for this reason, some vectors that rely on integration into replicating genomes have met with limited success. Furthermore, because macrophages are highly sensitive to "danger signals," some of the original viral vectors used for gene transfer induce strong antiviral responses in these cells, making them unsuitable for gene delivery. Additionally, bone marrow cells have the potential to differentiate into phenotypic variants with significantly different functionalities, which, under certain circumstances, may not be effective for their intended purpose in vivo. For example, bone marrow cells required for active migration to tumors and effective tumor-killing activity may require in vivo delivery at a cellular stage that retains their plasticity, based on the identification of certain cell surface markers. The plasticity of bone marrow cells may be affected by the nature of isolation, handling, and the method of ex vivo introduction of nucleic acid material into the cells to manipulate them.While bone marrow cells at various cellular stages (e.g., bone marrow cells at high or low stages of plasticity, or bone marrow cells with phenotypes that confer inflammatory or other immunomodulatory functions) each have particular value for their intended purpose for in vivo use, and all of these are within the scope of the present disclosure, in at least one aspect, the methods discussed herein focus on generating therapeutic bone marrow cell populations that retain a high degree of bone marrow cell plasticity prior to in vivo administration. In one aspect, the present disclosure presents novel methods and compositions that can successfully transfect or transduce, or otherwise induce genetic modifications in, bone marrow cells with the aim of enhancing functional aspects of the bone marrow cells, as well as not impairing the cells' differentiation potential, their maturation potential, and / or plasticity.

[0010]

[0010] In one aspect of the present specification, a therapeutic agent is provided that binds to an antigen expressed on a target cell, such as a diseased cell, e.g., a cancer cell. Binding of the therapeutic agent to the target antigen on the target cell can initiate a process of destruction of the target cell. In some embodiments, the therapeutic agent is a recombinant nucleic acid that can be expressed in a cell of a subject, such as a mammalian cell, a human cell, a bone marrow cell, or a monocyte. In some embodiments, the therapeutic agent is a recombinant protein that can bind to a target antigen on the target cell. In some embodiments, the therapeutic agent is a cell, e.g., a mammalian cell, a human cell, a bone marrow cell, or a monocyte, that is capable of targeting bone marrow cells to diseased cells that express the target antigen on their cell surface; the bone marrow cells contain the recombinant nucleic acid and / or express the recombinant protein such that the bone marrow cells lyse or phagocytose the diseased cells.

[0011]

[0011] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the chimeric fusion protein responds to an extracellular cue, and wherein upon receipt of the extracellular cue, the intracellular domain affects an intracellular mechanism of action and activation of myeloid cells. In one embodiment, the chimeric fusion protein is a chimeric receptor having an extracellular antigen-binding domain in addition to (a) the transmembrane domain and (b) the intracellular domain operably linked to the transmembrane domain, wherein engagement of the extracellular antigen-binding domain of the chimeric fusion protein with a target antigen bound by the extracellular binding domain provides the receptor with an extracellular cue for receptor-mediated activation of myeloid cells.

[0012]

[0012] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises an intracellular signaling domain having a Xaa1-Leu-Xaa3-Iso-Ser motif, where Xaa1 is a hydrophilic amino acid and Xaa3 is any amino acid. In some embodiments, Ser in Xaa1-Leu-Xaa3-Iso-Ser is a phosphorylation site.

[0013]

[0013] In some embodiments, Xaa1 is serine, threonine, tyrosine, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, or arginine. In some embodiments, Xaa1 is serine. In some embodiments, Xaa1 is threonine. In some embodiments, Xaa1 is tyrosine. In some embodiments, Xaa1 is histidine. In some embodiments, Xaa1 is asparagine, aspartic acid, glutamine, glutamic acid, lysine, or arginine. In some embodiments, Xaa1 is asparagine. In some embodiments, Xaa1 is aspartic acid. In some embodiments, Xaa1 is glutamine. In some embodiments, Xaa1 is glutamic acid. In some embodiments, Xaa1 is lysine. In some embodiments, Xaa1 is arginine.

[0014]

[0014] In some embodiments, Xaa1 is histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, or arginine. In some embodiments, Xaa1 is histidine. In some embodiments, Xaa1 is asparagine, aspartic acid, glutamine, glutamic acid, lysine, or arginine. In some embodiments, Xaa1 is asparagine. In some embodiments, Xaa1 is aspartic acid. In some embodiments, Xaa1 is glutamine. In some embodiments, Xaa1 is glutamic acid. In some embodiments, Xaa1 is lysine. In some embodiments, Xaa1 is arginine. In some embodiments, Xaa1 is not serine. In some embodiments, Xaa1 is not threonine. In some embodiments, Xaa1 is not tyrosine.

[0015]

[0015] In some embodiments, Xaa1 is serine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, or arginine. In some embodiments, Xaa1 is serine. In some embodiments, Xaa1 is asparagine. In some embodiments, Xaa1 is aspartic acid. In some embodiments, Xaa1 is glutamine. In some embodiments, Xaa1 is glutamic acid. In some embodiments, Xaa1 is lysine. In some embodiments, Xaa1 is arginine. In some embodiments, Xaa1 is not histidine. In some embodiments, Xaa1 is not threonine. In some embodiments, Xaa1 is not tyrosine.

[0016]

[0016] In some embodiments, Xaa1 is asparagine, aspartic acid, glutamine, glutamic acid, lysine, or arginine. In some embodiments, Xaa1 is asparagine. In some embodiments, Xaa1 is aspartic acid. In some embodiments, Xaa1 is glutamine. In some embodiments, Xaa1 is glutamic acid. In some embodiments, Xaa1 is lysine. In some embodiments, Xaa1 is arginine. In some embodiments, Xaa1 is not serine. In some embodiments, Xaa1 is not threonine. In some embodiments, Xaa1 is not tyrosine. In some embodiments, Xaa1 is not histidine.

[0017]

[0017] In some embodiments, Xaa1-Leu-Xaa3-Iso-Ser is SLHIS.

[0018]

[0018] In some embodiments, Xaa1-Leu-Xaa3-Iso-Ser is NLEIS.

[0019]

[0019] In some embodiments, Xaa1-Leu-Xaa3-Iso-Ser is DLAIS.

[0020]

[0020] In some embodiments, Xaa1-Leu-Xaa3-Iso-Ser is ELLIS.

[0021]

[0021] In some aspects, the chimeric fusion protein further comprises an extracellular domain comprising an antigen-binding domain, wherein the transmembrane domain is operably linked to the extracellular domain. In some embodiments, the intracellular domain comprises at least one additional intracellular signaling domain. In some embodiments, the at least one additional intracellular signaling domain is derived from an intracellular PI3 kinase recruitment domain, a phagocytic receptor intracellular domain, a pattern recognition receptor intracellular domain, a CD40 intracellular domain, an FcR intracellular domain, or a cytokine receptor intracellular domain or a chemokine receptor intracellular domain. In some embodiments, the intracellular signaling domain comprises an amino acid sequence derived from a cytosolic adaptor protein, a mitochondrial membrane protein, or an endoplasmic reticulum membrane protein. In one aspect, the intracellular signaling domain of the chimeric fusion protein comprises an amino acid sequence derived from a TRIF protein. In some embodiments, the intracellular signaling domain comprises a truncated TRIF intracellular domain. In some embodiments, the sequence of the truncated TRIF intracellular domain is about 150-300 amino acids in length, about 150-270 amino acids in length, or about 150-250 amino acids in length. In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 36. In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 41.

[0022]

[0022] In one aspect of the present specification, a recombinant nucleic acid is provided encoding a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises a signaling domain derived from G protein-coupled receptor 84 (GPR84), G protein subunit beta 2 (GNB2), or phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1). The intracellular signaling domain of GPR84, GNB2, or PREX1 can have pro-phagocytic activity. In some embodiments, the chimeric fusion protein further comprises an extracellular domain comprising an antigen-binding domain, wherein the transmembrane domain is operably linked to the extracellular domain. In some embodiments, the signaling domain derived from GPR84, GNB2, or PREX1 is an intracellular signaling domain derived from GPR84, GNB2, or PREX1.

[0023]

[0023] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises an intracellular signaling domain comprising a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 39.

[0024]

[0024] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises an intracellular signaling domain comprising a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 40.

[0025]

[0025] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises an intracellular signaling domain comprising a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 42.

[0026]

[0026] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises an intracellular signaling domain comprising a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 43.

[0027]

[0027] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises an intracellular signaling domain comprising a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 45.

[0028]

[0028] In one aspect of the present specification, there is provided a recombinant nucleic acid encoding a chimeric fusion protein comprising (a) a transmembrane domain, and (b) an extracellular domain comprising an antigen-binding domain, wherein the transmembrane domain is operably linked to the extracellular domain, and the transmembrane domain is derived from CD68 and comprises a mutation that inhibits or prevents homodimerization.

[0029]

[0029] In some embodiments, the chimeric fusion protein further comprises an intracellular domain operably linked to the transmembrane domain. In some embodiments, the transmembrane domain comprises SEQ ID NO:47.

[0030]

[0030] In one aspect herein, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an extracellular domain comprising an antigen-binding domain, wherein the transmembrane domain is operably linked to the extracellular domain, and the transmembrane domain is derived from CD64 or CD89, and the chimeric fusion protein lacks an intracellular signaling domain. In some embodiments, the transmembrane domain is derived from a CD16 protein, e.g., a CD16a protein. In some embodiments, the chimeric fusion protein forms a complex with the FcRγ chain when expressed intracellularly.

[0031]

[0031] In one aspect of the present specification, a recombinant nucleic acid is provided that encodes a chimeric fusion protein comprising (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain comprises an intracellular signaling domain comprising a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 48.

[0032] In some embodiments, the chimeric fusion protein further comprises a second intracellular signaling domain comprising a PI3 kinase recruitment domain. In some embodiments, the CFP further comprises an intracellular signaling domain comprising a fusion amino acid sequence comprising SEQ ID NO: 39 and the sequence of an IRF activation-enhancing motif. In some embodiments, the CFP further comprises an intracellular signaling domain comprising a fusion amino acid sequence comprising SEQ ID NO: 40 and the sequence of an IRF activation-enhancing motif.

[0033]

[0033] In one embodiment, the CFP further comprises an intracellular signaling domain comprising a fusion amino acid sequence comprising SEQ ID NO:39 and SEQ ID NO:40.

[0034] In some embodiments, the intracellular domain binds to an IRF protein. In some embodiments, the intracellular domain comprises a TRAF protein binding domain. In some embodiments, the intracellular domain comprises a TBK1 phosphorylation site. In some embodiments, the CFP further comprises a second, third, or fourth intracellular signaling domain. In some embodiments, the CFP further comprises an extracellular domain comprising an antigen-binding domain, wherein the transmembrane domain is operably linked to the extracellular domain.

[0035] In some embodiments, the antigen-binding domain comprises a domain having affinity for a tumor antigen. In some embodiments, the extracellular domain comprises a T lymphocyte antigen-binding domain. In some embodiments, the extracellular domain comprises a B lymphocyte antigen-binding domain. In some embodiments, the antigen to which the antigen-binding domain binds is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, CD56, 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, and fibroblast activity. FAP, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer 2D (NKG2D) group ligand, disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD70, CD56, CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin, integrin receptor, Claudin 3.0, Claudin 18.2, Trop-2, PRSS21, VEGFR2, PDGFR beta, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, and IGLL1. In some embodiments, the antigen-binding domain binds to CD5. In some embodiments, the antigen-binding domain binds to HER2.In some embodiments, the antigen-binding domain binds to CD137, CD70, Claudin 3.0, Claudin 18.2, or Trop-2. In some embodiments, the extracellular domain comprises a first target antigen-binding domain and a second target antigen-binding domain that is not identical to the first target binding domain. In some embodiments, the second target antigen-binding domain binds to CD47. In some embodiments, the antigen-binding domain comprises a domain with affinity for a microbial antigen. In some embodiments, the antigen-binding domain comprises a domain with affinity for a viral antigen. In some embodiments, the domain comprises an antibody or fragment thereof. In some embodiments, the domain comprises an scFv. In some embodiments, the transmembrane domain is derived from the transmembrane domain of CD8a, CD28, CD68, CD2, FcRγ, FcRα, FcRβ, FcRε, syntaxin 3, syntaxin 4, or syntaxin 5. In some embodiments, the extracellular domain comprises a hinge domain connecting the antigen-binding domain and the transmembrane domain. In some embodiments, when expressed intracellularly, the chimeric fusion protein activates the intracellular domain upon binding to its target antigen, inducing IRF activation. 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 associated with one or more lipids. In some embodiments, the recombinant nucleic acid is encapsulated in a liposome. In some embodiments, the liposome is a lipid nanoparticle.

[0036]

[0036] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 52. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 52. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 52. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 52. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 52 or a sequence having at least 80% sequence identity to SEQ ID NO: 52.

[0037]

[0037] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 53. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 53. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 53. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 53. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 53 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 53.

[0038]

[0038] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 54. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 54. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 54. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 54. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 54 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 54.

[0039]

[0039] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 55. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 55. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 55. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 55. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 55 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 55.

[0040]

[0040] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 56. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 56. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 56. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 56. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 56 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 56.

[0041]

[0041] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 57. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 57. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 57. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 57. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 57 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 57.

[0042]

[0042] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 58. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 58. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 58. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 58. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 58 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 58.

[0043]

[0043] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 59. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 59. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 59. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 59. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 59 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 59.

[0044]

[0044] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 60. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 60. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 60. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 60. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 60 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 60.

[0045]

[0045] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 61. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 61. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 61. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 61. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 61 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 61.

[0046]

[0046] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 62. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 62. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 62. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 62. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 62 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 62.

[0047]

[0047] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 63. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 63. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 63. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 63. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 63 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 63.

[0048]

[0048] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 64. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 64. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 64. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 64. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 64 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 64.

[0049]

[0049] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 65. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 65. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 65. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 65. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 65 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 65.

[0050]

[0050] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 66. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 66. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 66. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 66. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 66 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 66.

[0051]

[0051] Provided herein in one aspect is a recombinant nucleic acid encoding a sequence at least 80% identical to SEQ ID NO: 67. Provided herein in one aspect is a recombinant mRNA construct encoding a sequence at least 80% identical to SEQ ID NO: 67. Provided herein is a recombinant mRNA construct encoding a sequence at least 90% identical to SEQ ID NO: 67. Provided herein is a recombinant mRNA construct encoding a sequence identical to SEQ ID NO: 67. Provided herein in some embodiments is a cell comprising a recombinant nucleic acid encoding the sequence of SEQ ID NO: 67 or a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 67.

[0052]

[0052] In some embodiments, the recombinant nucleic acid is a plasmid or vector.

[0053]

[0053] In one aspect of the present specification, a cell is provided comprising a recombinant nucleic acid according to any one of the embodiments described above. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a myeloid cell, a lymphoid cell, a progenitor cell, a stem cell, or an induced pluripotent cell. In some embodiments, the cell is CD14+ / CD16-.

[0054]

[0054] In one aspect of the present specification, an engineered cell is provided that comprises a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain operably linked to the extracellular domain; and (c) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular signaling domain comprises an intracellular signaling domain that binds to an IFN transcription factor when activated.

[0055]

[0055] In one aspect of the present specification, an engineered cell is provided that comprises a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain operably linked to the extracellular domain; and (c) an intracellular domain operably linked to the transmembrane domain, and the intracellular signaling domain comprises an intracellular signaling domain derived from a mitochondrial receptor.

[0056] In some embodiments, the intracellular domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 39. In some embodiments, the intracellular domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 40.

[0057] In some embodiments, the intracellular domain is selected from the group consisting of TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, MAVS, TRIF, TASL, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), TNFR1, chemokine, MHC class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) receptor. These proteins contain intracellular signaling domains derived from proteins, 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 proteins, Dectin-1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily proteins, DCIR / CLEC4A, Dectin-2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), and Mincle (macrophage-inducible C-type lectin) (CLEC4E).

[0058]

[0058] In some embodiments, the intracellular domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:42.

[0059]

[0059] In some embodiments, the intracellular domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:43.

[0060]

[0060] In some embodiments, the intracellular domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:45.

[0061]

[0061] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain that binds to an IFN transcription factor upon activation.

[0062] In some embodiments, the IFN transcription factor is any one of the proteins IRF1-IRF9.

[0063] In some embodiments, the intracellular domain comprises a TRAF recruitment domain.

[0064] In some embodiments, the intracellular domain induces IKK phosphorylation.

[0065] In some embodiments, the intracellular domain induces TBK phosphorylation.

[0066]

[0066] In some embodiments, the antigen-binding domain binds to a tumor antigen or a microbial antigen.

[0067] In some embodiments, the antigen-binding domain is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, CD56, 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 Activator protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer 2D (NKG2D) group ligand, disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56, CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin, integrin receptor, Claudin 3.0, Claudin 18.2, Trop-2, PRSS21, VEGFR2, PDGFRbeta, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, and IGLL1.

[0068]

[0068] In some embodiments, the antigen-binding domain binds to a viral antigen.

[0069]

[0069] In some embodiments, the transmembrane domain is derived from the transmembrane domain of CD8a, CD28, CD68, CD2, FcRγ, FcRα, FcRβ, FcRε, syntaxin 3, syntaxin 4, or syntaxin 5.

[0070] In some embodiments, the intracellular domain further comprises an intracellular signaling domain derived from FcRγ, FcRα, or FcRε, hi some embodiments, the intracellular domain further comprises a PI3 kinase recruitment domain.

[0071]

[0071] In some embodiments, the intracellular signaling domain binds to an IRF transcription factor and activates the IRF transcription factor upon binding of the antigen binding domain to its cognate antigen.

[0072]

[0072] In some embodiments, the cell induces pro-inflammatory cytokines upon binding of the antigen-binding domain to its cognate antigen.

[0073]

[0073] In some embodiments, the pro-inflammatory cytokines include one or more of IL-1, IL-6, IL-12, IL-23, TNF, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, and interferon.

[0074] In some embodiments, the cell is a bone marrow cell, a lymphocyte, or a stem cell.

[0075] In some embodiments, the cells are bone marrow cells.

[0076]

[0076] Also provided herein, in one aspect of the disclosure, is a pharmaceutical composition comprising a recombinant nucleic acid according to any one of the embodiments described herein, a cell according to any one of the embodiments described herein, or an engineered cell according to any one of the embodiments described herein; and a pharmaceutically acceptable excipient.

[0077]

[0077] In some embodiments, the engineered cells are CD14+ / CD16-.

[0078]

[0078] Pharmaceutical compositions according to the embodiments described above include a population of cells, wherein at least 50% of the cells are CD14+ / CD16- and less than 10% of the cells are dendritic cells. In some embodiments, the cells exhibit high expression of CCR2. In some embodiments, the cells do not exhibit tonal signaling and de novo activation, and upon activation, exhibit M0, M1, or M2 differentiation.

[0079]

[0079] In some aspects of the present specification, there is provided a method for treating cancer or a viral infection in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of the above embodiments.

[0080]

[0080] In some aspects of the present specification, there is provided an engineered CD14+ / CD16- cell comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain operably linked to the extracellular domain; and (c) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular signaling domain comprises a domain derived from TRIF.

[0081]

[0081] In some embodiments, the intracellular domain comprising a domain derived from TRIF is a truncated TRIF domain.

[0082]

[0082] In some embodiments, the intracellular domain comprising the domain derived from TRIF comprises fewer than 300 amino acids.

[0083]

[0083] In some embodiments, the intracellular domain comprising a domain derived from TRIF comprises fewer than 250 amino acids.

[0084]

[0084] In some embodiments, the intracellular domain, which includes a domain derived from TRIF, elicits an IFN response upon engagement of the extracellular binding domain with a target.

[0085]

[0085] In one aspect of the present specification, there is provided a method for treating cancer in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of the embodiments described above.

[0086]

[0086] In one aspect of the present specification, there is provided a method for treating cancer in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of the embodiments described above.

[0087]

[0087] In some embodiments, the cancer is selected from the group consisting of gastric cancer, ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.

[0088] Incorporation by Reference

[0088] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0089] 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 of which: [Brief explanation of the drawings]

[0090] [Figure 1A]

[0090] Figure 1 depicts a schematic showing an exemplary CFP containing an extracellular binding domain and a transmembrane domain. The two figures on the left represent CFPs containing multiple intracellular signaling domains for enhancing intracellular signaling associated with phagocytosis or other domains for intracellular signaling for enhancing immune response or inflammatory signaling within cells expressing the CFP. The figure on the right illustrates another exemplary chimeric fusion protein containing a transmembrane domain derived from the Fc alpha R1 chain (FcaR1 chain or CD89) or the Fc gamma R1 chain (FcgR1 chain or CD64). Such a CFP, as shown in the figure on the right, may lack its own intracellular domain, and intracellular signaling will occur if and only if the CFP binds to an endogenous protein in the cell in which it is expressed and has an intracellular domain, or otherwise induces intracellular signaling. As described elsewhere in this disclosure, the exemplary CFP shown in the diagram on the right is capable of binding to endogenous Fc gamma receptors expressed on myeloid cells, after which the CFP becomes functional in intracellular signaling and activation of myeloid cells via activation of the Fc gamma receptor. The extracellular domain is the target binding domain. [Figure 1B]

[0091] 1 is a schematic representation of an exemplary myeloid cell that expresses the CFP receptor and engages cancer cells by binding to a target antigen on the cancer cell. Upon target engagement, activation of the intracellular signaling domain enhances phagocytosis. TM: transmembrane domain. [Figure 1C]

[0092] 1 is a diagrammatic representation of an exemplary CFP expressed on myeloid cells, which can target cancer antigens and activate type 1 interferon. It has an intracellular domain. FcR: Fc receptor. [Figure 1D]

[0093] FIG. 1 shows a schematic for an exemplary nucleic acid encoding an exemplary chimeric antigen receptor construct comprising an intracellular signaling domain (ICD) with a pLxIS motif that elicits a type I interferon response. [Figure 2A]

[0094] The upper panel of Figure 2A depicts a schematic diagram showing exemplary CFP construct designs with the indicated domains. Each construct was generated and used for functional characterization. Each construct has a hinge domain and a transmembrane domain derived from the CD8 molecule. The lower panel of Figure 2A shows flow cytometry data supporting the expression of each construct in the upper panel in THP-1 cells. [Figure 2B]

[0095] The upper panel of Figure 2B depicts a schematic diagram showing exemplary CFP construct designs with the indicated domains. Each construct is generated and used for functional characterization. Each construct has a hinge domain and a transmembrane domain derived from the CD68 molecule. The lower panel of Figure 2B shows flow cytometry data supporting the expression of each construct shown in the upper panel in THP-1 cells. [Figure 2C]

[0096] The upper panel of Figure 2C depicts a schematic showing exemplary CFP construct designs with the indicated domains. The construct on the left has the CD64 transmembrane domain, and the construct on the right has the CD89 transmembrane domain. The lower panel of Figure 2C shows flow cytometry data supporting the expression of each construct shown in the upper panel in THP-1 cells. [Figure 2D]

[0097] The upper panel of Figure 2D depicts a schematic diagram showing exemplary CFP construct designs with the indicated domains. The two constructs on the left each have a CD64 transmembrane domain and the following additional intracellular signaling domains: the first CFP from the left contains a PI3K recruitment domain and a signaling domain derived from MDA5; the second from the left contains a CD40 intracellular signaling domain and a PI3K recruitment domain. The two constructs on the right have a CD89 transmembrane domain and the following additional intracellular signaling domains: the third CFP from the left contains a PI3K recruitment domain and a signaling domain derived from MDA5; and the fourth from the left contains a CD40 intracellular signaling domain and a PI3K recruitment domain. The lower panel of Figure 2D shows flow cytometry data supporting the expression of each construct shown in the upper panel in THP-1 cells. [Figure 2E]

[0098] FIG. 1 shows quantitative data on the relative expression levels of each of the indicated constructs in THP-1 cells derived from analysis of flow cytometry results. [Figure 2F]

[0099] Figure 1 shows exemplary CFP designs (upper panel) and expression data (lower panel) for constructs in THP-1 cells. Respective domains are indicated on the figure. The HER-FcR-PI3K construct has the CD8 TM domain, and the other constructs have the CD68 TM domain. [Figure 2G]

[0100]

[0023] Figure 1 shows an exemplary CFP design (top panel) and expression data (bottom panel) for the construct in THP-1 cells. Each domain is indicated on the diagram. [Figure 3]

[0101] FIG. 1 shows an exemplary CFP design in which the extracellular domain contains one or more scFvs directed towards binding to a single target antigen or multiple targets, with each scFv having a different antigen specificity. [Figure 4A]

[0102] 1 shows an exemplary assay setup for quantitative evaluation of target cell phagocytosis by bone marrow cells expressing CFP.SKOV3 tumor cell line is an exemplary target cell labeled with a cytoplasmic dye.Phagocytosis of target cells results in the release of the dye within bone marrow cells, which is then analyzed by flow cytometry.Exemplary flow cytometry data from a representative experiment is also shown. [Figure 4B]

[0103] Figure 4 depicts representative quantitative data showing the phagocytic index of bone marrow cells expressing each of the indicated constructs. E:T ratio: effector (myeloid) cell:target cell ratio. Experimental results were analyzed according to the method for Figure 4A. [Figure 4C]

[0104] FIG. 1 depicts representative data showing the phagocytic index of bone marrow cells expressing each of the indicated constructs. [Figure 4D]

[0105] FIG. 1 depicts representative data showing the phagocytic index of bone marrow cells expressing each of the indicated constructs. [Figure 4E]

[0106]

[0023] Figure 1 depicts representative data showing the phagocytic index of bone marrow cells expressing each of the indicated constructs. Exemplary flow cytometry plots are shown in the upper panel, and quantitative data are shown in the lower panel. The data confirmed that cells expressing the HER2-FcR-PI3K-TRIF (short chain) construct exhibited a higher phagocytic index compared to other constructs. [Figure 5A]

[0107] 1 shows an exemplary assay setup for quantitative evaluation of phagocytic lysis of target cells by bone marrow cells expressing CFP, and a multiplexed assay for analyzing cytokines and chemokines. The target cells express the luciferase gene, and upon pinocytosis and lysis of the target cells, luciferase is released into the bone marrow cells, which can be used to detect and quantify direct target cell lysis. [Figure 5B]

[0108]

[0023] Figure 1 shows representative data for target cell lysis by each set of bone marrow cells expressing the indicated CFP constructs. Left panel: results from an experiment using a 1:1 effector:target cell ratio; right panel: results from an experiment using a 5:1 effector:target cell ratio. [Figure 5C]

[0109] Figure 1 shows data supporting the activation of NF-kappa B in THP-1 cells mock-electroporated or electroporated with the indicated CFP constructs and assayed 24 hours after transfection in the presence or absence of SKOV3 target cells. Cells expressing constructs with the CD64 intracellular domain alone demonstrate high activation of NF-kappa B in the presence of target cells. Constructs with both a PI3 kinase recruitment domain and an innate immune response signaling domain derived from CD40 exhibit tonic signaling for NF-kappa B activation (graph in right panel). A positive control using an NF-kappa B agonist is shown in the graph on the left. [Figure 5D]

[0110] Figure 5B shows data supporting the activation of IFN in THP-1 cells 24 hours after transfection with the CFP constructs as in Figure 5C or mock-transfected THP-1 cells. The HER2-CD64 construct does not activate the IFN response. The multidomain construct (right graph) does not activate IFN in the absence or presence of target cells. A positive control using a STING agonist is shown in the left graph. [Figure 5E]

[0111]

[0023] Figure 1 shows data supporting the activation of NF-kappa B in THP-1 cells that were mock-electroporated or electroporated with the indicated CFP constructs and assayed 24 hours after transfection in the presence or absence of SKOV3 target cells. Each construct contains a CD89 intracellular signaling domain. The CD89 intracellular domain alone is capable of activating NF-kappa B, but the additional intracellular domain did not induce NF-kappa B activation. [Figure 5F]

[0112] FIG. 5B shows data supporting activation of IFN in THP-1 cells 24 hours after transfection of CFP constructs as in FIG. 5E or mock-transfected THP-1 cells. [Figure 5G]

[0113]

[0023] Figure 1 shows data supporting activation of NF-kappa B in THP-1 cells expressing the individual constructs indicated. Cells expressing constructs with an FcR domain or an FcR domain together with an additional intracellular domain were examined and the results are shown. [Figure 5H]

[0114] FIG. 1 shows flow cytometry data confirming IFN activation in THP-1 cells 24 hours after electroporation with the indicated CFP constructs. [Figure 5I]

[0115] Figure 1 shows data supporting the expression of different HER2-CFP constructs with different intracellular domains, including the TLR domain. Flow cytometry analysis of cell surface expression of the HER2-binding agent CFP in mRNA-electroporated THP-1 cells using labeled antibody anti-HER2-AF647. The percentage of cells expressing the HER2-binding agent is indicated on the figure. [Figure 5J]

[0116]

[0023] Figure 1 shows flow cytometry data confirming expression of the indicated HER2-CFP constructs (upper panel) and activation of NF-kappa B in THP-1 cells 24 hours after electroporation with the indicated CFP constructs (lower panel). Expression of the HER2-FcR-PI3 kinase-MyD88 construct resulted in sustained NF-kappa B signaling (signaling in the absence of target SKOV3 cells, i.e., in the absence of engagement of the extracellular binding domain of CFP to antigen on SKOV3 cells). [Figure 5K]

[0117]

[0023] Figure 1 shows flow cytometry data confirming expression of the indicated HER2-CFP constructs and activation of NF-kappa B and IFN in THP-1 cells 24 hours after electroporation with the indicated HER2-CFP constructs. The data confirmed that sustained NF-kappa B / IFN gamma signaling was seen in multidomain constructs with the FcR-PI3K-RIG1 intracellular domain or the FcR-PI3K-MDA5 intracellular domain. [Figure 5L]

[0118] FIG. 1 shows flow cytometry data confirming expression of the indicated HER2-CFP constructs and activation of IFN in THP-1 cells 24 hours after electroporation with the indicated CFP constructs. [Figure 5M]

[0119] FIG. 1 shows flow cytometry data confirming expression of the indicated HER2-CFP constructs and activation of NF-kappa B in THP-1 cells 24 hours after electroporation with the indicated CFP constructs. [Figure 5N]

[0120] FIG. 1 shows flow cytometry data supporting expression of the indicated HER2-CFP constructs and cytokine expression by THP-1 cells 24 hours after electroporation with the indicated CFP mRNA constructs. [Figure 6A]

[0121] Figure 1 shows data supporting baseline cytokine (IL6 and TNFalpha) / chemokine (CCL3) expression by THP-1 cells expressing the indicated constructs compared to mock-transfected cells. A negative control (mock-transfected) graph is shown to the left of each CFP-transfected graph for each experimental set. [Figure 6B]

[0122] Figure 1 shows data supporting baseline cytokine (IL6 and TNFalpha) expression by THP-1 cells expressing the indicated constructs compared to mock-transfected cells. A negative control (mock-transfected) graph is shown to the left of each CFP-transfected graph for each experimental set. [Figure 6C]

[0123] Figure 1 shows data supporting baseline cytokine (IL6 and TNFalpha) expression by THP-1 cells expressing the indicated constructs compared to mock-transfected cells. A negative control (mock-transfected) graph is shown to the left of each CFP-transfected graph for each experimental set. [Figure 6D]

[0124] Figure 1 shows data supporting baseline cytokine (IL6, TNF-alpha, and IFN-gamma, as indicated on each graph) / chemokine (CCL3, CCL5, and IP10, as indicated) expression by THP-1 cells expressing the indicated constructs compared to mock-transfected cells. A negative control (mock-transfected) graph is shown to the left of each CFP-transfected graph for each experimental set. [Figure 6E]

[0125] FIG. 1 shows data supporting cytokine / chemokine expression in cells transfected with the indicated constructs compared to mock-transfected cells. [Figure 6F]

[0126] FIG. 1 shows data supporting cytokine / chemokine expression in cells transfected with the indicated constructs compared to mock-transfected cells. [Figure 7A]

[0127] FIG. 1 shows a schematic representation of an exemplary chimeric antigen receptor construct with the CD68 transmembrane domain. [Figure 7B]

[0128]

[0023] Figure 1 shows exemplary chimeric antigen receptors with the indicated domains. The top center and right panels show exemplary CFPs with CD64 transmembrane domains that oligomerize with endogenous Fc gamma and lack their own intracellular domains. The middle panel shows exemplary intracellular domains paired with various domain combinations in recombinant CFP construct designs. The bottom panel shows exemplary CFPs containing multiple domains. [Figure 8A]

[0129]

[0023] Figure 1 shows flow cytometry data confirming the expression of the indicated HER2-CFP constructs in primary human monocytes from a first donor after electroporation with the indicated HER2-CFP constructs. For example, HER2-CD8hTM-CD40-FcR-Pi3K refers to a construct having an anti-HER2 binding extracellular domain; a CD8 hinge domain and a CD8 transmembrane domain; and three distinct intracellular domains derived from the molecules CD40, FcR, and PI3 kinase. [Figure 8B]

[0130] FIG. 1 shows flow cytometry data confirming expression of the indicated HER2-CFP constructs in primary human monocytes from a second donor after electroporation with the indicated HER2-CFP constructs. [Figure 8C-1]

[0131] Figure 8 shows graphs of the percentage of cells positive and mean fluorescence intensity for the indicated HER2-CFP constructs using data from Figures 8A and 8B. Similar expression of the HER2-CFP constructs was observed in both donors. [Figure 8C-2] Continued from Figure 8C-1. [Figure 9A]

[0132] Figure 1 shows a graph of the killing activity of SKOV3 by primary human monocytes from two different donors electroporated with the indicated HER2-CFP constructs. 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were co-cultured with 20,000 SKOV3-GFP-luciferase tumor cells. After 72 hours of co-culture, luciferase activity in the tumor cells was detected, and killing was determined by a decrease in luciferase signal intensity. [Figure 9B]

[0133] Figure 1 shows a graph of the killing activity of SKBR3 by primary human monocytes from two different donors electroporated with the indicated HER2-CFP constructs. 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were co-cultured with 20,000 SKBR3-luciferase tumor cells. After 72 hours of co-culture, luciferase activity in the tumor cells was detected, and killing was determined by a decrease in luciferase signal intensity. [Figure 10A-1]

[0134] Figure 1 shows a graph of the induction of the pro-inflammatory cytokine IL-6 by primary human monocytes derived from human donors electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). For antigen-stimulated HER2-CFP primary monocyte samples, 96-well plates were coated with 2.5 μg / mL HER2-his protein, and 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were added per well. Supernatants were collected 48 hours after stimulation, and secreted cytokines were analyzed by Luminex. For tumor cell-stimulated HER2-CFP primary monocyte samples, 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were co-cultured with 20,000 tumor cells (SKOV3-GFP-luciferase cells or SKBR3-luciferase cells). Supernatants were collected 48 hours after stimulation, and secreted cytokines were analyzed by Luminex. Statistical significance was determined between primary monocyte samples electroporated with HER2-CFP and co-cultured with tumor cells and mock-electroporated and co-cultured with tumor cells. [Figure 10A-2] Continuation of Figure 10A-1. [Figure 10B-1]

[0135] Figure 10 shows a graph of the induction of the pro-inflammatory cytokine IL-1β by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analyses were performed as in Figure 10A. [Figure 10B-2] Continued from Figure 10B-1. [Figure 10C-1]

[0136] Figure 10 shows a graph of TNF-α induction by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 10A. [Figure 10C-2] Continued from Figure 10C-1. [Figure 10D-1]

[0137] Figure 10 shows a graph of IFN-α induction by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 10A. [Figure 10D-2] Continued from Figure 10D-1. [Figure 10E-1]

[0138] Figure 10 shows a graph of IP10 induction by primary human monocytes derived from human donors electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 10A. [Figure 10E-2] Continued from Figure 10E-1. [Figure 10F-1]

[0139] Figure 10 shows a graph of IL-12 induction by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 10A. [Figure 10F-2] Continued from Figure 10F-1. [Figure 11A]

[0140] FIG. 1 shows flow cytometry data confirming expression of the indicated HER2-CFP constructs in primary human monocytes from the first donor after electroporation with the indicated HER2-CFP constructs. [Figure 11B]

[0141] FIG. 1 shows flow cytometry data confirming expression of the indicated HER2-CFP constructs in primary human monocytes from a second donor after electroporation with the indicated HER2-CFP constructs. [Figure 11C-1]

[0142] FIG. 11 shows graphs of the percentage of cells positive and mean fluorescence intensity for the indicated HER2-CFP constructs using data from FIGS. 11A and 11B. [Figure 11C-2] Continued from Figure 11C-1. [Figure 12A]

[0143] Figure 1 shows a graph of the killing activity of SKOV3 by primary human monocytes from two different donors electroporated with the indicated HER2-CFP constructs. 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were co-cultured with 20,000 SKOV3-GFP-luciferase tumor cells. After 72 hours of co-culture, luciferase activity in the tumor cells was detected, and killing was determined by a decrease in luciferase signal intensity. [Figure 12B]

[0144] Figure 1 shows a graph of the killing activity of SKBR3 by primary human monocytes from two different donors electroporated with the indicated HER2-CFP constructs. 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were co-cultured with 20,000 SKBR3-luciferase tumor cells. After 72 hours of co-culture, luciferase activity in the tumor cells was detected, and killing was determined by a decrease in luciferase signal intensity. [Figure 13A-1]

[0145] Figure 1 shows a graph of the induction of the pro-inflammatory cytokine IL-6 by primary human monocytes derived from human donors electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). For antigen-stimulated HER2-CFP primary monocyte samples, 96-well plates were coated with 2.5 μg / mL HER2-his protein, and 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were added per well. Supernatants were collected 48 hours after stimulation, and secreted cytokines were analyzed by Luminex. For tumor cell-stimulated HER2-CFP primary monocyte samples, 100,000 primary human monocytes electroporated with the indicated HER2-CFP constructs were co-cultured with 20,000 tumor cells (SKOV3-GFP-luciferase cells or SKBR3-luciferase cells). Supernatants were collected 48 hours after stimulation, and secreted cytokines were analyzed by Luminex. Statistical significance was determined between primary monocyte samples electroporated with HER2-CFP and co-cultured with tumor cells and mock-electroporated and co-cultured with tumor cells. [Figure 13A-2] Continuation of Figure 13A-1. [Figure 13B-1]

[0146] Figure 13B shows a graph of the induction of the pro-inflammatory cytokine IL-1β by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analyses were performed as in Figure 13A. [Figure 13B-2] Continued from Figure 13B-1. [Figure 13C-1]

[0147] Figure 13B shows a graph of TNF-α induction by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 13A. [Figure 13C-2] Continued from Figure 13C-1. [Figure 13D-1]

[0148] Figure 13B shows a graph of IFN-α induction by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 13A. [Figure 13D-2] Continued from Figure 13D-1. [Figure 13E-1]

[0149] Figure 13B shows a graph of IP10 induction by primary human monocytes derived from human donors electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 13A. [Figure 13E-2] Continued from Figure 13E-1. [Figure 13F-1]

[0150] Figure 13B shows a graph of IL-12 induction by primary human monocytes derived from human donors that were electroporated with the indicated HER2-CFP constructs and stimulated with HER-2 antigen (top) or co-cultured with SKOV3 (bottom, left) or SKBR3 tumor cells (bottom, right). Experiments and analysis were performed as in Figure 13A. [Figure 13F-2] Continued from Figure 13F-1. [Figure 14-1]

[0151] FIG. 1 shows graphs indicating a comparison of tumor regression (left) and survival (right) in mice after administration of CFP constructs with the indicated intracellular domains. [Figure 14-2] Continued from Figure 14-1. DETAILED DESCRIPTION OF THE INVENTION

[0091]

[0152] T cell therapy has transformed cancer treatment for many patients. However, sustained clinical benefit has not been achieved for the majority of patients with advanced solid tumors. Unlike T cells, myeloid cells readily accumulate within tumors, sometimes contributing up to 50% of the tumor mass. Myeloid cells can be specifically engineered to become highly effective antitumor cells, termed Activate, Target, Attack & Kill (ATAK) cells, that specifically target, phagocytose, and lyse tumor cells and orchestrate in vivo immune activation against tumor cells.

[0092]

[0153] This application is based on an approach to enhance engineered myeloid cells that evade the immunosuppressive tumor microenvironment and exhibit high tumor cell lytic / immunogenic potency. Engagement of innate immune signaling sensors, such as Toll-like receptors and STING-cGAS, has been explored as a key pathway for upregulating pro-inflammatory antitumor immune responses and is associated with antitumor immunity. While these approaches, using synthetic agonists to activate these pathways, can be powerful, delivering localized, tumor-specific activation of innate immune signaling is difficult to achieve. Many conventional chemotherapeutics, targeted anticancer drugs, immune adjuvants, and oncolytic viruses are only fully effective in the presence of intact type I IFN signaling. Immunotherapy using vectors encoding recombinant type I interferons and IFNs has been attempted. For example, TLR-9 agonists have been combined with chemotherapy to treat untreated stage IIIb or stage IV non-small cell lung cancer (NSCLC) (Manegold, C. et al., "Addition of PF-3512676 (CpG 7909) to a taxane / platinum regimen for first-line treatment of unresectable non-small cell lung cancer (NSCLC) improves objective response - phase II clinical trial," Eur. J. Cancer., 3:326, 2005). Another study suggested that administration of IFN-producing iPSC-derived proliferative bone marrow cells combined with immune checkpoint blockade may be useful in overcoming resistance to single treatment modalities, for example, in cancers refractory to checkpoint blockade (Tsuchiya, N. et al., Cell Reports, 29, 162-175, October 2019).

[0093]

[0154] Disclosed herein is a method for creating a new class of chimeric antigen receptors (ATAK receptors) that couple tumor recognition with innate immune signaling. These recombinant chimeric receptors are expressed in myeloid cells described herein so that myeloid cells are targeted to cancer cells via the chimeric receptor, and then, upon tumor recognition, further activate the innate immune response in myeloid cells, leading to phagocytic lysis of tumor cells and further activation of lymphocytic immune responses. The combination of cancer recognition domains with intracellular signaling domains derived from innate immune receptors, such as FcRg, TLR, and cytokine receptors, as well as the chimeric antigen receptors designed and described herein, supports the idea that myeloid cells can be programmed to recognize cancer and elicit broad and fine-tuneable immune responses. The data demonstrate the versatility of engineering ATAK receptors by harnessing innate immune pathways and support their clinical development in cell therapy and direct in vivo therapy.

[0094]

[0155] All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise specified, 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 pertains.

[0095]

[0156] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0096]

[0157] 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 practiced in a single embodiment.

[0097]

[0158] 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, but not necessarily all embodiments, of the present disclosure.

[0098]

[0159] As used in this specification and claims, the terms "comprising" (and any form of "comprising," such as "comprise," "comprises," and "comprised"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "include" and "includes"), or "containing" (and any form of "containing," such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein may be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure may be used to achieve the methods of the disclosure. The methods of the present invention can be achieved.

[0099]

[0160] As used herein, the terms "about" or "approximately" when referring to a measurable value, such as a parameter, amount, time duration, etc., are intended to encompass variations of ±30% or less, ±20% or less, ±10% or less, ±5% or less, or ±1% or less of the specified value, and variations from the specified value, to the extent such variations are appropriate for practice in this disclosure. It is to be understood that values ​​to which the "about" or "approximately" modifier refers may also be specifically disclosed.

[0100]

[0161] An "agent" may refer to any cell, small molecule compound, antibody or fragment thereof, nucleic acid molecule, or polypeptide.

[0101]

[0162] "Alteration" or "change" can refer to an increase or decrease. For example, the alteration can be a 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90%, or 100% increase or decrease. For example, the alteration can be a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, or 40-fold, 50-fold, 60-fold, or even up to 70-fold, 75-fold, 80-fold, 90-fold, or 100-fold increase or decrease.

[0102]

[0163] As used herein, "antigen-presenting cells" or "APCs" include professional antigen-presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, and 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 MHC-complexed antigens on their surface, which can be recognized by T cells and induce T cell activation and immune responses. Professional antigen-presenting cells, particularly dendritic cells, play a key role in stimulating naive T cells. Non-professional antigen-presenting cells, such as fibroblasts, can also contribute to this process. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens on class I MHC molecules. Antigens that result in proteins that are recognized upon association with class I MHC molecules are generally proteins produced intracellularly, which are processed and associated with class I MHC molecules.

[0103]

[0164] A "biological sample" may refer to any tissue, cell, body fluid, or other material derived from an organism.

[0104]

[0165] The term "epitope" can refer to any protein determinant, such as a sequence or 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 groupings 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.

[0105]

[0166] Engineered cells, such as engineered bone marrow cells, may refer to cells that have at least one exogenous nucleic acid sequence within them, even if transiently expressed. Expression of the exogenous nucleic acid may be performed by a variety of methods, as described elsewhere, and encompasses methods known in the art. The present disclosure relates to the preparation and use of engineered cells, e.g., engineered bone marrow cells, such as engineered phagocytes. The present disclosure relates, inter alia, to engineered cells that contain an exogenous nucleic acid, e.g., encoding a chimeric fusion protein (CFP).

[0106]

[0167] The term "immune response" includes, but is not limited to, T cell-mediated immune responses, NK cell-mediated immune responses, 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, e.g., cytokine production and cell-mediated cytotoxicity. In addition, immune responses include immune responses indirectly influenced by NK cell activation, B cell activation, and / or T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages. Immune responses include adaptive immune responses. The adaptive immune system can react to foreign molecular structures, such as antigens of invading organisms. Unlike the innate immune system, the adaptive immune system is highly specific for pathogens. Adaptive immunity can also provide long-lasting protection. Adaptive immune responses include humoral and cell-mediated immune responses. In humoral immune responses, antibodies secreted by B cells into bodily fluids bind to pathogen-derived antigens and eliminate the pathogen through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells capable of destroying other cells are activated. For example, when disease-related proteins are present within cells, they are proteolytically fragmented into peptides within the cell. Specific cellular proteins then bind to the antigens or peptides thus formed and transport them to the cell surface, where they are presented by molecular defense mechanisms such as T cells. Cytotoxic T cells can recognize these antigens and kill cells bearing these antigens.

[0107]

[0168] A "ligand" may refer to a molecule capable of binding to or forming a complex with another molecule, such as a receptor. Ligands may include, but are not limited to, proteins, glycoproteins, carbohydrates, lipoproteins, hormones, fatty acids, phospholipids, or any entity that binds to a receptor. In some embodiments, a receptor has a specific ligand. In some embodiments, a receptor may bind to a ligand globally, in which case the receptor may bind to several ligands that share at least one similarity in structural organization, charge distribution, or any other physicochemical characteristic. A ligand may be a biomolecule. A ligand may be a non-biological material. For example, a ligand may be a ligand for a negatively charged particle, such as the scavenger receptor MARCO. For example, a ligand may be a ligand for TiO2, such as the scavenger receptor SRA1. In the context of the CFPs described herein, the extracellular binding domain may bind to a ligand, also referred to as the target of the binding domain. In some embodiments, the target is an antigen expressed on diseased cells such as cancer cells, which are target cells in the sense that the target cells express the target antigen that the extracellular antigen-binding domain of CFP binds to on their cell surface.In the present disclosure, anti-(target) binding domain, or anti-(target) binding extracellular domain, or anti-(target)CFP are often used interchangeably with terms such as (target) binding domain, or (target) binding extracellular domain, or (target)CFP, respectively.For example, HER2 expressed on cancer cells is the antigen (ligand) that the anti-HER2 binding extracellular domain of CFP binds to; or alternatively, it is the antigen (ligand) that is also stated to be bound by the HER2 binding extracellular domain of CFP.

[0108]

[0169] 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 may represent T cell epitopes. Human MHC is also sometimes referred to as an HLA complex. Thus, the terms "human leukocyte antigen (HLA)," "HLA molecule," or "HLA protein" are used interchangeably with the terms "major histocompatibility complex (MHC)," "MHC molecule," and "MHC protein." HLA proteins are sometimes classified as HLA class I or HLA class II. Although the proteins of the two HLA classes are structurally very similar, they have completely different functions. Class I HLA proteins are present on the surface of almost all cells in the body, including most tumor cells. Class I HLA proteins are usually loaded with antigens derived from endogenous proteins or pathogens present in cells, and then presented to naive or cytotoxic T-lymphocytes (CTLs).HLA class II proteins are present on antigen-presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages.HLA class II proteins mainly present peptides processed from external, for example, external antigen sources to helper T cells.

[0109]

[0170] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can engulf entities into phagosomes (although B cells exhibit a more general endocytosis into endosomes) that fuse with lysosomes, where acidic enzymes cleave the ingested protein into many different peptides. Autophagy is another source of HLA class II peptides. The most well-studied subclass II HLA genes are HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0110]

[0171] Presentation of peptides by HLA class II molecules to CD4+ helper T cells results in an immune response to foreign antigens. Upon activation, 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 an α chain and a β chain that interact to form a peptide-binding groove that is more open than the class I peptide-binding groove.

[0111]

[0172] HLA alleles are typically expressed in a codominant manner. For example, each person possesses two alleles of each of the three class I genes (HLA-A, HLA-B, and HLA-C), and thus can 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, encoding the α and β chains), HLA-DQ (DQA1 and DQB1 for the α and β chains), one gene, HLA-DRα (DRA1), and one or more genes, HLA-DRβ (DRB1 and DRB3, DRB4, or DRB5). HLA-DRB1, for example, has more than about 400 known alleles. This means that a heterozygous individual can inherit six or eight functional class II HLA alleles: three or more alleles from each parent. Thus, HLA genes are highly polymorphic; many different alleles exist in different individuals within a population. Genes encoding HLA proteins have many possible variations, allowing each person's immune system to respond to a wide range of foreign invaders. Hundreds of versions (alleles) of some HLA genes have been identified, each of which has a specific number. In some embodiments, class I HLA alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, and HLA-E*01:01 (non-classical). In some embodiments, class II HLA alleles are HLA-DRB*01:01, HLA-DRB*01:02, HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

[0112]

[0173] "Myeloid cells" broadly refers to cells of the myeloid lineage within the hematopoietic cell lineage, and may exclude, for example, the lymphoid lineage. Myeloid cells include, for example, cells of the granulocytic and monocytic lineages. Myeloid cells are a major cellular compartment of the immune system, including monocytes, dendritic cells, tissue macrophages, and granulocytes. Models of myeloid cell ontogeny, activation, differentiation, and tissue-specific functions have been reexamined over the past few years, yielding surprising results. However, their enormous plasticity and heterogeneity in both homeostasis and disease remain far from being understood. Myeloid cells possess many functions, including phagocytosis and their ability to activate T cells, yet harnessing these functions for therapeutic use remains elusive. Thus, novel approaches to use other cell types for the development of improved therapeutics, including, but not limited to, T cell malignancies, are needed.

[0113]

[0174] Myeloid cells differentiate from a common progenitor cell derived from hematopoietic stem cells in the bone marrow. Myeloid lineage commitment may be governed by the activation of distinct transcription factors; thus, myeloid cells may be characterized as cells with a level of plasticity, which can be described as 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 diverse chemokine receptors on their surface. Myeloid cells are responsive to a variety of cytokines and chemokines.

[0114]

[0175] Myeloid cells can be cells derived 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, within the bone marrow. In some embodiments, myeloid cells are progenitor cells. In some embodiments, myeloid cells can be cells with characteristics of common myeloid progenitor cells, or granulocyte progenitors, myeloblasts, or monocyte-dendritic cell progenitors, or combinations thereof. Bone marrow can contain granulocytes or monocytes, or their progenitors. Bone marrow can contain immature granulocytes, immature monocytes, immature macrophages, immature neutrophils, and immature dendritic cells. Bone marrow can contain monocytes or promonocytic cells or monocyte precursors. In some cases, myeloid cells, as used herein, can refer to monocytes with an M0 phenotype, an M1 phenotype, or an M2 phenotype. Bone marrow may contain dendritic cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, pre-dendritic cells, or DC precursors. Bone marrow may contain neutrophils, which may be mature neutrophils, neutrophil precursors, or polymorphonuclear cells (PMNs). Bone marrow may contain macrophages, monocyte-derived macrophages, tissue macrophages, and macrophages of the M0, M1, or M2 phenotype. Monocytes or macrophages exhibit polarization. As used herein, "polarization" may refer to the process by which macrophages exhibit distinct functional phenotypes in response to specific microenvironmental stimuli and signals, often referred to as physiological states. In some cases, macrophages may transition from one polarization state to another. For example, macrophages may be polarized into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages). M2 macrophages are divided into subtypes, M2a, M2b, M2c, and M2d, which differ in their cell surface markers, secreted cytokines, and biological functions. M1 macrophages are typically characterized by a phenotype in which the cells express TLR-2, TLR-4, CD80, CD86, iNOS, and MHC-II on their surface.These cells release a variety of cytokines and chemokines, such as TNF-α, IL-1α, IL-1β, IL-6, IL-12, CXCL9, and CXCL10, and typically exhibit activation of transcription factors such as NF-κB, STAT1, STAT5, IRF3, and IRF5, which regulate M1 gene expression. NF-κB and STAT1 are thought to be the two major pathways involved in M1 macrophage polarization. The M1 phenotype is associated with macrophage bactericidal and tumor-killing functions and exhibits high phagocytic and proinflammatory functions. On the other hand, tumor-associated macrophages in an immunosuppressive environment generally exhibit a greater degree of M2 polarization. Bone marrow can contain tumor-infiltrating monocytes (TIMs). Bone marrow can contain tumor-associated monocytes (TAMs). Bone marrow can contain myeloid-derived suppressor cells (MDSCs). Bone marrow can contain tissue-resident macrophages. Bone marrow may contain tumor-associated DCs (TADCs). Thus, bone marrow 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 can 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. In one embodiment, it is desired to activate M1 polarization of macrophages using the methods described herein.

[0115]

[0176] "Phagocytosis" is used interchangeably with "pinocytosis" 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 particles, such as cancer cells or infected cells, and / or remove them from the body. Phagocytic receptors can be involved in the phagocytic process. The phagocytic process can be closely linked to immune responses and antigen presentation. Processing of exogenous antigens follows their uptake into professional antigen-presenting cells through some types of endocytic events. Phagocytosis can also facilitate antigen presentation. For example, antigens derived from phagocytosed cells or pathogens, including cancer antigens, can be processed and presented on the cell surface of APCs.

[0116]

[0177] A "polypeptide" may refer to a molecule containing amino acids linked together via peptide bonds, such as a glycoprotein, lipoprotein, cellular 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 within 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.

[0117]

[0178] A "receptor" may refer to a chemical structure composed of a signal-transmitting polypeptide, such as a polypeptide that transmits an extracellular signal into a cell. Receptors are used to transmit information within a cell, the formation of a cell, or an organism. A receptor includes at least one receptor unit and may contain two or more receptor units, where each receptor unit includes a protein molecule, e.g., a glycoprotein molecule. A receptor may contain a structure that can bind to and form a complex with a ligand. Signaling information may be transmitted by a conformational change in the receptor following binding to a ligand on the surface of a cell.

[0118]

[0179] The term "antibody" refers to the class of proteins commonly known as immunoglobulins, including, but not limited to, IgG (including 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 Fab, Fab', and F(ab')2, Fd(V ... H and C H 1), single chain variable fragments (scFv), single chain antibodies, disulfide-linked variable fragments (dsFv), and V L Domain and / or V HAntibody fragments include, but are not limited to, fragments comprising the variable region(s). Antibodies can be derived from any animal. Antigen-binding antibody fragments, including single-chain antibodies, can comprise the variable region(s) alone or in combination with one or more of the hinge region, CH1 domain, CH2 domain, and CH3 domain. Any combination of the variable region(s) with the hinge region, CH1, CH2, and CH3 domain is also included. Antibodies can be, for example, monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind to an HLA-associated polypeptide or HLA-peptide complex.

[0119]

[0180] The term "recombinant nucleic acid" refers to a nucleic acid prepared, expressed, created, or isolated by recombinant means. Recombinant nucleic acids may contain a nucleotide sequence that is not naturally occurring. Recombinant nucleic acids may be synthesized in a laboratory. Recombinant nucleic acids may be prepared by using recombinant DNA techniques, such as enzymatic modification of DNA, such as enzymatic restriction 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, such as to produce messenger RNA (mRNA). Recombinant mRNA may be isolated, purified, and used to transfect cells. Recombinant nucleic acids may encode proteins or polypeptides. Throughout this specification, nucleic acid sequences are described that may include deoxyribonucleotides (DNA), ribonucleotides (RNA), or, in some embodiments, modified deoxyribonucleotides or modified ribonucleotides. For example, modified nucleotides can be 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 7-methylguanosine, pseudouridine, dihydrouridine, etc. Those skilled in the art can easily determine the RNA sequence, e.g., mRNA sequence, from a given polynucleotide sequence. The sequence may be codon-optimized.

[0120]

[0181] 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 adapted for plasmid DNA propagation, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence can be artificially induced in laboratory settings. 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.

[0121]

[0182] The term "vector" may refer to a nucleic acid molecule capable of autonomous replication in a host cell and allowing 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 transforming an exogenous gene 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, an expression vector or plasmid is in the form of a circular double-stranded DNA molecule. A vector or plasmid may or may not integrate 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, e.g., a gene or open reading frame carried by the plasmid, in a host cell. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that replicates autonomously in a host cell. 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 vector or an 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 which serve equivalent functions and are known to those skilled in the art, such as self-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 autonomous expression of nucleic acids to which they are linked.

[0122]

[0183] In some embodiments, nucleic acids can be delivered to biological systems in the form of nanoparticles. The nucleic acid sequences disclosed herein can be delivered in vivo via suitable nanoparticles, such as liposomes, lipid nanoparticles, or polymer nanoparticles. The lipid nanoparticles can comprise polar lipids. In some embodiments, the lipid nanoparticles comprise cationic lipids. In some embodiments, the lipid nanoparticles comprise cationic lipids and non-cationic lipids. In some embodiments, the lipid nanoparticles comprise neutral lipids. In some embodiments, the lipid nanoparticles comprise PEGylated lipids.

[0123]

[0184] Alternatively, in some embodiments, the nucleic acid may be electroporated into living cells ex vivo, in preparation for cell therapy, where the cells are bone marrow cells.

[0124]

[0185] 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 connecting protein or RNA sequences or preserving some minimum distance or other spatial relationship between the protein or RNA sequences. In some embodiments, the amino acids that are components of the spacer can be selected to affect some molecular property, such as molecular folding, flexibility, net charge, or hydrophobicity. Linkers suitable 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, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, a linker is used to separate two or more polypeptides, e.g., two antigenic peptides, by a distance sufficient to ensure proper folding of each antigenic peptide. Exemplary peptide linker sequences adopt a flexible, extended conformation and do not exhibit a tendency toward the development of ordered secondary structures. The amino acids in the flexible linker protein region can include any permutation of Gly, Asn, and Ser, or amino acid sequences containing Gly, Asn, and Ser. Other near-neutral amino acids, such as Thr and Ala, can also be used in the linker sequence.

[0125]

[0186] The terms "treat," "treated," "treating," "treatment," and the like are intended to refer to reducing, preventing, or ameliorating a disorder and / or its associated symptoms (e.g., a neoplasm or tumor, or an infectious agent, or an autoimmune disease). "Treating" can refer to administering a therapy to a subject after the onset or suspected onset of a disease (e.g., cancer, or infection by an infectious agent, or an autoimmune disease). "Treating" can refer to reducing the frequency or severity of any symptoms or other morbidity associated with a disease and / or the occurrence or recurrence of side effects associated with a treatment. The term "treating" also encompasses the concept of "alleviating." Included is the concept of "managing," which refers to reducing the severity of a disease or disorder in a patient, e.g., increasing the lifespan or survival of a patient with a disease, or delaying its recurrence, e.g., extending the period of remission in a patient who has previously suffered from a disease. It is understood that treating a disorder or condition does not require, but does not preclude, the complete disappearance of the disorder, condition, or symptoms associated therewith. 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 administration of an agent or compound begins. In certain embodiments, treatment of a subject or patient described herein includes administration of a therapeutic composition, such as a drug, metabolite, preventative component, nucleic acid, peptide, or protein, that encodes or otherwise forms the drug, metabolite, or preventative component. In some embodiments, treatment includes administering a cell or cell population to a subject in need thereof. In some embodiments, treatment includes administering to the subject one or more of the engineered cells described herein, e.g., bone marrow cells, such as one or more engineered phagocytes.Treatment includes treatment of a disease, condition, or syndrome, which may be a pathological disease, condition, or syndrome, or a latent disease, condition, or syndrome. In some cases, treatment 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, the engineered phagocytes may not result in or may reduce graft-versus-host disease (GVHD) or fratricide effects. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., have a match with an 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 an exemplary embodiment, the method includes determining genetic polymorphisms, generating an alignment of reads extracted from sequencing a dataset against a genetic reference set that includes allelic variants having the genetic polymorphisms; determining a first posterior probability or a 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; aligning the first allelic variant and one or more other allelic variants; the method may include steps of identifying one or more overlapping reads that have been identified using a weighting factor; determining a second posterior probability or a score derived from the posterior probability for one or more other allelic variants using a weighting factor; identifying the second allelic variant by selecting the allelic variant with the largest second posterior probability or score derived from the posterior probability, where the first allelic variant and the second allelic variant define a genotype of the polymorphism; and providing an output for the first allelic variant and the second allelic variant.

[0126]

[0187] A "fragment" may 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.

[0127]

[0188] The terms "isolated," "purified," "biologically pure," and their grammatical equivalents refer to material that is free, to varying degrees, from components that normally accompany it as found in its natural state. "Isolated from" refers to a degree of isolation from the original source or environment. "Purified from" refers to a degree of isolation that is greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials such that impurities do not substantially 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 when produced by recombinant DNA methodologies, or when chemically synthesized, when it is substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry methods, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can refer to a nucleic acid or protein that yields essentially one band in an electrophoretic gel. For proteins that can be subjected to modifications, such as phosphorylation or glycosylation, different modifications can yield different isolated proteins that can be individually purified.

[0128]

[0189] The term "neoplasm" 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 neoplasm or cancer. The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled growth, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells often take the form of tumors, but such cells can also exist alone in animals or be non-tumorigenic cancer cells, such as leukemia cells.

[0129]

[0190] The term "vaccine" should be 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 agent that includes a recombinant nucleic acid or a cell that contains and expresses a recombinant nucleic acid and is intended for use in humans or animals to generate specific defense and protection 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.

[0130]

[0191] The term "pharmaceutically acceptable" refers to approved or approvable by a federal or state regulatory agency for use in animals, including humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia. 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 its pharmacological activity, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0131]

[0192] The nucleic acid molecules useful in the method of the present disclosure include, but are not limited to, any nucleic acid molecules that have activity or encode polypeptides.Polynucleotides that have substantial identity to endogenous sequences can typically hybridize with at least one strand of a double-stranded nucleic acid molecule.The term "hybridize" refers to the situation where nucleic acid molecules pair with complementary polynucleotide sequences or their portions to form a double-stranded molecule under various stringency conditions (see, for example, Wahl, GM and S L. Berger (1987), Methods Enzymol., 152:399; Kimmel, AR (1987), Methods Enzymol., 152:507). For example, stringent salt concentrations can 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 can typically include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. Those skilled in the art are familiar with the variation of additional parameters, such as hybridization time, detergent concentration, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA. Various levels of stringency can be achieved by combining these various conditions as needed. In an exemplary embodiment, hybridization may occur in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In another exemplary embodiment, hybridization may occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C.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 in these conditions will be readily apparent to those of skill in the art. In most applications, the wash step following hybridization may also vary in stringency. Stringency conditions for washes may be defined by salt concentration and temperature. As noted above, wash stringency may be increased by decreasing salt concentration or by 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 steps may occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps may occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations in these conditions will be readily apparent to those of skill in the art.Hybridization methods 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.

[0132]

[0193] "Substantially homologous" 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 a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Such a sequence may be at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% 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 program, BESTFIT program, GAP program, or PILEUP / PRETTYBOX program). 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 may use the BLAST program, and a probability score between e-3 and e-m may indicate a closely related sequence. The "reference" is the basis for comparison. It will be understood that the numbering of specific positions or residues within each sequence will depend on the particular protein and the numbering scheme used. Numbering may differ, for example, between a precursor to a mature protein and the mature protein itself, and sequence differences between species may affect numbering.One skilled in the art will be able to identify the respective residues in any homologous proteins and in the nucleic acids encoding each by methods well known in the art, for example, by sequence alignment against a reference sequence and determination of homologous residues.

[0133]

[0194] The term "subject" or "patient" refers to an organism, such as an animal (e.g., a human), that 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 mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0134]

[0195] The term "therapeutic effect" refers to some degree of relief from one or more of the symptoms of a disorder (e.g., a neoplasm, tumor, or infection by an infectious agent, or an autoimmune disease) or its related pathologies. Alternatively, "therapeutic effect" may refer to a reduction in disease symptoms, e.g., a 10%, 20%, 30%, etc. reduction in tumor mass, following administration of a therapeutic composition. In another embodiment, "therapeutic effect" may relate to partial or complete remission of one or more symptoms, or amelioration of the disease. As used herein, "therapeutically effective amount" refers to the amount of agent, upon administration to a cell or subject, in a single or multiple doses, that is effective to prolong survival of a patient with such a disorder, alleviate one or more signs or symptoms of the disorder, prevent or delay the disorder beyond that expected in the absence of such treatment. "Therapeutically effective amount" is intended to quantify the amount required to achieve a therapeutic effect. An attending physician or veterinarian having ordinary skill in the medical arts can readily determine and prescribe the "therapeutically effective amount" (eg, ED50) of the pharmaceutical composition required.

[0135]

[0196] Provided herein are engineered myeloid cells (including, but not limited to, neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages) that are designed to specifically bind to a target antigen. The target antigen may be expressed only on target cells, such as infected cells, damaged cells, malignant cells, leukemia cells, or tumor cells. The engineered myeloid cells may attack and kill the 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.

[0136]

[0197] Although cancer is one exemplary embodiment described in detail in this disclosure, it is envisioned that the methods and techniques described herein will be useful in targeting infected or otherwise diseased cells within the body. Also described herein are therapeutic and vaccine compositions that use engineered cells.

[0137]

[0198] Myeloid effector cells can be isolated from human biological samples and modified ex vivo using methods to engineer such cells to prepare cells for therapeutic purposes, but the modifications do not alter the plasticity of these cells. Cells of the monocyte lineage are phagocytic and effective antigen-presenting cells. In one aspect, the present invention is based on the important finding that engineered myeloid cells can be a highly effective therapeutic modality in the treatment of numerous diseases, including cancer. Myeloid cells can be engineered to express chimeric antigen receptors (CARs), which enhance the immune function of myeloid cells; in this case, the cells are highly phagocytic and can attack and kill diseased or infected cells in the body. Chimeric antigen receptors are recombinant constructs designed and specifically engineered as described herein to be (a) highly target-specific, possessing an extracellular antigen-binding domain specifically oriented to bind to a target antigen, and (b) possessing a highly specialized intracellular domain to activate myeloid cells and achieve activation of the phagocyte phenotype. For example, a highly specialized intracellular domain is designed to create a chimeric receptor that, upon activation by target binding of the extracellular region of the receptor, can generate a signaling cue inside the cell that activates the intracellular interferon signaling cascade and transcription factors, i.e., directs the activation of transcription factors IRF (IFN regulatory factor). In addition, the methods and compositions described herein are also useful in gene therapy, in which a recombinant nucleic acid encoding a chimeric antigen receptor is administered locally or systemically to a subject in need thereof, so that the recombinant nucleic acid is specifically expressed in bone marrow cells in vivo, thereby generating activated bone marrow cells with therapeutic potential. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is delivered by LNP.

[0138]

[0199] Phagocytes are natural sentinels of the immune system and form the body's first line of defense. Phagocytes engulf pathogens, infected cells, foreign bodies, or cancerous cells and remove them from the body. Most potential pathogens are rapidly neutralized by the phagocytic system, for example, before they can cause significant infection or disease. This can involve pinocytosis, a receptor-mediated uptake via clathrin-coated pit systems, and in particular macropinocytosis, a consequence of membrane ruffling and engulfment. Thus, phagocytes are activated by a variety of non-self (and self) elements and exhibit a level of plasticity in recognizing their "targets." Most phagocytes express scavenger receptors on their surface, which are pattern recognition molecules that can bind to a wide range of foreign particles, as well as dead cells, cell debris, and unwanted particles within the body. In one embodiment, a recombinant nucleic acid encoding a chimeric antigen receptor (CAR) can be expressed intracellularly. CARs can be designed in various ways to attack specific tumor cells, and CAR-expressing myeloid effector cells can be activated to phagocytose and kill tumor cells. CARs can be designed to create phagocytic receptors that are activated specifically in response to target engagement, and the phagocytic potential of macrophages is enhanced by specifically engineered intracellular domains of the receptor. The CAR platform for myeloid cells described herein is designed so that tonic signaling is not detected in the myeloid cells at the time of administration into the body or at any time before the myeloid cells engage their target via the CAR. This is often examined ex vivo. At the same time, CAR-expressing myeloid cells can further differentiate into an M0, M1, or M2 phenotype in the presence of appropriate stimuli, and can retain cellular plasticity to differentiate at least at the time of administration. In addition, CAR-expressing myeloid effector cells can migrate to lymph nodes and cross-present antigens to naive T cells in the lymph nodes, thereby activating adaptive responses.

[0139]

[0200] In some embodiments, disclosed herein are compositions and methods for producing bone marrow cells that are isolated from a biological sample, engineered ex vivo to express a recombinant protein, and formulated into a pharmaceutical composition such that the bone marrow cells of the composition are "effector" bone marrow cells that are effective in inducing immune activation in vivo. In some embodiments, the bone marrow cells of the composition are referred to as "ATAK" bone marrow cells, which are bone marrow cells that are effective in attacking and destroying target cells. The ATAK bone marrow cells disclosed herein are engineered bone marrow recombinant proteins that express, for example, a chimeric receptor, e.g., a chimeric antigen receptor, that includes at least one intracellular signaling domain derived from an interferon-inducible protein in immune cells. In some embodiments, the methods and compositions described herein are directed to causing the engineered bone marrow cells to exhibit an effector phenotype. In some embodiments, the engineered bone marrow cells, e.g., monocytes, are monocytes of an M0 or M1 phenotype, and activation of the chimeric antigen receptor expressed in the bone marrow cells causes the cells to exhibit an M1 phenotype. The M1 phenotype exhibited by the engineered cells makes them highly tumoricidal when designed to be targeted to tumor cells.

[0140]

[0201] Provided herein are compositions and methods for treating diseases or conditions such as cancer. The compositions and methods provided herein utilize human bone marrow cells, including but not limited to neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages, to target diseased cells, such as cancer cells. The compositions and methods provided herein can be used to eliminate diseased cells, such as cancer cells, and / or diseased tissues through various mechanisms, including activation and recruitment of T cells, activation of effector immune cells (e.g., activation of CD8 T cells and NK cells), antigen cross-presentation, enhancement of inflammatory responses, reduction of regulatory T cells, and phagocytosis. For example, bone marrow cells can be used to maintain an immune response against cancer cells.

[0141]

[0202] The present applicants have previously described 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 can include an extracellular domain (ECD) comprising an antigen-binding domain that binds to an antigen on a target cell. The extracellular domain can be fused to a hinge domain or an extracellular domain derived from a receptor, such as a hinge domain or an extracellular domain. The CFPs encoded by the recombinant nucleic acids can further comprise a transmembrane domain, such as a transmembrane domain derived from CD2, CD8, CD28, 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 expression of a chemokine receptor), 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 / recruitment domains (CARDs).

[0142]

[0203] Provided herein are recombinant nucleic acids encoding improved immunogenic CAR compositions, such as chimeric fusion proteins (CFPs; alternatively referred to as CARs, chimeric antigen receptors), that contain an intracellular domain that activates an interferon response in cells expressing the CAR. Provided herein are immunogenic CFPs that contain at least one intracellular domain containing a pLxIS motif. The recombinant nucleic acid may be DNA or RNA. The recombinant nucleic acid encoding the CAR may be incorporated into a vector. When expressed in a cell, the recombinant CAR activates the production of type I interferon in the cell. Such cells are mammalian cells capable of type I interferon response. Such cells are immune cells, such as lymphocytes or myeloid cells.

[0143]

[0204] In some embodiments, the recombinant nucleic acid encoding the chimeric receptor comprises a specific sequence encoding the pro-inflammatory intracellular domain of the chimeric receptor. In some embodiments, the chimeric receptor proteins described herein comprise an intracellular domain that, upon engagement with its target in the extracellular domain, is capable of activating a signaling cascade that leads to the induction of interferon response genes or the production of type I interferon in cells expressing the chimeric antigen receptor. In some embodiments, the chimeric receptor proteins described herein comprise a domain derived from an innate immune pathway adaptor protein, such as mitochondrial antiviral signaling protein (MAVS), stimulator of interferon genes (STING), Toll / IL-1R domain-containing adaptor-inducing IFN (TRIF), and TLR adaptor interacting with endolysosomal SLC15A4 protein (TASL), or a portion thereof. In some embodiments, a domain or fragment of an innate immune pathway adaptor protein, e.g., a MAVS, STING, TRIF, or TASL protein, is incorporated by recombinant DNA techniques into the intracellular domain of a CFP or CAR described herein, where the domain or fragment contains a pLxIS motif (p represents a hydrophilic residue, x represents any residue, and S represents a phosphorylation site), which is phosphorylated by TBK1 or IKKε and mediates recruitment of IRF-3 to the signaling complex.

[0144]

[0205] In some embodiments, the chimeric receptor proteins described herein comprise an intracellular domain that, upon engagement with its target at the extracellular domain, is capable of activating a signaling cascade that results in a nuclear factor kappa B-responsive gene, or NF-kappa B response, in cells expressing the chimeric antigen receptor.

[0145] Effector myeloid cells and interferon activation

[0206] Type I and type II interferons (IFNs) play important roles in regulating immune responses in infection and cancer. Type I is represented by multiple subtypes, including numerous IFNα family members: IFNβ, IFNδ, IFNε, IFNκ, IFNτ, and IFNω, all of which utilize the same cell surface receptor, the heterodimeric IFNαR, composed of the IFNαR1 and IFNαR2 proteins. Type II IFNs are represented by IFNγ. These two IFN types are expressed by nearly all cells and bind to distinct cell surface receptors that trigger signaling events and elicit diverse cellular responses. Myeloid cells are key targets for interferons. In the early immune response to intracellular bacterial infection, activated natural killer (NK) cells and activated T cells are the source of IFNγ production. During the early stages of infection, production of the cytokines interleukin (IL) 12 and IL-18 drives antigen-nonspecific IFNγ production by these lymphocyte populations. Antigen-specific CD4+ and CD8+ T cells can also produce IFNγ in response to these pathogens. There are numerous distinct type I IFNs, including approximately 20 IFNα proteins and a single IFNβ. Each of these type I IFNs signals to host cells by binding to a conserved cell surface type I IFN receptor, the IFNαR. Ligation of the cell surface IFNαR induces the expression of numerous antiviral immune-stimulated gene (ISG) products, thereby protecting the host from certain viral infections (Sadler AJ, "Interferon-inducible antiviral effectors" (review), Nat Rev Immunol., July 2008, 8(7):559-68).However, responsiveness to type I IFNs also dramatically correlates with increased susceptibility to numerous intracellular bacterial infections, including Listeria monocytogenes, Mycobacterium tuberculosis, Fransicella tularensis, and other bacteria (Rayamajhi M. et al., "Antagonistic crosstalk between type I and II interferons and increased host susceptibility to bacterial infections." Virulence. September-October 2010, 1(5):418-22). IFNγ is secreted as a homodimer and acts on host cells by ligating cell surface receptors. Each IFNγ receptor is a heterodimer composed of two type I integral membrane subunits, IFNγR1 and IFNγR2. Binding of the IFNγ homodimer to cells triggers aggregation of the two receptor complexes, resulting in the presence of two IFNγR1 subunits and two IFNγR2 subunits, as well as additional signaling components. Although both subunits are required for signal transduction, the actual binding site for IFNγ is located on IFNγR1 (Kearney S. et al., "Differential effects of type I and II interferons on myeloid cells and resistance to intracellular bacterial infections." Immunol Res., March 2013, 55(0):187-200). When IFNγ interacts with the IFNγR1 subunit, it induces conformational changes that allow for tight association of the IFNγR1 and IFNγR2 subunits. These rearrangements within the receptor induce autophosphorylation and cross-phosphorylation of Janus-associated kinases (JAKs), which are constitutively associated with the receptor. IFNγR1 contains a binding motif for JAK1, and IFNγR2 contains a binding motif for JAK2.Phosphorylation of JAK proteins stimulates their catalytic activity, which then phosphorylates a tyrosine residue (Y440) in the C-terminus of IFNγR1. This phosphorylated tyrosine residue provides a docking site for the SH2 domain on the signal transducer and activator of transcription 1 (STAT-1) protein. Because each receptor complex contains two IFNγR1 subunits, two STAT-1 proteins can bind to the receptor. JAK1 and JAK2 maintain receptor association and phosphorylate each recruited STAT-1 protein at tyrosine residue 701 (Y701). This phosphorylation allows the release of STAT-1 monomers from the receptor and their subsequent formation into homodimers. STAT-1 homodimers translocate to the nucleus and bind to gamma-activating sequences (GAS) within the promoter DNA of IFN-inducible genes (ISGs), resulting in increased transcription. Type I IFNs, like those activated by IFNγ, signal through the canonical JAK / STAT pathway. Ligand binding to the IFNαR triggers dimerization of the two receptor subunits and transfer of phosphorylation to the associated TYK2 and JAK1 kinases. The kinases phosphorylate residues in the cytoplasmic tails of IFNαR1 and IFNαR2, recruiting the proteins STAT1 and STAT2 via their SH2 domains. Docking of these STAT proteins to the receptor subunits allows them to be phosphorylated by activated JAK proteins at Y701 on STAT-1 and Y690 on STAT-2. Phosphorylation of STAT monomers releases them from their docking sites, allowing them to dimerize and combine with IRF9 in homodimeric or heterodimeric forms to produce the transcription factor ISG factor 3 (ISGF3). ISGF3 translocates to the nucleus, identifies ISGs, and induces their transcription. ISGs induced by type I IFN signaling typically contain interferon-stimulated response elements (ISREs) or gamma-activated sequence (GAS) elements within their promoters, with a clear preference for genes containing ISREs.Some examples of ISGs that are transcribed as a result of type I IFNs are the ISRE containing genes ISG15, IP-10, IRF-7, and PKR

[66] , and the GAS containing genes IRF-1, IRF-2, IRF-8, and IRF-9 (Kearney S. et al., "Differential effects of type I and II interferons on myeloid cells and resistance to intracellular bacterial infections". Immunol Res., 2013 March;55(0):187-200).

[0146] Recombinant chimeric receptor proteins

[0207] Provided herein are classes of phagocytic or tethered receptor (PR) subunits (e.g., phagocytic receptor fusion proteins (PFPs)) that include (i) a transmembrane domain and (ii) an intracellular domain that includes a phagocytic receptor intracellular signaling domain; and an extracellular antigen-binding domain that is specific for an antigen, e.g., an antigen of a target cell or an antigen displayed on a target cell, wherein the transmembrane domain and the antigen-binding domain are operatively linked such that antigen binding to the target by the antigen-binding domain of the fusion receptor activates the intracellular signaling domain of the phagocytic receptor.

[0147]

[0208] 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 FCER1A extracellular domain. In some embodiments, the extracellular domain comprises the extracellular domain of FCGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, or FCGR3B. 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 domains: 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.

[0148]

[0209] In some embodiments, the CFP further comprises an extracellular domain comprising an 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 an immunoglobulin extracellular domain 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 scaffold for multimerization. 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 antigen-binding domain specifically binds to an antigen on a target cell. In some embodiments, the antigen-binding domain comprises an antibody domain. In some embodiments, the antigen-binding domain comprises a receptor domain, an antibody domain, where the antibody domain is a single-chain variable fragment (scFv), Fab, single-domain antibody (sdAb), nanobody, V H Domain, V L domain, VNAR domain, V HHIn some embodiments, the antigen-binding domain comprises a functional antibody fragment that is a domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof. In some embodiments, the antigen-binding domain comprises a ligand, an extracellular domain of a receptor, or an adaptor. In some embodiments, the antigen-binding domain comprises a single antigen-binding domain that is specific for a single antigen. In some embodiments, the antigen-binding domain comprises at least two antigen-binding domains, where each of the at least two antigen-binding domains is specific for a different antigen.

[0149]

[0210] 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 2D (NKG2D) family ligand, disialoganglioside 2 (GD2), CD2 , CD3, CD4, 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, 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.

[0150]

[0211] In some embodiments, the antigen-binding domain comprises a sequence of an antigen-binding domain presented herein, such as a sequence of an antigen-binding domain in the tables presented herein.

[0151]

[0212] In some embodiments, the target protein is CD70. In some embodiments, the antigen-binding domain comprises an anti-CD70 antibody or binding fragment thereof, wherein the antigen-binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain complementarity-determining region 3 (HC CDR3) that is the HC CDR3 of any one of the VH sequences selected from the group consisting of QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, the VH of the anti-CD70 antibody or binding fragment thereof comprises any one of the VH sequences selected from the group consisting of: QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS In some embodiments, the VH of the anti-CD70 antibody or binding fragment thereof further comprises a heavy chain complementarity determining region 1 (HC CDR1) that is HC CDR1. In some embodiments, the VH of the anti-CD70 antibody or binding fragment thereof further comprises a heavy chain complementarity determining region 2 (HC CDR2) that is HC CDR2 of any one of the VH sequences selected from the group consisting of QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, the VH of the anti-CD70 antibody or binding fragment thereof has 70 to 100% sequence identity to any one of the sequences selected from the group consisting of QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, the VH is a domain of a single-domain antibody. In some embodiments, the VH is a VHH.

[0152]

[0213] In some embodiments, the target protein is GPC3. In some embodiments, the antigen-binding domain comprises an anti-GPC3 antibody or a binding fragment thereof, wherein the antigen-binding domain is selected from the group consisting of ATACADTTQYAYDY, ATACADTTLYEYDY, ATACVDTTQYEYDY, ATACADATQHEYDY, ATACADTTQYDYDY, ATACADTTQYEYDY, ATACADTTHYEYDY, ATACVITTLYEYDY, ATACAETTLYEYDY, ATACADTTQHEYDY, ATACVDTTHYEYDY, ATACASTTLYEYDY, ATACVVTTLYEYDY, ATACGGATGPYDY, ATACAGAIGPYDY, ATACVVVGDQNDY, ATACVVVGDRNDY, ATDCAGGTSTPYDY, ATDCAGGTA The invention further comprises a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region 3 (HC CDR3) of any one of the sequences selected from the group consisting of: ATACGSLVGMYDY, ATACGSAVHEYDY, ATDCVGFGSNWFDY, ATACASPVIYEYDY, ATDCAGGVGHEYDY, ATDCSLHGSDYPYDY, and AVRIYSGSFDNTLAYDY. In some embodiments, the VH of the anti-GPC3 antibody or binding fragment thereof further comprises a heavy chain complementarity determining region 1 (HC CDR1) of any one of the sequences selected from the group consisting of GFPLAYYA, GFSLDYYA, GFPLDYYA, GFTLDYYA, GFSLNYYA, GFTLAYYA, GFTLGYYA, GFPLNYYA, GFPLHYYA, GFSLGYYA, GFPLGYYA, GFPLEYYA, GSDFRADA, GRTFSSYG, GFSLAYYA and GLTFRSVG. In some embodiments, the VH of the anti-GPC3 antibody or binding fragment thereof further comprises a heavy chain complementarity determining region 1 (HC CDR1) of any one of the sequences selected from the group consisting of ISNSDGST, ISASDGST,and a heavy chain complementarity determining region 2 (HC CDR2) of any one of the sequences selected from the group consisting of ISSSDGST, ISSSDGNT, ISSADGST, ISSSGGST, ISSGDGST, ISAGDGNT, ISSSDDST, ISSNDGST, ISSPDGST, ISSRTGGT, ISAGDGSST, ISSSDGSSSDGNT, ISSGDGNT, ISSGDGKT, ISSSDGGT, ISSRTGST, ISSRTGNT, ISSSDGHSST, ISSSSDGNT, ISASNGNT, ISSGSDGNT, ISASDGNT, IDSITSI, ISWSGGSTIAASVGST, ISSSDGSDGNT, and ASPSGVIT. In some embodiments, the VH of the anti-GPC3 antibody or binding fragment thereof is selected from the group consisting of: QVQLQESGGGLVHSGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS, QVQLQESGGGLVHSGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVHSGGSLRLSCAASGFTLDYYAIGWFRRAPGKER EGVSCISSGDGKTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAGAIGPYDYWGQGTQVTVSS, QVQLQESGGGLVPPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNS LGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVQAGGSLRLSCAASGFSLGYYAIGWFRQAPGKEREGVSCISSSDGHSSTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCATDCAGGTATPYDYWGQGTQVTVSS,QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSAWPAGPKHQVEYDYWGQGTQVTVSS、QVQLQESGGGLVQAGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVACISSRTGSTYYADSVKGRFTISRDNAKNTVALQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQDGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAETTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGESLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACANWSTLGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGESLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNRLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLKLSCAASGSDFRADAMGWYRQAPGKEREPVAIDSITSIYYVDSVEGRFTISRDNTKNTVYLQMTSLKPEDTAVYYCYARYSGRTYWGRGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCATACSDPRVYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTHYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADATQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRRAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISAGDGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACASTTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNAVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSPDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCSLHGSDYPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLEYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACSDPRVYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLGYYAIGWFRQAPGKEREGVSCISSSDDSTYYADSVKGRFTISRDNDKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATSCVVVTKNEYDYWGQGTQVTVSS、 QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVVADRNEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCATACVVADRNEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISASDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCASPEKYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGSAVHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLAYYAIGWFRQAPGKEREGVSCIAASVGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATDCAGGVGHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLNYYAIGWFRQAPGKEREGVSCISAGDGNTYYADSVKGRFTISRDNAANTVSLQMDSLKPEDTAVYYCATACVITTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPQDTAVYYCATACGSLVGMYDYWGQGTQVTVSP、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISASDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCASPEKYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISASNGNTYYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCATTCSGLTHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATHCGGSSWSNEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSNDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCATACVVTTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSP、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNMLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACASPVIYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACANWSSLGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCEGSGFSLDYYAIGWFRQAPGKEREGVSCISSGDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCVGFGSNWFDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVACISSRTGSTYYADSVKGRFTISRDNAKNTVALQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGGTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDRNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGNTYYADSVKGRFTISRDDAKNMVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQALGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFAISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCVASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、 QVQLQESGGGLVQPGGSLRLSCVASGFSLDYYAIGWFRQAPGKEREGVSCISNSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYAYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKEREGVSCISSGSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACSGLTHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKEREGVSCISSSDDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKEREGVSCISSSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCSGLTHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCVASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCVGSGFTLDYYAIGWFRQAPGKEREGVSCISSNDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQSGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAETTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQTGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGMVQAGESLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADATQHEYDYWGQGTQVTVSS、QVQLQESGGGSVQPGESLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS、QVQLQESGGGSVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTHYEYDYWGQGTQVTVSS、QVQLQESGGGSVQSGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGSVRPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGVAQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS、QVQLQESGGGVVQAGGSLKLSCAASGSDFRADAMGWYRQAPGKEREPVAIDSITSIYYVDSVEGRFTISRDNTKNTVYLQMTSLKPEDTAVYYCYARYSGRTYWGRGTQVTVSS、QVQLQESGGGVVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATHCGGTSWGTSYDYWGQGTQVTVSS、QVQLQESGGGVVQPGGSLRLSCAASGLTFRSVGMGWFRRAPGKEREFVATASPSGVITYYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAVRIYSGS FDNTLAYDYWGQGTQVTVSS, QVQLQESGGGVVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQM NSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, and QVQLQESGGGVVQSGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS. In some embodiments, the VH is a domain of a single domain antibody. In some embodiments, the VH is a VHH.

[0153]

[0214] In some embodiments, the antigen-binding domain comprises an autoantigen or a fragment thereof, such as Dsg1 or Dsg3. In some embodiments, the antigen-binding domain comprises a receptor domain or an antibody domain, where the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.

[0154]

[0215] In some embodiments, the transmembrane domain and the antigen-binding domain are operably linked via a linker, such as the hinge region of CD8α, IgG1, or IgG4.

[0155]

[0216] In some embodiments, the extracellular domain comprises a multimerization scaffold.

[0156]

[0217] 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 derived from a syntaxin, such as syntaxin 3 or syntaxin 4 or syntaxin 5. In some embodiments, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor when the CFP is expressed in a cell. In some embodiments, the transmembrane domain oligomerizes with the transmembrane domain of an exogenous receptor when the CFP is expressed in a cell. In some embodiments, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor when the CFP is expressed in a cell. In some embodiments, the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor when the CFP is expressed in a cell. 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.

[0157]

[0218] 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.

[0158]

[0219] 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, or 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 cells expressing PFP. In some embodiments, the intracellular domain comprises an F-actin disassembly 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, hi 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 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 but not more than 20 modifications thereof. In some embodiments, at least one ITAM domain comprises a phosphorylation site for a Src family kinase. 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.

[0159]

[0220] 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 integrins, such as the intracellular region of PSGL-1.

[0160]

[0221] In some embodiments, the intracellular signaling domain comprises a domain that activates the GTPase Rap1, such as domains derived 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 comprises a phagocytosis-enhancing domain.

[0161]

[0222] 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, NLRP family members 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.

[0162]

[0223] 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.

[0163]

[0224] 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 can comprise a signaling domain, such as an intracellular signaling domain derived from Siglec-1 (sialoadesin), 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.

[0164]

[0225] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a TLR protein. In some embodiments, the intracellular domain may comprise the intracellular signaling domain of an endolysosomal TLR, e.g., TLR3, TLR7, TLR8, or TLR9. In some embodiments, the intracellular signaling domain may be derived from a TLR3 protein. In some embodiments, the intracellular signaling domain may be derived from a TLR7 protein, a TLR8 protein, or a TLR9 protein. In some embodiments, the intracellular domain may comprise the intracellular signaling domain of cell surface TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10.

[0165]

[0226] In some embodiments, the intracellular signaling domain is specifically paired with another intracellular domain or transmembrane domain to maximize the efficacy and phagocytic potential of myeloid cells expressing the construct. For example, in some embodiments, a transmembrane domain comprising CD64 TM or a portion thereof may be specifically paired with an intracellular signaling domain comprising the ICD or PI3 kinase recruitment domain of an innate immune adaptor protein, or both. In some embodiments, the combination of domains with chimeric receptor intracellular domain(s) and / or transmembrane domain is directed toward maximizing the phagocytic index of the cells expressing the construct, e.g., myeloid cells. In some embodiments, the combination of domains with chimeric receptor intracellular domain(s) and / or transmembrane domain is directed toward maximizing the inflammatory potential of the cells expressing the construct, such that the cells can lyse target cells and activate immune response pathways to render the immune responsive over time. In some embodiments, the domain combinations of the chimeric receptor intracellular domain(s) and / or transmembrane domains are directed toward minimizing or eliminating tonic signaling by cells expressing the chimeric protein, hi some embodiments, the domain combinations of the chimeric receptor intracellular domain(s) and / or transmembrane domains are directed toward maximizing the specificity of the immune response.

[0166]

[0227] 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 that encodes 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 proteins, Dectin-1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily proteins, DCIR / CLEC4A, Dectin-2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), Mincle (macrophage inducible C type and / or any combination thereof.

[0167]

[0228] 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 the molecules IgCAM, cadherin, integrin, C-type lectin-like domain protein (CTLD), and / or proteoglycan. 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.

[0168]

[0229] In some embodiments, CFP does not comprise full-length intracellular signaling domain.In some embodiments, intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400 or 500 amino acid long.In some embodiments, intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400 or 500 amino acid long.

[0169]

[0230] 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, when expressed intracellularly, forms a complex with FcRγ. In some embodiments, the FcR α chain or FcR β chain, when expressed intracellularly, forms a complex with endogenous FcRγ. In some embodiments, the FcR α chain or FcR β chain is not incorporated into the membrane of cells that do 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.

[0170]

[0231] 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, e.g., ATP, ADP, UTP, UDP, and / or UDP-glucose, within the recombinant nucleic acid.

[0171] Intracellular interferon-responsive domain

[0232] Most TLRs activate an adaptor protein called MyD88, which activates the transcription factor protein NF-κB, which drives the expression of pro-inflammatory genes as part of the immune response. A subset of TLRs (TLR3 and TLR4) can engage the protein TRIF, which acts as a scaffold that allows kinase enzymes to add phosphate groups to the transcription factor IRF3. This phosphorylation activates IRF3, a member of a family of transcription factors called interferon regulatory factors (IRFs), which activate a wide range of gene expression programs. A manifestation of these programs is the production of type I interferon molecules. Because interferons are potent drivers of a branch of the immune system called the adaptive immune response, their presence risks contributing to autoimmunity. To prevent such attacks by the host's own immune system, the interferon response must be tightly regulated. As a safety valve, a specific sequence of amino acid residues within TRIF, the pLxIS motif, must be phosphorylated before IRF3 can be activated. This control mechanism is not specific to TRIF as an adaptor protein for TLR signaling, but rather involves IRF3 or the related protein IRF7, resulting in a "licensing step," a general manifestation of sensing pathways that drive interferon expression. With one exception, all identified innate sensing pathways that link nucleic acid recognition to type I interferon production have been shown to signal through one of three adaptor proteins known to contain the pLxIS motif: TRIF, MAVS, and STING. Thus, pLxIS motif-containing adaptor proteins specifically connect nucleic acid recognition to antiviral defense. In some embodiments, the intracellular signaling domain of a CFP comprises the ICD of an innate immune response protein.In some embodiments, the innate immune response protein is TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, MAVS, TRIF, TASL, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), TNFR1, chemokines, MHC class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) proteins. In some embodiments, the CFP comprises at least one intracellular signaling domain comprising the amino acid sequence motif pLxIS. In some embodiments, the intracellular interferon-inducible portion comprises an amino acid sequence selected from SEQ ID NOs: 36 to 41. Provided herein is a chimeric antigen receptor fusion protein for enhancing phagocytosis, the CFP comprising an antigen-binding domain and the MDA5 intracellular signaling domain of SEQ ID NO: 30. Provided herein is a chimeric antigen receptor fusion protein for enhancing phagocytosis, the CFP comprising an antigen-binding domain and the RIG1 intracellular signaling domain of SEQ ID NO: 36.Provided herein is a chimeric antigen receptor fusion protein for enhancing phagocytosis, the CFP comprising an antigen-binding domain and a Myd88 intracellular signaling domain of SEQ ID NO: 37. Provided herein is a chimeric antigen receptor fusion protein for enhancing phagocytosis, the CFP comprising an antigen-binding domain and an intracellular signaling domain comprising a STING portion of SEQ ID NO: 38. Provided herein is a chimeric antigen receptor fusion protein for enhancing phagocytosis, the CFP comprising an antigen-binding domain and an intracellular signaling domain comprising a MAVS portion of SEQ ID NO: 39. Provided herein is a chimeric antigen receptor fusion protein for enhancing phagocytosis, the CFP comprising an antigen-binding domain and an intracellular signaling domain comprising a TRIF portion of SEQ ID NO: 40. Provided herein is a chimeric antigen receptor fusion protein for enhancing phagocytosis, the CFP comprising an antigen-binding domain and an intracellular signaling domain comprising a TASL portion of SEQ ID NO: 41.

[0172]

[0233] 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, IL-8, 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.

[0173]

[0234] In some embodiments, an intracellular signaling domain derived from an intracellular adaptor protein known to be highly active in innate immune defense is incorporated into the chimeric receptor protein. In some embodiments, one or more mutations are introduced into one or more intracellular domains to reduce the responsiveness of the intracellular domain to intracellular stimuli characteristic of the natural intracellular adaptor protein domain without impairing the efficacy of the chimeric protein. In some embodiments, this efficacy is referred to as enhanced phagocytic potential compared to the same cells that do not express the chimeric protein. In some embodiments, this efficacy is referred to as enhanced inflammatory potential compared to the same cells that do not express the chimeric protein. In some embodiments, this efficacy is referred to as enhanced NF-kappa B activation or interferon activation in cells that express the chimeric protein compared to the same cells that do not express the chimeric protein.

[0174]

[0235] In some embodiments, bone marrow cells are specifically targeted for delivery. Bone marrow cells can be targeted using specialized biodegradable polymers such as PLGA (poly(lactic-co-glycolic acid)) and / or polyvinyl alcohol (PVA). In some embodiments, one or more compounds can be selectively incorporated into such polymer structures to affect bone marrow cell function. In some embodiments, the targeting structures are multilayered, e.g., with one or more PLGA layers and one or more PVA layers. In some embodiments, the targeting structures are assembled in an order for layering activity. In some embodiments, the targeting polymer structures are organized into specifically shaped components, such as flexible structures, that can attach to the bone marrow cell surface and deliver one or more components, such as growth factors and cytokines, to maintain the bone marrow cells in a microenvironment that confers specific polarization. In some embodiments, the polymer structures are configured such that they are not phagocytosed by bone marrow cells and can remain attached on the surface. In some embodiments, the one or more growth factors can be M1 polarization 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 IFNγ. 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 within a solid tumor.

[0175]

[0236] 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 within an untranslated region (UTR) of an mRNA. In some embodiments, the sequence within the UTR is a sequence that binds to an RNA-binding protein. In some embodiments, translation is inhibited or blocked upon binding of the RNA-binding protein to the sequence within the untranslated region (UTR). In some embodiments, the sequence within the UTR comprises the consensus sequence WWWU(AUUUA)UUUW, where W is A or U. In some embodiments, the recombinant nucleic acid is expressed on a bicistronic vector.

[0176]

[0237] 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 lymphocyte cancer cell. In some embodiments, the target cell is an ovarian cancer cell. In some embodiments, the target cell is a breast cell cancer cell. In some embodiments, the target cell is a pancreatic cell cancer cell. In some embodiments, the target cell is a glioblastoma cell cancer cell.

[0177]

[0238] 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 a circRNA. In some embodiments, the recombinant nucleic acid is a tRNA. In some embodiments, the recombinant nucleic acid is a microRNA.

[0178]

[0239] 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 a polycistronic vector. 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 a 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.

[0179]

[0240] Also provided herein are polypeptides encoded by the recombinant nucleic acids of the compositions described herein.

[0180]

[0241] Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP (e.g., a phagocytic receptor fusion protein (PFP)) comprising a phagocytic or tethered receptor (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, killing or phagocytosis of myeloid cells, such as neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, or macrophages, that express the CFP. Compositions are provided in which activity is increased by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or more than 1000% compared to cells that do not express CFP.

[0181]

[0242] Table 1 shows exemplary sequences of chimeric fusion protein domains and / or fragments thereof, which are intended to be non-limiting for the disclosure. The underlines indicate the CDR sequences according to the Kabat numbering system, in the order of CDR1, CDR2, and CDR3 for each heavy and light chain.

[0182] [Table 1-1]

[0183] [Table 1-2]

[0184] [Table 1-3]

[0185] [Table 1-4]

[0186] [Table 1-5]

[0187] [Table 1-6]

[0188] [Table 1-7]

[0189] [Table 1-8]

[0243] Provided herein are compositions comprising a recombinant nucleic acid sequence encoding a CFP (e.g., a phagocytic receptor fusion protein (PFP)) comprising a phagocytic or tethering receptor (PR) subunit, the composition comprising an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain, wherein the transmembrane domain and the extracellular domain are operably linked; and the CFP is expressed by neutrophils, monocytes, myeloid dendritic cells (MDCs), or myeloid dendritic cells (MDDs), which express the CFP upon binding of the CFP to the antigen of the target cell. C) Compositions are provided in which the killing or phagocytic activity of bone marrow cells, such as mast cells, or macrophages, is increased by at least 1.1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 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.

[0190]

[0244] Provided herein are recombinant nucleic acid sequences encoding the CFPs described in the immediately preceding paragraph, which include at least one innate immune activating intracellular domain, e.g., a pattern recognition receptor intracellular signaling domain, a TLR intracellular signaling domain, an FcR intracellular signaling domain, an intracellular adaptor protein signaling domain, or a fragment thereof, wherein the intracellular domain, upon contact with its target antigen, e.g., upon engagement of the antigen-binding domain with the target antigen, is capable of activating the innate immune response of myeloid cells, activating their phagocytic potential, activating inflammatory cytokine and chemokine responses, antigen presentation and T cell activation of myeloid cells expressing the CFP.

[0191]

[0245] 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, an IFN receptor, NLRP family members NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, or RANTES.

[0192]

[0246] In some embodiments, the CFP comprises an intracellular signaling domain comprising a sequence derived from a protein that activates interferon-responsive transcription factor IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, or IRF9.

[0193]

[0247] In some embodiments, the CFP comprises an intracellular signaling domain comprising a sequence derived from an intracellular adaptor protein. In some embodiments, the adaptor protein may comprise a transmembrane protein that anchors the CFP to an organelle, such as a mitochondrial compartment, an endoplasmic reticulum compartment, or a lysosomal compartment. In some embodiments, the intracellular adaptor protein is a cytosolic protein.

[0194] [Table 2-1]

[0195] [Table 2-2]

[0248] The CFPs described herein can comprise any one of the sequences listed in Table 1 combined with an intracellular domain listed in Table 2.

[0196]

[0249] In some embodiments, for example, of a CFP described herein, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 30, or an intracellular signaling domain derived from the MDA5 intracellular signaling domain having at least 85% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 86%, or at least 87%, or at least 88%, or at least 89% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the MDA5 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 91%, or at least 92%, or at least 93%, or at least 94% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the MDA5 intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular domain of a CFP comprises an intracellular signaling domain derived from an MDA5 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 30; wherein the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from an MDA5 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 30; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the MDA5 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 30; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:30.

[0197]

[0250] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a RIG-1 intracellular domain having the amino acid sequence of SEQ ID NO: 36, or at least 85% sequence identity to SEQ ID NO: 36. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a RIG-1 intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:36.

[0198]

[0251] In some embodiments, the intracellular signaling domain of the CFP comprises an intracellular signaling domain derived from the MyD88 intracellular signaling domain having the amino acid sequence of SEQ ID NO: 37, or at least 85% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the MyD88 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular signaling domain comprises a sequence having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the MyD88 intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a MyD88 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 37; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a MyD88 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 37; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the MyD88 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 37; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:37.

[0199]

[0252] In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 38, or an intracellular signaling domain derived from a STING intracellular signaling domain having at least 85% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular signaling domain comprises a sequence with at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a STING intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38; in this case, the CFP comprises an extracellular binding domain that is capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:38.

[0200]

[0253] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a MAVS intracellular signaling domain having an amino acid sequence of SEQ ID NO: 39 or 40, or an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 39 or 40. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 39 or 40. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a MAVS intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 39 or 40. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 39 or 40; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 39 or 40; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a hinge domain.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 39 or 40; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:39 or 40.

[0201]

[0254] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain having the amino acid sequence of SEQ ID NO: 45, or a sequence with at least 85% sequence identity to SEQ ID NO: 45. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain with at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain with at least 95% sequence identity to SEQ ID NO: 45. In some embodiments, the intracellular signaling domain comprises a sequence with at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 45; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 45; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 45; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:45.

[0202]

[0255] In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:41, or an intracellular signaling domain derived from the TRIF intracellular domain having at least 85% sequence identity to SEQ ID NO:41. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 86%, or at least 87%, or at least 88%, or at least 89% sequence identity to SEQ ID NO:41. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO:41. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 91%, or at least 92%, or at least 93%, or at least 94% sequence identity to SEQ ID NO:41. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 95% sequence identity to SEQ ID NO:41. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 41; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 41; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 41; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:41.

[0203]

[0256] In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 43, or an intracellular signaling domain derived from the TRIF intracellular domain having at least 85% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 86%, or at least 87%, or at least 88%, or at least 89% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 91%, or at least 92%, or at least 93%, or at least 94% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 43; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 43; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 43; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:43.

[0204]

[0257] In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 44, or an intracellular signaling domain derived from the TRIF intracellular domain having at least 85% sequence identity to SEQ ID NO: 44. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 86%, or at least 87%, or at least 88%, or at least 89% sequence identity to SEQ ID NO: 44. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, the intracellular signaling domain comprises a sequence having at least at least 91%, or at least 92%, or at least 93%, or at least 94% sequence identity to SEQ ID NO: 44. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 44.

[0205]

[0258] In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 44; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 44; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 44; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:44.

[0206]

[0259] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a TASL intracellular domain having the amino acid sequence of SEQ ID NO: 42, or at least 85% sequence identity to SEQ ID NO: 42. In some embodiments, the intracellular domain of a CFP comprises an intracellular signaling domain derived from a TASL intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 42; wherein the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a TASL intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 42; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, and a hinge domain.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a TASL intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 42; in this case, the CFP comprises an extracellular binding domain capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell, together with one or more additional intracellular signaling domains, such as a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain. For example, an exemplary CFP molecule disclosed herein comprises an extracellular CD5-binding domain having an amino acid sequence comprising SEQ ID NO:8 or SEQ ID NO:9, or both, or SEQ ID NO:10 or SEQ ID NO:11; and an intracellular domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:42.

[0207]

[0260] In some embodiments, an exemplary anti-CD5 binding CFP described herein comprises an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a heavy chain variable domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a light chain variable domain comprising the CDR3 sequence of QQYDESPWT (SEQ ID NO: 100); and further comprises an intracellular domain of SEQ ID NO: 41. In some embodiments, an exemplary CFP described herein comprises an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprises an intracellular domain of SEQ ID NO: 43. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprise an intracellular domain of SEQ ID NO: 44. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprise an intracellular domain of SEQ ID NO: 30. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprise an intracellular domain of SEQ ID NO: 36. In some embodiments, an exemplary CFP described herein comprises an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprises an intracellular domain of SEQ ID NO: 37.In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprise an intracellular domain of SEQ ID NO: 38. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprise an intracellular domain of SEQ ID NO: 39. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprise an intracellular domain of SEQ ID NO: 40. In some embodiments, an exemplary CFP described herein comprises an extracellular antigen-binding domain having the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or a VH domain comprising at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 100; and further comprises an intracellular domain of SEQ ID NO: 45.

[0208]

[0261] In some embodiments, exemplary anti-HER2 binding CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a heavy chain variable domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a light chain variable domain comprising the CDR3 sequence of QQHYTTPPT (SEQ ID NO: 102); and further comprise an intracellular domain of SEQ ID NO: 41. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 43. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 44. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 30. In some embodiments, an exemplary CFP described herein comprises an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprises an intracellular domain of SEQ ID NO: 36.In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 37. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 38. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 39. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 40. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 14 or SEQ ID NO: 94, or a VH domain comprising at least the CDR3 sequence of WGGDGFYAMDV (SEQ ID NO: 101), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 102; and further comprise an intracellular domain of SEQ ID NO: 45.

[0209]

[0262] In some embodiments, exemplary anti-TROP2-binding CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a heavy chain variable domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a light chain variable domain comprising the CDR3 sequence of QQHYITPLT (SEQ ID NO: 104); and further comprise an intracellular domain of SEQ ID NO: 41. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprise an intracellular domain of SEQ ID NO: 43. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprise an intracellular domain of SEQ ID NO: 44. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprise an intracellular domain of SEQ ID NO: 30. In some embodiments, an exemplary CFP described herein comprises an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprises an intracellular domain of SEQ ID NO: 36.In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprise an intracellular domain of SEQ ID NO: 37. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprise an intracellular domain of SEQ ID NO: 38. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprise an intracellular domain of SEQ ID NO: 39. In some embodiments, exemplary CFPs described herein comprise an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprise an intracellular domain of SEQ ID NO: 40. In some embodiments, an exemplary CFP described herein comprises an extracellular antigen-binding domain having the sequence of SEQ ID NO: 34 or SEQ ID NO: 35, or a VH domain comprising at least the CDR3 sequence of GGFGSSYWYFDV (SEQ ID NO: 103), and / or a VL domain comprising the CDR3 sequence of SEQ ID NO: 104; and further comprises an intracellular domain of SEQ ID NO: 45.

[0210] IRF-induced proteins and IRF-activated pathways

[0263] Type I IFNs are key cytokines mediating innate antiviral immunity and thus driving proinflammatory responses. They are readily induced by cGMP-AMP synthase, retinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors, which recognize microbial double-stranded (ds) DNA, dsRNA, and LPS. These signaling pathways converge in the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor-inducing IFN-β) mediate IRF-3 recruitment via a conserved pLxIS motif. While the pLxIS motifs of phosphorylated STING, MAVS, and TRIF generally bind IRF-3 in a similar manner, residues upstream of the motif confer specificity. Type I IFNs, such as IFN-α and IFN-β, are a major cytokine family mediating antiviral immunity. Microbial dsDNA in the cytosol binds to and activates cGAS (cGMP-AMP synthase), an enzyme that catalyzes the synthesis of the cyclic dinucleotide cGAMP (cyclic [G(2',5')pA(3',5')p]). As a second messenger, cGAMP binds to the adaptor protein STING (stimulator of IFN genes) located on the endoplasmic reticulum (ER) membrane, directing the activation of the transcription factor IRF-3 (IFN regulatory factor 3) via the protein kinase TBK1 (TANK-binding kinase 1). Phosphorylated IRF-3 dimerizes, translocates to the nucleus, and initiates IFN-β gene transcription. In contrast, in the cytosol, viral dsDNA is sensed by RLRs [retinoic acid-inducible protein 1 (RIG-I)-like receptors], which activate IRF-3 through the adaptor protein MAVS (mitochondrial antiviral signaling).Furthermore, the Toll-like receptors (TLRs), TLR3 and TLR4, which recognize viral dsRNA in endosomes and LPS, a bacterial cell wall component, also mediate the induction of type I IFN and proinflammatory cytokines, respectively (1). These two TLRs use the adaptor protein TRIF (TIR domain-containing adaptor-inducing IFN-β) to mediate the recruitment and activation of IRF-3. Notably, the signaling pathways of these three innate immune sensor families converge upon the activation of TBK1 and IRF-3. Mechanistically, the adaptor proteins STING, MAVS, and TRIF contain a conserved motif, pLxIS (p represents a hydrophilic residue, x represents any residue, and S represents the phosphorylation site), which is phosphorylated by TBK1 or IKKε and mediates the recruitment of IRF-3 to the signaling complex. The induction of proximity between TBK1 and IRF-3 results in the phosphorylation and activation of IRF-3. Furthermore, IRF-3 itself also contains a pLxIS motif, which is crucial for phosphorylation-induced dimerization / activation of IRF-3. Mutation of the serine phosphorylation site within the pLxIS motif of STING, MAVS, and TRIF abolishes the induction of type I IFN in their respective signaling pathways. However, the precise molecular mechanism of IRF-3 recruitment and activation remains unknown. To elucidate the structural basis of IRF-3 recruitment by phosphorylated STING (pSTING), MAVS (pMAVS), and TRIF (pTRIF), we expressed peptides containing the pLxIS motif from the three adaptor proteins, phosphorylated them with TBK1 in vitro, and determined the crystal structure of their complex with the C-terminal domain (CTD) of IRF-3.

[0211] Mechanism of IRF-3 recruitment by pMAVS and pTRIF.

[0212]

[0264] In contrast to dsDNA sensing via the cGAS-STING pathway, RLRs sense dsDNA in the cytosol and activate IRF-3 through the adaptor MAVS, whereas TLR3 and TLR4 recruit IRF-3 using the adaptor TRIF. Phosphorylation of the pLxIS motif of MAVS or TRIF is required for IRF-3 recruitment and activation.

[0213] TRAF-interacting proteins:

[0265] The presence of a TRAF domain, a protein-interaction domain of approximately 180 amino acids, is a distinctive feature of TRAF family proteins. In mammals, six of the seven TRAF proteins in the family (TRAF1-TRAF6) conform to this criterion and have been identified as members of the TRAF family. The TRAF domain can be subdivided into two distinct regions: the TRAF-N domain and the TRAF-C domain. Diverse receptors bind to the TRAF-C domain, whereas diverse intracellular signaling molecules bind to the TRAF-N domain. Despite the structural similarity of the TRAF domains, each TRAF protein has a distinct biological function, with specificity for its interaction partners: upstream receptors and downstream effector molecules. The structure of the TRAF2 TRAF domain was first reported by Dr. Wu's research group around 1999, and the structure of the TRAF6 TRAF domain was reported by the same research group three years later. Since then, the structures of the TRAF domains of TRAF3, TRAF5, TRAF4, and TRAF1 have also been reported. The TRAF structures revealed that the TRAF-N domain is a coiled-coil structure, while TRAF-C is composed of seven to eight antiparallel β-sheet folds. Structural alignment of all six TRAF family members indicates that the TRAF-C domains are well aligned, whereas the position and length of TRAF-N vary among TRAF family members. Sequence analysis indicates that while the length of TRAF-N varies within the family, the length of the TRAF-C domain is conserved: TRAF-N in TRAF4 and TRAF6 is relatively short, whereas TRAF-N in TRAF3 and TRAF5 is relatively long. Although the overall structures are nearly identical, clear structural differences were observed. For example, the length and position of some loops within the TRAF domains of TRAF4 and TRAF6 differ from those in other TRAF family members. TRAF4 contains a more negatively charged surface within the receptor-binding region, whereas TRAF6 contains a more positively charged surface within the receptor-binding region.Because surface features often determine their mode of interaction with partners, the electrostatic surface of TRAF domains, which is similar among TRAF1, TRAF2, TRAF3, and TRAF5, i.e., the diverse charged surfaces, have been shown to be important for accepting diverse receptors into the same binding pocket in a similar interaction mode. In contrast, the different features on the binding surfaces of functionally distinct TRAFs, TRAF4 and TRAF6, indicate that TRAF4 and TRAF6 accept different receptors in different interaction modes.

[0214]

[0266] For the purposes of this disclosure, any pathway, signal transduction intermediate or activation part discussed in the above paragraph can be considered to be activatable or functional when the CFP disclosed herein is induced, depending on application.Similarly, the CFP disclosed herein can be useful in targeting any applicable target described in the pathway discussed.It is understood that any pathway or part thereof that is readily known to those skilled in the art at the date of existing literature regarding signal transduction domain, signal transduction pathway, signal transduction intermediate, transcription factor of activated gene is within the scope of the present disclosure.

[0215] Chimeric proteins with TLR intracellular domains, TLR intracellular signaling pathways, and activation of NF-kappa B:

[0267] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a TLR protein. In some embodiments, a CFP designed to comprise an intracellular signaling domain derived from a TLR intracellular signaling domain can activate NF-kappaB upon engagement of the receptor's extracellular domain with its target. In some embodiments, the intracellular domain can comprise the intracellular signaling domain of an endolysosomal TLR, e.g., TLR3, TLR7, TLR8, or TLR9. In some embodiments, the intracellular signaling domain can be derived from a TLR3 protein. In some embodiments, the intracellular signaling domain can be derived from a TLR7 protein, a TLR8 protein, or a TLR9 protein. In some embodiments, the intracellular domain can comprise the intracellular signaling domain of cell surface TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10. 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.

[0216]

[0268] 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.

[0217]

[0269] In some embodiments, the cytoplasmic portion of the chimeric receptor (e.g., phagocytic receptor (PR) fusion protein (PFP)) comprises a cytoplasmic domain derived 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).

[0218]

[0270] In general, TLRs have diverse subcellular localizations, developmental pathways, activation, recognition, and modes of action. TLRs are expressed in innate immune cells such as dendritic cells (DCs) and macrophages, as well as non-immune cells such as fibroblasts and epithelial cells. Cell surface TLRs primarily recognize microbial membrane components, such as lipids, lipoproteins, and proteins. TLR4 recognizes bacterial lipopolysaccharide (LPS). TLR2, together with TLR1 or TLR6, recognizes a wide variety of PAMPs, including lipoproteins, peptidoglycan, lipoteichoic acid, zymosan, mannan, and tGPI-mucin. TLR5 recognizes bacterial flagellin. TLR10 is a pseudogene in mice due to the insertion of a stop codon, but human TLR10 recognizes ligands derived from Listeria monocytogenes in cooperation with TLR2. TLR10 also senses viral infection by influenza A virus.

[0219]

[0271] Intracellular TLRs recognize nucleic acids derived from bacteria and viruses, and in disease states such as autoimmunity, they also recognize self-nucleic acids. TLR3 recognizes viral double-stranded RNA (dsRNA), small interfering RNA, and self-RNA derived from damaged cells. TLR7 is primarily expressed in plasmacytoid DCs (pDCs) and recognizes single-stranded (ss) RNA derived from viruses. TLR7 also recognizes RNA derived from group B streptococcus in conventional DCs (cDCs). Human TLR8 responds to viral and bacterial RNA. Structural analysis revealed that unstimulated human TLR8 exists as a preformed dimer, and the Z loop between LRR14 and LRR15 is cleaved into N- and C-terminal halves, but maintains association with each other and participates in ligand recognition and dimerization. Ligand binding induces reorganization of the dimer, bringing the two C-termini into close proximity. TLR13 recognizes bacterial 23S rRNA and an unknown component of vesicular stomatitis virus. TLR9 recognizes bacterial and viral DNA rich in unmethylated CpG-DNA motifs; it also recognizes hemozoin, an insoluble crystalline by-product produced by Plasmodium falciparum during the detoxification process after host hemoglobin is digested. TLR11 is localized within endolysosomes and recognizes flagellin or an unknown protein component of uropathogenic Escherichia coli (UPEC), as well as a profilin-like molecule from Toxoplasma gondii. TLR12 is expressed primarily in myeloid cells and recognizes profilin from T. gondii, which is highly similar to TLR11. TLR12 functions as a homodimer or heterodimer with TLR11. All TLRs are synthesized in the ER, transported to the Golgi apparatus, and recruited to the cell surface or intracellular compartments such as endosomes. The intracellular localization of TLRs is thought to be crucial for ligand recognition as well as for preventing TLRs from coming into contact with self-nucleic acids, which can lead to autoimmunity.

[0220]

[0272] Individual TLRs differentially recruit members of a set of TIR domain-containing adaptors, such as MyD88, TRIF, TIRAP / MAL, or TRAM. MyD88 is utilized by all TLRs and activates NF-κB and MAPK for the induction of proinflammatory cytokine genes. TIRAP is a selective adaptor that recruits MyD88 to cell surface TLRs, such as TLR2 and TLR4. TIRAP also participates in signaling through endosomal TLRs, such as TLR9. The lipid-binding domain of TIRAP binds PI(4,5)P2 at the plasma membrane and PI(3)P on endosomes, mediating the formation of functional TLR4 / TLR9 signaling complexes at the respective sites. Thus, TIRAP associates with both cell surface and endosomal TLRs by binding to different lipids. TRIF is recruited to TLR3 and TLR4, promoting an alternative pathway that leads to the activation of IRF3, NF-κB, and MAPK for the induction of type I IFN and proinflammatory cytokine genes. TRAM is selectively recruited to TLR4, but not to TLR3, to link TRIF and TLR4. TLR3 interacts directly with TRIF, and this interaction requires phosphorylation of two tyrosine residues in the cytoplasmic domain of TLR3 by epidermal growth factor ErbB1 and Btk. After TLR engagement, MyD88 forms a complex with members of the IRAK kinase family. IRAK4 activates IRAK1, which is then autophosphorylated at several sites and released from MyD88. IRAK1 associates with TRAF6, a RING domain E3 ubiquitin ligase. TRAF6, together with the ubiquitin-conjugating enzymes UBC13 and UEV1A, promotes K63-linked polyubiquitination of both TRAF6 itself and the TAK1 protein kinase complex. TAK1 is a member of the MAPKKK family and forms a complex with the regulatory subunits TAB1, TAB2, and TAB3, which interact with TRAF6-generated polyubiquitin chains and drive TAK1 activation.TAK1 then activates two distinct pathways, the IKK complex-NF-κB pathway and the IKK complex-MAPK pathway. The IKK complex is composed of catalytic subunits IKKα and IKKβ and a regulatory subunit, NEMO (also called IKKγ). TAK1 binds to the IKK complex via ubiquitin chains, allowing the IKK complex to phosphorylate and activate IKKβ. The IKK complex phosphorylates the NF-κB inhibitor protein IκBα, which undergoes proteasomal degradation, allowing NF-κB to translocate to the nucleus and induce pro-inflammatory gene expression. TAK1 activation also results in the activation of MAPK family members, such as ERK1 / 2, p38, and JNK, which mediate the activation of AP-1 family transcription factors or mRNA stabilization, thereby regulating inflammatory responses.

[0221]

[0273] In some embodiments, the intracellular domains described herein can be specifically paired with another domain, for example, a structural domain such as another intracellular domain, a transmembrane domain, or an extracellular domain; or a functional domain such as a signaling domain. In some embodiments, the intracellular domains described in this section, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, can be adjusted or modified to pair with another domain or its components. Pairing is intended to mean the incorporation of a portion of one or more domains under consideration, or a fragment or component thereof, into the CFP design as any part of the molecular structure of the CFP protein. In some embodiments, adjustment can be the structural alignment or positioning of domains within a CFP molecule, for example, two domains under consideration are juxtaposed, separated by one or more domains between them, or separated by one or more amino acids between them. One or more amino acids can be a linker. The one or more amino acids may provide structural separation between two adjacent domains, may provide flexibility between two adjacent domains, or may confer more three-dimensional orientation to a molecular structure that includes a domain without one or more amino acids. In some embodiments, the adjustment or alteration may include a modification within a domain, such as a mutation.

[0222]

[0274] In some embodiments, an intracellular domain described in this section, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, can be adjusted or modified for pairing with or incorporation into another intracellular domain in a CFP, such as a kinase recruitment domain, such as a PI3 kinase recruitment domain. In some embodiments, the PI3 kinase recruitment domain is modified to block tonic signaling. In some embodiments, one of the other intracellular domains, such as any one or more of the domains MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or fragments thereof, is modified to reduce or eliminate tonic signaling.

[0223]

[0275] In some embodiments, the intracellular domains described in this section, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, may be adjusted or modified for pairing with or incorporation into another structural domain, such as a transmembrane domain. In some embodiments, the transmembrane domain is a CD68 domain. In some embodiments, the transmembrane domain is a CD64 domain. In some embodiments, the transmembrane domain is a CD89 domain. In some embodiments, a mutant CD68 domain, e.g., SEQ ID NO: 46, may be used.

[0224]

[0276] For the purposes of this disclosure, any pathway, signal transduction intermediate, or activation portion discussed in the above paragraphs may be considered activatable or functional, depending on the application, upon induction of the disclosed CFPs, including the TLR intracellular signaling domains disclosed herein.Similarly, the CFPs disclosed herein may be useful in targeting any of the applicable targets described within the pathways discussed.It is understood that any pathway or part thereof that is readily known to those skilled in the art as of the date of existing literature regarding signal transduction domains, signal transduction pathways, signal transduction intermediates, and transcription factors of activated genes is within the scope of the present disclosure.

[0225] therapeutic composition

[0277] In one aspect of the present disclosure, bone marrow cells are provided, such as CD14+ cells, CD14+ / CD16- cells, CD14+ / CD16+ cells, CD14- / CD16+ cells, CD14- / CD16- cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages, or mosaic bone marrow cells / macrophages / dendritic cells. In some embodiments, therapeutic compositions are provided comprising at least 20%, at least 30%, at least 40%, or at least 50% CD14+ cells. In some embodiments, therapeutic compositions comprise at least 20%, at least 30%, at least 40%, or at least 50% CD14+ / CD16- cells. In some embodiments, therapeutic compositions are provided comprising less than 20%, less than 15%, less than 10%, or less than 5% dendritic cells. The bone marrow cells for the therapeutic compositions described herein comprise a recombinant nucleic acid encoding a chimeric fusion protein, the recombinant nucleic acid encoding a CFP receptor protein or an engager protein described herein. The bone marrow cells for the therapeutic compositions described herein express the CFP encoded by the recombinant nucleic acid or express the engager protein encoded by the recombinant nucleic acid described herein.

[0226]

[0278] Some embodiments herein include chimeric fusion proteins, such as chimeric fusion receptor proteins (CFPs), wherein the CFP comprises: (a) (i) an scFv that specifically binds to any one of the targets disclosed herein, 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) 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, (ii) a second intracellular signaling domain derived from FcRγ or FcRε, ) Therapeutic compositions are provided that include a second intracellular signaling domain, e.g., an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, an IFN receptor, NLRP family members NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, or RANTES, and a PI3K recruitment domain, or a domain derived from CD40.

[0227]

[0279] In some embodiments, the pharmaceutical composition comprises a therapeutically effective dose of a cell population comprising bone marrow cells. In some embodiments, the cell population differentiates into effector cells in a subject after administration; infiltrates or migrates to an affected site in a subject after administration; and / or has a lifespan of at least 5 days in a subject after administration.

[0228]

[0280] In some embodiments, bone marrow cells can be further modified or engineered to generate therapeutically effective bone marrow cells. The isolated cells can be engineered by expressing genes, or fragments thereof, within the cells without altering their functional / developmental plasticity, differentiation potential, and cell viability.

[0229]

[0281] In some embodiments, bone marrow cells can be further modified or engineered to generate therapeutically effective bone marrow cells by expressing a non-endogenous polynucleotide into the cells. The non-endogenous polynucleotide encodes a protein or peptide. Alternatively, the non-endogenous polypeptide can be a non-coding sequence, such as an inhibitory RNA or a morpholino.

[0230]

[0282] In some embodiments, bone marrow cells can be further modified or engineered to generate therapeutically effective bone marrow cells by stably altering the genomic sequence of the cells. In some embodiments, bone marrow cells can be engineered by editing the bone marrow cell genome using a CRISPR-CAS system. In some embodiments, one or more genes can be edited to silence gene expression. In some embodiments, bone marrow cells can be engineered to delete genes. In some embodiments, one or more genes can be edited to enhance genes. In some embodiments, genetic material is introduced into bone marrow cells in the form of messenger RNA, where the messenger RNA encodes a protein or peptide, thereby making the bone marrow cells therapeutically effective. In some embodiments, naked DNA or messenger RNA (mRNA) can be used to introduce nucleic acids into bone marrow cells. In some embodiments, DNA or mRNA encoding a chimeric antigen receptor is introduced into phagocytic cells by encapsulation in lipid nanoparticles (LNPs). The mRNA can be single-stranded and codon-optimized. In some embodiments, the mRNA may include one or more modified or unnatural bases, such as 5'-methylcytosine, or pseudouridine or methylpseudouridine. In some embodiments, about 50% or more of the uridine ("U") residues in the mRNA may be converted to methylpseudouridine. In some embodiments, the mRNA may be 50-10,000 bases in length. In one aspect, the transgene is delivered as mRNA. The mRNA may contain more than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 bases. In some embodiments, the mRNA may be more than 10,000 bases in length. In some embodiments, the mRNA may be about 11,000 bases in length. In some embodiments, the mRNA may be about 12,000 bases in length.In some embodiments, the mRNA comprises a transgene sequence encoding a fusion protein. DNA or RNA encapsulated in LNPs may be used to transfect macrophages and administered to a subject. In some embodiments, the mRNA is incorporated into effector myeloid cell populations by transient transfection. In some embodiments, the transient transfection method comprises electroporation of the mRNA. In some embodiments, the transient transfection comprises chemical transfection. In some embodiments, 1 to 5,000 micrograms of mRNA per ml may be used for transfection using protocols suitable for the methods described above. In some embodiments, 1 to 2,000 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1 to 1,000 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1 to 1,000 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1-500 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1-250 micrograms of mRNA per ml may be used for transfection. In some embodiments, about 500 micrograms or less of mRNA per ml may be used for transfection. In some embodiments, about 250 micrograms or less of mRNA per ml may be used for transfection. In some embodiments, about 10 micrograms of mRNA per ml is used. In some embodiments, about 20 micrograms of mRNA per ml is used. In some embodiments, about 30 micrograms of mRNA per ml is used. In some embodiments, about 40 micrograms of mRNA per ml is used. In some embodiments, about 50 micrograms of mRNA per ml is used. In some embodiments, about 60 micrograms of mRNA per ml is used.In some embodiments, about 80 micrograms of mRNA per ml is used. In some embodiments, about 100 micrograms of mRNA per ml is used. In some embodiments, about 150 micrograms of mRNA per ml is used. In some embodiments, about 200 micrograms of mRNA per ml is used. In some embodiments, about 20, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 micrograms of mRNA per ml is used. An appropriate cell density is selected for transfection based on the manufacturer's instructions for the method and instrument and / or reagent, or as would be known to one of skill in the art.

[0231]

[0283] In some embodiments, the recombinant nucleic acid is mRNA. The mRNA construct can be thawed on ice and gently pipetted into monocytes and premixed. In some embodiments, the mRNA is electroporated into the cells. After elutriation, the cells can be pooled, centrifuged, and electroporated with the mRNA using a MaxCyte ATX system optimized for this purpose. In some embodiments, electroporation buffer, cell density, and / or mRNA concentration optimized for each protocol for each construct are used.

[0232]

[0284] In some embodiments, polynucleotides can be introduced into bone marrow cells in the form of circular RNA (circRNA), in which the 3' and 5' ends are covalently linked. CircRNAs can be delivered into cells using LNPs.

[0233]

[0285] In some embodiments, stable integration of transgenes into macrophages and other phagocytes can be achieved through the use of transposases and transposable elements, particularly mRNA-encoded transposases. In one embodiment, long interspersed element-1 (L1) RNA can be envisioned for retrotransposition of transgenes and stable integration into macrophages or phagocytes. Retrotransposons can be used for stable integration of recombinant nucleic acids encoding phagocytic or tethering receptor (PR) fusion proteins (PFPs).

[0234]

[0286] In some embodiments, bone marrow cells can be modified by expressing a transgene via incorporation of the transgene into a transient expression vector. In some embodiments, expression of the transgene can be temporally regulated from outside the cell by a regulatory factor. Examples include the Tet-on / Tet-off system, in which transgene expression is regulated via the presence or absence of tetracycline.

[0235]

[0287] In some embodiments, bone marrow cells can be modified to produce therapeutically effective cells by contacting the cells with a compound, which may be a protein, which may be an enzyme, which may be an inhibitor, or which may be an activator, within the bone marrow cells.

[0236]

[0288] In some embodiments, a polynucleotide encoding a chimeric antigen receptor may be introduced into isolated bone marrow cells obtained by the methods described in the preceding paragraph, where the chimeric antigen receptor, upon expression in the bone marrow cells, enhances the innate immune response function of the bone marrow cells. In some embodiments, expression of the chimeric antigen receptor may direct bone marrow cells to specific targets in vivo or in vitro. In some embodiments, the chimeric antigen receptor may increase the phagocytic potential of bone marrow cells. In some embodiments, the chimeric antigen receptor increases the immunogenicity of bone marrow cells. In some embodiments, the chimeric antigen receptor may enhance intracellular signaling. In some embodiments, the chimeric antigen receptor may function cooperatively with one or more proteins within the cell. In some embodiments, the chimeric antigen receptor may dimerize or multimerize with a second receptor or transmembrane protein within the bone marrow cells, where the second receptor or transmembrane protein is an endogenous protein. In some embodiments, the cells are cultured ex vivo immediately after thawing or after nucleic acid incorporation. In some embodiments, ex vivo culturing is performed in the presence of an appropriate medium, which may include regulated serum components, such as human serum albumin (HSA). In some embodiments, ex vivo culturing and manipulation may be performed in a medium with a low serum content. In some embodiments, the serum is specifically treated to inactivate complement. In some embodiments, bone marrow cells may be cultured ex vivo in the presence of M-CSF, as described above. In some embodiments, bone marrow cells may be cultured ex vivo in the presence of GM-CSF, as described above. In some embodiments, bone marrow cells may be cultured in the presence of one or more cytokines. In some embodiments, bone marrow cells may be cultured or manipulated ex vivo for a period of time in the absence of growth factors or cytokines.In some embodiments, the methods provided herein involve the separation or enrichment and manipulation of bone marrow cells in less than 72 hours, 70 hours, 65 hours, 60 hours, 55 hours, 50 hours, 45 hours, 40 hours, 35 hours, 30 hours, 28 hours, 26 hours, or 24 hours. In some embodiments, the bone marrow cells may be cultured for less than 24 hours, or less than 20 hours, or less than 16 hours, or less than 14 hours, or less than 12 hours, or less than 10 hours, or less than 8 hours, or less than 6 hours, or less than about 4 hours. After separation or enrichment and manipulation, the bone marrow cells may be cultured for a short period of time and frozen until further use. In some embodiments, the bone marrow cells are thawed once or at most twice.

[0237]

[0289] In some embodiments, therapeutically competent cells are cells electroporated with a recombinant nucleic acid encoding a polypeptide, frozen and thawed, and the culture stabilized for less than 24 hours, wherein the cells in the cell population at the time of administration exhibit (i) greater than 70% viability, (ii) at least greater than 50% CD14+ / CD16− cells; and / or greater than 50% CD11b+ / CD14+ / CD16− cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, and less than about 10% CD42b+ cells; and (iv) greater than 50% of cells expressing the polypeptide encoded by the electroporated nucleic acid. In some embodiments, therapeutically competent cells are cells electroporated with a recombinant nucleic acid encoding a polypeptide, the culture stabilized for less than 24 hours, frozen, and thawed, wherein the cells in the cell population at the time of administration exhibit (i) greater than 70% viability, (ii) at least greater than 50% CD14+ / CD16− cells; and / or greater than 50% CD11b+ / CD14+ / CD16− cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, and less than about 10% CD42b+ cells; and (iv) greater than 50% cells expressing the polypeptide encoded by the electroporated nucleic acid. In some embodiments, therapeutically competent cells are cells whose culture has been stabilized for less than 24 hours, electroporated with a recombinant nucleic acid encoding a polypeptide, and then frozen and thawed, wherein the cells in the cell population at the time of administration exhibit (i) at least 70% viability, (ii) at least 50% CD14+ / CD16- cells; and / or more than 50% CD11b+ / CD14+ / CD16- cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, and less than about 10% CD42b+ cells; and (iv) more than 50% cells expressing the polypeptide encoded by the electroporated nucleic acid. The cells must be pathogen-free.In the above embodiments, the therapeutically competent cells may be frozen and thawed no more than twice, preferably once, and may be administered within 24 hours of thawing, within 18 hours of thawing, within 8 hours of thawing, or within 2 hours of thawing. Prior to administration, the cells are inspected for quality assurance to meet the standards described herein in this disclosure.

[0238]

[0290] Provided herein is a method for treating cancer in a subject, using a pharmaceutical composition comprising engineered phagocytes, particularly macrophages, expressing recombinant nucleic acid encoding phagocytic receptor (PR) fusion protein (PFP), which is specifically designed to target, attack and kill cancer cells.PFP is also referred to as chimeric antigen receptor for phagocytosis (CAR-P), and in this specification, either term can be used interchangeably.In this specification, engineered phagocytes are also referred to as CAR-P cells.

[0239]

[0291] Cancers include, but are not limited to, B-cell cancers (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), heavy chain diseases (e.g., alpha chain diseases, gamma chain diseases, and mu chain diseases), benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic prostate cancer, hormone refractory prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematopoietic tissue cancer, and the like. Other non-limiting examples of cancer types amenable to methods encompassed by the present disclosure include human sarcomas and human carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular carcinoma, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia, e.g., acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancer may also be characterized in various other forms, including, but not limited to, serous carcinoma, endometrial carcinoma, mucinous carcinoma, clear cell carcinoma, or undifferentiated carcinoma. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or subtypes thereof, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.

[0240]

[0292] Generally, cellular immunotherapy involves administering to a patient a medicine containing live cells. In some embodiments, a patient or subject with cancer is treated with autologous cells, the method comprising isolating or enriching PBMC-derived macrophages, modifying the macrophages ex vivo by introducing a recombinant nucleic acid encoding a chimeric antigen receptor for phagocytosis, which is a phagocytic receptor fusion protein (PFP), into the macrophages to generate highly phagocytic macrophages capable of tumor lysis, and administering the modified macrophages to the patient or subject.

[0241]

[0293] In one embodiment, a subject is administered one or more doses of a pharmaceutical composition containing therapeutic phagocytic cells, where the cells are allogeneic cells. HLA can be matched to the subject for compatibility and to prevent the cells from causing graft-versus-host disease (GVHD). Subjects who visit the clinic are subjected to HLA genotyping to determine the HLA antigens expressed by the subject before determining the therapeutic agent or treatment regimen.

[0242]

[0294] In some embodiments, a therapeutically effective dose is 10 cells / infusion. 7 10 bone marrow cells 12The number of cells may vary according to age, weight, and other parameters related to the subject and can be determined by a medical professional. In some embodiments, a therapeutically effective dose ranges between about 10 bone marrow cells and about 10 7 In some embodiments, the therapeutically effective dose is about 2 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 3 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 4 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 6 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 7 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 8 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 9 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 2 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 3 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 4 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 8 In some embodiments, a therapeutically effective dose is about 6 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 7 x 10 bone marrow cells. 8 In some embodiments, a therapeutically effective dose is about 8 x 10 bone marrow cells. 8 In some embodiments, a therapeutically effective dose is about 9 x 10 bone marrow cells. 8 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 2 x 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 3 x 10 bone marrow cells. 9In some embodiments, the therapeutically effective dose is about 4 x 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 6 x 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 7 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 8 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 9 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 10 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 10 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 11 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 11 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 12 There are individuals.

[0243]

[0295] In one aspect of the present specification, one or more recombinant polynucleic acids are provided that encode one or more recombinant proteins, which may be chimeric fusion proteins such as receptors or engagers, as described herein. In some embodiments, the recombinant polynucleic acid is an mRNA. In some embodiments, the recombinant polynucleic acid comprises a circRNA. In some embodiments, the recombinant polynucleic acid is incorporated into a viral vector. In some embodiments, the recombinant polynucleic acid is delivered via a viral vector.

[0244]

[0296] In some embodiments, therapeutic compositions are provided herein that comprise a chimeric fusion protein, such as a chimeric fusion receptor protein (CFP), wherein the CFP comprises: (a) (i) an scFv that specifically binds to any one of the targets disclosed herein, 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) 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, an interferon-inducible domain, and / or (ii) an intracellular domain comprising a third intracellular signaling domain, wherein (A) comprises a PI3K recruitment domain or (B) is derived from CD40.

[0245]

[0297] In some embodiments, therapeutic compositions are provided herein that include recombinant nucleic acids encoding the bispecific or trispecific engagers disclosed herein.

[0246] Other Therapeutic Compositions for Co-Administration

[0298] In some embodiments, the therapeutic composition further comprises 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 GTPase, an agent that promotes F-actin disassembly, an agent that promotes the recruitment of PI3K to PFPs, an agent that promotes the activity of PI3K, an agent that promotes the production of phosphatidylinositol 3,4,5-trisphosphate, an agent that promotes the activity of ARHGAP12, an agent that promotes the activity of ARHGAP25, an agent that promotes the activity of SH3BP1, an agent that promotes the sequestration of lymphocytes 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.

[0247]

[0299] In some embodiments, the bone marrow 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 bone marrow cells that express CFP compared to bone marrow cells that do not express CFP.

[0248]

[0300] In some embodiments, the bone marrow cells exhibit (i) increased effector activity, cross-presentation, respiratory burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4,5-triphosphate production, engagement with 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 , IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES, interferon, MHC class I proteins, MHC class II proteins, CD40, CD48, CD58, CD80, CD86, CD112, CD155, death receptors of the TRAIL / TNF family, TGFβ, B7-DC, B7-H2, LIGHT, HVEM, TL1A, 41BBL, OX40L, GITRL, CD30L, TIM1, TIM4, SLAM, PDL1, MMPs (e.g., MMP2, MMP7, and MMP9), or any combination thereof.

[0249]

[0301] In some embodiments, the intracellular signaling domain is derived from a phagocytic or tethering receptor (PR), 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 derived 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.

[0250]

[0302] In some embodiments, the intracellular signaling domain is derived from a receptor that contains an ITAM domain.

[0251]

[0303] Provided herein are compositions comprising a recombinant nucleic acid encoding a CFP, such as a phagocytic or tethered receptor (PR) fusion protein (PFP), comprising 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 to 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 a phagocytic receptor selected from Megf10, MerTk, FcRα, or Bai1.

[0252]

[0304] In some embodiments, upon binding of the CFP to an antigen on a target cell, the killing activity of cells expressing the CFP is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% greater than that of cells not expressing the CFP. , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000%. In some embodiments, the CFP is functionally integrated into the cell membrane of the cell when the CFP is expressed in the cell. In some embodiments, upon binding of the CFP to an antigen on a target cell, the killing activity of cells expressing the CFP is increased by at least 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 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 the CFP.

[0253]

[0305] 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.

[0254]

[0306] As used herein, a PR subunit includes a transmembrane domain and an intracellular domain including an intracellular signaling domain; and an extracellular domain including an antigen-binding domain specific to an antigen on 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, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCA1, and the like. Compositions are provided that include a recombinant nucleic acid encoding a CFP, such as a phagocytic or tethered receptor (PR) fusion protein (PFP), derived from a receptor, such as a phagocytic receptor selected from the group consisting of RF2, 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.

[0255]

[0307] In some embodiments, upon binding of the CFP to an antigen on a target cell, the killing activity of cells expressing the CFP is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% greater than that of cells not expressing the CFP. , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% increased. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, or 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.

[0256]

[0308] In some embodiments, cells expressing CFP exhibit increased phagocytosis of target cells expressing antigen compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit at least 1.1-fold increased phagocytosis of target cells expressing antigen compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit at least 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 increased phagocytosis of target cells expressing antigen compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased cytokine production compared to cells not expressing 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, CFP-expressing cells exhibit increased expression of CD86 compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of MHC class I proteins compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of death receptors of the TRAIL / TNF family compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of B7-H2 compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of LIGHT compared to cells that do not express 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 a ligand for TIM1 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 that express CFP exhibit an increased respiratory burst compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit an increased production of ROS 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 iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased extracellular vesicle produ...

Claims

1. A polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen-binding domain that specifically binds to glypican 3 (GPC3); and (b) a transmembrane domain operably linked to an extracellular domain; Including, the transmembrane domain is a transmembrane domain derived from a protein that dimerizes with an endogenous FcRγ receptor; the CFP is expressed on the surface of a myeloid cell that expresses the FcRγ receptor; and the antigen binding domain comprises a heavy chain comprising a heavy chain variable domain, the heavy chain variable domain comprising a heavy chain complementarity determining region 1 (HCDR1) of DYEMH (amino acid residues 31-35 of SEQ ID NO:97), a HCDR2 of ALDPKTGDTAYSQKFKG (amino acid residues 50-66 of SEQ ID NO:97), and a HCDR3 of FYSYTY (amino acid residues 99-104 of SEQ ID NO:97).

2. 2. The polynucleic acid of claim 1, wherein the antigen binding domain comprises a light chain comprising a light chain variable domain, the light chain variable domain comprising a light chain complementarity determining region 1 (LCDR1) of RSSQSLVHSNRNTYLH (amino acid residues 24-39 of SEQ ID NO:98), a LCDR2 of KVSNRFS (amino acid residues 55-61 of SEQ ID NO:98), and a LCDR3 of SQNTHVPPT (amino acid residues 94-102 of SEQ ID NO:98).

3. 3. The polynucleic acid of claim 1 or 2, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), a Fab, a single domain antibody (sdAb), a nanobody, or a diabody.

4. The polynucleic acid of claim 3 , wherein the antigen-binding domain comprises an scFv domain.

5. The polynucleic acid according to any one of claims 1 to 4, wherein the heavy chain variable domain comprises a sequence having at least 80% identity to SEQ ID NO:

97.

6. The polynucleic acid according to any one of claims 2 to 5, wherein the light chain variable domain comprises a sequence having at least 80% identity to SEQ ID NO:

98.

7. The polynucleic acid according to any one of claims 1 to 6, wherein the antigen-binding domain comprises a sequence having at least 80% sequence identity to SEQ ID NO:

106.

8. The polynucleic acid according to any one of claims 1 to 7, wherein the transmembrane domain comprises a transmembrane domain derived from CD16a, CD64, CD68 or CD89.

9. The polynucleic acid according to claim 8, wherein the transmembrane domain is a transmembrane domain derived from CD89.

10. 10. The polynucleic acid of claim 9, wherein the transmembrane domain comprises the sequence LIRMAVAGLVLVALLAILV (amino acid residues 278-296 of SEQ ID NO:58).

11. The polynucleic acid according to any one of claims 1 to 10, wherein the extracellular domain further comprises an extracellular domain derived from CD89, CD16a, CD64, or CD68, or a fragment thereof.

12. The polynucleic acid of claim 11 , wherein the extracellular domain comprises an extracellular domain derived from CD89 or a fragment thereof.

13. The polynucleic acid according to claim 12, wherein the extracellular domain comprises the sequence DSIHQDYTTQN (amino acid residues 267-277 of SEQ ID NO:58).

14. The polynucleic acid of any one of claims 1 to 13, wherein the CFP further comprises an intracellular domain comprising one or more intracellular signaling domains, the one or more intracellular signaling domains comprising an intracellular signaling domain derived from CD89, CD16a, CD64, CD68, FCERIG, CD40, or CD3ζ.

15. The polynucleic acid of claim 14, wherein the one or more intracellular signaling domains comprise an intracellular signaling domain derived from CD89.

16. 16. The polynucleic acid of claim 15, wherein the one or more intracellular signaling domains comprise the sequence ENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (amino acid residues 297-337 of SEQ ID NO:58).

17. The polynucleic acid according to any one of claims 1 to 16, wherein the CFP further comprises a GMCSF signal peptide.

18. 18. The polynucleic acid of claim 17, wherein the GMCSF signal peptide comprises the sequence MWLQSLLLLGTVACSIS (SEQ ID NO: 7).

19. The polynucleic acid according to any one of claims 1 to 18, wherein the polynucleic acid is RNA.

20. 20. The polynucleic acid of claim 19, wherein the RNA is mRNA.

21. A polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) (i) an antigen-binding domain that specifically binds glypican 3 (GPC3), and (ii) an extracellular domain comprising the sequence DSIHQDYTTQN (amino acid residues 267-277 of SEQ ID NO:58); (b) a transmembrane domain operably linked to an extracellular domain, where the transmembrane domain comprises the sequence of LIRMAVAGLVLVALLAILV (amino acid residues 278-296 of SEQ ID NO:58); and (c) an intracellular domain operably linked to a transmembrane domain, wherein the intracellular domain comprises the sequence ENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (amino acid residues 297-337 of SEQ ID NO:58); Including, The antigen-binding domain is a single chain variable fragment (scFv) comprising a heavy chain comprising a heavy chain variable domain and a light chain comprising a light chain variable domain, the heavy chain variable domain comprising a heavy chain complementarity determining region 1 (HCDR1) of DYEMH (amino acid residues 31-35 of SEQ ID NO:97), a HCDR2 of ALDPKTGDTAYSQKFKG (amino acid residues 50-66 of SEQ ID NO:97), and a HCDR3 of FYSYTY (amino acid residues 51-52 of SEQ ID NO:97). and a light chain variable domain comprising a light chain complementarity determining region 1 (LCDR1) of RSSQSLVHSNRNTYLH (amino acid residues 24-39 of SEQ ID NO:98), a LCDR2 of KVSNRFS (amino acid residues 55-61 of SEQ ID NO:98), and a LCDR3 of SQNTHVPPT (amino acid residues 94-102 of SEQ ID NO:98).

22. A composition comprising the polynucleic acid according to any one of claims 1 to 21.

23. The composition of claim 22, wherein the polynucleic acid is encapsulated in a lipid nanoparticle (LNP).

24. 24. A pharmaceutical composition comprising the composition of claim 22 or 23 and a pharma- ceutically acceptable excipient.

25. 25. Use of the pharmaceutical composition of claim 24 in the manufacture of a medicament for treating cancer.

26. 25. The pharmaceutical composition of claim 24 for use in the treatment of cancer.

27. 27. The use according to claim 25, or the pharmaceutical composition according to claim 26, wherein the cancer is selected from the group consisting of ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer and lung cancer.

28. 28. The use according to claim 27, wherein the cancer is liver cancer.

29. The use according to any one of claims 25 to 28, wherein the cancer is GPC3 positive.

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