Manipulated chimeric fusion protein composition and method 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 of related applications

[0001] This application claims the benefits 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 herein by reference in whole. [Background technology]

[0002]

[0002] Cellular immunotherapy is a promising new technology for combating difficult-to-treat diseases such as cancer and persistent infections, and for combating certain diseases that are refractory to other forms of treatment. The discovery of CAR-T cells and their potential use in immunotherapy has represented a major breakthrough. CAR-T cells are T lymphocytes that express chimeric antigen receptors, which help T cells target specific affected cells, such as cancer cells, and may induce a cytotoxic response intended to kill targeted cancer cells, or they may induce immunosuppression and / or immune tolerance, depending on the intracellular domains used and the immunosuppressive cytokines co-expressed. CAR-T cells remain a promising tool for cancer treatment, but several limitations along the way have slowed progress in CAR-T cells and diminished their potential in clinical trials.

[0003]

[0003] Understanding the limitations of CAR-T cells is key to leveraging the technology and sustaining innovation in good immunotherapy models. Specifically, in T-cell malignancies, CAR-T cells are thought to face significant problems. Because CAR-T cells and malignant T cells share surface antigens in most T-cell lymphomas (TCLs), CAR-T cells are subjected to cytotoxicity, similar to cancer cells. In some cases, CAR-T products may be contaminated by malignant T cells. In addition, T cell aplasia is a potential problem for extending the survival of CAR-T cells. Other limitations include the poor ability of CAR-T cells to penetrate solid tumors and potent tumor microenvironments, which act to downmodulate their antitumor potential. CAR-T cell function is also negatively affected by immunosuppressive tumor microenvironments (TMEs), which result in the inactivation and exhaustion of endogenous T cells.

[0004]

[0004] Myeloid cells, including macrophages, are cells derived from the myeloid lineage and belong to the innate immune system. Myeloid cells are derived from myeloid stem cells that migrate into the bloodstream and into tissues. Some of their major functions include phagocytosis, activation of T cell responses, and removal of cytolytica and extracellular matrix. Myeloid cells also play an important role in maintaining homeostasis and inducing and resolving inflammation. Furthermore, myeloid cells can differentiate into a number of downstream cells, including macrophages, which can present different responses ranging from pro-inflammatory to anti-inflammatory depending on the type of stimulus they receive from the surrounding microenvironment. In addition, tissue macrophages have been shown to play a broad regulatory and activating role for other immune cell types, including CD8+ T effector cells and CD4+ T effector cells, NK cells, and regulatory T cells. Macrophages have been shown to be the major immune infiltrators in malignant tumors and have also been shown to exert broad immunosuppressive effects on effector immune infiltration and immune function. [Overview of the project]

[0005]

[0005] The diverse functionalities of bone marrow cells make them ideal candidates for cell therapies, which can be manipulated to exert numerous therapeutic effects. This disclosure relates to immunotherapy using bone marrow cells of the immune system (e.g., CD14+ cells), in particular phagocytic cells. Numerous therapeutic indications may be envisioned using bone marrow cells. For example, bone marrow cell immunotherapy may be crucial in cancer, autoimmune diseases, fibrotic diseases, and infections. This disclosure relates to immunotherapy using bone marrow cells, including phagocytic cells of the immune system, in particular monocytes. Utilizing one or more of these functions of bone marrow cells for therapeutic use is an object of the invention disclosed herein. For example, utilizing the phagocytic activity of bone marrow cells, including manipulated bone marrow cells, for therapeutic use is an object of the invention disclosed herein. For example, utilizing the ability of bone marrow cells, including manipulated bone marrow cells, to promote T cell activation is an object of the invention disclosed herein. For example, utilizing the ability of bone marrow cells, including manipulated bone marrow cells, to promote the secretion of tumor-killing molecules is an object of the invention disclosed herein. For example, utilizing the ability of myeloid cells, including engineered myeloid cells, to facilitate the recruitment and transport of immune cells and immune molecules is an object of the invention disclosed herein. In one aspect, the disclosure provides novel and useful chimeric constructs that, when expressed in myeloid cells, drive targeted attack and phagocytosis of molecules, molecular assemblies, objects, or targets, such as cells containing a target antigen on their surface. One of the many aspects of the disclosure is to (i) enhance the phagocytic ability of myeloid cells (e.g., engineered myeloid cells expressing a novel improved chimeric construct); and to help evoke a systematic and sustained immune response against a target (e.g., a target antigen). In addition to enhancing the functional aspects of myeloid cells, the disclosure presents novel methods and compositions that may successfully transfect or transduce myeloid cells, or otherwise induce genetic modification within myeloid cells, without impairing the differentiation potential, maturation potential, and / or plasticity of the cells. The resulting cells may be referred to as therapeutically effective, engineered bone marrow cells or effector bone marrow cells.One strategy for the improvements described herein is to induce an inflammatory phenotype in myeloid cells to generate effector myeloid cells. One strategy is to create effector myeloid cells that are capable of exhibiting an inflammatory phenotype upon engagement with a target. In one embodiment, the effector myeloid cells are capable of inducing or activating a type I interferon response within the myeloid cells upon engagement with their target. Another strategy is to enhance the type I interferon response within the myeloid cells to generate these cells into effector myeloid cells. One strategy for inducing an inflammatory phenotype is to induce or activate an NF-κB response within the effector myeloid cells upon engagement with their target. One strategy for generating myeloid cells isolated from a biological sample into effector myeloid cells for the immunotherapy described herein is to induce and / or enhance a type I interferon response or an NF-kappa B response or both within the myeloid cells.

[0006]

[0006] This disclosure involves the creation and use of engineered bone marrow cells (e.g., CD14+ cells), such as macrophages or other phagocytic cells (ATAK), which can directly and / or indirectly attack and kill diseased cells, such as cancer cells and infected cells. Engineered bone marrow cells, such as macrophages and other phagocytic cells, can be prepared by incorporating nucleic acid sequences (e.g., mRNA, DNA, plasmids, viral constructs) encoding chimeric fusion proteins (CFPs) having extracellular binding domains specific to disease-associated antigens (e.g., cancer antigens) 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. Bone marrow cells have been found to be engineered to exert a broad and diverse range of activity. For example, bone marrow cells can be engineered to express chimeric fusion proteins (CFPs) containing antigen-binding domains to exert a broad and diverse range of activity. For instance, bone marrow cells can be engineered to enhance phagocytic activity so that, upon binding of CFPs to antigens on target cells, the cells exhibit increased phagocytosis of target cells. Furthermore, bone marrow cells can be engineered to promote T cell activation, such as promoting the activation of T cells within the tumor microenvironment, upon binding of CFPs to antigens on target cells. Engineered bone marrow cells can be engineered to promote the secretion of tumor-killing molecules, such as promoting the secretion of tumor-killing molecules from nearby cells, upon binding of CFPs to antigens on target cells. Engineered bone marrow cells can be engineered to promote the recruitment and transport of immune cells and immune molecules, such as promoting the recruitment and transport of immune cells and immune molecules to target cells or the tumor microenvironment, upon binding of CFPs to antigens on target cells.

[0007]

[0007] This disclosure is an important discovery in which engineered bone marrow cells overcome at least some of the limitations of CAR-T cells, including being easily mobilized to solid tumors; having an operable survival period, and thus a low risk of extended survival resulting in aplasia and immunodeficiency; not being likely to be contaminated with T cells; being able to avoid fratricide because, for example, they do not express the same antigens as malignant T cells; and having a large number of anti-tumor functions that can be deployed. In some respects, engineered bone marrow-derived cells could be a safer immunotherapy tool for targeting and destroying diseased cells.

[0008]

[0008] Furthermore, bone marrow cells such as macrophages have been found to be ubiquitous within the tumor microenvironment (TME), and are, in particular, the most abundant cells in some tumor types. As part of their role in the immune system, bone marrow cells such as macrophages are naturally involved in the removal of diseased cells. The present invention relates to the utilization of bone marrow cell functions, and in particular to the targeting, killing, and direct and / or indirect removal of diseased cells, as well as to the delivery of payloads such as antigens and cytokines.

[0009]

[0009] Manipulated bone marrow cells are also often found to be short-lived, phenotypically diverse, highly sensitive, and plastic in vivo, and difficult to manipulate in vitro. For example, the expression of exogenous genes in monocytes is more difficult than the expression of exogenous genes in non-hematopoietic cells. Significant technical difficulties exist associated with transfecting bone marrow cells (e.g., monocytes / macrophages). As professional phagocytic cells, bone marrow cells such as monocytes / macrophages contain many potent degrading enzymes that can destroy nucleic acid integrity, making gene transfer into these cells an inefficient process. This is especially true for activated macrophages, which undergo dramatic physiological changes after exposure to immune or inflammatory stimuli. Since macrophages are generally non-dividing final-stage cells, viral transduction into these cells is not ideal; therefore, some vectors that rely on integration into a replicable genome have limited success. Furthermore, because macrophages are highly responsive to "danger signals," some of the original viral vectors used for gene delivery induce a strong antiviral response within these cells, rendering these vectors unsuitable for gene delivery. In addition, myeloid cells have the potential to differentiate into phenotypic variants with significantly different functionalities, and under certain circumstances, may not be effective for the intended purpose for in vivo use. For example, myeloid cells required for active migration to tumors and effective tumor-killing activity may need to be delivered in vivo at a cellular stage that retains their plasticity, based on the identification of certain cell surface markers. The plasticity of myeloid cells may be affected by factors such as the nature of isolation, handling, and the method of introducing nucleic acid material into the cells, both ex vivo and for manipulating the cells.Bone marrow cells at various cell stages (e.g., bone marrow cells at high or low levels of plasticity, or bone marrow cells with a phenotype conferring inflammatory or other immunomodulatory functions) each have a particular value for the intended purpose for in vivo use, and all of these are within the scope of the present disclosure. In at least one aspect, however, the methods discussed herein focus on the generation of a therapeutic bone marrow cell population that retains a high degree of bone marrow cell plasticity prior to in vivo administration. In one aspect, the present disclosure provides novel methods and compositions for transfecting or transducing bone marrow cells, or otherwise inducing genetic modification within bone marrow cells, such that, in addition to enhancing the functional aspects of the bone marrow cells, the differentiation ability, the potential for its maturation, and / or the plasticity of the cells are not impaired.

[0010]

[0010] In one aspect of the present specification, a therapeutic agent that binds to an antigen expressed on a target cell, such as a diseased cell, for example, a cancer cell, is provided. 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 within 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 the target antigen on the target cell. In some embodiments, the therapeutic agent is a cell, such as a mammalian cell, a human cell, a bone marrow cell, or a monocyte, where the cell is capable of targeting diseased cells that express the target antigen on the cell surface; the bone marrow cell contains a recombinant nucleic acid and / or expresses a recombinant protein such that the bone marrow cell lyses or phagocytoses the diseased cell.

[0011]

[0011] In one aspect of this 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 the intracellular domain, upon receiving the extracellular cue, influences an intracellular mechanism of action and myeloid activation. In one embodiment, the chimeric fusion protein is a chimeric receptor having an extracellular antigen-binding domain in addition to (a) a transmembrane domain and (b) an intracellular domain operably linked to the transmembrane domain, wherein the engagement of the extracellular antigen-binding domain of the chimeric fusion protein with a target antigen to which the extracellular binding domain binds results in an extracellular cue for receptor-mediated activation of the receptor by myeloid cells.

[0012]

[0012] In one aspect of this 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 embodiments, the chimeric fusion protein further comprises an extracellular domain containing an antigen-binding domain, in which case the transmembrane domain is operably linked to the extracellular domain. In some embodiments, the intracellular domain comprises at least one further intracellular signaling domain. In some embodiments, the at least one further intracellular signaling domain is derived from an intracellular PI3 kinase mobilization 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 adapter protein, a mitochondrial membrane protein, or an endoplasmic reticulum membrane protein. In one embodiment, 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 long, about 150–270 amino acids long, or about 150–250 amino acids long. In some embodiments, the intracellular signaling domain includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 36. In some embodiments, the intracellular signaling domain includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular signaling domain includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular signaling domain includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 41.

[0022]

[0022] In one aspect of this specification, recombinant nucleic acids are provided that encode 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 may have phagocytic activity. In some embodiments, the chimeric fusion protein further comprises an extracellular domain comprising an antigen-binding domain, in which case 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 this specification, a recombinant nucleic acid is provided which 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 at least 80% amino acid sequence identity to SEQ ID NO: 39.

[0024]

[0024] In one aspect of this specification, a recombinant nucleic acid is provided which 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 at least 80% amino acid sequence identity to SEQ ID NO: 40.

[0025]

[0025] In one aspect of this specification, a recombinant nucleic acid is provided which 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 at least 80% amino acid sequence identity to SEQ ID NO: 42.

[0026]

[0026] In one aspect of this specification, a recombinant nucleic acid is provided which 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 at least 80% amino acid sequence identity to SEQ ID NO: 43.

[0027]

[0027] In one aspect of this specification, a recombinant nucleic acid is provided which 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 at least 80% amino acid sequence identity to SEQ ID NO: 45.

[0028]

[0028] In one aspect of this specification, a recombinant nucleic acid is provided which 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 CD68 and comprises a mutation that inhibits or blocks 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 of this specification, recombinant nucleic acids are provided that encode a chimeric fusion protein comprising (a) a transmembrane domain and (b) an extracellular domain including 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 lacks an intracellular signaling domain. In some embodiments, the transmembrane domain is derived from a CD16 protein, for example, a CD16a protein. In some embodiments, the chimeric fusion protein forms a complex with an FcRγ chain when expressed in a cell.

[0031]

[0031] In one aspect of this specification, a recombinant nucleic acid is provided which 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 at least 80% amino acid sequence identity to SEQ ID NO: 48.

[0032]

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

[0033]

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

[0034]

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

[0035]

[0035] In some embodiments, the antigen-binding domain includes a domain having affinity for a tumor antigen. In some embodiments, the extracellular domain includes a T lymphocyte antigen-binding domain. In some embodiments, the extracellular domain includes a B lymphocyte antigen-binding domain. In some embodiments, the antigens to which the antigen-binding domain binds include 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), follicular-stimulating hormone receptor, and fibroblast activity. Facial protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer 2D (NKG2D) group ligands, disiaroganglioside 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 The antigens are selected from the group consisting of antigens derived from 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 includes a first target antigen-binding domain and a second target antigen-binding domain that is not identical to the first target antigen-binding domain. In some embodiments, the second target antigen-binding domain binds to CD47. In some embodiments, the antigen-binding domain includes a domain having affinity for a microbial antigen. In some embodiments, the antigen-binding domain includes a domain having affinity for a viral antigen. In some embodiments, the domain includes an antibody or a fragment thereof. In some embodiments, the domain includes 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 includes a hinge domain connecting the antigen-binding domain and the transmembrane domain. In some embodiments, when expressed intracellularly, the chimeric fusion protein activates its intracellular domain upon binding to its target antigen, thereby 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 associates with one or more lipids. In some embodiments, the recombinant nucleic acid is encapsulated within liposomes. In some embodiments, the liposomes are lipid nanoparticles.

[0036]

[0036] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 52. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 52. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 52. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 52 or a sequence having at least 80% sequence identity to Sequence ID No. 52.

[0037]

[0037] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 53. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 53. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 53. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 53 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 53.

[0038]

[0038] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 54. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 54. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 54. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 54 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 54.

[0039]

[0039] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to sequence number 55. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to sequence number 55. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to sequence number 55. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is identical to sequence number 55. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of sequence number 55 or a sequence that has at least 80% sequence identity to the sequence of sequence number 55.

[0040]

[0040] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to sequence number 56. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to sequence number 56. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to sequence number 56. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is identical to sequence number 56. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of sequence number 56 or a sequence that has at least 80% sequence identity to the sequence of sequence number 56.

[0041]

[0041] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 57. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 57. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 57. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 57 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 57.

[0042]

[0042] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 58. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 58. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 58. In some embodiments of this specification, a cell is provided that contains a recombinant nucleic acid that codes for the sequence of Sequence ID No. 58 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 58.

[0043]

[0043] In one aspect of this specification, a recombinant nucleic acid encoding a sequence that is at least 80% identical to sequence number 59 is provided. In one aspect of this specification, a recombinant mRNA construct encoding a sequence that is at least 80% identical to sequence number 59 is provided. In this specification, a recombinant mRNA construct encoding a sequence that is at least 90% identical to sequence number 59 is provided. In this specification, a recombinant mRNA construct encoding a sequence that is identical to sequence number 59 is provided. In some embodiments of this specification, a cell is provided comprising a recombinant nucleic acid encoding the sequence of sequence number 59 or a sequence having at least 80% sequence identity to the sequence of sequence number 59.

[0044]

[0044] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to sequence number 60. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to sequence number 60. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to sequence number 60. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is identical to sequence number 60. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of sequence number 60 or a sequence that has at least 80% sequence identity to the sequence of sequence number 60.

[0045]

[0045] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 61. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 61. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 61. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 61 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 61.

[0046]

[0046] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 62. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 62. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 62. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 62 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 62.

[0047]

[0047] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 63. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 63. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 63. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 63 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 63.

[0048]

[0048] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 64. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 64. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 64. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 64 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 64.

[0049]

[0049] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 65. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 65. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 65. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 65 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 65.

[0050]

[0050] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 66. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 66. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 66. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 66 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 66.

[0051]

[0051] In one aspect of this specification, a recombinant nucleic acid is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 67. In one aspect of this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 80% identical to Sequence ID No. 67. In this specification, a recombinant mRNA construct is provided that codes for a sequence that is at least 90% identical to Sequence ID No. 67. In some embodiments of this specification, a cell is provided that comprises a recombinant nucleic acid that codes for the sequence of Sequence ID No. 67 or a sequence having at least 80% sequence identity to the sequence of Sequence ID No. 67.

[0052]

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

[0053]

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

[0054]

[0054] In one aspect of the Specified, there is an engineered cell comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain including 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, the intracellular signaling domain comprising an intracellular signaling domain that, when activated, binds to an IFN transcription factor.

[0055]

[0055] In one aspect of this specification, an engineered cell is provided 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, the intracellular signaling domain comprising an intracellular signaling domain derived from a mitochondrial receptor.

[0056]

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

[0057]

[0057] In some embodiments, the intracellular domain 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, chemokine, MHC class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) It contains 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 immune receptor (DCIR) subfamily proteins, DCIR / CLEC4A, dectin 2 / CLEC6A, serum DC antigen 2 (BDCA2) (CLEC4C), and Mincle (macrophage-inducible C-type lectin) (CLEC4E).

[0058]

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

[0059]

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

[0060]

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

[0061]

[0061] In some embodiments, the intracellular signaling domain includes an intracellular signaling domain that, when activated, binds to an IFN transcription factor.

[0062]

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

[0063]

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

[0064]

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

[0065]

[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]

[0067] In some embodiments, the antigen-binding domain is 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), follicular-stimulating hormone receptor, fibroblast Activated protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer 2D (NKG2D) group ligands, disiaroganglioside 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 It binds to one or more of the following: 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.

[0068]

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

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

[0071]

[0071] In some embodiments, the intracellular signaling domain binds to an IRF transcription factor and activates the IRF transcription factor when the antigen-binding domain binds to its cognitive antigen.

[0072]

[0072] In some embodiments, cells induce pro-inflammatory cytokines when the antigen-binding domain binds to its cognitive 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 interferons.

[0074]

[0074] In some embodiments, the cells are bone marrow cells, lymphocytes, or stem cells.

[0075]

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

[0076]

[0076] In one aspect of the present disclosure, a pharmaceutical composition is also presented 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 manipulated cells are CD14+ / CD16-.

[0078]

[0078] The pharmaceutical compositions according to the embodiments described above include a population of cells in which 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 this specification, a method is provided for treating cancer or a viral infection in a subject, the method comprising the step of administering to the subject a pharmaceutical composition according to any one of the embodiments described above.

[0080]

[0080] In some aspects of this specification, engineered CD14+ / CD16- cells are provided, comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain including 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, the intracellular signaling domain comprising a domain derived from TRIF.

[0081]

[0081] In some embodiments, the intracellular domain containing the TRIF-derived domain is a shortened TRIF domain.

[0082]

[0082] In some embodiments, the intracellular domain, which includes a domain derived from TRIF, contains fewer than 300 amino acids.

[0083]

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

[0084]

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

[0085]

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

[0086]

[0086] In one aspect of this specification, a method for treating cancer in a subject is provided, comprising the step of 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 stomach 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] Embedding by reference

[0088] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0089]

[0089] Novel features of the present invention are described in detail in the appended claims. A good understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings illustrating exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0090] [Figure 1A]

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

[0091] This is a schematic representation of exemplary myeloid cells that express the CFP receptor and engage with cancer cells by binding to target antigens on those cells. Activation of the intracellular signaling domain upon target engagement enhances phagocytosis. TM: Transmembrane domain. [Figure 1C]

[0092] This is a schematic representation of exemplary CFP expressed on bone marrow cells. CFP is capable of targeting cancer antigens and activating type 1 interferons. It has an intracellular domain. FcR: Fc receptor. [Figure 1D]

[0093] This figure shows a schematic representation of an exemplary nucleic acid encoding an exemplary chimeric antigen receptor construct, which includes an intracellular signaling domain (ICD) containing a pLxIS motif that induces a type I interferon response. [Figure 2A]

[0094] The upper panel of Figure 2A illustrates a schematic diagram showing exemplary CFP construct designs with the indicated domains. Each construct is created 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 in THP-1 cells supporting the expression of each construct in the upper panel. [Figure 2B]

[0095] The upper panel of Figure 2B illustrates a schematic diagram showing exemplary CFP construct designs with the indicated domains. Each construct is created 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 in THP-1 cells supporting the expression of each construct shown in the upper panel. [Figure 2C]

[0096] The upper panel of Figure 2C shows a schematic diagram illustrating exemplary CFP construct designs with the indicated domains. The construct on the left has a CD64 transmembrane domain, and the construct on the right has a CD89 transmembrane domain. The lower panel of Figure 2C shows flow cytometry data in THP-1 cells supporting the expression of each construct shown in the upper panel. [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 further intracellular signaling domains: the first CFP from the left contains a PI3K mobilizing domain and a signaling domain derived from MDA5; the second from the left contains a CD40 intracellular signaling domain and a PI3K mobilizing domain. The two constructs on the right have a CD89 transmembrane domain and the following further intracellular signaling domains: the third CFP from the left contains a PI3K mobilizing domain and a signaling domain derived from MDA5; the fourth from the left contains a CD40 intracellular signaling domain and a PI3K mobilizing domain. The lower panel of Figure 2D shows flow cytometry data in THP-1 cells supporting the expression of each construct shown in the upper panel. [Figure 2E]

[0098] This figure shows quantitative data on the relative expression levels of each construct shown in the figure within THP-1 cells, derived from the analysis of flow cytometry results. [Figure 2F]

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

[0100] This figure shows an exemplary CFP design (upper panel) and expression data (lower panel) for a construct within THP-1 cells. Each domain is indicated in the figure. [Figure 3]

[0101] This figure shows an exemplary CFP design in which the extracellular domain is directed to bind to a single target antigen or multiple targets, and each scFv has a different antigen specificity. [Figure 4A]

[0102] This figure shows an exemplary assay setup for the quantitative evaluation of target cell phagocytosis by CFP-expressing bone marrow cells. The SKOV3 tumor cell line is an exemplary target cell line labeled with a cytoplasmic dye. Phagocytosis of the target cells resulted in the release of the dye into the bone marrow cells, which was then analyzed by flow cytometry. Exemplary flow cytometry data from representative experiments are also shown. [Figure 4B]

[0103] This figure illustrates representative quantitative data showing the phagocytic index of bone marrow cells expressing each of the indicated constructs. The E:T ratio represents the ratio of effector (bone marrow) cells to target cells. The experimental results were analyzed according to the method shown in Figure 4A. [Figure 4C]

[0104] This figure illustrates representative data showing the phagocytic index of bone marrow cells expressing each of the indicated constructs. [Figure 4D]

[0105] This figure illustrates representative data showing the phagocytic index of bone marrow cells expressing each of the indicated constructs. [Figure 4E]

[0106] This figure illustrates 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 supported cells expressing the HER2-FcR-PI3K-TRIF (short chain) construct, which showed a high phagocytic index compared to other constructs. [Figure 5A]

[0107] This figure shows an exemplary assay configuration for the quantitative evaluation of phagocytic lysis of target cells by myeloid cells expressing CFP, as well as a multiplexed assay for the analysis of cytokines and chemokines. The target cells express the luciferase gene, and upon action on the target cells and upon target cell lysis, luciferase is released into the myeloid cells, which can be used to detect and quantify direct target cell lysis. [Figure 5B]

[0108] This figure shows representative data on target cell lysis by various sets of bone marrow cells expressing the CFP construct shown. The left figure shows results from experiments using a 1:1 effector cell:target cell ratio; the right figure shows results from experiments using a 5:1 effector cell:target cell ratio. [Figure 5C]

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

[0110] Figure 5C shows data supporting IFN activation in THP-1 cells or mock-transfected THP-1 cells 24 hours after transfection with the CFP constructs shown. 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] This figure shows data supporting NF kappa B activation in THP-1 cells, which were electroporated using mock cells or the CFP constructs shown, and assayed 24 hours after transfection in the presence or absence of SKOV3 target cells. Each construct possesses a CD89 intracellular signaling domain. The CD89 intracellular domain alone was capable of activating NF kappa B, but further intracellular domains did not induce NF kappa B activation. [Figure 5F]

[0112] Figure 5E shows data supporting IFN activation in THP-1 cells or mock-transfected THP-1 cells 24 hours after transfection with the CFP construct shown in the diagram. [Figure 5G]

[0113] This figure shows data supporting the activation of NF kappa B in THP-1 cells expressing each of the indicated constructs. The results are shown for cells expressing constructs that have an FcR domain, or constructs that have an FcR domain along with additional intracellular domains. [Figure 5H]

[0114] This figure shows flow cytometry data supporting IFN activation in THP-1 cells 24 hours after electroporation with the CFP construct shown. [Figure 5I]

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

[0116] The figures show the expression of the indicated HER2-CFP construct in THP-1 cells (upper panel) and flow cytometry data supporting NF kappa B activation 24 hours after electroporation with the indicated CFP construct (lower panel). Expression of the HER2-FcR-PI3 kinase-MyD88 construct resulted in sustained NF kappa B signaling (signaling in the absence of the target cell SKOV3, i.e., signaling in the absence of engagement of the extracellular binding domain of CFP with the antigen on SKOV3 cells). [Figure 5K]

[0117] This figure shows flow cytometry data in THP-1 cells 24 hours after electroporation with the indicated HER2-CFP construct, supporting the expression of the indicated HER2-CFP construct and the activation of NF kappa B and IFN. The data support that persistent NF kappa B / IFN gamma signaling is observed within multi-domain constructs having either the FcR-PI3K-RIG1 intracellular domain or the FcR-PI3K-MDA5 intracellular domain. [Figure 5L]

[0118] This figure shows flow cytometry data supporting the expression of the indicated HER2-CFP construct and IFN activation within THP-1 cells 24 hours after electroporation with the indicated CFP construct. [Figure 5M]

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

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

[0121] This figure shows data supporting baseline cytokine (IL6 and TNF-alpha) / chemokine (CCL3) expression in THP-1 cells expressing the indicated construct, compared to mock-transfected cells. Negative control (mock-transfected) graphs are shown to the left of each CFP-transfected graph for each experimental set. [Figure 6B]

[0122] This figure shows data supporting baseline cytokine (IL6 and TNF-alpha) expression in THP-1 cells expressing the indicated construct, compared to mock-transfected cells. Negative control (mock-transfected) graphs are shown to the left of each CFP-transfected graph for each experimental set. [Figure 6C]

[0123] This figure shows data supporting baseline cytokine (IL6 and TNF-alpha) expression in THP-1 cells expressing the indicated construct, compared to mock-transfected cells. Negative control (mock-transfected) graphs are shown to the left of each CFP-transfected graph for each experimental set. [Figure 6D]

[0124] This figure shows data supporting baseline cytokine (IL6, TNF-alpha, and IFN-gamma, shown in their respective graphs) / chemokine (CCL3, CCL5, and IP10, shown) expression in THP-1 cells expressing the indicated constructs, compared to mock-transfected cells. Negative control (mock-transfect) graphs are shown to the left of each CFP-transfect graph for each experimental set. [Figure 6E]

[0125] This figure shows data supporting cytokine / chemokine expression in cells transfected with the shown construct, compared to mock-transfected cells. [Figure 6F]

[0126] This figure shows data supporting cytokine / chemokine expression in cells transfected with the shown construct, compared to mock-transfected cells. [Figure 7A]

[0127] This figure shows a schematic representation of an exemplary chimeric antigen receptor construct containing a CD68 transmembrane domain. [Figure 7B]

[0128] This figure shows exemplary chimeric antigen receptors with the indicated domains. The center and right figures in the upper panel show exemplary CFPs with CD64 transmembrane domains oligomerized with endogenous Fc gamma and lacking their own intracellular domains. The middle panel shows exemplary intracellular domains paired with diverse domain combinations in recombinant CFP construct designs. The bottom panel shows exemplary CFPs containing multiple domains. [Figure 8A]

[0129] This figure shows flow cytometry data supporting the expression of the HER2-CFP construct in primary human monocytes derived from a first donor, after electroporation with the HER2-CFP construct. 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] This figure shows flow cytometry data confirming the expression of the HER2-CFP construct in primary human monocytes derived from a second donor, following electroporation with the HER2-CFP construct. [Figure 8C-1]

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

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

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

[0134] This figure shows a graph of the induction of the pro-inflammatory cytokine IL-6 by primary human monocytes derived from human donors, either electroporated with the indicated HER2-CFP construct and stimulated with the HER-2 antigen (top), or co-cultured with SKOV3 tumor cells (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 of HER2-his protein, and 100,000 electroporated primary human monocytes containing the indicated HER2-CFP construct were added to each well. The supernatant was collected 48 hours after stimulation, and the secreted cytokines were analyzed using Luminex. For HER2-CFP primary monocyte samples stimulated with tumor cells, 100,000 primary human monocytes electroporated with the indicated HER2-CFP construct were co-cultured with 20,000 tumor cells (SKOV3-GFP-luciferase cells or SKBR3-luciferase cells). The supernatant was collected 48 hours after stimulation, and secreted cytokines were analyzed using Luminex. Statistical significance was determined between primary monocyte samples electroporated with HER2-CFP and co-cultured with tumor cells, and primary monocyte samples electroporated with mock cells and co-cultured with tumor cells. [Figure 10A-2] Continuation of Figure 10A-1. [Figure 10B-1]

[0135] This figure shows graphs of the induction of the pro-inflammatory cytokine IL-1β by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 10A. [Figure 10B-2] Continuation of Figure 10B-1. [Figure 10C-1]

[0136] This figure shows graphs of TNF-α induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 10A. [Figure 10C-2] Continuation of Figure 10C-1. [Figure 10D-1]

[0137] This figure shows graphs of IFN-α induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 10A. [Figure 10D-2] Continuation of Figure 10D-1. [Figure 10E-1]

[0138] This figure shows graphs of IP10 induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 10A. [Figure 10E-2] Continuation of Figure 10E-1. [Figure 10F-1]

[0139] This figure shows graphs of IL-12 induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 10A. [Figure 10F-2] Continuation of Figure 10F-1. [Figure 11A]

[0140] This figure shows flow cytometry data confirming the expression of the HER2-CFP construct in primary human monocytes derived from the first donor, following electroporation with the HER2-CFP construct. [Figure 11B]

[0141] This figure shows flow cytometry data confirming the expression of the HER2-CFP construct in primary human monocytes derived from a second donor, following electroporation with the HER2-CFP construct. [Figure 11C-1]

[0142] This figure shows graphs of the percentage of cells positive for the HER2-CFP construct shown, and the mean fluorescence intensity, using data derived from Figures 11A and 11B. [Figure 11C-2] Continuation of Figure 11C-1. [Figure 12A]

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

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

[0145] This figure shows a graph of the induction of the pro-inflammatory cytokine IL-6 by primary human monocytes derived from human donors, either electroporated with the indicated HER2-CFP construct and stimulated with the HER-2 antigen (top), or co-cultured with SKOV3 tumor cells (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 of HER2-his protein, and 100,000 electroporated primary human monocytes containing the indicated HER2-CFP construct were added to each well. The supernatant was collected 48 hours after stimulation, and the secreted cytokines were analyzed using Luminex. For HER2-CFP primary monocyte samples stimulated with tumor cells, 100,000 primary human monocytes electroporated with the indicated HER2-CFP construct were co-cultured with 20,000 tumor cells (SKOV3-GFP-luciferase cells or SKBR3-luciferase cells). The supernatant was collected 48 hours after stimulation, and secreted cytokines were analyzed using Luminex. Statistical significance was determined between primary monocyte samples electroporated with HER2-CFP and co-cultured with tumor cells, and primary monocyte samples electroporated with mock cells and co-cultured with tumor cells. [Figure 13A-2] Continuation of Figure 13A-1. [Figure 13B-1]

[0146] This figure shows graphs of the induction of the pro-inflammatory cytokine IL-1β by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 13A. [Figure 13B-2] Continuation of Figure 13B-1. [Figure 13C-1]

[0147] This figure shows graphs of TNF-α induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 13A. [Figure 13C-2] Continuation of Figure 13C-1. [Figure 13D-1]

[0148] This figure shows graphs of IFN-α induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 13A. [Figure 13D-2] Continuation of Figure 13D-1. [Figure 13E-1]

[0149] This figure shows graphs of IP10 induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 13A. [Figure 13E-2] Continuation of Figure 13E-1. [Figure 13F-1]

[0150] This figure shows graphs of IL-12 induction by primary human monocytes derived from human donors, either by electroporating the HER2-CFP construct shown and stimulating it with the HER-2 antigen (top), or by co-culturing it with SKOV3 tumor cells (bottom left) or SKBR3 tumor cells (bottom right). The experiments and analyses were performed as shown in Figure 13A. [Figure 13F-2] Continuation of Figure 13F-1. [Figure 14-1]

[0151] This figure shows a graph comparing tumor regression (left) and survival (right) in mice after administration of a CFP construct containing the intracellular domain shown. [Figure 14-2] Continuation of Figure 14-1. [Modes for carrying out the invention]

[0091]

[0152] T-cell therapy has revolutionized cancer treatment for many patients. However, sustained clinical benefits have not been achieved for the majority of patients with advanced solid tumors. Unlike T cells, bone marrow cells readily accumulate within tumors, sometimes contributing up to 50% of the tumor mass. Bone marrow cells can be specifically engineered to become highly effective anti-tumor cells, known as Activate, Target, Attack & Kill (ATAK) cells, which specifically target, phagocytose, and lyse tumor cells, organizing in vivo immune activation against tumor cells.

[0092]

[0153] This application is based on a method for enhancing myeloid cells engineered to circumvent the immunosuppressive tumor microenvironment and exhibit high oncolytic / immunogenic efficacy. Engagement with innate immune signaling sensors such as Toll-like receptors and STING-cGAS has been explored as a key pathway for upmodulating pro-inflammatory antitumor immune responses and is associated with antitumor immunity. While methods using synthetic agonists to activate these pathways can be potent, the delivery of localized, tumor-specific activation of innate immune signaling is difficult to achieve. Many conventional chemotherapeutic agents, targeted anticancer agents, immunoadjuvants, and oncolytic viruses are only sufficiently effective in the presence of complete type I IFN signaling. Immunotherapy using recombinant type I interferon and IFN-encoding vectors has been attempted. For example, TLR-9 agonists have been used in combination 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 myeloid cells in combination with immune checkpoint blockade may be useful in overcoming resistance to single-treatment modalities in cancers that are refractory to checkpoint blockade, for example (Tsuchiya, N. et al., Cell Reports, 29, 162-175, October 2019).

[0093]

[0154] The method disclosed herein is for creating a novel class of chimeric antigen receptors (ATAK receptors) that couple tumor recognition with innate immune signaling. Such recombinant chimeric receptors are expressed in myeloid cells, as described herein, so that, via the chimeric receptor, myeloid cells target cancer cells, and then, upon tumor recognition, further activate the innate immune response within the myeloid cells, leading to phagocytic lysis of tumor cells and further activation of the lymphocytic immune response. The combination of the cancer recognition domain with intracellular signaling domains derived from innate immune receptors such as FcRg, TLR, and cytokine receptors, and the chimeric antigen receptors designed and described herein, supports the idea that myeloid cells can be programmed to recognize cancer and induce a broad and finely tuned immune response. The data demonstrate the versatility of constructing ATAK receptors by leveraging innate immune pathways, supporting their clinical development in cell therapies and direct in vivo therapies.

[0094]

[0155] All terms are intended to be understood as they are understood by those skilled in the art. Unless otherwise specified, technical and scientific terms used herein have the same meanings as they are generally understood by those skilled in the art in which this disclosure relates.

[0095]

[0156] Section headings used herein are for structural purposes only and should not be construed as limiting the subjects described.

[0096]

[0157] The various features of this disclosure may be described in the context of a single embodiment, but the features may also be provided individually or in any suitable combination. Conversely, for clarity, this disclosure may be described in the context of individual embodiments, but this disclosure may also be implemented in a single embodiment.

[0097]

[0158] References in this specification to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” mean that any features, structures, or characteristics described in relation to an embodiment are included in at least some embodiments of this disclosure, but not necessarily in all embodiments.

[0098]

[0159] As used herein and in the claims, the terms “comprising” (and any form of “comprising,” such as “comprise,” “comprises,” and “comprised”), “having” (and any form of “has,” 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 further, unlisted elements or method steps. 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] When referring to measurable values ​​such as parameters, quantities, or temporal durations, the terms “about” or “approximately” as used herein are intended to encompass variations of ±30% or less, ±20% or less, ±10% or less, ±5% or less, or ±1% or less of a specified value, and such variations from the specified value, to the extent that such variations are appropriate to implement in this disclosure. It is understood that the values ​​referred to by the modifiers “about” or “approximately” may also be specifically disclosed.

[0100]

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

[0101]

[0162] "Change" or "alteration" may refer to an increase or decrease. For example, a change could be an increase or decrease of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even 70%, 75%, 80%, 90%, or 100%. For example, a change could be an increase or decrease of 1x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, or 40x, 50x, 60x, or even 70x, 75x, 80x, 90x, or 100x.

[0102]

[0163] As used herein, “antigen-presenting cells” or “APCs” include professional antigen-presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells). APCs are capable of expressing major histocompatibility complex (MHC) molecules and presenting MHC-complexed antigens on their surface that can be recognized by T cells and can induce T cell activation and an immune response. Professional antigen-presenting cells, particularly dendritic cells, play a key role in naive T cell stimulation. 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 IMHC molecules. Antigens that yield proteins recognized upon association with class I MHC molecules are generally intracellularly produced proteins, which are processed and then associate with class I MHC molecules.

[0103]

[0164] The term "biological sample" may refer to any tissue, cell, bodily fluid, or other material derived from a living organism.

[0104]

[0165] The term "epitope" may refer to any protein determinant, such as a sequence, structure, or amino acid residue, that can bind to an antibody or its binding fragment, a T cell receptor, and / or an antibody-like molecule. Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acid or sugar side chains, and generally possess specific three-dimensional structural features as well as specific charge features. A "T cell epitope" may 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 still possess at least one exogenous nucleic acid sequence within the cell, even if transiently expressed. Expression of exogenous nucleic acids may be carried out by a variety of methods, as described elsewhere, encompassing methods known in the art. This disclosure relates to the preparation and use of engineered cells, such as engineered bone marrow cells, including engineered phagocytic cells. In particular, this disclosure relates to engineered cells containing exogenous nucleic acids, such as those encoding chimeric fusion proteins (CFPs).

[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 may be influenced by T cell costimulation and modulation of 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 cytokine-responsive cells, e.g., macrophage activation. 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 to pathogens. Adaptive immunity can also provide long-lasting protection. Adaptive immune responses include humoral immune responses and cell-mediated immune responses. In humoral immune responses, antibodies secreted into the body fluid by B cells bind to pathogen-derived antigens and lead to the elimination of pathogens through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells capable of destroying other cells are activated. For example, if disease-related proteins are present within cells, they are fragmented into peptides through proteolysis within the cell. Then, specific cellular proteins conjugate to the thus formed antigens or peptides and transport them to the cell surface, where the antigens or peptides are presented by molecular defense mechanisms such as T cells. Cytotoxic T cells can recognize these antigens and kill the cells that possess them.

[0107]

[0168] The term "ligand" may refer to a molecule that can bind to or form 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 other component that binds to a receptor. In some embodiments, the receptor has a specific ligand. In some embodiments, the receptor may bind broadly to ligands, in which case the receptor may bind to several ligands that share at least one similarity in structural composition, charge distribution, or any other physicochemical features. Ligands may be biomolecules. Ligands may be non-biomaterials. For example, the ligand may be a ligand for negatively charged particles, such as the scavenger receptor MARCO. For example, the ligand may be a ligand for TiO2, such as the scavenger receptor SRA1. In the context of CFP as described herein, the extracellular binding domain may bind to ligands, which are also referred to as targets of the binding domain. In some embodiments, the target is an antigen expressed on a diseased cell, such as a cancer cell, which is a target cell in the sense that the target cell expresses a target antigen on its cell surface to which the extracellular antigen-binding domain of CFP binds. In this disclosure, the terms anti(target)binding domain, anti(target)binding extracellular domain, or anti(target)CFP are often used interchangeably with terms such as (target)binding domain, (target)binding extracellular domain, or (target)CFP, respectively. For example, HER2 expressed on cancer cells is an antigen (ligand) to which the anti-HER2 binding extracellular domain of CFP binds; or alternatively, an antigen (ligand) to which the HER2 binding extracellular domain of CFP also binds.

[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 called the HLA complex. Therefore, the terms "human leukocyte antigen (HLA)," "HLA molecule," or "HLA protein" are used interchangeably with the terms "major histocompatibility complex (MHC)," "MHC molecule," and "MHC protein." HLA proteins may be classified as HLA class I or HLA class II. Although the structures of the two HLA class proteins are 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 typically loaded with endogenous proteins or pathogen-derived antigens present within the cell, and then presented to naive or cytotoxic T lymphocytes (CTLs). Class II HLA proteins are present on antigen-presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages. Class II HLA proteins primarily present peptides processed from external antigen sources, such as extracellular antigen sources, to helper T cells.

[0109]

[0170] In the HLA class II system, phagocytic cells such as macrophages and immature dendritic cells can take up substances through phagocytosis, and their acidic enzymes can cleave the ingested proteins into many different peptides, forming phagosomes (although B cells exhibit more common endocytosis into endosomes). Autophagy is another source of HLA class II peptides. The most studied subclass II HLA genes are HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0110]

[0171] The presentation of peptides to CD4+ helper T cells by HLA class II molecules triggers 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 consisting of α-chains and β-chains that interact to form peptide bond grooves that are more open than those of class I peptide bond grooves.

[0111]

[0172] HLA alleles are typically expressed in a codominant manner. For example, each person can express six different types of class II HLA, as they possess two alleles for each of the three class I genes (HLA-A, HLA-B, and HLA-C). At the class II HLA locus, each person inherits pairs of HLA-DP genes (DPA1 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 over 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. Therefore, HLA genes are highly polymorphic; many different alleles exist in different individuals within a population. The genes encoding HLA proteins have many possible variations, allowing each person's immune system to respond to a wide range of foreign invaders. Some HLA genes have been identified with hundreds of versions (alleles), each of which is given a specific number. In some embodiments, the 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, the 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] The term "bone marrow cells" broadly refers to cells of the myeloid lineage within the hematopoietic cell system, sometimes excluding, for example, lymphocyte lineages. Bone marrow cells include, for example, granulocyte and monocyte lineages. Bone marrow cells, including monocytes, dendritic cells, tissue macrophages, and granulocytes, constitute a major cellular compartment of the immune system. Models of myeloid cell development, activation, differentiation, and tissue-specific functions have been re-examined over the past few years, yielding surprising results. However, their immense plasticity and heterogeneity in both homeostasis and disease remain far from fully understood. While myeloid cells possess many functions, including their ability to phagocytose and activate T cells, the utilization of these functions for therapeutic use remains elusive. Thus, novel avenues for using other cell types are needed towards the development of improved therapeutic agents, including but not limited to T-cell malignancies.

[0113]

[0174] Myeloid cells differentiate from common progenitor cells derived from hematopoietic stem cells in the bone marrow. Constraint to myeloid cell lineages may be controlled by the activation of distinct transcription factors; therefore, myeloid cells can be characterized as cells with a level of plasticity that 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 may be cells derived from hematopoietic stem cells, for example, within the bone marrow under the influence of one or more cytokines and chemokines, such as G-CSF, GM-CSF, Flt3L, CCL2, VEGF, and S100A8 / 9. In some embodiments, myeloid cells are progenitor cells. In some embodiments, myeloid cells may be common myeloid progenitor cells, or cells having the characteristics of granulocyte progenitor cells, myeloblasts, or monocyte-dendritic cell progenitor cells, or combinations thereof. Bone marrow may contain granulocytes or monocytes, or their progenitor cells. Bone marrow may contain immature granulocytes, immature monocytes, immature macrophages, immature neutrophils, and immature dendritic cells. Bone marrow may contain monocytes or promonocyte cells or monocyte precursors. Where applicable, myeloid cells as used herein may refer to monocytes having an M0 phenotype, M1 phenotype, or M2 phenotype. Bone marrow may contain dendritic cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, predendritic 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 with M0, M1, or M2 phenotypes. Monocytes or macrophages exhibit polarization. As used herein, “polarization” may refer to the process by which macrophages exhibit significantly different 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 polarize into classically activated macrophages (M1 macrophages) and surrogate activated macrophages (M2 macrophages). M2 macrophages are further divided into subtypes: M2a, M2b, M2c, and M2d. These macrophages differ in their cell surface markers, secreted cytokines, and biological functions. M1 macrophages are typically characterized by a phenotype in which cells express TLR-2, TLR-4, CD80, CD86, iNOS, and MHC-II on their surface.These cells release a diverse range 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 two major pathways involved in the polarization of M1 macrophages. The M1 phenotype is associated with the bactericidal and tumor-killing functions of macrophages and exhibits high levels of phagocytic and inflammatory function. On the other hand, tumor-associated macrophages under immunosuppressive conditions generally exhibit a greater degree of M2 polarization. Bone marrow may contain tumor-infiltrating monocytes (TIMs). Bone marrow may contain tumor-associated monocytes (TAMs). Bone marrow may contain myeloid-derived suppressor cells (MDSCs). Bone marrow may contain tissue-resident macrophages. Bone marrow may contain tumor-associated DCs (TADCs). Therefore, 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. Depending on the circumstances, bone marrow cells may be characterized by high or low expression of one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, or combinations thereof. In one embodiment, it is desirable to activate M1 polarization of macrophages using the method described herein.

[0115]

[0176] "Phagocytosis" is sometimes used interchangeably with "ingestion" and refers 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 particles. This process can be used to take in 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 is followed by their uptake into professional antigen-presenting cells by certain types of endocytosis 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 contain one or more subunits of a protein. A polypeptide may be encoded by recombinant nucleic acid. In some embodiments, a polypeptide may contain one or more peptide sequences within a single amino acid chain, which may be separated by spacers, linkers, or peptide cleavage sequences. A polypeptide may be a fusion polypeptide. A polypeptide may contain one or more domains, modules, or parts.

[0117]

[0178] The term "receptor" can refer to a chemical structure composed of signaling polypeptides, such as polypeptides that transmit extracellular signals to cells. Receptors are used to transmit intracellular information and the formation of cells or organisms. A receptor contains at least one receptor unit, and may contain two or more receptor units, in which case each receptor unit contains a protein molecule, such as a glycoprotein molecule. Receptors may contain structures that can bind to a ligand and form a complex with the ligand. Signaling information may be transmitted by conformational changes of the receptor following binding to a ligand on the cell surface.

[0118]

[0179] The term "antibody" refers to a 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 not limited to full-length antibodies, single-chain antibodies, single-domain antibodies (sdAb), and their antigen-binding fragments. Antigen-binding antibody fragments include Fab, Fab', and F(ab')2, Fd(V H and C H A single-chain variable fragment (scFv), a single-chain antibody, a disulfide-linked variable fragment (dsFv), and V L Domain and / or V HAntibodies may include, but are not limited to, fragments containing domains. Antibodies may originate from any animal. Antigen-binding antibody fragments, including single-chain antibodies, may contain a variable region(s) alone, or a variable region(s) combined with one or more of the hinge region, CH1 domain, CH2 domain, and CH3 domain. They may also include any combination of a variable region(s) with the hinge region, CH1, CH2, and CH3 domains. Antibodies may be, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and human monoclonal and human polyclonal antibodies that specifically bind to HLA-related polypeptides or HLA-peptide complexes.

[0119]

[0180] The term “recombinant nucleic acid” refers to nucleic acids that are prepared, expressed, created, or isolated by recombinant means. Recombinant nucleic acids may contain nucleotide sequences that do not occur spontaneously. Recombinant nucleic acids may be synthesized in the laboratory. Recombinant nucleic acids may be prepared by using recombinant DNA technologies, such as enzyme restriction digestion, ligation, and DNA cloning, which involve enzymatic modification of DNA. Recombinant nucleic acids may be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA may be transcribed ex vivo or in vitro, for example, 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 that may contain deoxyribonucleotides (DNA), ribonucleotides (RNA), or, in some embodiments, modified deoxyribonucleotides or modified ribonucleotides are described. For example, modified nucleotides may be 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 7-methylguanosine, pseudouridine, dihydrouridine, etc. Those skilled in the art can easily determine an RNA sequence, such as an 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 mediated through transformation, transfection, or transduction. Transformation is the process by which bacterial cells take up foreign nucleic acids. This process is adapted to the propagation of plasmid DNA, the production of proteins, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take in 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 may also refer to the introduction of bacteriophages 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 that is capable of autonomous replication within a host cell and enables the cloning of nucleic acid molecules. As is known to those skilled in the art, vectors include, but are not limited to, plasmids, cosmids, phagemids, viral vectors, phage vectors, yeast vectors, and mammalian vectors. For example, a vector for transforming an exogenous gene may be a plasmid. In certain embodiments, a vector includes a nucleic acid sequence containing an origin of replication, as well as other elements necessary for the replication and / or maintenance of the nucleic acid sequence within 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 that they are operatively linked to. In some embodiments, expression vectors or plasmids are in the form of circular double-stranded DNA molecules. Vectors or plasmids may or may not be incorporated into the genome of a host cell. In some embodiments, the nucleic acid sequence of a plasmid is not incorporated into the genome or chromosome of a host cell after introduction. For example, a plasmid may include elements for the transient or stable expression of a nucleic acid sequence within a host cell, such as a gene or open reading frame held by the plasmid. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that autonomously replicates within the host cell. In some embodiments, the nucleic acid sequence of the plasmid is integrated into the host cell's genome or chromosome upon introduction into the host cell. Expression vectors that may 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 may 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 lentiviral vector, which can be integrated into the host cell's genome (e.g., via reverse transcription) upon introduction into the host cell.Other forms of expression vectors performing equivalent functions, known to those skilled in the art, such as self-replicating extrachromosomal vectors or vectors capable of integration into the host genome, may also be used. Exemplary vectors are those to which the ligated nucleic acids are capable of autonomous replication and / or autonomous expression.

[0122]

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

[0123]

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

[0124]

[0185] The terms “spacer” or “linker,” as used in reference to fusion proteins, refer to peptide sequences that connect two other peptide sequences in a fusion protein. In some embodiments, the linker or spacer has no specific bioactivity other than connecting protein or RNA sequences or preserving some minimum distance or other spatial relationship between protein or RNA sequences. In some embodiments, the amino acids that make up the spacer may be selected to influence certain molecular properties, such as molecular folding, flexibility, net charge, or hydrophobicity. Linkers suitable for use in embodiments of this disclosure include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers, which are well known to those skilled in the art. In some embodiments, the linker is used to separate two or more polypeptides, for example, two antigenic peptides, by a distance sufficient to ensure that each antigenic peptide folds properly. Exemplary peptide linker sequences adopt a flexible, unfolded conformation and do not exhibit a tendency toward the development of ordered secondary structures. The amino acids within the flexible linker protein region may include Gly, Asn, and Ser, or any rearrangement of amino acid sequences containing Gly, Asn, and Ser. Other near-neutral amino acids, such as Thr and Ala, may also be used within the linker sequence.

[0125]

[0186] Terms such as “to treat,” “treated,” “treating,” and “treatment” are intended to mean reducing, preventing, or improving a disorder and / or symptoms associated therewith (e.g., a neoplasm or tumor, or an infectant, or an autoimmune disease). “To treat” may mean administering treatment to a subject after the onset or suspected onset of a disease (e.g., cancer, or an infectant infection, or an autoimmune disease). “To treat” may mean reducing the frequency or severity of any symptoms or effects of other diseases associated with the disease and / or side effects associated with the treatment; the concept of “alleviating” also includes the term “to treat.” This includes the concept of “managing,” which means reducing the severity of a disease or disorder in a patient, e.g., extending the lifespan or survival of a patient with the disease, or delaying its relapse, e.g., extending the remission period in a patient who has had the disease before. It is understood that treatment of a disorder or condition does not, but does not require, that the disorder, condition, or associated symptoms be completely eliminated. As used herein, the terms “prevent,” “prevent,” and “prevent” and their grammatical equivalents may refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who is not exhibiting such symptoms at the time administration of the drug or compound begins. In certain embodiments, treatment of a subject or patient as described herein includes the administration of a therapeutic composition, such as a drug, metabolite, preventive component, nucleic acid, peptide, or protein, which encodes or otherwise forms a drug, metabolite, or preventive component. In some embodiments, treatment includes the step of administering cells or a population of cells to a subject in need thereof. In some embodiments, treatment includes the step of administering to a subject one or more of the manipulated cells described herein, such as myeloid cells, such as one or more manipulated phagocytic cells.The treatment may be a pathological disease, condition, or syndrome, or a potential disease, condition, or syndrome, and includes treatment of a disease, condition, or syndrome. Where applicable, the treatment used herein may include a step of administering a therapeutic vaccine. In some embodiments, engineered phagocytic cells are administered to a patient or subject. In some embodiments, the cells administered to a human subject result in reduced immunogenicity. For example, engineered phagocytic cells may not cause or reduce graft-versus-host disease (GVHD) or fructoliside effects. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., they have a match with the naturally expressed HLA subtype in the subject). The subject-specific HLA allele, or the subject's HLA genotype, may be determined by any method known in the art. In an exemplary embodiment, the method comprises the steps of determining a genetic polymorphism, generating an alignment of reads extracted from sequencing a dataset against a gene reference set including allele variants having the genetic polymorphism; determining a first posterior probability or a score derived from the posterior probability for each allele variant in the alignment; identifying the allele variant with the highest first posterior probability or score derived from the posterior probability as the first allele variant; and aligning the first allele variant with one or more other allele variants. The method may include the steps of: identifying one or more duplicate reads; determining a second posterior probability or a score derived from the posterior probability for one or more other allele variants using weighting coefficients; identifying a second allele variant by selecting the allele variant that has the highest second posterior probability or score derived from the posterior probability when the first and second allele variants define the genotype of a genetic polymorphism; and providing an output for the first and second allele variants.

[0126]

[0187] The term "fragment" may refer to a portion of a protein or nucleic acid. In some embodiments, the fragment retains at least 50%, 75%, 80%, 90%, 95%, or even 99% of the bioactivity of the reference protein or reference nucleic acid.

[0127]

[0188] The terms “isolated,” “purified,” and “biologically pure,” and their grammatical equivalents, refer to materials that are, to varying degrees, free of the components typically associated with them as they would be found in their natural state. “Isolating” describes the degree of isolation from the original source or environment. “Purifying” describes a higher degree of isolation than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that impurities do not substantially affect the protein’s biological properties or cause other harmful consequences. That is, the nucleic acids or peptides of this disclosure are purified if they are produced by recombinant DNA methods, if they are substantially free of cell material, viral material, or culture medium, or if they are chemically synthesized, if they are 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" may refer to a nucleic acid or protein that essentially yields a single band in an electrophoresis gel. For proteins that can be modified, such as phosphorylation or glycosylation, different modifications may result in different isolated proteins that can be purified individually.

[0128]

[0189] The terms “neoplasm” or “cancer” refer to any disease caused by, or resulting from, an inappropriately high level of cell division, an inappropriately low level of apoptosis, or both. Glioblastoma is one non-definite example of a neoplasm or cancer. The terms “cancer” or “tumor” or “hyperproliferative disorder” refer to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled growth, immortality, potential for metastasis, rapid growth and growth rate, as well as certain characteristic morphological features. While cancer cells are often in tumor form, such cells can also exist alone in animals and may be non-tumorogenic cancer cells, such as leukemia cells.

[0129]

[0190] The term “vaccine” should be understood to mean a composition for inducing immunity for the prevention and / or treatment of a disease (e.g., neoplasm / tumor / infectious agent / autoimmune disease). Therefore, as used herein, vaccine refers to a recombinant nucleic acid, or a recombinant nucleic acid comprising cells expressing it, intended for use in humans or animals to produce specific protective substances upon vaccination. “Vaccine composition” may include pharmaceutically acceptable excipients, carriers, or diluents. Aspects of this disclosure relate to the use of techniques in the preparation of phagocytic cell-based vaccines.

[0130]

[0191] The term "pharmaceutically acceptable" means that an excipient is approved or eligible for approval by a U.S. federal or state regulatory agency for use in animals, including humans, or is listed in the United States Pharmacopeia or any other generally recognized pharmacopoeia. A "pharmaceutically acceptable excipient, carrier, or diluent" means 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 dose of the drug.

[0131]

[0192] Nucleic acid molecules useful in the methods of this disclosure include, but are not limited to, any nucleic acid molecules that are active or encode polypeptides. Polynucleotides having substantial identity to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means, under varying degrees of strictness, that nucleic acid molecules pair with complementary polynucleotide sequences or parts thereof to form a double-stranded molecule (see, for example, Wahl, GM and S. L. Berger (1987), Methods Enzymol., 152:399; Kimmel, AR (1987), Methods Enzymol., 152:507). For example, the exact salt concentration may typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low-strict hybridization can be obtained in the absence of an organic solvent, such as formamide, while high-strict hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. The exact temperature conditions may typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Those skilled in the art are also familiar with variations in further parameters, such as hybridization time, the concentration of a washing agent, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of the carrier DNA. By combining these various conditions as needed, various levels of strictness can be achieved. In an exemplary embodiment, hybridization may occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization may occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA).In another exemplary embodiment, hybridization may occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Those skilled in the art will readily see the useful variations under these conditions. In most applications, the washing step following hybridization may also vary in strictness. Washing strictness conditions can be defined by salt concentration and temperature. As described above, washing strictness may be increased by decreasing the salt concentration or by increasing the temperature. For example, a strict salt concentration for the washing step 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. Strict temperature conditions for the washing step 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 washing step may occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the washing step may occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another exemplary embodiment, the washing step would occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations in these conditions will readily become apparent to those skilled 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" means 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%, or more identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence homology 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 a variety of 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. Exemplary methods for determining the degree of identity may involve the use of the BLAST program, where probability scores between e-3 and em may indicate closely related sequences. "Reference" refers to the criterion for comparison. It should be understood that the numbering of specific positions or residues within each sequence depends on the specific protein and the numbering scheme used. Numbering may differ, for example, between the precursor of a mature protein and the mature protein itself, and interspecies sequence differences can affect numbering.Those skilled in the art will be able to identify individual residues within any homologous protein and within the nucleic acids encoding them 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 being treated, observed, or experimented on. For example, subjects include, but are not limited to, mammals, including, but not limited to, humans or non-human mammals, such as non-human primates, rodents, bovines, equids, canids, sheep, or felines.

[0134]

[0195] The term “therapeutic effect” refers to a degree of relief of one or more symptoms of a disorder (e.g., neoplasm, tumor, or infection by an infectious agent, or autoimmune disease) or a related condition. On the other hand, “therapeutic effect” may refer to a reduction in the symptoms of a disease after administration of a therapeutic composition, for example, a reduction of 10%, 20%, 30%, etc., of a tumor mass. In another embodiment, “therapeutic effect” may relate to partial or complete remission of one or more symptoms, or improvement of the disease. As used herein, “therapeutic effective dose” refers to the amount of drug, in a single-dose or multi-dose dose, that, upon administration to cells or subjects, is effective in extending the survival of a patient with such disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying the disorder beyond what would be expected in the absence of such treatment. “Therapeutic effective dose” is intended to quantify the amount required to achieve a therapeutic effect. A physician or veterinarian skilled in the art of medical technology can easily determine and prescribe the required "therapeutic effective dose" (e.g., ED50) of a pharmaceutical composition.

[0135]

[0196] This specification provides engineered myeloid cells (including, but not limited to, neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages) designed to specifically bind to target antigens. Target antigens 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 / or indirectly attack and / or kill target cells, either directly (e.g., by phagocytosis) or / or indirectly (e.g., by activating T cells). In some embodiments, the target cells are cancer cells.

[0136]

[0197] While cancer is one exemplary embodiment described in detail in this disclosure, the methods and techniques described herein are intended to be useful in targeting infected cells or other diseased cells in the body. Similarly, therapeutic and vaccine compositions using manipulated cells are described herein.

[0137]

[0198] Bone marrow effector cells are isolated from human biological samples and modified ex vivo using methods for manipulating such cells to prepare cells for therapeutic purposes, but the modification may be derived from bone marrow cells modified so as not to alter the plasticity of these cells. Monocyte-lineage cells are phagocytic and effective antigen-presenting cells. In one embodiment, the present invention is based on the important finding that manipulated bone marrow cells can be a highly effective therapeutic modality in the treatment of numerous diseases, including cancer. Bone marrow cells may be manipulated to express chimeric antigen receptors that enhance the immune function of the bone marrow cells, in which 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 modified as described herein to (a) be highly target-specific, specifically oriented to bind to a target antigen, and possess an extracellular antigen-binding domain, and (b) possess an intracellular domain highly specialized to activate bone marrow cells to achieve activation of a phagocytic cell phenotype. For example, highly specialized intracellular domains are designed to create chimeric receptors in which, upon activation of the extracellular domain of the receptor by binding to a target, the extracellular domain can generate a signaling queue within the cell that activates the intracellular interferon signaling cascade and transcription factors, i.e., a transcription factor that directs the activation of IRF (IFN regulator). In addition, the methods and compositions described herein are also useful in gene therapy in which recombinant nucleic acids encoding chimeric antigen receptors are administered locally or systemically to a target requiring them, so that the recombinant nucleic acids are specifically expressed in vivo in bone marrow cells, thereby generating therapeutically active bone marrow cells. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is delivered by LNP.

[0138]

[0199] Phagocytic cells are the natural outposts of the immune system and form the front line of the body's defense. They engulf pathogens, infected cells, foreign substances, or cancerous cells, removing them from the body. Most potential pathogens are rapidly neutralized by the phagocytic system before they can cause significant infection or disease. This can involve pinocytosis, a receptor-mediated uptake via clathrin-coated pit systems, particularly macropinocytosis as a consequence of membrane undulation and phagocytosis. Thus, phagocytic cells are activated by various non-self (and self) elements and exhibit a degree of plasticity in recognizing their "targets." Most phagocytic cells express scavenger receptors on their surface, which are pattern-recognition molecules capable of binding to a wide range of foreign particles, as well as dead cells, cell lysates, and unwanted particles within the body. In one embodiment, recombinant nucleic acids encoding chimeric antigen receptors (CARs) may be expressed intracellularly. CARs can be designed in various ways to attack specific tumor cells, and bone marrow effector cells expressing CARs can be activated to phagocytose and kill tumor cells. CARs can be designed to produce phagocytic receptors that are activated specifically in response to engagement with a target, and the phagocytic potential of macrophages is enhanced by the receptor's specifically engineered intracellular domain. The CAR platforms for bone marrow cells described herein are designed so that tonic signaling is not detected within the bone marrow cells at the time of administration or at any point before the bone marrow cells engage with their target via the CAR. This is often investigated ex vivo. Simultaneously, bone marrow cells expressing CARs can further differentiate into M0, M1, or M2 phenotypes in the presence of appropriate stimulation, and can retain cellular plasticity to differentiate at least at the time of administration. In addition, CAR-expressing bone marrow effector cells can migrate to lymph nodes and cross-present antigens to naive T cells within the lymph nodes, thereby activating adaptive responses.

[0139]

[0200] In some embodiments, this specification discloses compositions and methods for producing myeloid cells that are isolated from a biological sample, engineered ex vivo to express recombinant proteins, and formulated into a pharmaceutical composition such that the myeloid cells of the composition become “effector” myeloid cells that are effective in inducing immune activation in vivo. In some embodiments, the myeloid cells of the composition are referred to as “ATAK” myeloid cells, in which the cells are myeloid cells that are effective in attacking and destroying target cells. The ATAK myeloid cells disclosed herein are engineered myeloid recombinant proteins that express a chimeric antigen receptor, which includes, for example, a chimeric receptor, for example, an interferon-inducible protein in an immune cell, comprising at least one intracellular signaling domain. In some embodiments, the methods and compositions described herein aim to cause the engineered myeloid cells to exhibit an effector phenotype. In some embodiments, the engineered myeloid cells, for example monocytes, are M0 phenotypic or M1 phenotypic monocytes, and activation of the chimeric antigen receptor expressed in the myeloid cells causes the cells to exhibit the M1 phenotype. The M1 phenotype exhibited by the manipulated cells makes the cells highly tumor-killing when designed to target tumor cells.

[0140]

[0201] This specification provides 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, bone marrow dendritic cells (mDCs), mast cells, and macrophages, to target affected cells such as cancer cells. The compositions and methods provided herein can be used to eliminate affected cells, such as cancer cells, and / or affected tissues, through various mechanisms including T cell activation and recruitment, activation of effector immune cells (e.g., 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 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-mobilizing receptor (caspase-CAR) fusion proteins. CFPs encoded by recombinant nucleic acids may include an extracellular domain (ECD) containing an antigen-binding domain that binds to an antigen on a target cell. The extracellular domain may be fused to a hinge domain or other extracellular domain derived from a receptor, such as a hinge domain or an extracellular domain. CFPs encoded by recombinant nucleic acids may further include transmembrane domains, such as CD2, CD8, CD28, CD68, a transmembrane domain derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. In some embodiments, CFPs encoded by recombinant nucleic acids further include an intracellular domain containing 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 phagocytic receptors, scavenger receptors, or integrin receptors. For example, the intracellular domain may include one or more intracellular signaling domains that promote phagocytic activity, inflammatory response, nitric oxide production, integrin activation, enhancement of effector cell migration (e.g., via chemokine receptor expression), antigen presentation, and / or enhancement of cross-presentation. In some embodiments, CFP is a phagocytic receptor fusion protein (PFP). In some embodiments, CFP is a phagocytic scavenger receptor fusion protein (PFP). In some embodiments, CFP is an integrin receptor fusion protein (IFP). In some embodiments, CFP is an inflammatory receptor fusion protein. In some embodiments, CFP encoded by recombinant nucleic acid further includes an intracellular domain containing 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] This specification provides improved immunogenic CAR compositions, such as recombinant nucleic acids encoding chimeric fusion proteins (CFPs; synonymous with CARs being chimeric antigen receptors), comprising intracellular domains that activate the interferon response in cells expressing the CAR. This specification provides immunogenic CFPs comprising 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 intracellularly, the recombinant CAR activates the production of intracellular type I interferon. Such cells are mammalian cells capable of a 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 contains a specific sequence encoding the pro-inflammatory intracellular domain of the chimeric receptor. In some embodiments, the chimeric receptor protein described herein includes an intracellular domain capable of activating a signaling cascade in cells expressing the chimeric antigen receptor, which, upon engagement with its target at the extracellular domain, leads to the induction of interferon-responsive genes or type I interferon production. In some embodiments, the chimeric receptor protein described herein includes a domain derived from innate immune pathway adapter proteins, such as mitochondrial antiviral signaling protein (MAVS), interferon gene stimulator (STING), Toll / IL-1R domain-containing adapter-inducible IFN (TRIF), and TLR adapter (TASL) that interacts with the endolysosomal SLC15A4 protein, or a portion thereof. In some embodiments, a domain or fragment of an innate immune pathway adapter protein, such as MAVS, STING, TRIF, or TASL protein, is incorporated into the intracellular domain of a CFP or CAR described herein by recombinant DNA technology, in which case the domain or fragment contains a pLxIS motif (where p represents a hydrophilic residue, x represents any residue, and S represents a phosphorylation site) that is phosphorylated by TBK1 or IKKε and mediates the recruitment of IRF-3 to a signaling complex.

[0144]

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

[0145] Activation of effector bone marrow cells and interferon

[0206] Type I and Type II interferons (IFNs) play crucial roles in regulating immune responses in infection and cancer. Type I IFNs are represented by multiple subtypes, including numerous IFNα family members: IFNβ, IFNδ, IFNε, IFNκ, IFNτ, and IFNω. All of these utilize the same cell surface receptor, IFNαR, a heterodimer composed of IFNαR1 and IFNαR2 proteins. Type II IFNs are represented by IFNγ. These two IFN types are expressed by almost all cells and bind to remarkably different cell surface receptors, triggering signaling events and eliciting diverse cellular responses. Bone marrow cells are a key target for interferons. In the initial immune response to intracellular bacterial infection, activated natural killer (NK) cells and activated T cells are sources of IFNγ production. In the early stages of infection, the production of the cytokines interleukin (IL) 12 and IL-18 drives antigen-nonspecific IFNγ production by these lymphocyte populations. Antigen-specific CD4+ T cells and CD8+ T cells can also produce IFNγ in response to these pathogens. There are numerous individual 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 the conserved cell surface type I IFN receptor, IFNαR. Ligation of cell surface IFNαR induces the expression of numerous 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 those caused by 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 intrinsic membrane subunits, IFNγR1 and IFNγR2. Binding of the IFNγ homodimer to the cell causes aggregation of the two receptors into a complex, with two IFNγR1 subunits and two IFNγR2 subunits, as well as further signaling components. Both subunits are required for signal transduction, but 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, IFNγ induces a conformational change that allows for close association between the IFNγR1 and IFNγR2 subunits. These rearrangements within the receptor induce autophosphorylation and crossphosphorylation of Janus-associated kinases (JAKs) that constitutively associate 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 the tyrosine residue (Y440) at the C-terminus of IFNγR1. This phosphorylated tyrosine residue provides a docking site for the SH2 domain on the transcriptional signaling and activator 1 (STAT-1) protein. Since each receptor complex contains two IFNγR1 subunits, two STAT-1 proteins are able to bind to the receptor. JAK1 and JAK2 maintain their association with the receptor and phosphorylate each recruited STAT-1 protein at tyrosine residue 701 (Y701). This phosphorylation allows for the release of STAT-1 monomers from the receptor and, as a result, the formation of homodimers. The STAT-1 homodimers translocate to the nucleus and bind to the gamma-activating sequence (GAS) in the promoter DNA of IFN-inducible genes (ISGs), resulting in increased transcription. Type I IFN, as with that activated by IFNγ, signals via the canonical JAK / STAT pathway. Ligand binding to IFNαR induces dimerization of the two receptor subunits, as well as phosphate transfer to the associated TYK2 and JAK1 kinases. The kinases phosphorylate residues in the cytoplasmic tails of IFNαR1 and IFNαR2 via their SH2 domains to recruit the proteins STAT1 and STAT2. The docking of these STAT proteins to the receptor subunits allows for phosphorylation 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 form to produce the transcription factor ISG factor 3 (ISGF3). ISGF3 translocates to the nucleus to identify ISGs and induce their transcription. ISGs induced by type I IFN signaling typically contain either an interferon-stimulated response element (ISRE) or a gamma-activating element (GAS) within their promoters, but genes containing ISREs are clearly preferred.Some examples of ISGs transcribed as a result of type I IFNs include ISREs containing the genes ISG15, IP-10, IRF-7, and PKR

[66] , and GASs containing the 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., March 2013, 55(0):187~200).

[0146] Recombinant chimeric receptor protein

[0207] This specification provides a class of phagocytic or tethering receptor (PR) subunits (e.g., phagocytic receptor fusion proteins (PFPs)) comprising (i) a transmembrane domain and (ii) an intracellular domain comprising a phagocytic receptor intracellular signaling domain; and an extracellular antigen-binding domain that is specific to an antigen, e.g., an antigen of a target cell or an antigen presented 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 CFP includes an Ig-binding domain. In some embodiments, the extracellular domain includes IgA, IgD, IgE, IgG, IgM, FcRγI, FcRγIIA, FcRγIIB, FcRγIIC, FcRγIIIA, FcRγIIIB, FcRn, TRIM21, and FcRL5-binding domains. In some embodiments, the extracellular domain of CFP includes an FcR extracellular domain. In some embodiments, the extracellular domain of CFP includes an FcRα, FcRβ, FcRε, or FcRγ extracellular domain. In some embodiments, the extracellular domain includes an FcRα (FCAR) extracellular domain. In some embodiments, the extracellular domain includes an FcRβ extracellular domain. In some embodiments, the extracellular domain includes an FCER1A extracellular domain. In some embodiments, the extracellular domain includes the extracellular domain of FCGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, or FCGR3B. In some embodiments, the extracellular domain includes an integrin domain or an integrin receptor domain. In some embodiments, the extracellular domain includes one or more integrin domains, namely 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 containing an antigen-binding domain operatively linked to a transmembrane domain. In some embodiments, the extracellular domain further comprises the extracellular domain, hinge, spacer, and / or linker of a receptor. 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 originates. In some embodiments, the extracellular domain comprises the extracellular domain of a scavenger receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises the extracellular domain of an immunoglobulin or the hinge region of an immunoglobulin. In some embodiments, the extracellular domain comprises a phagocytic domain. In some embodiments, the extracellular domain comprises a structure capable of multimerization. In some embodiments, the extracellular domain comprises a base 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 long. In some embodiments, the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids long. In some embodiments, the antigen-binding domain specifically binds to the antigen of the target cell. In some embodiments, the antigen-binding domain includes an antibody domain. In some embodiments, the antigen-binding domain includes a receptor domain and an antibody domain, in which case 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 HHThe functional antibody fragment comprises 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 adapter. In some embodiments, the antigen-binding domain comprises a single antigen-binding domain that is specific to a single antigen. In some embodiments, the antigen-binding domain comprises at least two antigen-binding domains, in which case each of the at least two antigen-binding domains is specific to 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 includes 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 antigens include 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), follicular stimulating hormone receptor, fibroblast-activating protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer 2D (NKG2D) group ligands, disialoganglioside 2 (GD2), and CD2. The antigens are selected from the group consisting of 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 receptor, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and antigens derived from 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 may bind to two or more different antigens.

[0150]

[0211] In some embodiments, the antigen-binding domain includes sequences of antigen-binding domains presented herein, such as the sequences of antigen-binding domains in the table 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 a binding fragment thereof, in which case the antigen-binding domain comprises a heavy chain variable domain (VH) which includes a heavy chain complementarity determining region 3 (HC CDR3) which is 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 its binding fragment is one of the VH sequences selected from the group consisting of QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS, The present embodiment further comprises a heavy chain complementation determining region 1 (HC CDR1), which is HC CDR1. In some embodiments, the VH of the anti-CD70 antibody or its binding fragment further comprises a heavy chain complementation determining region 2 (HC CDR2), which is HC CDR2, which is 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 its binding fragment has 70-100% sequence identity to any one of the sequences selected from the group consisting of QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, VH is the domain of a single-domain antibody. In some embodiments, VH is 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, in which case the antigen-binding domain is ATACADTTQYAYDY, ATACADTTLYEYDY, ATACVDTTQYEYDY, ATACADATQHEYDY, ATACADTTQYDYDY, ATACADTTQYEYDY, ATACADTTHYEYDY, ATACVITTLYEYDY, ATACAETTLYEYDY, ATACADTTQHEYDY, ATACVDTTHYEYDY, ATACASTTLYEYDY, ATACVVTTLYEYDY, ATACGGATGPYDY, ATACAGAIGPYDY, ATACVVVGDQNDY, ATACVVVGDRNDY, ATDCAGGTSTPYDY, ATDAGGTA The sequence contains a heavy chain variable domain (VH) which includes a heavy chain complementarity determining region 3 (HC CDR3), selected from the group consisting of TPYDY, ATACVVADRNEYDY, ATSCVVVTKNEYDY, ATACSGLTHEYDY, ATTCSGLTHEYDY, ATACANWSSLGPYDY, ATACANWSTLGPYDY, ATACSDPRVYEYDY, ATTCASPEKYEYDY, ATHCGGTSWGTSYDY, ATHCGGSSWSNEYDY, YARYSGRTY, ASSAWPAGPKHQVEYDY, ATACGSLVGMYDY, ATACGSAVHEYDY, ATDCVGFGSNWFDY, ATACASPVIYEYDY, ATDAGGVGHEYDY, ATDCSLHGSDYPYDY, and AVRIYSGSFDNTLAYDY. In some embodiments, the VH of the anti-GPC3 antibody or its binding fragment further comprises one heavy chain complementation determining region 1 (HC CDR1) 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 its binding fragment further comprises ISNSDGST, ISASDGST,It further includes one heavy chain complementarity-determining region 2 (HC CDR2) 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 its binding fragment is 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 It has 70-100% sequence identity to any one of the sequences selected from the group consisting of NSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS and QVQLQESGGGVVQSGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS. In some embodiments, VH is the domain of a single-domain antibody. In some embodiments, VH is VHH.

[0153]

[0214] In some embodiments, the antigen-binding domain includes an autoantigen or a fragment thereof, such as Dsg1 or Dsg3. In some embodiments, the antigen-binding domain includes a receptor domain or an antibody domain, in which case 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 operatively linked via a linker. In some embodiments, the transmembrane domain and the antigen-binding domain are operatively linked via a linker such as a hinge region of CD8α, IgG1, or IgG4.

[0155]

[0216] In some embodiments, the extracellular domain includes a polymerization base.

[0156]

[0217] In some embodiments, the transmembrane domain includes a CD8 transmembrane domain. In some embodiments, the transmembrane domain includes a CD28 transmembrane domain. In some embodiments, the transmembrane domain includes a CD68 transmembrane domain. In some embodiments, the transmembrane domain includes a CD2 transmembrane domain. In some embodiments, the transmembrane domain includes an FcR transmembrane domain. In some embodiments, the transmembrane domain includes an FcRγ transmembrane domain. In some embodiments, the transmembrane domain includes an FcRα transmembrane domain. In some embodiments, the transmembrane domain includes an FcRβ transmembrane domain. In some embodiments, the transmembrane domain includes an FcRε transmembrane domain. In some embodiments, the transmembrane domain includes a transmembrane domain derived from a syntaxin such as syntaxin 3, syntaxin 4, or syntaxin 5. In some embodiments, the transmembrane domain oligomerizes with the endogenous receptor transmembrane domain when CFP is expressed intracellularly. In some embodiments, the transmembrane domain oligomerizes with the exogenous receptor transmembrane domain when CFP is expressed intracellularly. In some embodiments, the transmembrane domain dimerizes with the endogenous receptor transmembrane domain when CFP is expressed intracellularly. In some embodiments, the transmembrane domain dimerizes with the exogenous receptor transmembrane domain when CFP is expressed intracellularly. In some embodiments, the transmembrane domain originates from a different protein than the intracellular signaling domain. In some embodiments, the transmembrane domain originates from a different protein than the extracellular domain. In some embodiments, the transmembrane domain includes the transmembrane domain of the phagocytic receptor. In some embodiments, the transmembrane and extracellular domains originate from the same protein. In some embodiments, the transmembrane domain originates from the same protein as the intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes the DAP12 mobilization domain. In some embodiments, the transmembrane domain includes 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 long. In 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 long.

[0158]

[0219] In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a phagocytic receptor other than Megf10, MerTk, FcRα, or Bai1. In some embodiments, the intracellular signaling domain includes 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 The intracellular signaling domain includes 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 includes a PI3K mobilization domain. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a scavenger receptor. In some embodiments, the intracellular domain includes a CD47 inhibitory domain. In some embodiments, the intracellular domain includes a Rac inhibitory domain, a Cdc42 inhibitory domain, or a GTPase inhibitory domain. In some embodiments, the Rac inhibitory domain, Cdc42 inhibitory domain, or GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase in the phagocytic cup of PFP-expressing cells. In some embodiments, the intracellular domain includes an F-actin disassembly activating domain, an ARHGAP12 activating domain, an ARHGAP25 activating domain, or an SH3BP1 activating domain. In some embodiments, the intracellular domain includes a phosphatase inhibitory domain. In some embodiments, the intracellular domain includes an ARP2 / 3 inhibitory domain.In some embodiments, the intracellular domain includes at least one ITAM domain. In some embodiments, the intracellular domain includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ITAM domains. In some embodiments, the intracellular domain includes at least one ITAM domain selected from CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, Fc epsilon receptor 1, Fc epsilon receptor 2, Fc gamma receptor 1, Fc gamma receptor 2a, Fc gamma receptor 2b1, Fc gamma receptor 2b2, Fc gamma receptor 3a, Fc gamma receptor 3b, Fc beta receptor 1, 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 thereto. In some embodiments, the at least one ITAM domain includes a phosphorylation site of a Src family kinase. In some embodiments, at least one ITAM domain includes a Syk recruitment domain. In some embodiments, the intracellular domain includes 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 an integrin-activating domain, such as the intracellular region of PSGL-1.

[0160]

[0221] In some embodiments, the intracellular signaling domain includes 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 adhesion plaque kinase. In some embodiments, the intracellular signaling domain is derived from a single phagocytic receptor. In some embodiments, the intracellular signaling domain is derived from a single scavenger receptor. In some embodiments, the intracellular domain includes a phagocytic enhancement domain.

[0161]

[0222] In some embodiments, the intracellular domain includes a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain includes a kinase activation domain or a kinase-binding domain. In some embodiments, the pro-inflammatory signaling domain includes an IL-1 signaling cascade activation domain. In some embodiments, the pro-inflammatory signaling domain includes 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, pyrine, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), caspase domain, procaspase-binding domain, or any combination thereof.

[0162]

[0223] In some embodiments, the intracellular domain includes a signaling domain, such as an intracellular signaling domain derived from a connexin (Cx) protein. For example, the intracellular domain may include 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 include a signaling domain, such as an intracellular signaling domain derived from Cx43.

[0163]

[0224] In some embodiments, the intracellular domain includes a signaling domain such as an intracellular signaling domain derived from a SIGLEC protein. For example, the intracellular domain may include a signaling domain such as an intracellular signaling domain derived from Siglec-1 (sialoadesine), 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 includes a signaling domain, such as an intracellular signaling domain derived from a TLR protein. In some embodiments, the intracellular domain may include the intracellular signaling domain of an endolysosomal TLR, such as TLR3, TLR7, TLR8, or TLR9. In some embodiments, the intracellular signaling domain may be derived from the TLR3 protein. In some embodiments, the intracellular signaling domain may be derived from the TLR7 protein, TLR8 protein, or TLR9 protein. In some embodiments, the intracellular domain may include the intracellular signaling domains of cell surface TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10.

[0165]

[0226] In some embodiments, an intracellular signaling domain is specifically paired with another intracellular or transmembrane domain to maximize the efficacy and phagocytic potential of myeloid cells expressing the construct. For example, in some embodiments, a transmembrane domain containing CD64™ or a portion thereof may be specifically paired with an intracellular signaling domain containing the ICD or PI3 kinase mobilization domain of an innate immune adapter protein, or both. In some embodiments, the combination of domains, consisting of a chimeric receptor intracellular domain(s) and / or transmembrane domain, is directed to maximize the phagocytic index of cells expressing the construct, such as myeloid cells. In some embodiments, the combination of domains, consisting of a chimeric receptor intracellular domain(s) and / or transmembrane domain, is directed to maximize the inflammatory potential of cells expressing the construct, so that the cells can activate immune response pathways to lyse target cells and make immunity responsive over time. In some embodiments, the combination of domains, consisting of intracellular domains and / or transmembrane domains of the chimeric receptor, is directed to minimize or eliminate tonic signaling by cells expressing the chimeric protein. In some embodiments, the combination of domains, consisting of intracellular domains and / or transmembrane domains of the chimeric receptor, is directed to maximize the specificity of the immune response.

[0166]

[0227] In some embodiments, the intracellular domain includes a signaling domain such as an intracellular signaling domain derived from a type C lectin protein. For example, the intracellular domain may include a signaling domain such as an intracellular signaling domain derived from a mannose receptor protein. For example, the intracellular domain may include a signaling domain such as an intracellular signaling domain derived from an asialoglycoprotein receptor protein. For example, the intracellular domains include macrophage galactose 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 immune receptor (DCIR) subfamily proteins, DCIR / CLEC4A, dectin 2 / CLEC6A, serum DC antigen 2 (BDCA2) (CLEC4C), and Mincle (macrophage-inducible C-type This may include signaling domains such as intracellular signaling domains derived from lectin (CLEC4E), NOD-like receptor protein, NOD-like receptor MHC class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) protein, RIG-I, MDA5, LGP2, NAIP5 / Birc1e, NLRP protein, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP14, NLR protein, NOD1 or NOD2, or any combination thereof.

[0167]

[0228] In some embodiments, the intracellular domain includes signaling domains such as intracellular signaling domains derived from cell adhesion molecules. For example, the intracellular domain may include signaling domains such as intracellular signaling domains derived from molecules such as IgCAM, cadherins, integrins, C-type lectin-like domain proteins (CTLDs), and / or proteoglycans. For example, the intracellular domain may include signaling domains such as intracellular signaling domains derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin. For example, the intracellular domain may include signaling domains such as intracellular signaling domains derived from selectins such as E-selectin, L-selectin, or P-selectin.

[0168]

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

[0169]

[0230] In some embodiments, recombinant nucleic acids encode the extracellular domain of the FcRα chain, the transmembrane domain of the FcRα chain, and / or the intracellular domain of the FcRα chain. In some embodiments, recombinant nucleic acids encode the extracellular domain of the FcRβ chain, the transmembrane domain of the FcRβ chain, and / or the intracellular domain of the FcRβ chain. In some embodiments, when expressed intracellularly, the FcRα or FcRβ chain forms a complex with FcRγ. In some embodiments, when expressed intracellularly, the FcRα or FcRβ chain forms a complex with endogenous FcRγ. In some embodiments, the FcRα or FcRβ chain is not incorporated into a cell membrane that does not express FcRγ. In some embodiments, CFP does not contain the intracellular signaling domain of the FcRα chain. In some embodiments, CFP does not contain the intracellular signaling domain of the FcRβ chain. In some embodiments, recombinant nucleic acids encode the TREM extracellular domain, the TREM transmembrane domain, and / or the TREM intracellular domain. In some embodiments, TREM is TREM1, TREM2, or TREM3.

[0170]

[0231] In some embodiments, the recombinant nucleic acid includes a sequence encoding a pro-inflammatory polypeptide. In some embodiments, the composition further includes pro-inflammatory nucleotides or nucleotides within the recombinant nucleic acid, such as ATP, ADP, UTP, UDP, and / or UDP-glucose.

[0171] Intracellular interferon-responsive domain

[0232] Most TLRs activate an adapter protein called MyD88, which activates the transcription factor protein NF-κB, driving the expression of pro-inflammatory genes as part of the immune response. Subgroups of TLRs (TLR3 and TLR4) can engage with the protein TRIF, which acts as a foundation for kinase enzymes to add a phosphate group to the transcription factor IRF3. This phosphorylation activates IRF3, a member of a family of transcription factors called interferon regulators (IRFs), which activate a wide range of gene expression programs. The manifestation of these programs is the production of type I interferon molecules. Since interferons are potent drivers of a branch of the immune system called the adaptive immune response, their presence carries the risk of 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, the pLxIS motif, a specific sequence of amino acid residues within TRIF, must be phosphorylated before IRF3 is activated. This control mechanism is not specific solely to TRIF as an adapter protein for TLR signaling, but involves IRF3 or its analogue, IRF7, to result in a "licensing step," a general characteristic of sensing pathways that drive interferon expression. All identified innate sensing pathways linking nucleic acid recognition to type I interferon production have been shown to signal via one of three adapter proteins known to contain the pLxIS motif: TRIF, MAVS, and STING, with one exception. Thus, pLxIS motif-containing adapter proteins specifically circuit nucleic acid recognition to antiviral defense. In some embodiments, the intracellular signaling domain of CFP contains the innate immune response protein ICD.In some embodiments, innate immune response proteins include 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, and RIG-I-like receptor (RLR) proteins. The intracellular signaling domains are selected from the following proteins: macrophage galactose 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 immune receptor (DCIR) subfamily proteins, DCIR / CLEC4A, dectin 2 / CLEC6A, serum DC antigen 2 (BDCA2) (CLEC4C), and Mincle (macrophage-inducible C-type lectin) (CLEC4E). In some embodiments, CFP includes at least one intracellular signaling domain comprising the amino acid sequence motif, pLxIS. In some embodiments, the intracellular interferon-inducible moiety includes an amino acid sequence selected from SEQ ID NOs: 36-41. This specification provides a CFP for enhancing phagocytosis, comprising an antigen-binding domain and the MDA5 intracellular signaling domain of SEQ ID NO: 30. This specification provides a CFP for enhancing phagocytosis, comprising an antigen-binding domain and the RIG1 intracellular signaling domain of SEQ ID NO: 36.This specification provides a CFP for enhancing phagocytosis, comprising a chimeric antigen-receptor fusion protein comprising an antigen-binding domain and the Myd88 intracellular signaling domain of SEQ ID NO: 37. This specification provides a CFP for enhancing phagocytosis, comprising an antigen-binding domain and the intracellular signaling domain comprising the STING portion of SEQ ID NO: 38. This specification provides a CFP for enhancing phagocytosis, comprising an antigen-binding domain and the intracellular signaling domain comprising the MAVS portion of SEQ ID NO: 39. This specification provides a CFP for enhancing phagocytosis, comprising an antigen-binding domain and the intracellular signaling domain comprising the TRIF portion of SEQ ID NO: 40. This specification provides a CFP for enhancing phagocytosis, comprising an antigen-binding domain and the intracellular signaling domain comprising the 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 adapter 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 innate intracellular adapter protein domain, without impairing the efficacy of the chimeric protein. In some embodiments, such efficacy is referred to as enhanced phagocytic potential compared to identical cells that do not express the chimeric protein. In some embodiments, such efficacy is referred to as enhanced inflammatory potential compared to identical cells that do not express the chimeric protein. In some embodiments, such efficacy is referred to as enhanced NF kappa B activation or interferon activation in cells expressing the chimeric protein, compared to identical cells that do not express the chimeric protein.

[0174]

[0235] In some embodiments, bone marrow cells are specifically targeted for delivery. Bone marrow cells may be targeted using specialized biodegradable polymers such as PLGA (poly(lactic acid-coglycolic 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 targeted structure is multilayered, for example, by one or more PLGA layers and one or more PVA layers. In some embodiments, the targeted structure is assembled into an order for stratification activity. In some embodiments, the targeted polymer structure is organized into components of a specific shape, such as a flexible structure, capable of delivering one or more components, such as growth factors and cytokines, to adhere to the surface of bone marrow cells and maintain adhesion to the cells within a microenvironment that confers specific polarization. In some embodiments, the polymer structure is such that it is not phagocytosed by bone marrow cells and can maintain adhesion to the surface. In some embodiments, one or more growth factors may be M1 polarization factors such as cytokines. In some embodiments, one or more growth factors may be M2 polarization factors such as cytokines. In some embodiments, one or more growth factors may be macrophage-activating cytokines such as IFNγ. In some embodiments, the polymer structure is capable of sustained release of 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 includes a sequence encoding an inflammation homeostasis regulator. In some embodiments, the inflammation homeostasis regulator is a sequence within the untranslated region (UTR) of 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 includes a consensus sequence WWWU(AUUUA)UUUW [wherein 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 cells are mammalian cells. In some embodiments, the target cells are human cells. In some embodiments, the target cells include cells infected with a pathogen. In some embodiments, the target cells are cancer cells. In some embodiments, the target cells are cancer cells that are lymphocytes. In some embodiments, the target cells are cancer cells that are ovarian cancer cells. In some embodiments, the target cells are cancer cells that are mammary gland cells. In some embodiments, the target cells are cancer cells that are pancreatic cells. In some embodiments, the target cells are cancer cells that are glioblastoma cells.

[0177]

[0238] In some embodiments, recombinant nucleic acid is DNA. In some embodiments, recombinant nucleic acid is RNA. In some embodiments, recombinant nucleic acid is mRNA. In some embodiments, recombinant nucleic acid is unmodified mRNA. In some embodiments, recombinant nucleic acid is modified mRNA. In some embodiments, recombinant nucleic acid is circRNA. In some embodiments, recombinant nucleic acid is tRNA. In some embodiments, recombinant nucleic acid is microRNA.

[0178]

[0239] This specification also provides vectors comprising recombinant nucleic acid sequences encoding the CFPs 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 ligated 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 sequences is operably ligated to a separate promoter. In some embodiments, the vector further comprises one or more internal ribosome entry sites (IRESs). In some embodiments, the vector further comprises a 5'UTR and / or 3'UTR adjacent to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.

[0179]

[0240] This specification also provides polypeptides encoded by recombinant nucleic acids of the compositions described herein.

[0180]

[0241] This specification describes a composition 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 including a transmembrane domain and an intracellular domain including an intracellular signaling domain; and an extracellular domain including an antigen-binding domain specific to the antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operatively linked; and upon binding of the CFP to the antigen of a target cell, the CFP is killed or phagocytosed by myeloid cells such as neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, or macrophages that express the CFP. A composition is provided in which the 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 disclosures. Underlined CDR sequences are shown for each heavy and light chain in the order CDR1, CDR2, and CDR3, according to the Kabat numbering system.

[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] This specification describes a composition comprising a recombinant nucleic acid sequence encoding a CFP (e.g., a phagocytic receptor fusion protein (PFP)) containing a phagocytic or tethering receptor (PR) subunit, comprising an extracellular domain containing an antigen-binding domain specific to the antigen of a target cell; a transmembrane domain; and an intracellular domain containing an intracellular signaling domain, wherein the transmembrane domain and the extracellular domain are operatively linked; and upon binding of the CFP to the antigen of a target cell, the CFP is expressed by neutrophils, monocytes, bone marrow dendritic cells (mD C) A composition is provided in which the killing or phagocytic activity of myeloid cells such as mast cells or macrophages is increased by at least 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 50 times, 75 times, or 100 times compared to cells that do not express CFP.

[0190]

[0244] This specification provides recombinant nucleic acid sequences encoding CFP, as described in the preceding paragraph, which include at least one innate immune-activating intracellular domain, such as a pattern recognition receptor intracellular signaling domain, a TLR intracellular signaling domain, an FcR intracellular signaling domain, an intracellular adapter protein signaling domain, or a fragment thereof, in which the intracellular domain can activate the innate immune response of myeloid cells, activate their phagocytic potential, and activate inflammatory cytokine and chemokine responses, antigen presentation by CFP-expressing myeloid cells, and T cell activation, upon contact with its target antigen, for example, upon engagement of the antigen-binding domain with the target antigen.

[0191]

[0245] In some embodiments, the pro-inflammatory signaling domain includes intracellular signaling domains derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, NLRP family members NLRP1-14, NOD1, NOD2, pyrine, 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 includes an intracellular signaling domain comprising a sequence derived from a protein that activates the interferon-responsive transcription factors IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, or IRF9.

[0193]

[0247] In some embodiments, CFP includes an intracellular signaling domain containing a sequence derived from an intracellular adapter protein. In some embodiments, the adapter protein may include 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 adapter protein is a cytosolic protein.

[0194]

Table 2-1

[0195]

Table 2-2

[0248] The CFP described in this specification may include any one of the sequences listed in Table 1, combined with the intracellular domains listed in Table 2.

[0196]

[0249] In some embodiments, for example, the intracellular signaling domain of the CFP described herein includes an intracellular signaling domain derived from the amino acid sequence of SEQ ID NO: 30, or an MDA5 intracellular signaling domain having at least 85% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain includes a sequence having 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 includes an intracellular signaling domain derived from an MDA5 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain includes a sequence having 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 includes an intracellular signaling domain derived from an MDA5 intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular signaling domain includes a sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30. In some embodiments, the intracellular domain of CFP includes 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, CFP includes an extracellular binding domain capable of binding to the CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells.In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5, HER2, CD19, TROP2, GPC3, CD70, CD137, CD7, Claudin, CD22, or GP75 molecules on target cells, along with a CD8 transmembrane domain or CD28 transmembrane domain, or CD68 transmembrane domain or CD64 transmembrane domain or CD16 transmembrane domain, or CD89 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5, HER2, CD19, TROP2, GPC3, CD70, CD137, CD7, Claudin, CD22, or GP75 molecules on target cells, along with one or more further intracellular signaling domains, such as a CD8 or CD28 transmembrane domain, or a CD64 or CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase mobilization domain, or a CD40 intracellular signaling domain. For example, the exemplary CFP molecules disclosed herein include an extracellular CD5-binding domain having an amino acid sequence comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or both thereof, or the sequence of 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 includes an intracellular signaling domain derived from the RIG-1 intracellular domain having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 36, or to SEQ ID NO: 36. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from the RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from the RIG-1 intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, the intracellular domain of CFP comprises an intracellular signaling domain derived from the RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36; in this case, CFP comprises an extracellular binding domain capable of binding to the CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells. In some embodiments, the intracellular domain of CFP comprises an intracellular signaling domain derived from the RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36; in this case, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, together with a CD8 transmembrane domain or CD28 transmembrane domain, or CD68 transmembrane domain, or CD64 transmembrane domain, or CD16 transmembrane domain, or CD89 transmembrane domain, and a hinge domain.In some embodiments, the intracellular domain of CFP comprises an intracellular signaling domain derived from the RIG-1 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 36; in this case, CFP comprises an extracellular binding domain capable of binding to a CD5 molecule, a HER2 molecule, a CD19 molecule, a TROP2 molecule, a GPC3 molecule, a CD70 molecule, a CD137 molecule, a CD7 molecule, a Claudin molecule, a CD22 molecule, or a GP75 molecule on a target cell, along with one or more further 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 mobilization domain, or a CD40 intracellular signaling domain. For example, the exemplary CFP molecules disclosed herein include an extracellular CD5-binding domain having an amino acid sequence comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or both thereof, or the sequence of 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 CFP includes an intracellular signaling domain derived from the amino acid sequence of SEQ ID NO: 37, or a MyD88 intracellular signaling domain having at least 85% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a MyD88 intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular signaling domain includes 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 includes an intracellular signaling domain derived from a MyD88 intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 37. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to the CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells.In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, together with a CD8 transmembrane domain or CD28 transmembrane domain, or CD68 transmembrane domain, or CD64 transmembrane domain, or CD16 transmembrane domain, or CD89 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, along with one or more further intracellular signaling domains, such as a CD8 transmembrane domain or CD28 transmembrane domain, or a CD64 transmembrane domain or CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase mobilization domain, or a CD40 intracellular signaling domain. For example, the exemplary CFP molecules disclosed herein include an extracellular CD5-binding domain having an amino acid sequence comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or both thereof, or the sequence of 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 includes an intracellular signaling domain derived from the STING intracellular signaling domain having at least 85% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from the STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular signaling domain includes 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: 38. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from the STING intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 38. In some embodiments, the intracellular domain of CFP comprises an intracellular signaling domain derived from the STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38; in this case, CFP comprises an extracellular binding domain capable of binding to the CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells.In some embodiments, the intracellular domain of CFP comprises an intracellular signaling domain derived from the STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38; in this case, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, together with a CD8 transmembrane domain or CD28 transmembrane domain, or CD68 transmembrane domain or CD64 transmembrane domain or CD16 transmembrane domain, or CD89 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of CFP comprises an intracellular signaling domain derived from the STING intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 38; in this case, CFP comprises an extracellular binding domain capable of binding to a CD5 molecule, a HER2 molecule, a CD19 molecule, a TROP2 molecule, a GPC3 molecule, a CD70 molecule, a CD137 molecule, a CD7 molecule, a Claudin molecule, a CD22 molecule, or a GP75 molecule on a target cell, along with one or more further 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 mobilization domain, or a CD40 intracellular signaling domain. For example, the exemplary CFP molecules disclosed herein include an extracellular CD5-binding domain having an amino acid sequence comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or both thereof, or the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and an intracellular domain having at least 90% sequence identity to SEQ ID NO: 38.

[0200]

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

[0201]

[0254] In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a MAVS intracellular signaling domain having the amino acid sequence of SEQ ID NO: 45, or a sequence having at least 85% sequence identity to SEQ ID NO: 45. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a MAVS intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a MAVS intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 45. In some embodiments, the intracellular signaling domain includes 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: 45. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to the CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells.In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, together with a CD8 transmembrane domain or CD28 transmembrane domain, or CD68 transmembrane domain, or CD64 transmembrane domain, or CD16 transmembrane domain, or CD89 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to a CD5 molecule, a HER2 molecule, a CD19 molecule, a TROP2 molecule, a GPC3 molecule, a CD70 molecule, a CD137 molecule, a CD7 molecule, a Claudin molecule, a CD22 molecule, or a GP75 molecule on a target cell, along with one or more further 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 mobilization domain, or a CD40 intracellular signaling domain. For example, the exemplary CFP molecules disclosed herein include an extracellular CD5-binding domain having an amino acid sequence comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or both thereof, or the sequence of 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 includes an intracellular signaling domain derived from a TRIF intracellular domain having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 41. In some embodiments, the intracellular signaling domain includes a sequence having at least 86%, at least 87%, at least 88%, or at least 89% sequence identity to SEQ ID NO: 41. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 41. In some embodiments, the intracellular signaling domain includes a sequence having at least 91%, at least 92%, at least 93%, or at least 94% sequence identity to SEQ ID NO: 41. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a TRIF intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 41. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to the CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells.In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, together with a CD8 transmembrane domain or CD28 transmembrane domain, or CD68 transmembrane domain or CD64 transmembrane domain or CD16 transmembrane domain, or CD89 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to a CD5 molecule, a HER2 molecule, a CD19 molecule, a TROP2 molecule, a GPC3 molecule, a CD70 molecule, a CD137 molecule, a CD7 molecule, a Claudin molecule, a CD22 molecule, or a GP75 molecule on a target cell, along with one or more further 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 mobilization domain, or a CD40 intracellular signaling domain. For example, the exemplary CFP molecules disclosed herein include an extracellular CD5-binding domain having an amino acid sequence comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or both thereof, or the sequence of 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 includes an intracellular signaling domain derived from a TRIF intracellular domain having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the intracellular signaling domain includes a sequence having at least 86%, at least 87%, at least 88%, or at least 89% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a TRIF intracellular signaling domain having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular signaling domain includes a sequence having at least 91%, at least 92%, at least 93%, or at least 94% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular signaling domain includes an intracellular signaling domain derived from a TRIF intracellular signaling domain having at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to the CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells.In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, together with a CD8 transmembrane domain or CD28 transmembrane domain, or CD68 transmembrane domain, or CD64 transmembrane domain, or CD16 transmembrane domain, or CD89 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of 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, CFP comprises an extracellular binding domain capable of binding to CD5 molecule, HER2 molecule, CD19 molecule, TROP2 molecule, GPC3 molecule, CD70 molecule, CD137 molecule, CD7 molecule, Claudin molecule, CD22 molecule, or GP75 molecule on target cells, along with one or more further intracellular signaling domains, such as a CD8 transmembrane domain or CD28 transmembrane domain, or a CD64 transmembrane domain or CD16 transmembrane domain, or a CD89 transmembrane domain, and a PI3 kinase mobilization domain, or a CD40 intracellular signaling domain. For example, the exemplary CFP molecules disclosed herein include an extracellular CD5-binding domain having an amino acid sequence comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 9, or both thereof, or the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and an intracellular domain having at least 90% sequence identity to SEQ ID NO: 43.

[0204]

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

[0205]

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

[0206]

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

[0207]

[0260] In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a light chain variable domain containing the CDR3 sequence of QQYDESPWT (SEQ ID NO: 100); further comprising the intracellular domain of SEQ ID NO: 41. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; further comprising the intracellular domain of SEQ ID NO: 43. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; further comprising an intracellular domain of SEQ ID NO: 44. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; further comprising an intracellular domain of SEQ ID NO: 30. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; further comprising an intracellular domain of SEQ ID NO: 36. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; and further comprises an intracellular domain of SEQ ID NO: 37.In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; further comprising an intracellular domain of SEQ ID NO: 38. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; further comprising an intracellular domain of SEQ ID NO: 39. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; further comprising an intracellular domain of SEQ ID NO: 40. In some embodiments, the 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 containing at least the CDR3 sequence of RGYDWYFDV (SEQ ID NO: 99), and / or a VL domain containing the CDR3 sequence of SEQ ID NO: 100; and further comprises an intracellular domain of SEQ ID NO: 45.

[0208]

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

[0209]

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

[0210] IRF-inducible proteins and IRF activation pathways

[0263] Type I IFNs are key cytokines that mediate innate antiviral immunity and thus drive pro-inflammatory responses. Type I IFNs are readily induced by cGMP-AMP synthase, retinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors that recognize microbial double-stranded (ds)DNA, dsRNA, and LPS. These signaling pathways converge with the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). Adapter proteins STING (IFN gene stimulator), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adapter-inducible IFN-β) mediate IRF-3 recruitment via conserved pLxIS motifs. While the pLxIS motifs of phosphorylated STING, MAVS, and TRIF generally bind to IRF-3 in a similar manner, upstream residues of the motif confer specificity. Type I IFNs, such as IFN-α and IFN-β, are a major cytokine family that mediates 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 adapter protein STING (IFN gene stimulator) 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 and translocates to the nucleus, inducing transcription of the IFN-β gene. In contrast, viral dsDNA in the cytosol is sensed by RLR [retinoic acid-inducible protein 1 (RIG-I)-like receptor], which activates IRF-3 via the adapter protein MAVS (mitochondrial antiviral signaling).Furthermore, TLR3 and TLR4, Toll-like receptors (TLRs) that recognize viral dsRNA in endosomes and LPS, a component of bacterial cell walls, also mediate the induction of type I IFN and inflammatory cytokines, respectively (1). These two TLRs mediate the recruitment and activation of IRF-3 using the adapter protein TRIF (TIR domain-containing adapter-inducible IFN-β). Notably, the signaling pathways of these three innate immune sensor families converge upon the activation of TBK1 and IRF-3. Mechanistically, the adapter proteins STING, MAVS, and TRIF are phosphorylated by TBK1 or IKKε and contain pLxIS (where p represents a hydrophilic residue, x represents any residue, and S represents a phosphorylation site), a conserved motif that mediates the recruitment of IRF-3 into the signaling complex. Induction of proximity between TBK1 and IRF-3 results in the phosphorylation and activation of IRF-3. Furthermore, IRF-3 itself also contains the pLxIS motif, which is crucial for the phosphorylation-induced dimerization / activation of IRF-3. Mutations in the serine phosphorylation site within the pLxIS motifs of STING, MAVS, and TRIF invalidate the induction of type I IFN in their respective signaling pathways. However, the precise molecular mechanisms of IRF-3 recruitment and activation remain 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 derived from the three adapter proteins, phosphorylated them in vitro with TBK1, and determined the crystal structures of their complexes with the C-terminal domain (CTD) of IRF-3.

[0211] The mechanism for mobilizing IRF-3s by pMAVS and pTRIF.

[0212]

[0264] In contrast to dsDNA sensing via the cGAS-STING pathway, RLR senses dsDNA in the cytosol and activates IRF-3 via the adapter MAVS, while TLR3 and TLR4 recruit IRF-3 using the adapter TRIF. Phosphorylation of the pLxIS motif in MAVS or TRIF is required for IRF-3 recruitment and activation.

[0213] TRAF-interacting proteins:

[0265] The presence of the TRAF domain, a protein-interacting domain of approximately 180 amino acids, is a distinguishing feature of the TRAF family proteins. In mammals, six of the seven TRAF proteins in the family (TRAF1-TRAF6) that adhere to this criterion have been identified as part of the TRAF family. The TRAF domain can be subdivided into two distinct regions: the TRAF-N domain and the TRAF-C domain. A variety of receptors bind to the TRAF-C domain, while a variety of intracellular signaling molecules bind to the TRAF-N domain. Despite the structural similarity of the TRAF domains, each TRAF protein has a specific biological function, accompanied by specificity for its interaction partners: upstream receptors and downstream effector molecules. The structure of the TRAF domain of TRAF2 was first reported around 1999 by Dr. Wu's research group, and the structure of the TRAF domain of TRAF6 was reported three years later by the same group. Since then, the structures of the TRAF domains of TRAF3, TRAF5, TRAF4, and TRAF1 have also been reported. The TRAF structure revealed that the TRAF-N domain has a coiled-coil structure, and TRAF-C is composed of 7-8 antiparallel β-sheet folds. Structural alignment of all six TRAF family members showed that the TRAF-C domain was well aligned, while the position and length of TRAF-N varied among TRAF family members. Sequence analysis indicated that while the length of TRAF-N varied within the family, the length of the TRAF-C domain was conserved: the TRAF-N lengths of TRAF4 and TRAF6 were relatively short, while those of TRAF3 and TRAF5 were relatively long. The overall structure was nearly identical, but clear structural differences were observed. For example, the length and position of some loops within the TRAF domains of TRAF4 and TRAF6 differed from those of other TRAF family members. TRAF4 contains a more strongly negatively charged surface within its receptor-binding region, while TRAF6 contains a more strongly positively charged surface within its receptor-binding region.Surface features often determine their mode of interaction with partners. Thus, the electrostatic surfaces of the TRAF domains, namely TRAF1, TRAF2, TRAF3, and TRAF5, which are similar among diverse charged surfaces, have been shown to be important for accepting diverse receptors within the same binding pocket in a similar mode of interaction. In contrast, TRAF4 and TRAF6, which are distinct features on the binding surfaces of functionally different TRAFs, indicate that TRAF4 and TRAF6 accept different receptors in different modes of interaction.

[0214]

[0266] For the purposes of the present disclosure, any pathway, signaling intermediate, or activating moiety discussed in the above paragraphs can be considered to be activatable or functional, as appropriate, upon induction of CFP as disclosed herein. Similarly, the CFP disclosed herein can be useful in targeting any of the applicable targets described within the discussed pathways. It is understood that any pathway or part thereof that is readily known to those skilled in the art as of the date of the present document with respect to signal transduction domains, signal transduction pathways, signal transduction intermediates, transcription factors of activated genes, is within the purview 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 includes an intracellular signaling domain derived from a TLR protein. In some embodiments, a CFP designed to include an intracellular signaling domain derived from a TLR intracellular signaling domain may activate NF kappa B upon engagement of the extracellular domain of the receptor with its target. In some embodiments, the intracellular domain may include an 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 the TLR3 protein. In some embodiments, the intracellular signaling domain may be derived from the TLR7 protein, TLR8 protein, or TLR9 protein. In some embodiments, the intracellular domain may include the intracellular signaling domains of cell surface TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10. In some embodiments, the cytoplasmic domain for the inflammatory response includes the intracellular signaling domains of TLR3, TLR4, TLR9, MYD88, TRIF, RIG-1, MDA5, CD40, 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) contains a pro-inflammatory cytoplasmic domain for activation of the IL-1 signaling cascade.

[0217]

[0269] In some embodiments, the cytoplasmic portion of a chimeric receptor (e.g., a phagocytic receptor (PR) fusion protein (PFP)) includes cytoplasmic domains derived from Toll-like receptors, such as the intracellular signaling domains of Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TLR8), and Toll-like receptor 9 (TLR9).

[0218]

[0270] Generally, TLRs have diverse intracellular localizations, developmental pathways, activations, recognition mechanisms, and modes of action. TLRs are expressed in innate immune cells such as dendritic cells (DCs) and macrophages, as well as in 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, along with TLR1 or TLR6, recognizes a wide variety of PAMPs, including lipoproteins, peptidoglycans, lipoteichoic acid, zymosan, mannan, and tGPI-mucin. TLR5 recognizes bacterial flagellin. In mice, TLR10 is a pseudogene due to the insertion of a stop codon, but human TLR10, in cooperation with TLR2, recognizes ligands derived from Listeria monocytogenes. TLR10 also senses viral infection by influenza A virus.

[0219]

[0271] Intracellular TLRs recognize nucleic acids derived from bacteria and viruses, and also recognize self-nucleotides in disease states such as autoimmunity. TLR3 recognizes viral double-stranded RNA (dsRNA), small interfering RNA, and self-RNA derived from damaged cells. TLR7 is mainly expressed in plasmacytoid DCs (pDCs) and recognizes viral single-stranded (ss)RNA. TLR7 also recognizes RNA derived from Streptococcus bacterium in conventional DCs (cDCs). Human TLR8 responds to viral and bacterial RNA. Structural analysis revealed that unstimulated human TLR8 exists as a pre-formed dimer, and the Z-loop between LRR14 and LRR15 is cleaved into an N-terminal half and a C-terminal half, but maintains association with each other and participates in ligand recognition and dimerization. Ligand binding induces dimer reorganization so that the two C-terminuses are brought into close proximity. TLR13 recognizes bacterial 23S rRNA and unknown vesicular stomatitis virus components. TLR9 recognizes bacterial and viral DNA rich in unmethylated CpG-DNA motifs; it also recognizes hemozoin, an insoluble crystalline byproduct produced by Plasmodium falciparum during the detoxification process after the digestion of host hemoglobin. TLR11 is localized within endolysosomes and recognizes flagellin or unknown protein components of urinary tract pathogenic Escherichia coli (UPEC), as well as profilin-like molecules derived from Toxoplasma gondii. TLR12 is mainly expressed in bone marrow cells, is highly similar to TLR11, and recognizes profilin derived from T. gondii. 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. Intracellular localization of TLRs is considered extremely important not only for ligand recognition but also for preventing TLRs from coming into contact with the body's own nucleic acids, which can trigger autoimmunity.

[0220]

[0272] Individual TLRs differentially recruit members of a set of TIR domain-containing adapters, such as MyD88, TRIF, TIRAP / MAL, or TRAM. MyD88 is utilized by all TLRs and activates NF-κB and MAPK for the induction of inflammatory cytokine genes. TIRAP is a preparative adapter that recruits MyD88 to cell surface TLRs such as TLR2 and TLR4. TIRAP also participates in signaling via endosomal TLRs such as TLR9. The lipid-binding domain of TIRAP binds to PI(4,5)P2 on the cell membrane and to PI(3)P on endosomes, which mediate the formation of functional TLR4 / TLR9 signaling complexes at their respective sites. Thus, TIRAP associates with both cell surface TLRs 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 genes and inflammatory cytokine genes. TRAM is selectively recruited to TLR4 rather than TLR3 to link TRIF with TLR4. TLR3 directly interacts with TRIF, and this interaction requires phosphorylation of two tyrosine residues in the cytoplasmic domain of TLR3 by epidermal growth factor ErbB1 and Btk. Following 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, along 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 regulatory subunits TAB1, TAB2, and TAB3, which interact with the polyubiquitin chain generated by TRAF6 to drive TAK1 activation.Subsequently, TAK1 activates two distinct pathways, resulting in the activation of the IKK complex-NF-κB pathway and the IKK complex-MAPK pathway. The IKK complex consists of catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO (also called IKKγ). TAK1 binds to the IKK complex via ubiquitin chains, which allows the IKK complex to be phosphorylated and activate IKKβ. The IKK complex phosphorylates the NF-κB inhibitory protein IκBα, which undergoes proteasomal degradation, allowing NF-κB to translocate to the nucleus and induce pro-inflammatory gene expression. Activation of TAK1 also results in the activation of MAPK family members, such as ERK1 / 2, p38, and JNK, which regulate the inflammatory response by mediating the activation of AP-1 family transcription factors or mRNA stabilization.

[0221]

[0273] In some embodiments, intracellular domains described herein may be specifically paired with other domains, such as structural domains like other intracellular domains, transmembrane domains, or extracellular domains; or functional domains like signaling domains. In some embodiments, intracellular domains described in sections herein, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domains referred herein, may be modified or altered 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 fragments or components thereof, into the CFP design as any part of the molecular structure of the CFP protein. In some embodiments, modification may be a structural alignment or arrangement of domains within the CFP molecule, for example, two domains under consideration may be juxtaposed, separated by one or more domains between them, or separated by one or more amino acids. One or more amino acids may be linkers. One or more amino acids may introduce a structural suspension between two adjacent domains, introduce flexibility between two adjacent domains, or confer a more three-dimensional orientation to the molecular structure, including domains without one or more amino acids. In some embodiments, modifications or alterations may include modifications within domains, such as mutations.

[0222]

[0274] In some embodiments, intracellular domains described in the sections herein, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domains referred herein, may be modified or altered for pairing with or incorporation with another intracellular domain within the CFP, such as a kinase mobilization domain, such as a PI3 kinase mobilization domain. In some embodiments, the PI3 kinase mobilization domain is modified to block tonic signaling. In some embodiments, one of the other intracellular domains, such as the domain MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any one or more of these fragments, is modified to reduce or eliminate tonic signaling.

[0223]

[0275] In some embodiments, intracellular domains described in the sections herein, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, and TASL, or any other domains referred herein, may be modified or altered for pairing with or incorporation with 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, such as SEQ ID NO: 46, may be used.

[0224]

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

[0225] therapeutic composition

[0277] In one embodiment of this specification, bone marrow cells 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 are provided. In some embodiments of this specification, therapeutic compositions are provided that contain at least 20%, at least 30%, at least 40%, or at least 50% CD14+ cells. In some embodiments of this specification, therapeutic compositions are provided that contain at least 20%, at least 30%, at least 40%, or at least 50% CD14+ / CD16- cells. In some embodiments of this specification, therapeutic compositions are provided that contain 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 are recombinant nucleic acids encoding a chimeric fusion protein, comprising recombinant nucleic acids encoding either a CFP receptor protein or an engager protein as described herein. The bone marrow cells for the therapeutic compositions described herein express CFP encoded by the recombinant nucleic acid or an engager protein encoded by the recombinant nucleic acid as described herein.

[0226]

[0278] In some embodiments of this specification, a chimeric fusion protein such as a chimeric fusion receptor protein (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, the at least two intracellular signaling domains comprising (i) a first intracellular signaling domain derived from FcRγ or FcRε, (ii A therapeutic composition is provided comprising a second intracellular signaling domain, for example, an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, NLRP family members NLRP1-14, NOD1, NOD2, pyrine, 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 mobilization domain or a domain derived from CD40.

[0227]

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

[0228]

[0280] In some embodiments, bone marrow cells may be further modified or manipulated to produce therapeutically effective bone marrow cells. Isolated cells may be manipulated intracellularly by expressing genes or fragments thereof without altering their functional / developmental plasticity, differentiation potential, and cell viability.

[0229]

[0281] In some embodiments, bone marrow cells may be further modified or manipulated to produce therapeutically effective bone marrow cells by expressing non-endogenous polynucleotides into the cells. Non-endogenous polynucleotides encode proteins or peptides. Alternatively, non-endogenous polypeptides may be non-coding sequences, such as inhibitory RNA or morpholino.

[0230]

[0282] In some embodiments, bone marrow cells may be further modified or manipulated to produce therapeutically effective bone marrow cells by stably altering the cellular genome sequence. In some embodiments, bone marrow cells may be manipulated by editing the bone marrow cell genome using the CRISPR-CAS system. In some embodiments, one or more genes may be edited to silence gene expression. In some embodiments, bone marrow cells may be manipulated to delete genes. In some embodiments, one or more genes may be edited to enhance genes. In some embodiments, genetic material is introduced into bone marrow cells in the form of messenger RNA, in which case the messenger RNA codes for a protein or peptide, thereby making the bone marrow cells therapeutically effective. In some embodiments, naked DNA or messenger RNA (mRNA) may 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). mRNA is single-stranded and may be codon-optimized. In some embodiments, the mRNA may contain one or more modified or non-native bases, such as 5'-methylcytosine, or pseudouridine or methylpseudridine. In some embodiments, about 50% or more of the uridine ("U") residues of the mRNA may be converted to methylpseudridine. In some embodiments, the mRNA may be 50 to 10,000 base pairs long. In one embodiment, the transgene is delivered as mRNA. mRNA may contain approximately 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, and more than 10,000 bases. In some embodiments, mRNA may exceed 10,000 bases in length. In some embodiments, mRNA may be approximately 11,000 bases in length. In some embodiments, mRNA may be approximately 12,000 bases in length.In some embodiments, the mRNA contains a transgene sequence encoding a fusion protein. The DNA or RNA encapsulated in the LNP may be used to transfect macrophages or administered to a subject. In some embodiments, the mRNA is incorporated into an effector bone marrow cell population by transient transfection. In some embodiments, the transient transfection method includes electroporation of the mRNA. In some embodiments, transient transfection includes chemical transfection. In some embodiments, 1 to 5,000 micrograms of mRNA per ml may be used for transfection using a protocol suitable for the method 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 to 500 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1 to 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, approximately 80 micrograms of mRNA per ml are used. In some embodiments, approximately 100 micrograms of mRNA per ml are used. In some embodiments, approximately 150 micrograms of mRNA per ml are used. In some embodiments, approximately 200 micrograms of mRNA per ml are used. In some embodiments, approximately 20, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 micrograms of mRNA per ml are used. An appropriate cell density is selected for transfection based on the manufacturer's instructions for the method, as well as for the measuring instruments and / or reagents, or as is well known to those skilled in the art.

[0231]

[0283] In some embodiments, the recombinant nucleic acid is mRNA. The mRNA construct may be thawed on ice, gently pipetteed into monocytes, and pre-mixed. In some embodiments, the mRNA is electropermeated into the cells. After elutriation, the cells may be pooled, centrifuged, and subjected to mRNA electropermeation using a MaxCyte ATX system optimized for the aforementioned purposes. In some embodiments, electropermeation 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). Within the circular RNA (circRNA), the 3' and 5' ends are covalently linked. The circRNA can be delivered into the cell using LNPs.

[0233]

[0285] In some embodiments, the stable integration of transgenes into macrophages and other phagocytic cells may be achieved through the use of transposases and transposition factors, particularly mRNA-coding transposases. In one embodiment, long-chain scattered element-1 (L1) RNA may be used for the retrotransposition of transgenes and their stable integration into macrophages or phagocytic cells. Retrotransposons may be used for the stable integration of recombinant nucleic acids encoding phagocytic or tethering receptor (PR) fusion proteins (PFPs).

[0234]

[0286] In some embodiments, bone marrow cells may be modified by expressing a transgene via the incorporation of the transgene into a transient expression vector. In some embodiments, transgene expression may be temporally regulated from outside the cell by a regulator. An example is a 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 may be modified to produce therapeutically effective cells by contacting them with compounds that may be proteins, enzymes, inhibitors, or activators within the bone marrow cells.

[0236]

[0288] In some embodiments, the polynucleotide encoding the chimeric antigen receptor may be introduced into isolated myeloid cells obtained by the method described in the preceding section, in which case the chimeric antigen receptor enhances the innate immune response function of myeloid cells upon expression within the myeloid cells. In some embodiments, the expression of the chimeric antigen receptor may direct myeloid cells to specific targets in vivo or in vitro. In some embodiments, the chimeric antigen receptor may increase the phagocytic potential of myeloid cells. In some embodiments, the chimeric antigen receptor may increase the immunogenicity of myeloid 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 intracellular proteins. In some embodiments, the chimeric antigen receptor may dimerize or multimerize within myeloid cells with a second receptor or transmembrane protein, in which case the second receptor or transmembrane protein is an endogenous protein. In some embodiments, cells are cultured ex vivo immediately after thawing or after nucleic acid integration. In some embodiments, ex vivo culture is carried out in the presence of a suitable medium which may contain a modified serum component, such as human serum albumin (HSA). In some embodiments, ex vivo culture and manipulation may be carried out in a medium with a low serum concentration. In some embodiments, serum is specifically treated for complement inactivation. 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 presented herein include isolation or concentration and manipulation of bone marrow cells for 72 hours, 70 hours, 65 hours, 60 hours, 55 hours, 50 hours, 45 hours, 40 hours, or 35 hours, or 30 hours, or 28 hours, or 26 hours, or less than 24 hours. In some embodiments, 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 isolation or concentration and manipulation, bone marrow cells may be cultured for a short period and frozen until further use. In some embodiments, bone marrow cells are thawed once or up to twice.

[0237]

[0289] In some embodiments, therapeutically competent cells are cells that have been electroporated with recombinant nucleic acids encoding polypeptides, frozen and thawed, and whose culture has been stabilized for less than 24 hours, in which case the cells in the cell population at administration exhibit (i) a viability of at least 70% or more, (ii) at least 50% or more 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 approximately 10% CD56+ cells, less than approximately 10% CD42b+ cells; and (iv) more than 50% of cells expressing polypeptides encoded by the electroporated nucleic acids. In some embodiments, therapeutically competent cells are cells that have been electroporated with recombinant nucleic acids encoding polypeptides, and the culture has been stabilized for less than 24 hours, followed by freezing and thawing, in which case the cells in the cell population at administration exhibit (i) a viability of at least 70% or more, (ii) at least 50% or more 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 approximately 10% CD56+ cells, less than approximately 10% CD42b+ cells; and (iv) more than 50% of cells express polypeptides encoded by the electroporated nucleic acids. In some embodiments, therapeutically competent cells are cells whose culture has been stabilized for less than 24 hours, electroporated with recombinant nucleic acids encoding polypeptides, and frozen and thawed, in which case the cells in the cell population at administration exhibit (i) a viability of at least 70% or more, (ii) at least 50% or more 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, less than about 10% CD42b+ cells; and (iv) more than 50% cells expressing polypeptides encoded by electroporated nucleic acids. The cells must be pathogen-free.In the embodiments described above, therapeutically competent cells may be frozen and thawed more than twice, preferably once, and may be administered within 24 hours, 18 hours, 8 hours, or 2 hours of thawing. Prior to administration, the cells are inspected for quality assurance to meet the criteria described herein.

[0238]

[0290] This specification provides a method for treating cancer in a subject using a pharmaceutical composition comprising engineered phagocytic cells, particularly macrophages, that express recombinant nucleic acids encoding phagocytic receptor (PR) fusion proteins (PFPs) specifically designed to target, attack, and kill cancer cells. PFPs are also referred to as chimeric antigen receptors for phagocytosis (CAR-Ps), and both terms may be used interchangeably herein. In this specification, engineered phagocytic cells are also referred to as CAR-P cells.

[0239]

[0291] Cancer includes, but is not limited to, B-cell cancers (e.g., multiple myeloma, Waldenstrom macroglobulinemia), heavy chain diseases (e.g., alpha chain disease, gamma chain disease, and muon chain disease, etc.), benign monoclonal immunoglobulinemia, and immunocellular 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 cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer or endometrial cancer, oral cancer or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine cancer or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematopoietic tissue cancer, etc. Other non-limiting examples of cancer types applicable to the methods encompassed by this disclosure include human sarcomas and human carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, 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 cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminomas, embryonic carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testis Cancer, including lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia, e.g., acute lymphoblastic 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); as well as polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, and heavy chain disease. In some embodiments, cancer is an epithelial cancer, including but not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney 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. The epithelial cancer can also be characterized in other diverse forms including, but not limited to, serous cancer, endometrial cancer, mucinous cancer, clear cell cancer, or undifferentiated cancer. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphomas or subtypes thereof, including but not limited to mantle cell lymphoma. Lymphoproliferative disorders are also considered proliferative diseases.

[0240]

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

[0241]

[0293] In one aspect, the subject is administered a dose of a pharmaceutical composition for one or more administrations containing therapeutic phagocytic cells, where the cells are allogeneic cells. The HLA can be matched for compatibility with the subject and so that the cells do not cause graft-versus-host disease (GVHD), which is a transplant rejection. Subjects presenting at a clinic undergo HLA genotyping to determine the HLA antigens expressed by the subject prior to determining a therapeutic agent or regimen.

[0242]

[0294] In some embodiments, the therapeutically effective dose is from 10 7 cells to 10 12This is a range between individuals. The cell count may vary depending on age, weight, and other parameters relevant to the subject, and may be determined by a healthcare professional. In some embodiments, the therapeutically effective dose is approximately 10 myeloid cells. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 2 × 10⁶ cells of bone marrow. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 3 × 10⁶ cells of bone marrow. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 4 × 10⁶ cells of bone marrow. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 5 × 10⁶ bone marrow cells. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 6 × 10⁶ cells of bone marrow. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 7 × 10⁶ cells of bone marrow. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 8 × 10⁶ cells of bone marrow. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 9 × 10⁶ bone marrow cells. 7 It is one. In some embodiments, the therapeutically effective dose is approximately 10 myeloid cells. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 2 × 10⁶ cells of bone marrow. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 3 × 10⁶ cells of bone marrow. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 4 × 10⁶ cells of bone marrow. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 5 × 10⁶ bone marrow cells. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 6 × 10⁶ cells of bone marrow. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 7 × 10⁶ cells of bone marrow. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 8 × 10⁶ cells of bone marrow. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 9 × 10⁶ bone marrow cells. 8 It is one. In some embodiments, the therapeutically effective dose is approximately 10 myeloid cells. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 2 × 10⁶ cells of bone marrow. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 3 × 10⁶ cells of bone marrow. 9It is one. In some embodiments, the therapeutically effective dose is approximately 4 × 10⁶ cells of bone marrow. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 5 × 10⁶ bone marrow cells. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 6 × 10⁶ cells of bone marrow. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 7 × 10⁶ cells of bone marrow. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 8 × 10⁶ cells of bone marrow. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 9 × 10⁶ bone marrow cells. 9 It is one. In some embodiments, the therapeutically effective dose is approximately 10 myeloid cells. 10 It is one. In some embodiments, the therapeutically effective dose is approximately 5 × 10⁶ bone marrow cells. 10 It is one. In some embodiments, the therapeutically effective dose is approximately 10 myeloid cells. 11 It is one. In some embodiments, the therapeutically effective dose is approximately 5 × 10⁶ bone marrow cells. 11 It is one. In some embodiments, the therapeutically effective dose is approximately 10 myeloid cells. 12 It is an individual.

[0243]

[0295] In one aspect of this 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 mRNA. In some embodiments, the recombinant polynucleic acid comprises 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 of this specification, a therapeutic composition is provided comprising 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) a PI3K mobilization domain or (B) a third intracellular signaling domain derived from CD40.

[0245]

[0297] In some embodiments of this specification, therapeutic compositions are provided that include recombinant nucleic acids encoding a dispecific or trispecific engager as disclosed herein.

[0246] Other therapeutic compositions for co-administration

[0298] In some embodiments, the therapeutic composition further comprises a further therapeutic agent selected from the group consisting of CD47 agonists, Rac inhibitors, Cdc42 inhibitors, GTPase inhibitors, agents that promote F-actin disassembly, agents that promote the recruitment of PI3K to PFP, agents that promote PI3K activity, agents that promote the production of phosphatidylinositol 3,4,5-triphosphate, agents that promote the activity of ARHGAP12, agents that promote the activity of ARHGAP25, agents that promote the activity of SH3BP1, agents that promote the blockade of lymphocytes in primary and / or secondary lymphoid organs, agents that increase 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 endogenous protein that dimerizes with CFP; (b) a non-endogenous peptide or non-endogenous protein that dimerizes with CFP; and / or (c) a second recombinant polynucleic acid sequence, in which case the second recombinant polynucleic acid sequence comprises a sequence encoding a peptide or protein that interacts with CFP; the dimerization or interaction enhances phagocytosis by CFP-expressing bone marrow cells compared to bone marrow cells that do not express CFP.

[0248]

[0300] In some embodiments, bone marrow cells are (i) increased effector activity, cross-presentation, respiratory burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4,5-triphosphate production, gnawing 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 IL-1, IL-3, IL-6, IL-10, IL-12, IL-13, IL-23, TNFα, TNF family cytokines, CCL2, CXCL9, CXCL10, CXCL11 It exhibits increased expression of 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, TRAIL / TNF family death receptors, 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 includes 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 The receptors are derived from proteins such as receptors selected from the group consisting of 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., phagocytic receptors). In some embodiments, the intracellular signaling domain includes a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain includes a pro-inflammatory signaling domain that is not a PI3K mobilization domain.

[0250]

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

[0251]

[0303] This specification provides a composition comprising a PR subunit including a transmembrane domain and an intracellular domain including an intracellular signaling domain; and an extracellular domain including an antigen-binding domain specific to the antigen of a target cell; the transmembrane domain and the extracellular domain being operetically linked; and the intracellular signaling domain comprising a recombinant nucleic acid encoding a CFP such as a phagocytic or tethering receptor (PFP) fusion protein, which 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 CFP to the antigen on target cells, the toxic activity of CFP-expressing cells is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, and 45% compared to cells that do not express CFP. The levels are increased to over 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, CFP is functionally incorporated into the cell membrane once it is expressed within a cell. In some embodiments, upon binding of CFP to the antigen of target cells, the toxic activity of CFP-expressing cells is increased by at least 1.1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, or 100 times compared to cells that do not express CFP.

[0253]

[0305] In some embodiments, the intracellular signaling domains include 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, and SRCR. The receptors are derived from phagocytic receptors 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 CD169. In some embodiments, the intracellular signaling domain includes a pro-inflammatory signaling domain.

[0254]

[0306] This specification includes 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 the antigen of the target cell; the transmembrane domain and the extracellular domain are operetically linked; and the intracellular signaling domain includes TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCA A composition is provided comprising recombinant nucleic acids encoding CFPs, such as phagocytic or tethering receptor (PR) fusion proteins (PFPs), derived from receptors such as phagocytic receptors 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 CFP to the antigen on target cells, the toxic activity of CFP-expressing cells is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, and 45% compared to cells that do not express CFP. , are increased to 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%. 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 includes a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain includes a PI3K mobilization domain, such as a PI3K mobilization domain derived from CD19. In some embodiments, the intracellular signaling domain includes a pro-inflammatory signaling domain that is not a PI3K mobilization domain.

[0256]

[0308] In some embodiments, CFP-expressing cells exhibit increased phagocytosis of antigen-expressing target cells compared to CFP-non-expressing cells. In some embodiments, CFP-expressing cells exhibit at least a 1.1-fold increase in phagocytosis of antigen-expressing target cells compared to CFP-non-expressing cells. In some embodiments, CFP-expressing cells exhibit at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, or 50-fold increase in phagocytosis of antigen-expressing target cells compared to CFP-non-expressing cells. In some embodiments, CFP-expressing cells exhibit increased cytokine production compared to CFP-non-expressing cells. In some embodiments, cytokines are selected from the group consisting of IL-1, IL-3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferon, and combinations thereof. In some embodiments, cells expressing CFP exhibit increased effector activity compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased cross-presentation compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class II proteins compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD80 compared to cells not expressing CFP. In some embodiments, cells expressing CFP show increased CD86 expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased MHC class I protein expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased TRAIL / TNF family death receptor expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased B7-H2 expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased LIGHT expression compared to cells that do not express CFP.In some embodiments, cells expressing CFP show increased HVEM expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased CD40 expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased TL1A expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased 41BBL expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased OX40L expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased GITRL death receptor expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased CD30L expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased TIM4 expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of TIM1 ligand compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of SLAM compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of CD48 compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of CD58 compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of CD155 compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of CD112 compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of PDL1 compared to cells that do not express CFP. In some embodiments, cells expressing CFP show increased expression of B7-DC compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased respiratory bursts compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased ROS 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 iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased extracellular vesicle production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased gnawing activity with antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased resistance to CD47-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased resistance to LILRB1-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased production of phosphatidylinositol 3,4,5-triphosphate.

[0257]

[0309] This specification also presents pharmaceutical compositions comprising compositions described herein, such as recombinant nucleic acids, vectors, polypeptides, or cells described herein, and pharmaceutically acceptable excipients. The manipulated cells are myeloid cells. In one embodiment, a pharmaceutical composition is disclosed comprising recombinant nucleic acid encoding or containing any one of the sequences of SEQ ID NOs: 1 to 51, or a cell comprising recombinant nucleic acid encoding or containing any one of the sequences of SEQ ID NOs: 1 to 51, or a manipulated cell comprising recombinant nucleic acid encoding or containing any one of the sequences of SEQ ID NOs: 1 to 51, and pharmaceutically acceptable excipients. In one embodiment, the cell comprises recombinant nucleic acid encoding an amino acid sequence containing at least one of the sequences selected from SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45. In one embodiment, the cell is a myeloid cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a primary human cell. In one embodiment, the cells are primary human immune cells. In some embodiments, the cells are progenitor cells, stem cells, or undifferentiated cells. In some embodiments, the cells are obtained from a biological sample of a human subject. In some embodiments, the cells are isolated from a biological sample of a human subject and selected for their phenotypic features, such as the expression of cell surface markers. In one embodiment, the isolated cells are progenitor cells, myeloid progenitor cells, or cells characterized as CD14+ / CD16-.

[0258]

[0310] In one embodiment, the isolated or manipulated cells are CD14+ / CD16-.

[0259]

[0311] In one embodiment, the pharmaceutical composition comprises engineered cells, in which case the engineered cells are CD14+ / CD16-.

[0260]

[0312] In one embodiment, the pharmaceutical composition comprises a population of cells in which at least 50% of the cells are CD14+ / CD16- and less than 10% of the cells are dendritic cells. ...

Claims

1. A polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is: (a) (i) an antigen-binding domain that specifically binds to glypican 3 (GPC3), and (ii) an extracellular domain comprising the sequence DSIHQDYTTQN (amino acid residues 267-277 of SEQ ID NO: 58), and (b) Transmembrane domain operably linked to the extracellular domain, Includes, The transmembrane domain is derived from a protein that dimerizes with the endogenous FcRγ receptor; CFP is expressed on the surface of myeloid cells expressing the FcRγ receptor; the antigen-binding domain includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain being the heavy chain complementarity determining region 1 (HCDR1) sequence of DYEMH (amino acid residues 31-35 of SEQ ID NO: 97) and ALDPKTGDTAYSQKFKG (amino acid residues 50-66 of SEQ ID NO: 97). The polynucleic acid comprises the HCDR2 sequence and the HCDR3 sequence of FYSYTY (amino acid residues 99-104 of SEQ ID NO: 97), and the light chain variable domain comprises the light chain complementarity determining region 1 (LCDR1) sequence of RSSQSLVHSNRNTYLH (amino acid residues 24-39 of SEQ ID NO: 98), the LCDR2 sequence of KVSNRFS (amino acid residues 55-61 of SEQ ID NO: 98), and the LCDR3 sequence of SQNTHVPPT (amino acid residues 94-102 of SEQ ID NO: 98).

2. The polynucleic acid according to claim 1, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv).

3. The polynucleic acid according to claim 1 or 2, wherein the heavy chain variable domain includes a sequence having at least 80% identity with SEQ ID NO:

97.

4. The polynucleic acid according to any one of claims 1 to 3, wherein the light chain variable domain includes a sequence having at least 80% identity with SEQ ID NO:

98.

5. The polynucleic acid according to any one of claims 1 to 4, wherein the antigen-binding domain includes a sequence having at least 80% sequence identity with respect to sequence number 106.

6. The polynucleic acid according to any one of claims 1 to 5, wherein the transmembrane domain comprises a transmembrane domain derived from CD16a, CD64, CD68, or CD89.

7. The polynucleic acid according to claim 6, wherein the transmembrane domain is a transmembrane domain derived from CD89.

8. The polynucleic acid according to claim 7, wherein the transmembrane domain comprises the sequence LIRMAVAGLVLVALLAILV (amino acid residues 278-296 of SEQ ID NO: 58).

9. The polynucleic acid according to any one of claims 1 to 8, wherein the CFP further comprises an intracellular domain comprising one or more intracellular signaling domains, and the one or more intracellular signaling domains comprising an intracellular signaling domain derived from CD89, CD16a, CD64, CD68, FCERIG, CD40, or CD3ζ.

10. The polynucleic acid according to claim 9, wherein one or more intracellular signaling domains include an intracellular signaling domain derived from CD89.

11. The polynucleic acid according to claim 10, wherein one or more intracellular signaling domains include the sequence ENWHSHTALNKEASADVAEPWSQQMCQPGLTFARTPSVCK (amino acid residues 297-337 of SEQ ID NO: 58).

12. The polynucleic acid according to any one of claims 1 to 11, wherein the CFP further comprises a GMCSF signal peptide.

13. The polynucleic acid according to claim 12, wherein the GMCSF signal peptide comprises the sequence MWLQSLLLLLGTVACSIS (SEQ ID NO: 7).

14. The polynucleic acid according to any one of claims 1 to 13, wherein the polynucleic acid is RNA.

15. The polynucleic acid according to claim 14, wherein RNA is mRNA.

16. The polynucleic acid according to any one of claims 1 to 15, wherein the polynucleic acid is unmodified.

17. A polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is: (a) (i) an antigen-binding domain that specifically binds to 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 the extracellular domain, wherein the transmembrane domain contains the sequence LIRMAVAGLVLVALLAILV (amino acid residues 278-296 of SEQ ID NO: 58); and (c) an intracellular domain operably linked to the transmembrane domain, wherein the intracellular domain contains the sequence ENWHSHTALNKEASADVAEPWSQQMCQPGLTFARTPSVCK (amino acid residues 297-337 of SEQ ID NO: 58); Includes, The antigen-binding domain is a single-chain variable fragment (scFv) containing a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain includes heavy-chain complementarity-determining region 1 (HCDR1) of DYEMH (amino acid residues 31-35 of SEQ ID NO: 97), HCDR2 of ALDPKTGDTAYSQKFKG (amino acid residues 50-66 of SEQ ID NO: 97), and HCDR3 of FYSYTY (amino acid residues 99-104 of SEQ ID NO: 97), and the light-chain variable domain includes light-chain complementarity-determining region 1 (LCDR1) of RSSQSLVHSNRNTYLH (amino acid residues 24-39 of SEQ ID NO: 98), LCDR2 of KVSNRFS (amino acid residues 55-61 of SEQ ID NO: 98), and LCDR3 of SQNTHVPPT (amino acid residues 94-102 of SEQ ID NO: 98), as described above for the polynucleic acid.

18. A composition comprising the polynucleic acid described in any one of claims 1 to 17.

19. The composition according to claim 18, wherein polynucleic acids are encapsulated in lipid nanoparticles (LNPs).

20. A pharmaceutical composition comprising the composition according to claim 18 or 19 and a pharmaceutically acceptable excipient.

21. A pharmaceutical composition according to claim 20 for use in the treatment of cancer.

22. The pharmaceutical composition according to claim 21, wherein the cancer is selected from the group consisting of ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer.

23. The pharmaceutical composition according to claim 21 or 22, wherein the cancer is liver cancer.

24. The pharmaceutical composition according to any one of claims 21 to 23, wherein the cancer is GPC3 positive.

25. Use of the pharmaceutical composition according to claim 20 in the manufacture of a pharmaceutical for treating cancer.

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

27. The use according to claim 26, wherein the cancer is liver cancer.

28. The use according to any one of claims 25 to 27, wherein the cancer is GPC3 positive.

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