Engineered chimeric fusion protein compositions and methods of use thereof

Abstract

To provide compositions and methods for making and using engineered phagocytic cells that express a chimeric antigen receptor having enhanced phagocytic activity for immunotherapy in cancer or infection.SOLUTION: There is provided a composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP). The CFP comprises: (a) a phagocytic or tethering receptor (PR) subunit including (i) a transmembrane domain and (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising an antigen binding domain specific to an antigen of a target cell; wherein the transmembrane domain and the extracellular domain are operatively linked, the antigen is not a CD19 or CD22 antigen, and upon binding of the CFP to the antigen of the target cell, killing or phagocytosis activity of a cell expressing the CFP is increased by at least 20% compared to a cell not expressing the CFP, wherein killing or phagocytosis activity is measured by flow cytometry.SELECTED DRAWING: Figure 1A

Description

Related Applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 841,190, filed April 30, 2019, U.S. Provisional Patent Application No. 62 / 841,183, filed April 30, 2019, U.S. Non-Provisional Patent Application No. 16 / 827,381, filed March 23, 2020, and U.S. Non-Provisional Patent Application No. 16 / 827,302, filed March 23, 2020, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002]

[0002] Cellular immunotherapy is a promising new technology that combats the challenges of treating diseases such as cancer and persistent infections, as well as certain diseases refractory to other forms of treatment. A major breakthrough has been the discovery of CAR-T cells and their potential use in immunotherapy. CAR-T cells are T lymphocytes that express chimeric antigen receptors that help target T cells to specific diseased cells, such as cancer cells, and can induce a cytotoxic response intended to kill the targeted cancer cells or immunosuppression and / or tolerance through the intracellular domains of co-expressed immunosuppressive cytokines. However, to date, several limitations have slowed the progress of CAR-T cells and diminished their promise in clinical trials.

[0003] Understanding the limitations of CAR-T cells is key to leveraging the technology and continuing to innovate toward better immunotherapy models. Particularly in T-cell malignancies, CAR-T cells appear to have faced numerous challenges. CAR-T cells and malignant T cells share surface antigens in most T-cell lymphomas (TCLs), and thus, CAR-T cells are subject to cytotoxicity in the same manner as cancer cells. In some cases, CAR-T products can contaminate malignant T cells. Furthermore, T-cell aplasia may be an issue with prolonged persistence of CAR-T cells. Other limitations include the poor ability of CAR-T cells to penetrate solid tumors and the powerful tumor microenvironment, which acts to downregulate their antitumor potential. CAR-T cell function is also negatively affected by the immunosuppressive tumor microenvironment (TME), which leads to the inactivation and depletion of endogenous T cells.

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

[0005] Myeloid cells are the major intracellular compartment of the immune system, including monocytes, dendritic cells, tissue macrophages, and granulocytes. Models of myeloid cell ontogeny, activation, differentiation, and tissue-specific function have been reexamined for many years with surprising results. However, their significant plasticity and heterogeneity during both homeostasis and disease remain far from being understood. Myeloid cells have many functions, including phagocytosis and their ability to activate T cells, yet inhibiting these functions for therapeutic use remains elusive. Therefore, current approaches are being considered to use other cell types towards the development of improved therapies, including but not limited to T cell malignancies.

[0006]

[0006] Engineered myeloid cells can be short-lived, phenotypically diverse, sensitive, and plastic in vivo, and they are often difficult to manipulate in vitro. For example, exogenous gene expression in monocytes is more challenging than exogenous gene expression in non-hematopoietic stem cells. There are significant technical challenges associated with transfecting myeloid cells (e.g., monocytes / macrophages). As professional phagocytes, myeloid cells such as monocytes / macrophages contain many potent degradative enzymes that can disrupt the integrity of nucleic acids and transfer genes into these cells in an inefficient process. This is particularly true for activated macrophages, which undergo dramatic changes in their physiology after exposure to immune or inflammatory stimuli. Viral transduction of these cells is hampered because macrophages are generally terminally differentiated cells; therefore, some vectors that rely on integration into replicative genomes face limited success. Furthermore, macrophages are sensitive to "danger signals," and therefore, some of the original viral vectors used for gene transfer induce strong antiviral responses in these cells, making these vectors unsuitable for gene delivery. Summary of the Invention

[0007] This specification includes the disclosure of the following inventions. [Item 1] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP is (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; the antigen is not a CD19 or CD22 antigen; when the CFP binds to the antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 20% compared to cells not expressing the CFP; and the killing or phagocytic activity is measured by flow cytometry. [Item 2] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP is (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and when the CFP binds to an antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 10-fold compared to cells not expressing the CFP, wherein the killing or phagocytic activity is calculated as phagocytosis per 100 cells expressing the CFP. [Item 3] The composition of items 1 or 2, wherein the intracellular signaling domain is derived from a phagocytic or tethering receptor, or the intracellular signaling domain comprises a phagocytic activation domain. [Item 4] The composition according to any one of Items 1 to 3, wherein the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR, or Bai1. [Item 5] The composition according to any one of Items 1 to 4, wherein the intracellular signaling domain is derived from a receptor selected from the group consisting of receptors listed in Table 2. [Item 6] The composition according to any one of Items 1 to 5, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain. [Item 7] The composition described in Item 6, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain. [Item 8] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP is (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein said transmembrane domain and said extracellular domain are operably linked; A composition, wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, and Bai1. [Item 9] The composition described in Item 8, wherein when the CFP binds to an antigen on the target cell, the killing activity of cells expressing the CFP increases by at least 20% compared to cells not expressing the CFP; the killing or phagocytic activity of cells expressing the CFP increases by at least 20% compared to cells not expressing the CFP; and the killing or phagocytic activity is measured by flow cytometry. [Item 10] The intracellular signaling domain is selected from the group consisting of lectin, dectin 1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, and CD 209, the composition of item 8 or 9, wherein the protein is derived from a protein selected from the group consisting of RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha receptor I, CR1, CD35, CD3zeta, CR3, CR4, Tim-1, Tim-4, TNFR1, MDA5, CD40, and CD169. [Item 11] The composition described in any one of Items 8 to 10, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain. [Item 12] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP is (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is selected from the group consisting of 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 SRCRB4. D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha receptor I, CR1, CD35, CD3zeta, CR3, CR4, Tim-1, Tim-4, TNFR1, MDA5, CD40, and CD169. [Item 13] The composition described in Item 12, wherein when the CFP binds to an antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 55% compared to cells not expressing the CFP. [Item 14] The composition described in Item 12 or 13, wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, or Bai1. [Item 15] The composition described in any one of Items 12 to 14, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain. [Item 16] The composition described in Item 15, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain. [Item 17] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP is (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain. [Item 18] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a signaling domain derived from a TFNR1 receptor. [Item 19] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP is (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a signaling domain derived from a CD40 receptor. [Item 20] A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP is (a)(i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain; a phagocytic or tethering receptor (PR) subunit, including (b) an extracellular domain containing an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a signaling domain derived from MDA5. [Item 21] The composition described in any one of Items 17 to 20, wherein when the CFP binds to an antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 20% compared to cells not expressing CFP, and the killing or phagocytic activity is measured by flow cytometry. [Item 22] The composition described in any one of Items 17 to 21, wherein the intracellular signaling domain is derived from a phagocytic receptor. [Item 23] The composition described in any one of Items 17 to 22, wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR, or Bai1. [Item 24] The intracellular signaling domain is selected from the group consisting of lectin, dectin 1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, 24. The composition of any one of items 17 to 23, wherein the phagocytic receptor is selected from the group consisting of RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha receptor I, CR1, CD35, CD3zeta, CR3, CR4, Tim-1, Tim-4, TNFR1, MDA5, CD40, and CD169. [Item 25] The composition described in any one of Items 1 to 15, wherein the intracellular signaling domain comprises a PI3K recruitment domain. [Item 26] The composition of any one of the preceding items, wherein the intracellular signaling domain comprises an FcRγ intracellular domain. [Item 27] ​​The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and a CD40 receptor intracellular signaling domain in N to C order. [Item 28] The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and a CD40 receptor intracellular signaling domain in C to N order. [Item 29] The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and a TNFR1 receptor intracellular signaling domain in N to C order. [Item 30] The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and a TNFR1 receptor intracellular signaling domain in C to N order. [Item 31] The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and an MDA5 signaling domain in N to C order. [Item 32] The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and an MDA5 signaling domain in C to N order. [Item 33] The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and a PI3K recruitment domain in N to C order. [Item 34] The composition described in Item 26, wherein the intracellular signaling domain comprises an FcRγ intracellular domain and a PI3K recruitment domain in C to N order. [Item 35] The composition of any one of the preceding items, wherein the CFP is functionally integrated into the cell membrane of the cell when expressed in the cell. [Item 36] The cells expressing the CFP express the antigen more effectively than cells not expressing the CFP. The composition of any one of the preceding items exhibits increased phagocytosis of target cells expressing the composition. [Item 37] The composition described in Item 36, wherein cells expressing the CFP exhibit at least a 1.1-fold increase in phagocytosis of target cells expressing the antigen compared to cells not expressing the CFP, and phagocytosis is measured by flow cytometry. [Item 38] The composition of any one of the preceding items, wherein the cells expressing the CFP exhibit at least a 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold increase in phagocytosis of target cells expressing the antigen compared to cells not expressing the CFP, and the phagocytosis is measured by flow cytometry. [Item 39] The composition described in any one of the preceding items, wherein the target cells expressing the antigen are cancer cells. [Item 40] The composition of any one of the preceding items, wherein the target cells expressing the antigen are at least 0.8 microns in diameter. [Item 41] The composition described in any one of the preceding items, wherein the intracellular signaling domain is derived from a scavenger receptor. [Item 42] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased cytokine production compared to cells not expressing the CFP. [Item 43] The composition of Item 42, wherein the cytokine is selected from the group consisting of IL-1, IL3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferon, and combinations thereof. [Item 44] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased effector activity compared to cells not expressing the CFP. [Item 45] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased cross-presentation compared to cells not expressing the CFP. [Item 46] A composition described in any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of MHC class II proteins compared to cells not expressing the CFP. [Item 47] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of CD80 compared to cells not expressing the CFP. [Item 48] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of CD86 compared to cells not expressing the CFP. [Item 49] A composition described in any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of MHC class I proteins compared to cells not expressing the CFP. [Item 50] The method according to any one of the preceding items, wherein the cells expressing the CFP exhibit increased expression of TRAIL / TNF family death receptors compared to cells not expressing the CFP. composition. [Item 51] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of B7-H2 compared to cells not expressing the CFP. [Item 52] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of LIGHT compared to cells not expressing the CFP. [Item 53] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of HVEM compared to cells not expressing the CFP. [Item 54] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of CD40 compared to cells not expressing the CFP. [Item 55] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of TL1A compared to cells not expressing the CFP. [Item 56] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of 41BBL compared to cells not expressing the CFP. [Item 57] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of OX40L compared to cells not expressing the CFP. [Item 58] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of the GITRL death receptor compared to cells not expressing the CFP. [Item 59] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of CD30L compared to cells not expressing the CFP. [Item 60] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of TIM4 compared to cells not expressing the CFP. [Item 61] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of a TIM1 ligand compared to cells not expressing the CFP. [Item 62] A composition described in any one of the preceding items, wherein cells expressing the CFP show increased expression of SLAM compared to cells not expressing the CFP. [Item 63] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of CD48 compared to cells not expressing the CFP. [Item 64] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of CD58 compared to cells not expressing the CFP. [Item 65] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of CD155 compared to cells not expressing the CFP. [Item 66] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased expression of CD112 compared to cells not expressing the CFP. [Item 67] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of PDL1 compared to cells not expressing the CFP. [Item 68] The composition of any one of the preceding items, wherein cells expressing the CFP show increased expression of B7-DC compared to cells not expressing the CFP. [Item 69] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit an increased respiratory burst compared to cells not expressing the CFP. [Item 70] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased ROS production compared to cells not expressing the CFP. [Item 71] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased iNOS production compared to cells not expressing the CFP. [Item 72] The composition of any one of the preceding items, wherein cells expressing the CFP exhibit increased iNOS production compared to cells not expressing the CFP. [Item 73] A composition described in any one of the preceding items, wherein cells expressing the CFP exhibit increased extracellular vesicle production compared to cells not expressing the CFP. [Item 74] A composition described in any one of the preceding items, wherein cells expressing the CFP exhibit increased trogocytosis by target cells expressing the antigen compared to cells not expressing the CFP. [Item 75] A composition described in any one of the preceding items, wherein cells expressing the CFP exhibit increased resistance to CD47-mediated inhibition of phagocytosis compared to cells not expressing the CFP. [Item 76] A composition described in any one of the preceding items, wherein cells expressing the CFP exhibit increased resistance to LILRB1-mediated inhibition of phagocytosis compared to cells not expressing the CFP. [Item 77] The composition described in any one of the preceding items, wherein the intracellular domain comprises a Rac inhibitory domain, a Cdc42 inhibitory domain, or a GTPase inhibitory domain. [Item 78] The composition described in Item 64, wherein the Rac inhibitory domain, the Cdc42 inhibitory domain, or the GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase in the phagocytic cup of a cell expressing the CFP. [Item 79] The composition described in any one of the preceding items, wherein the intracellular domain comprises an F-actin dissociation activation domain, an ARHGAP12 activation domain, an ARHGAP25 activation domain, or an SH3BP1 activation domain. [Item 80] The composition described in any one of the preceding items, wherein the cells expressing the CFP exhibit increased production of phosphatidylinositol 3,4,5-triphosphate. [Item 81] The composition described in any one of the preceding items, wherein the extracellular domain comprises an Ig-binding domain. [Item 82] The composition of any one of the preceding items, wherein the extracellular domain comprises an IgA, IgD, IgE, IgG, IgM, FcRγI, FcRγIIA, FcRγIIB, FcRγIIC, FcRγIIIA, FcRγIIIB, FcRn, TRIM21, or FcRL5 binding domain. [Item 83] The composition described in any one of the preceding items, wherein the extracellular domain comprises an FcR extracellular domain. [Item 84] The composition of any one of the preceding items, wherein the extracellular domain comprises an FcR-alpha, GcRβ, FcRε, or FcRγ extracellular domain. [Item 85] The composition described in any one of the preceding items, wherein the extracellular domain comprises an FcRα (FCAR) extracellular domain. [Item 86] The composition described in any one of the preceding items, wherein the extracellular domain comprises an FcRβ extracellular domain. [Item 87] The composition described in any one of the preceding items, wherein the extracellular domain comprises an FcRε (FCER1A) extracellular domain. [Item 88] The composition of any one of the preceding items, wherein the extracellular domain comprises an FcRγ (FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, FCGR3B) extracellular domain. [Item 89] The composition described in any one of the preceding items, wherein the extracellular domain comprises an integrin domain. [Item 90] The composition of any one of the preceding items, wherein the extracellular domain comprises one or more integrin α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, or β8 domains. [Item 91] The composition described in any one of the preceding items, wherein the intracellular domain comprises a CD47 inhibitory domain. [Item 92] The composition of any one of the preceding items, wherein the PR subunit further comprises an extracellular domain and an extracellular antigen-binding domain operably linked to the transmembrane domain. [Item 93] The composition described in Item 92, wherein the extracellular domain further comprises a receptor extracellular domain, a hinge, a spacer, or a linker. [Item 94] The composition described in Item 93, wherein the extracellular domain comprises the extracellular portion of PR. [Item 95] The composition described in Item 94, wherein the extracellular portion of the PR is derived from the same PR as the PR intracellular signaling domain. [Item 96] The composition described in any one of Items 92 to 95, wherein the extracellular domain comprises an extracellular domain of a scavenger receptor or an immunoglobulin domain. [Item 97] The immunoglobulin domain is an extracellular domain of an immunoglobulin or an immunoglobulin 97. The composition of item 96, comprising a hinge region. [Item 98] The composition described in any one of Items 92 to 97, wherein the extracellular domain comprises a phagocytosis marker. [Item 99] The composition described in any one of Items 92 to 98, wherein the extracellular domain comprises a structure capable of multimeric assembly. [Item 100] The composition described in any one of Items 92 to 98, wherein the extracellular domain comprises a scaffold for multimerization. [Item 101] The composition of any one of the preceding items, wherein the extracellular domain is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 amino acids in length. [Item 102] The composition of any one of the preceding items, wherein the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids in length. [Item 103] The composition of any one of the preceding items, wherein the extracellular antigen-binding domain specifically binds to an antigen on a target cell. [Item 104] The composition of any one of the preceding items, wherein the extracellular antigen-binding domain comprises an antibody domain. [Item 105] The extracellular antigen-binding domain comprises a receptor domain, an antibody domain, and the antibody domain is a functional antibody fragment, a single-chain variable fragment (scFv), a Fab, a single-domain antibody (sdAb), a nanobody, a V H Domain, V L domain, VNAR domain, V HH The composition of any one of the preceding items, comprising a domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof. [Item 106] The composition of any one of the preceding items, wherein the extracellular antigen-binding domain comprises an extracellular domain of a ligand, receptor, or adapter. [Item 107] The composition of any one of the preceding items, wherein the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain specific for a single antigen. [Item 108] The composition of any one of the preceding items, wherein the extracellular antigen-binding domain comprises at least two extracellular antigen-binding domains, each of which is specific for a different antigen. [Item 109] The composition described in any one of the preceding items, wherein the antigen is a cancer antigen, a pathogenic antigen, or an autoimmune antigen. [Item 110] The composition described in any one of the preceding items, wherein the antigen comprises a viral antigen. [Item 111] The composition described in any one of the preceding items, wherein the antigen is a T lymphocyte antigen. [Item 112] The composition described in any one of the preceding items, wherein the antigen is an extracellular antigen. [Item 113] The composition described in any one of the preceding items, wherein the antigen is an intracellular antigen. [Item 114] The antigen is selected from the group consisting of thymidine kinase (TK1), hypoxanthine-guanine phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-1, mucin-16 (MUC16), MUC1, epidermal growth factor receptor vIII (EGFRvIII), mesothelin, human epidermal growth factor receptor 2 (HER2), mesothelin, EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B-cell maturation antigen (BCMA), glypican 3 (GPC3), follicle-stimulating hormone receptor, fibroblast-activating protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer group 2D (NKG2D) ligand, disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, CD8, and CD1. 9, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptors, PRSS21, VEGFR2, PDGFR-β, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, IGLL1, cutaneous lymphocyte-associated antigen (CLA), and combinations thereof. [Item 115] The composition of any one of the preceding items, wherein the antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CXCR4, CD8, CD30, CD45, CD56 and cutaneous lymphocyte-associated antigen (CLA). [Item 116] The composition described in Item 114, wherein the antigen is a CD5 antigen. [Item 117] The composition described in Item 114, wherein the antigen is a HER2 antigen. [Item 118] The composition described in any one of the preceding items, wherein the antigen is an ovarian cancer antigen or a T lymphoma antigen. [Item 119] The composition described in any one of the preceding items, wherein the antigen is an integrin receptor. [Item 120] The composition of any one of the preceding items, wherein the antigen is an integrin receptor 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. [Item 121] The composition of any one of the preceding items, wherein the antigen comprises two or more antigens. [Item 122] The composition of any one of the preceding items, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked via a linker. [Item 123] The composition of any one of the preceding items, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked through a linker such as the hinge region of CD8α, IgG1, or IgG4. [Item 124] The composition of any one of the preceding items, wherein the extracellular domain comprises a multimerized scaffold. [Item 125] The composition of any one of the preceding items, wherein the transmembrane domain comprises an FcR transmembrane domain. [Item 126] The composition of any one of the preceding items, wherein the transmembrane domain comprises an FcR-ε having 20, 10, or 5 or fewer modified transmembrane domains. [Item 127] The composition of any one of the preceding items, wherein the transmembrane domain comprises a transmembrane domain from a syntaxin such as syntaxin 3, syntaxin 4, or syntaxin 5. [Item 128] The composition described in any one of the preceding items, wherein the transmembrane domain comprises a CD8 transmembrane domain. [Item 129] The composition described in any one of Items 1 to 127, wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD2 transmembrane domain. [Item 130] The composition described in any one of Items 1 to 127, wherein the transmembrane domain comprises a CD68 transmembrane domain. [Item 131] A composition described in any one of the preceding items, wherein when the CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor. [Item 132] A composition described in any one of the preceding items, wherein when the CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an exogenous receptor. [Item 133] A composition described in any one of the preceding items, wherein when the CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor. [Item 134] A composition described in any one of the preceding items, wherein when the CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor. [Item 135] The composition of any one of the preceding items, wherein the transmembrane domain is derived from a protein different from the protein from which the intracellular signaling domain is derived. [Item 136] The composition of any one of the preceding items, wherein the transmembrane domain is derived from a protein different from the protein from which the extracellular domain is derived. [Item 137] The composition described in any one of the preceding items, wherein the transmembrane domain comprises the transmembrane domain of a phagocytic receptor. [Item 138] The composition described in any one of the preceding items, wherein the transmembrane domain and the extracellular domain are derived from the same protein. [Item 139] The composition of any one of the preceding items, wherein the transmembrane domain is derived from the same protein as the intracellular signaling domain. [Item 140] The composition described in any one of the preceding items, wherein the recombinant nucleic acid encodes a DAP12 recruitment domain. [Item 141] The composition described in any one of the preceding items, wherein the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12. [Item 142] The composition of any one of the preceding items, wherein the transmembrane domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length. [Item 143] The composition of any one of the preceding items, wherein the transmembrane domain is at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length. [Item 144] The composition described in any one of the preceding items, wherein the intracellular domain comprises a phosphatase inhibitory domain. [Item 145] The composition described in any one of the preceding items, wherein the intracellular domain comprises an ARP2 / 3 inhibitory domain. [Item 146] The composition described in any one of the preceding items, wherein the intracellular domain comprises at least one ITAM domain. [Item 147] The composition of any one of the preceding items, wherein the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains. [Item 148] The composition described in any one of the preceding items, wherein the intracellular domain further comprises at least one ITAM domain. [Item 149] The composition of any one of the preceding items, wherein the intracellular domain further comprises at least one ITAM domain selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one to 20 or less modifications. [Item 150] The composition described in Item 149, wherein at least one ITAM domain contains a Src family kinase phosphorylation site. [Item 151] The composition described in Item 149, wherein the at least one ITAM domain includes a Syk recruitment domain. [Item 152] The composition described in any one of the preceding items, wherein the intracellular domain comprises an F-actin depolymerization activation domain. [Item 153] The composition of any one of the preceding items, wherein the intracellular domain lacks enzymatic activity. [Item 154] The intracellular domain does not contain a domain derived from the CD3 zeta intracellular domain. The composition described in any one of the preceding items. [Item 155] The composition described in any one of the preceding items, wherein the intracellular domain comprises a CD47 inhibitory domain. [Item 156] The composition of any one of the preceding items, wherein the intracellular signaling domain comprises a domain that activates an integrin, such as the intracellular region of PSGL-1. [Item 157] The intracellular signaling domain is Rap1, such as those derived from EPAC and C3G. The composition of any one of the preceding items, comprising a domain that activates a GTPase. [Item 158] The composition described in any one of the preceding items, wherein the intracellular signaling domain is derived from paxillin. [Item 159] The composition described in any one of the above items, wherein the intracellular signaling domain activates focal adhesion kinase. [Item 160] The composition described in any one of the preceding items, wherein the intracellular signaling domain is derived from a single phagocytic receptor. [Item 161] The composition described in any one of the preceding items, wherein the intracellular signaling domain is derived from a single scavenger receptor. [Item 162] The composition described in any one of the preceding items, wherein the intracellular domain further comprises a phagocytosis-enhancing domain. [Item 163] The composition of any one of the preceding items, wherein the intracellular domain comprises a pro-inflammatory signaling domain. [Item 164] The composition described in Item 163, wherein the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain. [Item 165] The composition described in Item 163 or 164, wherein the pro-inflammatory signaling domain includes an IL-1 signaling cascade activation domain. [Item 166] The composition of any one of Items 163 to 165, wherein the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, an IFN receptor, an NLRP family member, NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank-binding kinase (TNK), a caspase domain, or a pro-caspase binding domain, or any combination thereof. [Item 167] The composition described in any one of the preceding items, wherein the CFP does not contain a full-length intracellular signaling domain. [Item 168] The composition of any one of the preceding items, wherein the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. [Item 169] The composition of any one of the preceding items, wherein the intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. [Item 170] The composition described in any one of the preceding items, wherein the recombinant nucleic acid encodes the FcRα chain extracellular domain, the FcRα chain transmembrane domain, and / or the FcRα chain intracellular domain. [Item 171] The composition described in any one of the preceding items, wherein the recombinant nucleic acid encodes an FcR β chain extracellular domain, an FcR β chain transmembrane domain, and / or an FcR β chain intracellular domain. [Item 172] The composition described in Item 170 or 171, wherein the FcRα chain or the FcRβ chain forms a complex with FcRγ when expressed in a cell. [Item 173] The composition described in Item 172, wherein the FcRα chain or FcRβ chain forms a complex with endogenous FcRγ when expressed in a cell. [Item 174] The composition described in any one of Items 170 to 173, wherein the FcRα chain or the FcRβ chain is not incorporated into the cell membrane of a cell that does not express FcRγ. [Item 175] The composition described in any one of Items 170 to 174, wherein the CFP does not contain an FcRα chain intracellular signaling domain. [Item 176] The composition described in any one of Items 170 to 175, wherein the CFP does not contain an FcR β chain intracellular signaling domain. [Item 177] A composition described in any one of the preceding items, wherein the recombinant nucleic acid encodes a TREM extracellular domain, a TREM transmembrane domain and / or a TREM intracellular domain. [Item 178] The composition described in Item 177, wherein the TREM is TREM1, TREM2 or TREM3. [Item 179] A composition comprising a recombinant nucleic acid encoding a phagocytic or anchoring receptor (PR) fusion protein (CFP), wherein the CFP is N-terminally to C-terminally (a) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; (b)(i) the CD8 transmembrane domain, and (ii) an intracellular domain comprising an Fcγ intracellular signaling domain and a CD40 intracellular signaling domain; and PR subunits containing wherein the transmembrane domain and the extracellular domain are operably linked by a linker. [Item 180] A composition comprising a recombinant nucleic acid encoding a phagocytic or anchoring receptor (PR) fusion protein (CFP), wherein the CFP is (a) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; (b)(i) the CD8 transmembrane domain, and (ii) an intracellular domain comprising an Fcγ intracellular signaling domain and a TNFR1 intracellular signaling domain; and PR subunits containing wherein the transmembrane domain and the extracellular domain are operably linked by a linker. [Item 181] A composition comprising a recombinant nucleic acid encoding a phagocytic or anchoring receptor (PR) fusion protein (CFP), wherein the CFP is N-terminally to C-terminally (a) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; (b)(i) the CD8 transmembrane domain, and (ii) an intracellular domain comprising an Fcγ intracellular signaling domain and an MDA5 signaling domain; and PR subunits containing wherein the transmembrane domain and the extracellular domain are operably linked by a linker. [Item 182] The composition described in any one of Items 179 to 181, wherein the antigen is a CD5 antigen. [Item 183] The composition described in any one of Items 179 to 181, wherein the antigen is a HER2 antigen. [Item 184] The composition described in any one of Items 179 to 183, wherein the CFP further comprises a signal peptide. [Item 185] The composition described in Item 184, wherein the signal peptide is a GMCSF signal peptide. [Item 186] The composition described in Item 184, wherein the CFP comprises the amino acid sequence of SEQ ID NO: 24. [Item 187] The composition described in Item 184, wherein the CFP comprises the amino acid sequence of SEQ ID NO: 25. [Item 188] The composition described in Item 184, wherein the CFP comprises the amino acid sequence of SEQ ID NO: 26. [Item 189] The composition described in any one of the preceding items, wherein the recombinant nucleic acid comprises a pro-inflammatory nucleotide or polynucleotide sequence encoding a pro-inflammatory polypeptide. [Item 190] The composition described in any one of the preceding items, wherein the composition further comprises a pro-inflammatory polypeptide. [Item 191] The composition described in Item 189 or 190, wherein the pro-inflammatory polypeptide is a chemokine or cytokine. [Item 192] The composition described in Item 191, wherein the chemokine is selected from the group consisting of IL-1, IL3, IL5, IL-6, IL8, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon. [Item 193] The composition according to Item 191, wherein 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. thing. [Item 194] The composition described in Item 189, wherein the nucleotide is selected from ATP, ADP, UTP, UDP, and / or UDP-glucose. [Item 195] The composition described in any one of the preceding items, wherein the recombinant nucleic acid comprises a sequence encoding a homeostatic regulator of inflammation. [Item 196] The composition described in Item 195, wherein the inflammatory homeostatic regulator is a sequence of an untranslated region (UTR) of an mRNA. [Item 197] The composition described in Item 196, wherein the sequence of the UTR is a sequence that binds to an RNA-binding protein. [Item 198] The composition described in Item 196 or 197, wherein translation is inhibited or prevented when the RNA-binding protein binds to a sequence in the untranslated region (UTR). [Item 199] The composition described in Item 197 or 198, wherein the sequence of the UTR comprises a consensus sequence of WWWU(AUUUA)UUUW, where W is A or U. [Item 200] The composition described in any one of the preceding items, wherein the recombinant nucleic acid is expressed in a bicistronic vector. [Item 201] The composition described in any one of the preceding items, wherein the target cell is a mammalian cell. [Item 202] The composition described in any one of the above items, wherein the target cells are human cells. [Item 203] The composition of any one of the preceding items, wherein the target cells include cells infected by a pathogen. [Item 204] The composition described in any one of the preceding items, wherein the target cells are cancer cells. [Item 205] The composition described in any one of the preceding items, wherein the target cells are lymphocyte cancer cells. [Item 206] The composition described in any one of the preceding items, wherein the target cells are cancer cells that are ovarian cancer cells. [Item 207] The composition described in any one of the preceding items, wherein the target cells are cancer cells that are ovarian pancreatic cells. [Item 208] The composition described in any one of the above items, wherein the target cells are cancer cells that are glioblastoma cells. [Item 209] The composition described in any one of the preceding items, wherein the recombinant nucleic acid is DNA. [Item 210] The composition described in any one of the preceding items, wherein the recombinant nucleic acid is RNA. [Item 211] The composition described in any one of the preceding items, wherein the recombinant nucleic acid is mRNA. [Item 212] The composition described in any one of the preceding items, wherein the recombinant nucleic acid is a circRNA. [Item 213] The composition described in any one of the preceding items, wherein the recombinant nucleic acid is a tRNA. [Item 214] The composition described in any one of the preceding items, wherein the recombinant nucleic acid is a microRNA. [Item 215] A vector comprising a recombinant nucleic acid of the composition described in any one of Items 1 to 214. [Item 216] The vector described in Item 215, which is a viral vector. [Item 217] The vector described in Item 216, wherein the viral vector is a retroviral vector or a lentiviral vector. [Item 218] A vector described in any one of Items 215 to 217, further comprising a promoter operably linked to at least one nucleic acid sequence encoding one or more polypeptides. [Item 219] A vector described in any one of Items 215 to 218, which is polycistronic. [Item 220] A vector described in Item 218 or 219, wherein each of the at least one nucleic acid sequence is operably linked to a separate promoter. [Item 221] A vector described in any one of items 215 to 220, further comprising one or more internal ribosome entry sites (IRES). [Item 222] A vector described in any one of items 215 to 221, further comprising a 5'UTR and / or a 3'UTR adjacent to at least one nucleic acid sequence encoding one or more polypeptides. [Item 223] A vector described in any one of Items 215 to 222, further comprising one or more regulatory regions. [Item 224] A polypeptide encoded by a recombinant nucleic acid of the composition described in any one of Items 1 to 214. [Item 225] A cell comprising the composition described in any one of Items 1 to 214, the vector described in any one of Items 216 to 223, or the polypeptide described in Item 224. [Item 226] The cell according to Item 225, which is an immature myeloid cell. [Item 227] The cell of Item 225, which is a non-polarized or undifferentiated myeloid cell. [Item 228]CD14+ / CD16 low 226. The cell of item 225, which is a cell. [Item 229]CD14+ / CD16 - cells, CD14 - / CD16 + 226. The cell of item 225, which is a cell. [Item 230] The cell according to Item 225, which is a phagocyte. [Item 231] The cell of Item 225, which is a stem cell-derived cell, a myeloid cell, a macrophage, a dendritic cell, a lymphocyte, a mast cell, a monocyte, a neutrophil, a microglia, an eosinophil, a basophil, a myeloid progenitor cell, a mosaic phenotype cell, or an astrocyte. [Item 232] The cell according to Item 225, which is an M1 macrophage cell. [Item 233] The cell according to Item 225, which is an M2 macrophage cell. [Item 234] The cells described in any one of Items 225 to 233, which are autologous cells. [Item 235] The cell according to any one of Items 225 to 233, which is an allogeneic cell. [Item 236] A population of modified cells, wherein a plurality of the populations of modified cells comprises the composition described in any one of Items 1 to 214, the vector described in any one of Items 216 to 223, or the polypeptide described in Item 224. [Item 237] The population of modified cells described in Item 236, wherein the plurality comprises at least 80% of the population of modified cells. [Item 238] The population of modified cells described in Item 237, wherein the population of cells is not concentrated. [Item 239] The population of cells is CD14+ / CD16- cells, CD14- / CD16+ cells, or CD14+ / CD16 low 239. The population of modified cells of items 237 or 238, which are cells. [Item 240] The population of modified cells described in Item 237 or 239, wherein the population of cells is phagocytes. [Item 241] (a) a composition according to any one of Items 1 to 214, a vector according to any one of Items 216 to 223, or a polypeptide according to Item 224, a cell according to any one of Items 225 to 235, or a population of cells according to any one of Items 236 to 240, and (b) a pharmaceutically acceptable excipient 10. A pharmaceutical composition comprising: [Item 242] The pharmaceutical composition according to Item 241, further comprising an additional therapeutic agent. [Item 243] The pharmaceutical composition of Item 241 or 242, wherein the additional therapeutic agent is selected from the group consisting of a CD47 agonist, a drug that inhibits Rac, a drug that inhibits Cdc42, a drug that inhibits GTPase, a drug that promotes F-actin dissociation, a drug that promotes PI3K recruitment to CFP, a drug that promotes PI3K activity, a drug that promotes the production of phosphatidylinositol 3,4,5-trisphosphate, a drug that promotes ARHGAP12 activity, a drug that promotes ARHGAP25 activity, a drug that promotes SH3BP1 activity, and any combination thereof. [Item 244] The pharmaceutical composition according to any one of Items 241 to 243, wherein the pharmaceutically acceptable excipient comprises a serum-free medium, a lipid, or a nanoparticle. [Item 245] A method for treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to any one of Items 241 to 244. [Item 246] The method described in Item 245, wherein the disease is cancer. [Item 247] The method described in Item 246, wherein the cancer is a solid cancer. [Item 248] The method according to Item 247, wherein the solid cancer is selected from the group consisting of ovarian cancer, suitable cancers including ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer. [Item 249] The method described in Item 246, wherein the cancer is a liquid cancer. [Item 250] The method described in Item 249, wherein the liquid cancer is leukemia or lymphoma. [Item 251] The method described in Item 249, wherein the liquid cancer is T-cell lymphoma. [Item 252] The method described in Item 245, wherein the disease is a T-cell malignancy. [Item 253] The method described in any one of Items 245 to 252, further comprising the step of administering an additional therapeutic agent to the subject. [Item 254] The method of Item 253, wherein the additional therapeutic agent is selected from the group consisting of a CD47 agonist, a drug that inhibits Rac, a drug that inhibits Cdc42, a drug that inhibits GTPase, a drug that promotes F-actin dissociation, a drug that promotes PI3K recruitment to CFP, a drug that promotes PI3K activity, a drug that promotes the production of phosphatidylinositol 3,4,5-trisphosphate, a drug that promotes ARHGAP12 activity, a drug that promotes ARHGAP25 activity, a drug that promotes SH3BP1 activity, and any combination thereof. [Item 255] The method according to any one of Items 245 to 254, wherein the administering step includes an infusion step or an injection step. [Item 256] The method according to any one of Items 245 to 255, wherein the administering step comprises a step of directly administering to a solid tumor. [Item 257] The method described in any one of Items 245 to 256, wherein the administering step includes a step of administering a circRNA, mRNA, viral vector, particle, nanoparticle, liposome, exosome or cell. [Item 258] The method of any one of items 245 to 257, wherein a CD4+ T cell response or a CD8+ T cell response is induced in the subject. [Item 259] A method for preparing a cell, comprising the step of contacting the cell with a composition described in any one of Items 1 to 214, a vector described in any one of Items 216 to 223, or a polypeptide described in Item 224. [Item 260] The method described in Item 259, wherein the contacting step includes a transduction step. [Item 261] The method of Item 260, wherein the transducing step comprises chemical transfection, electroporation, nucleofection, or viral infection. [Item 262] A method for preparing a pharmaceutical composition, comprising: mixing a lipid with the composition according to any one of Items 1 to 214, the vector according to any one of Items 216 to 223, or the vector according to Item 224. The method comprises contacting a mammalian cell with the polypeptide described above. [Item 263] The method described in Item 262, wherein the contacting step includes forming lipid nanoparticles. [Item 264] A method for preparing a pharmaceutical composition, comprising a step of contacting an antibody with a composition described in any one of Items 1 to 214 or a vector described in any one of Items 216 to 223. [Item 265] The method described in Item 264, wherein the contacting step includes a step of forming lipid nanoparticles. The diverse functionalities of myeloid cells make them ideal candidates for cell therapy, which can be engineered to have numerous therapeutic effects. The present disclosure relates to immunotherapy using myeloid cells of the immune system (e.g., CD14+ cells), particularly phagocytes. Many therapeutic indications could be explored using myeloid cells. For example, myeloid cell immunotherapy could be crucial in cancer, autoimmunity, fibrosis, and infection. The present disclosure relates to immunotherapy using myeloid cells, including phagocytes of the immune system, particularly macrophages. It is an object of the inventions disclosed herein to inhibit one or more of these functions of myeloid cells for therapeutic use. For example, it is an object of the inventions disclosed herein to inhibit the phagocytic activity of myeloid cells, including engineered myeloid cells, for therapeutic use. For example, it is an object of the inventions disclosed herein to inhibit the ability of myeloid cells, including engineered myeloid cells, to promote T cell activation. For example, it is an object of the inventions disclosed herein to inhibit the ability of myeloid cells, including engineered myeloid cells, to promote the secretion of tumoricidal molecules. For example, inhibiting the ability of myeloid cells, including engineered myeloid cells, to promote the recruitment and trafficking of immune cells and molecules is an objective of the inventions disclosed herein. The present disclosure provides innovative methods and compositions that can induce genetic modifications in myeloid cells with the goal of successfully transfecting or transducing the myeloid cells or otherwise increasing their functional aspects, without further compromising the cells' differentiation potential, maturation potential, and / or their plasticity.

[0008]

[0008] The present disclosure involves the production and use of engineered myeloid cells, such as macrophages or other phagocytes (e.g., CD14+ cells), that can directly or indirectly attack and kill (ATAK) diseased cells, such as cancer cells and infected cells. Engineered myeloid cells, such as macrophages and other phagocytes, can be prepared by incorporating into cells, for example, using recombinant nucleic acid technology, synthetic nucleic acid, gene editing technology (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, or nucleofection, a nucleic acid sequence (e.g., mRNA, plasmid, viral construct) encoding a chimeric fusion protein (CFP) having an extracellular binding domain specific for a disease-associated antigen (e.g., cancer antigen). It has been discovered that myeloid cells can be engineered to have a broad and diverse range of activities. For example, it has been discovered that myeloid cells can be engineered to express chimeric fusion proteins (CFPs) containing antigen-binding domains to have a broad and diverse range of activities. For example, when the CFP binds to an antigen on the target cell, the cells bind to the target cell. It has been found that bone marrow cells can be engineered to have enhanced phagocytic activity, exhibiting increased phagocytosis of vesicles. It has also been found that bone marrow cells can be engineered to promote T cell activation, such that when CFP binds to an antigen on a target cell, the cells promote the activation of T cells, for example, T cells in a tumor microenvironment. Engineered bone marrow cells can be engineered to promote the secretion of tumoricidal molecules, such that when CFP binds to an antigen on a target cell, the cells promote the secretion of tumoricidal molecules from neighboring cells. Engineered bone marrow cells can be engineered to promote the recruitment and transport of immune cells and molecules, such that when CFP binds to an antigen on a target cell, the cells promote the recruitment and transport of immune cells and molecules to the target cell or tumor microenvironment.

[0009]

[0009] The present disclosure is based on the important discovery that engineered myeloid cells overcome at least some of the limitations of CAR-T cells, including being easily mobilized to solid tumors; having a manageable survival time, thus reducing the risk of prolonged persistence resulting in aplasia and immunodeficiency; myeloid cells cannot contaminate T cells; for example, myeloid cells can avoid fratricide because they do not express the same antigens as malignant T cells; and myeloid cells have a plethora of anti-tumor functions that can be deployed. In some respects, engineered bone marrow-derived cells may be a safer immunotherapeutic means of targeting and destroying diseased cells.

[0010]

[0010] Furthermore, myeloid cells such as macrophages are ubiquitously found in the tumor environment (TME), and are particularly the most abundant cells in some tumor types. As part of their role in the immune system, myeloid cells such as macrophages are naturally involved in the elimination of diseased cells. The present invention relates to inhibiting myeloid cell function, particularly targeting, killing, and directly and / or indirectly eliminating diseased cells, as well as delivering payloads such as antigens and cytokines.

[0011] Engineered myeloid cells can be short-lived, phenotypically diverse, sensitive, and plastic in vivo, and they are often difficult to manipulate in vitro. For example, exogenous gene expression in monocytes is more challenging than exogenous gene expression in non-hematopoietic stem cells. There are significant technical challenges associated with transfecting myeloid cells (e.g., monocytes / macrophages). As professional phagocytes, myeloid cells such as monocytes / macrophages contain many potent degradative enzymes that can disrupt the integrity of nucleic acids and inefficiently transfer genes into these cells. This is particularly true for activated macrophages, which undergo dramatic changes in their physiology after exposure to immune or inflammatory stimuli. Viral transduction of these cells is hampered because macrophages are generally terminally differentiated cells; therefore, some vectors that rely on integration into replicative genomes face limited success. Furthermore, macrophages are sensitive to "danger signals," and thus some original viral vectors used for gene transfer induce strong antiviral responses in these cells, making these vectors unsuitable for gene delivery. The present disclosure provides innovative methods and compositions that can induce genetic modifications in myeloid cells with the aim of successfully transfecting or transducing them or otherwise increasing their functional aspects, without further compromising the cells' differentiation potential, maturation potential, and / or their plasticity.

[0012] Incorporation by Reference

[0012] Each individual publication, patent, or patent application is specifically and individually incorporated by reference. All publications, patents, and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0014] [Figure 1A]

[0014] Figure 1 illustrates a diagram showing some of the potentially manipulable functions of myeloid cells. [Figure 1B]

[0015] FIG. 1 illustrates a diagram indicating the presence of various cell types in various types of cancer. Macrophages are the most abundant cells in the cancer types schematized. [Figure 2A]

[0016]

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

[0017]

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

[0018] FIG. 1 illustrates a schematic showing an exemplary CFP dimer containing an anti-CD5 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain containing an intracellular domain derived from FcRγ fused to a PI3K recruitment domain. [Figure 2D]

[0019] FIG. 1 illustrates a schematic showing an exemplary CFP dimer containing an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain containing a phagocytic domain, a PI3K recruitment domain, and a pro-inflammatory domain. [Figure 3]

[0020] Schematic diagram illustrating an exemplary CFP homodimer (left) in which each subunit contains an extracellular domain fused to an scFv that binds a single target, and an exemplary CFP heterodimer (right) in which a first subunit of the heterodimer contains an extracellular domain fused to an scFv that binds a first target and a second subunit of the heterodimer subunit contains an extracellular domain fused to an scFv that binds a second target. [Figure 4A]

[0021] FIG. 1 is a schematic diagram illustrating an exemplary recombinant nucleic acid encoding a CFP containing a signal peptide fused to an antigen-specific scFv fused to the extracellular domain (ECD), transmembrane domain (TM), and intracellular domain of a scavenger receptor. [Figure 4B]

[0022] 4B is a schematic diagram illustrating the CFP of FIG. 4A incorporated into the cell membrane of a myeloid cell. The illustrated CFP contains an scFv bound to a cancer antigen of a cancer cell. The extracellular, transmembrane, and intracellular domains can be derived from one or more scavenger receptors. [Figure 4C]

[0023]

[0023] Figure 1 is an exemplary graph illustrating expected results regarding relative phagocytosis in human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP co-cultured with dye-loaded tumor cells. Phagocytosis is quantified using flow cytometry. [Figure 4D]

[0024] FIG. 1 is an exemplary graph illustrating the expected results regarding the percent specific lysis of tumor cells when incubated in the presence of an empty vector (control) or human primary myeloid cells (effector cells) transduced with a vector encoding CFP co-cultured with luciferase-expressing tumor cells (target cells) at the indicated effector cell:target cell ratio (E:T ratio). [Figure 4E]

[0025] FIG. 1 is an exemplary graph illustrating expected results in terms of percent survival in a mouse xenograft tumor model following treatment with cells transduced with an empty vector (control) or a vector encoding CFP. [Figure 5A]

[0026] FIG. 1 is a schematic diagram illustrating an exemplary recombinant nucleic acid encoding a CFP (M1-CAR) containing a signal peptide fused to an antigen-specific scFv fused to a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular domain containing a phagocytic activation domain and a pro-inflammatory domain. [Figure 5B]

[0027] FIG. 5B is a schematic diagram illustrating the CFP (M1-CAR) of FIG. 5A incorporated into the cell membrane of a myeloid cell. The schematized CFP contains an scFv bound to a cancer antigen on a cancer cell. [Figure 5C]

[0028]

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

[0029] FIG. 10 is an exemplary graph illustrating expected results in terms of fold increase in production of schematized cytokines in myeloid cells transduced with vector control or vector encoding CFP(M1-CAR). [Figure 5E]

[0030] FIG. 1 is an exemplary graph illustrating expected results in terms of fold increase in production of schematized M1 markers in human primary myeloid cells transduced with vector control or vector encoding CFP(M1-CAR). [Figure 5F]

[0031]

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

[0032] FIG. 1 is an exemplary graph illustrating expected results in terms of percent survival in a mouse xenograft tumor model following treatment with human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (M1-CAR). [Figure 6A]

[0033] FIG. 1 is a schematic diagram illustrating an exemplary recombinant nucleic acid encoding a CFP (integrin-CAR) containing a signal peptide fused to an antigen-specific scFv fused to a CD8 hinge domain, a CD8 transmembrane domain, and intracellular phagocytosis and internalization activation domains. [Figure 6B]

[0034] 6B is a schematic diagram illustrating the CFP (integrin-CAR) of FIG. 6A integrated into the cell membrane of a myeloid cell. The schematized CFP contains an scFv bound to a cancer antigen on a cancer cell. [Figure 6C]

[0035]

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

[0036] FIG. 1 is an exemplary graph illustrating expected results in terms of percent specific lysis of tumor cells when incubated in the presence of an empty vector (control) or human primary myeloid cells (effector cells) transduced with a vector encoding CFP (integrin-CAR) co-cultured with luciferase-expressing tumor cells (target cells) at the indicated effector cell:target cell ratio (E:T ratio). [Figure 6E]

[0037] FIG. 1 is an exemplary graph illustrating expected results regarding the relative infiltration of human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (integrin-CAR). [Figure 6F]

[0038] FIG. 1 is an exemplary graph illustrating expected results in terms of percent survival in a mouse xenograft tumor model following treatment with human primary myeloid cells transduced with an empty vector (control) or a vector encoding CFP (integrin-CAR). [Figure 7]

[0039] 1 is a schematic diagram illustrating a CFP (cross-presentation-CAR) integrated into the cell membrane of a myeloid cell. The illustrated cross-presentation-CAR contains an scFv that binds to a cancer antigen of a cancer cell fused to a CD8 hinge domain, a CD8 transmembrane domain, an intracellular phagocytosis activation domain, and an intracellular cross-presentation activation domain. The cross-presentation-CAR can direct antigens into the cross-presentation pathway. [Figure 8-1]

[0040] FIG. 1 illustrates exemplary flow cytometry data (side scatter (SSC) vs. CD5+) following mock expression or expression of various constructs with an extracellular domain (ECD) comprising an anti-CD5 scFv in myeloid cells. Schematic constructs include: an ECD containing an anti-CD5 scFv fused to an FcRγ intracellular domain, fused to a CD40 intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-CD40-FcR); an ECD containing an anti-CD5 scFv fused to a CD40 intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR-CD40); an ECD containing an anti-CD5 scFv fused to a PI3K recruitment domain, fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR-PI3K); an anti-CD5 fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain ECD containing an scFv (CD5-CD8h-CD8tm-FcR); an ECD containing an anti-CD5 scFv fused to a CD8 transmembrane domain and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-no ICD); an ECD containing an anti-CD5 scFv fused to a PI3K recruitment domain, fused to an FcRγ intracellular domain, fused to a CD28 transmembrane domain, and fused to a CD28 hinge domain (CD5-CD28h-CD28tm-FcR-PI3K); an ECD containing an anti-CD5 scFv fused to a CD8 hinge domain, fused to a CD68 transmembrane domain, fused to an FcRγ intracellular domain, and fused to a PI3K recruitment domain (CD5-CD8h-CD68tm-FcR-PI3K); an anti-CD5 fused to a CD8 transmembrane domain, fused to an FcRγ intracellular domain, fused to a PI3K recruitment domain ECD containing scFv (CD5-CD8tm-FcR-PI3K);An ECD containing an anti-CD5 scFv fused to a CD28 transmembrane domain, fused to an FcRγ intracellular domain, fused to a PI3K recruitment domain (CD5-CD28tm-FcR-PI3K); and an ECD containing an anti-CD5 scFv fused to a CD68 transmembrane domain, fused to an FcRγ intracellular domain, fused to a PI3K recruitment domain (CD5-CD68tm-FcR-PI3K). [Figure 8-2] Continued from Figure 8-1. [Figure 9]

[0041] FIG. 1 illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) following mock expression or expression of various constructs with an extracellular domain (ECD) comprising an anti-CD5 scFv in myeloid cells. Schematic constructs include: an ECD containing an anti-CD5 scFv fused to a PI3K recruitment domain, fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR-PI3K); an ECD containing an anti-CD5 scFv fused to a CD8 transmembrane domain and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-FcR); an ECD containing an anti-CD5 scFv fused to a CD8 transmembrane domain and fused to a CD8 hinge domain (CD5-CD8h-CD8tm-no ICD); an anti-CD5 fused to a CD40 intracellular domain, fused to an FcRγ intracellular domain, fused to a CD8 transmembrane domain, and fused to a CD8 hinge domain ECD containing an scFv (CD5-CD8h-CD8tm-FcR-CD40); and ECD containing an anti-CD5 scFv fused to the TNFR2 intracellular domain, fused to the FcRγ intracellular domain, fused to the CD8 transmembrane domain, or fused to the CD8 hinge domain (CD5-CD8h-CD8tm-FcR-TNFR2). [Figure 10A]

[0042] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using coated FITC-labeled beads for antigen targeting FarRed fluorophore-labeled CFP expressed in THP-1 cells. [Figure 10B]

[0043] FIG. 10B illustrates exemplary flow cytometry data (side scatter (SSC) vs. CSFE-FarRed) following mock expression or expression of anti-CD5 CFP using the experimental design of FIG. 10A. [Figure 10C]

[0044] FIG. 10B illustrates an exemplary graph showing relative phagocytosis in human primary myeloid cells transduced with an empty vector (mock) or a vector encoding the schematized CFP co-cultured with FITC-labeled beads coated with BSA or CD5, using the experimental design of FIG. 10A. [Figure 10D-1]

[0045] FIG. 10B depicts an exemplary bar graph of the concentration (pg / mL) of the indicated proteins after mock expression or expression of the indicated anti-CD5 CFPs using the experimental design of FIG. 10A. Each CFP contained an ECD containing an anti-CD5 scFv fused to a CD8 hinge domain fused to a CD8 transmembrane domain fused to the indicated intracellular domain. [Figure 10D-2] Continued from Figure 10D-1. [Figure 10E]

[0046]

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

[0047]

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

[0048]

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

[0049]

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

[0050] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using CFSE-labeled target cells targeting FarRed fluorophore-labeled CFP expressed in THP-1 cells. [Figure 11B]

[0051] FIG. 11B illustrates exemplary flow cytometry data (side scatter (SSC) vs. forward scatter (FSC); CSFE vs. FarRed; and cell count CSFE) following mock expression or expression of anti-CD5 CFP in THP-1 cells using the experimental design of FIG. 11A. Myeloid cell lines were electroporated with anti-CD5 CFP and labeled with intracellular FarRed dye. These cells were incubated with CFSE-prelabeled H9 T-cell cancer cells at a 1:3 myeloid cell:tumor cell ratio. After 4 hours, phagocytosis was measured by flow cytometry. [Figure 11C]

[0052] Figure 11B illustrates an exemplary graph showing relative phagocytosis in myeloid cell lines electroporated with an empty vector (mock) or a vector encoding the schematized CFP and labeled with intracellular FarRed dye, using the experimental design of Figure 11A. These cells were incubated with H9 T cell cancer cells prelabeled with CFSE at a myeloid cell:tumor cell ratio of 1:3. After 4 hours, phagocytosis was measured by flow cytometry. [Figure 12A]

[0053] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using pHRod-labeled target cells targeting FarRed fluorophore-labeled CFP expressed in primary human monocytic cells. [Figure 12B]

[0054] Figure 12B illustrates exemplary flow cytometry data (pHRodo vs. FarRed) following mock expression or expression of anti-CD5 CFP in primary human monocytic cells using the experimental design of Figure 12A. Primary human monocytic cells were electroporated with anti-CD5 CFP and labeled with intracellular FarRed dye. These cells were incubated with H9 T cell cancer cells prelabeled with pHRodo. After incubation, phagocytosis was measured by flow cytometry. [Figure 12C]

[0055] FIG. 12B illustrates an exemplary graph quantifying the results of FIG. 12B showing relative phagocytosis after mock expression or expression of a diagrammed anti-CD5 CFP in primary human monocytic cells using the experimental design of FIG. 12A. [Figure 12D-1]

[0056] FIG. 1 illustrates an exemplary bar graph of the concentration (pg / mL) of the indicated proteins after mock expression or expression of the indicated anti-CD5 CFP in monocytic cells after performing a bead-based phagocytosis assay. [Figure 12D-2] Continued from Figure 12D-1. [Figure 13]

[0057]

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

[0058]

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

[0059]

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

[0060] FIG. 15B illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) following expression of anti-CD5 CFP in human primary monocytic cells from the experiment shown in FIG. 15A. [Figure 15C]

[0061]

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

[0062] Figure 15B depicts a graph of relative tumor size over time from the results of Figure 15C. Tumor burden was measured using IVIS imaging of luciferase fluorescence. [Figure 16A]

[0063]

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

[0064] FIG. 16B illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) following expression of anti-CD5 CFP in human primary monocytic cells from the experiment shown in FIG. 16A. The data demonstrate the achievement of 95% transfection efficiency. [Figure 16C]

[0065] Figure 16B depicts a graph of relative tumor size over time from the experiment shown in Figure 16A. Tumor burden was measured using IVIS imaging of luciferase fluorescence. [Figure 16D]

[0066] FIG. 16B illustrates a graph of relative tumor size over time from the experiment shown in FIG. 16A. Tumor burden was measured using caliper measurements. The data demonstrate that treatment was associated with a delay in tumor progression and a statistically significant reduction in tumor burden in a compromised mouse model. Statistical significance was determined using the Bonferroni-Dunn method at alpha=0.5. Each column was analyzed individually, without assuming a constant SD. Number of t-tests: 8 or 4. [Figure 17A]

[0067] FIG. 1 illustrates a schematic showing an exemplary CFP containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from MDA5. [Figure 17B]

[0068] FIG. 1 depicts exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) showing expression in untransfected primary monocytes (top) and primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing an extracellular CD5-binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from MDA5. [Figure 17C]

[0069] FIG. 1 depicts an exemplary bar graph of the concentrations (pg / mL) of the indicated cytokines secreted in untransfected primary monocytes and primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing a CD5 domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from MDA5. [Figure 18A]

[0070] FIG. 1 illustrates a schematic showing an exemplary chimeric receptor fusion protein (CFP) containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR1 or TNFR2. [Figure 18B]

[0071] FIG. 1 illustrates exemplary flow cytometry data (side scatter (SSC) vs. anti-CD5 CFP+) showing expression in untransfected primary monocytes (left); primary monocytes transfected with in vitro-transcribed mRNA encoding a CFP containing an extracellular CD5-binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR1 (center); and primary monocytes transfected with in vitro-transcribed mRNA encoding a CFP containing an extracellular CD5-binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR2 (right). [Figure 18C-1]

[0072] FIG. 1 depicts an exemplary bar graph of the concentrations (pg / mL) of the indicated cytokines / chemokines secreted in untransfected primary monocytes; primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing an extracellular CD5 binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR1; and primary monocytes transfected with in vitro transcribed mRNA encoding a CFP containing an extracellular CD5 binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from TNFR2. [Figure 18C-2] Continued from Figure 18C-1. [Figure 19A]

[0073] FIG. 1 illustrates a schematic showing a CFP containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain derived from FcRγ, and a second intracellular signaling domain derived from CD40, a PI3K recruitment domain, or TNFR2. [Figure 19B]

[0074]

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

[0075]

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

[0076]

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

[0077] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay. [Figure 20C]

[0078]

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

[0079] 20B depicts exemplary flow cytometry data (FarRed vs. PE) showing phagocytosis after performing the phagocytosis assay depicted in FIG. 20B. Transduced THP-1 cells, activated with or without PMA, were incubated overnight with FarRed-labeled SKOV3 tumor cells. [Figure 20E]

[0080] FIG. 20B illustrates exemplary flow cytometry data (SSC vs. FSC and FarRed vs. PE) after performing the phagocytosis assay depicted in FIG. 20B. Transduced THP-1 cells, activated with or without PMA, were incubated overnight with FarRed-labeled SKOV3 tumor cells.

number

[0081] FIG. 1 illustrates a schematic showing an exemplary experimental flow diagram of a phagocytosis assay using CD14+ cells isolated from a healthy donor Leukopak and transduced with a lentiviral vector encoding a CFP containing an extracellular HER2-binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain. [Figure 21B]

[0082] 21A illustrates an exemplary bar graph of the percentage of phagocytosis of CD14+ cells transduced with a lentiviral vector encoding a CFP isolated from a healthy donor Leukopak, which contains an extracellular HER2 binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain, using the phagocytosis assay illustrated in FIG. 21A. The transduced cells were incubated overnight with target cells (Jurkat (HER2-) or SKOV3 (HER2+)). Also illustrated is an exemplary fluorescent microscopy image of cells showing the phagocytosis of SKOV3 cells but not Jurkat cells. [Figure 21C-1]

[0083]

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

[0084] FIG. 1 illustrates a schematic diagram showing an exemplary experimental flow diagram of an MSTO mesothelioma animal model experiment to investigate the ability of CFP-expressing cells to penetrate tumor sites and to evaluate the activation of CFP-expressing cells after penetration. [Figure 22B]

[0085]

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

[0086] Figure 1 shows fluorescent microscopy images of spleen samples removed 24 hours after administration of CFSE-labeled CD14+ cells isolated from a healthy donor Leukopak and transduced with a lentiviral vector encoding a CFP containing an extracellular HER2-binding domain (scFv), an extracellular Flag tag, an extracellular hinge domain derived from CD8, a CD8 transmembrane domain, and a first intracellular signaling domain derived from FcRγ and a second intracellular signaling domain containing a PI3K recruitment domain into MSTO tumor-bearing NSG mice. The transduced cells were observed to migrate to the spleen. CFSE-labeled cells isolated from the spleen 24 hours after cell injection were also examined by flow cytometry. The CFSE-labeled cells in the spleen maintained expression of HLA, CD14, and CD303. Interestingly, CCR2 expression was observed to decrease with a concomitant increase in CD370 (CLEC9A), potentially suggesting that the cells migrate to the spleen and develop into professional APCs capable of priming T cell responses. Interestingly, CD206 (mannose) expression was observed to decrease as well as CD45. The decrease in mannose receptor expression can be associated with differentiation towards an M1 phenotype. [Figure 23]

[0087]

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

[0088] Figure 24 illustrates a graph of tumor size over time from the experiment shown in Figure 23. Infusion of human primary monocytes expressing anti-HER2CFP was associated with delayed tumor progression compared to control-treated animals. [Figure 25]

[0089] FIG. 1 illustrates a diagram showing the inhibition of phagocytic receptors by target cell CD47 receptor SIRP-alpha (SIRPα)-mediated signaling. [Figure 26A]

[0090]

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

[0091] FIG. 1 shows exemplary expected results for relative phagocytosis by control and dominant-negative CFP-transduced cells. [Figure 26C]

[0092] FIG. 1 shows exemplary expected results for target cell lysis (E:T, effector:target) by control and dominant-negative CFP-transduced cells. [Figure 26D]

[0093] FIG. 1 shows an example of expected outcomes of mouse survival in a tumor model after treatment with dominant-negative CFP-transduced macrophages. [Figure 27A]

[0094]

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

[0095] FIG. 1 shows examples of expected results regarding relative phagocytosis by control and SIRPα-PI3K CFP-transduced cells. [Figure 27C]

[0096] FIG. 1 shows examples of expected results for relative Akt phosphorylation by control and SIRPα-PI3K CFP transduced cells. [Figure 27D]

[0097] FIG. 1 shows the expected results of increased tumor cell lysis by cells expressing CFP (integrin-CAR) compared to control (empty vector-transduced) macrophages. [Figure 27E]

[0098] FIG. 1 shows predicted survival curves in a mouse xenograft model of tumor after treatment with SIRPα-PI3K CFP-transduced macrophages or untreated controls. [Figure 28A]

[0099] The top panel illustrates a diagrammatic representation of the design of a recombinant CFP, (SIRPα-M1) (top panel), that contains a SIRPα extracellular domain capable of binding CD47, a SIRPα TM domain, but lacks an intracellular SIRPα signaling domain. CFP contains an intracellular signaling domain with a pro-inflammatory domain. The bottom panel illustrates a diagrammatic representation showing the inhibition of endogenous SIRPα by recombinant CFP protein when expressed in myeloid cells (e.g., macrophages). The pro-inflammatory domain can induce M1 polarization. [Figure 28B]

[0100] FIG. 1 shows examples of expected results for flow cytometry assays showing increased M1 state marker expression when myeloid cells (e.g., macrophages) are transduced with SIRPα-M1 compared to vector controls. [Figure 28C]

[0101] FIG. 1 shows examples of expected results for flow cytometry assays showing an increase in pro-inflammatory markers when myeloid cells (e.g., macrophages) are transduced with SIRPα-M1 compared to vector controls. [Figure 28D]

[0102] FIG. 1 shows the expected results of increased tumor cell lysis by cells expressing SIRPα-M1 compared to control (empty vector-transduced) myeloid cells (eg, macrophages). [Figure 28E]

[0103] FIG. 1 shows predicted survival curves in a mouse xenograft model of tumor following treatment with SIRPα-M1 transduced myeloid cells (eg, macrophages) or untreated controls. [Figure 29A]

[0104] The top panel illustrates an exemplary schematic diagram of a receptor-based CFP, SIRPαβ, containing an extracellular scFv specific for a cancer antigen fused to the SIRPαβ chain. The extracellular portion of the CD47 receptor SIRPα is fused to the cancer antigen-specific scFv. The ECD of SIRPα is fused to the transmembrane domain of SIRPβ. The intracellular domain of the CFP contains the intracellular domain derived from SIRPβ. Activation of the CFP by binding of the scFv to a target ligand activates the SIRPβ intracellular domain, inducing phagocytosis of the target cell through activation of DAP12. The bottom panel illustrates a diagrammatic representation of recombinant SIRPαβ protein expressed in myeloid cells (e.g., macrophages). [Figure 29B]

[0105] FIG. 1 shows a graphical representation of the phagocytic receptor fusion protein SIRPα□ compared to a vector control. [Figure 29C]

[0106] FIG. 1 shows the expected results of increased lysis of target cells by SIRPα□-transduced macrophages compared to control (empty vector-transduced) macrophages. [Figure 29D]

[0107] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with SIRPαβ-transduced macrophages or untreated controls. [Figure 30A]

[0108] FIG. 1 illustrates an exemplary schematic diagram of a nucleic acid construct comprising a regulatory element sequence, a CFP-encoding sequence, a T2A-encoding sequence, and a sialidase-encoding sequence. The T2A sequence allows for cleavage of the sialidase from the CFP during translation. [Figure 30B]

[0109] FIG. 1 illustrates a diagrammatic representation of enhanced phagocytic engulfment of target cells in the presence of secreted sialidase. [Figure 30C]

[0110] FIG. 1 illustrates predicted results showing enhanced lysis of target cells by engineered myeloid cells expressing CFP in the presence of sialidase. [Figure 30D]

[0111] FIG. 1 illustrates an exemplary schematic diagram of a nucleic acid construct encoding a sialidase with regulatory elements for expression in activated monocytes (eg, macrophages). [Figure 30E]

[0112] 1 illustrates a diagrammatic representation of enhanced phagocytic engulfment of target cells as a result of NF-kappa B (NF-κB) activation in phagocytes. NF-kappa B activation activates expression of a nucleic acid construct encoding a sialidase. [Figure 30F]

[0113]

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

[0114] FIG. 6C is a diagrammatic representation of the enhanced phagocytic engulfment of target cells as a result of expressing the sialidase construct shown in FIG. 6F. [Figure 31A]

[0115]

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

[0116] FIG. 1 illustrates a graphical representation of the relative phagocytic activity of cells expressing CFP(FcRα-CAR) compared to vector control. [Figure 31C]

[0117] FIG. 1 shows the expected results for increased lysis of target cells by CFP (FcRα-CAR)-transduced myeloid cells (e.g., macrophages) compared to control (empty vector-transduced) myeloid cells (e.g., macrophages). [Figure 31D]

[0118] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with CFP (FcRα-CAR)-transduced myeloid cells (e.g., macrophages) or untreated controls. [Figure 32A]

[0119]

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

[0120] FIG. 1 illustrates a graphical representation of the relative phagocytic activity of cells expressing CFP(TREM-CAR) compared to vector control. [Figure 32C]

[0121] FIG. 1 shows expected results for increased lysis of target cells by CFP (TREM-CAR)-transduced myeloid cells (e.g., macrophages) compared to control (empty vector-transduced) myeloid cells (e.g., macrophages). [Figure 32D]

[0122] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with CFP (TREM-CAR)-transduced myeloid cells (e.g., macrophages) or untreated controls. [Figure 33A]

[0123] Figure 1 shows an exemplary schematic diagram of a caspase-recruiting CFP (caspase-CAR). The construct consists of a signal peptide from the N-terminus to the C-terminus, an antigen-specific scFv, a hinge region (e.g., from CD8), a TM (e.g., from CD8), an ITAM (e.g., FcRγ) containing a phagocytic signaling domain, a T2A sequence for bicistronic expression, an SH2 domain, a caspase cleavage sequence, and a pro-caspase (upper panel). When transduced into macrophages, this construct co-expresses CFP and SH2-procaspase. The pro-caspase is autoinhibited in a quiescent state. Binding of tumor surface antigens to the CAR receptor triggers phosphorylation of the ITAM tyrosine motif, leading to recruitment of the SH2-fused pro-caspase. Clustering of the pro-caspase triggers its autocleavage and activation. The linker between the SH2 and pro-caspase is also cleaved at the recognition site. Activated caspases 1, 4, and 5 mediate potent inflammation (lower panel). [Figure 33B]

[0124]

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

[0125] FIG. 1 shows expected flow cytometry results illustrating increased pro-inflammatory cell surface marker expression in cells expressing caspase-recruiting CFP (caspase-CAR) compared to empty vector when human primary myeloid cells (e.g., macrophages) are co-cultured with target tumor cells. [Figure 33D]

[0126] FIG. 1 shows the expected results for increased lysis of target tumor cells by caspase-recruiting CFP (caspase-CAR)-transduced myeloid cells (e.g., macrophages) compared to control (empty vector-transduced) myeloid cells (e.g., macrophages). [Figure 33E]

[0127] FIG. 1 shows expected results illustrating survival curves in a mouse xenograft model of tumor after treatment with caspase-recruiting CFP (caspase-CAR)-transduced macrophages or untreated controls. [Figure 34A]

[0128] FIG. 1 illustrates a diagrammatic illustration of an exemplary modular design of a CFP construct. [Figure 34B]

[0129] FIG. 1 illustrates a diagrammatic illustration of an exemplary modular design of a CFP construct. [Figure 34C]

[0130] FIG. 1 illustrates a diagrammatic illustration of an exemplary modular design of a CFP construct. DETAILED DESCRIPTION OF THE INVENTION

[0015]

[0131] It is intended that all terms be understood as would be understood by a person skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0016]

[0132] The section headings used herein are for organizational purposes only and do not limit the subject matter described. shall not be construed as limiting.

[0133] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be used separately. Conversely, although for clarity the disclosure may be described herein in the context of separate embodiments, the disclosure may also be implemented in a single embodiment.

[0017]

[0134] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a feature, structure, or characteristic described in connection with an embodiment may be It is intended to be included in at least some, but not necessarily all, embodiments of the present disclosure.

[0018]

[0135] As used in this specification and claim(s), the term "comprising ("comprise" and "comprises" are examples of "comprising" "having" (either form) "including" (any form of including, such as "includes" and "include") or " containing ("contains" and "contain") Any form of "containing," such as "containing," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, compositions of the disclosure can be used to achieve the methods of the disclosure.

[0019]

[0136] As used herein, the terms "about" or "approximately" when referring to a measurable value, e.g., a parameter, amount, temporal period, etc. To the extent that such variations are appropriate to the practice of this disclosure, specific values ​​and specific "about" or "approximately" is meant to encompass variations of + / -30% or less, + / -20% or less, + / -10% or less, + / -5% or less, or + / -1% or less from the value. It is understood that values ​​preceded by the modifier "about" or "approximately" are themselves also specifically disclosed.

[0020]

[0137] Herein, engineered bone marrow cells are engineered to specifically bind to target cells. Myeloid cells (including, but not limited to, neutrophils, monocytes, myeloid dendritic cells (mDCs), adipocytes, and macrophages) are provided. The engineered myeloid cells are capable of attacking and killing target cells directly (e.g., by phagocytosis) and / or indirectly (e.g., by activating T cells). In some embodiments, the target cells are cancer cells.

[0021]

[0138] Cancer is an exemplary embodiment described in detail in this disclosure, but the The methods and techniques described are believed to be useful for targeting infected or otherwise diseased cells within the body. Similarly, therapeutic and vaccine compositions using the engineered cells are described herein.

[0022]

[0139] Provided herein are compositions and methods for treating diseases or conditions such as cancer. The compositions and methods provided herein utilize human myeloid cells, including but not limited to neutrophils, monocytes, myeloid dendritic cells (mDCs), adipocytes, and macrophages, to target diseased cells, such as cancer cells. The compositions and methods provided herein eliminate diseased cells, such as cancer cells, and / or diseased tissues through a variety of mechanisms, including T cell activation and recruitment, effector immune cell activation (e.g., CD8 T cell and NK cell activation), antigen cross-presentation, enhanced inflammatory responses, reduction of regulatory T cells, and phagocytosis. For example, myeloid cells can be used to sustain an immune response against cancer cells.

[0023]

[0140] As used herein, phagocytic receptor (PR) fusion proteins (PFPs), scavenging Compositions are provided that include recombinant nucleic acids encoding chimeric fusion proteins (CFPs), such as scavenger receptor (SR) fusion proteins (SFPs), integrin receptor (IR) fusion proteins (IFPs), or caspase-recruiting receptor (caspase-CAR) fusion proteins. The CFPs encoded by the recombinant nucleic acids may include an extracellular domain (ECD) that includes an antigen-binding domain that binds to an antigen on a target cell. The extracellular domain may be fused to a hinge domain or extracellular domain derived from a receptor, such as CD2, CD8, CD28, CD68, a phagocytic receptor, a scavenger receptor, or an integrin receptor. The CFPs encoded by the recombinant nucleic acids may further include a transmembrane domain, such as a transmembrane domain derived from CD2, CD8, CD28, CD68, a phagocytic receptor, a scavenger receptor, or an integrin receptor. In some embodiments, the CFPs encoded by the recombinant nucleic acids further include an intracellular domain that includes an intracellular signaling domain, such as an intracellular signaling domain derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain may include one or more intracellular signaling domains derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain may comprise one or more intracellular signaling domains that promote phagocytic activity, an inflammatory response, nitric oxide production, integrin activation, enhanced effector cell migration (e.g., via chemokine receptor expression), antigen presentation, and / or enhanced cross-presentation. In some embodiments, the CFP is a phagocytic receptor fusion protein (PFP). In some embodiments, the CFP is a phagocytic scavenger receptor fusion protein (PFP). In some embodiments, the CFP is an integrin receptor fusion protein (IFP). In some embodiments, the CFP is an inflammatory receptor fusion protein. In some embodiments, the CFP encoded by the recombinant nucleic acid further comprises an intracellular domain comprising a recruitment domain. For example, the intracellular domain may comprise one or more PI3K recruitment domains, caspase recruitment domains, or a combination thereof. domain or caspase activation and recruitment domain (CARD).

[0024]

[0141] Provided herein are compositions comprising recombinant nucleic acids encoding CFPs, provides a composition comprising a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)) comprising (i) a transmembrane domain, and (ii) an intracellular domain comprising a phagocytic receptor intracellular signaling domain, and an extracellular antigen-binding domain specific for an antigen, e.g., an antigen of a target cell or an antigen presented on a target cell, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked such that antigen binding to the target by the extracellular antigen-binding domain of the fusion receptor activates the intracellular signaling domain of the phagocytic receptor.

[0025]

[0142] Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP, the composition comprising a CFP-encoding nucleic acid sequence, the CFP-encoding nucleic acid sequence ... the FP comprises a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), which comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and upon binding of the CFP to the antigen of the target cell, the killing or phagocytic activity of a myeloid cell, such as a neutrophil, monocyte, myeloid dendritic cell (mDC), mast cell, or macrophage, that expresses the CFP is enhanced; The compositions provide an increase of 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.

[0026]

[0143] Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP, the composition comprising a CFP-encoding nucleic acid sequence, the CFP-encoding nucleic acid sequence ... The FP comprises a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), which comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; when the CFP binds to the antigen of the target cell, the CFP-expressing neutrophils, monocytes, myeloid dendritic cells (mDCs), Compositions are provided that increase the killing or phagocytic activity of myeloid cells, such as mast cells or macrophages, by at least 1.1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 75 fold, or 100 fold compared to cells that do not express CFP.

[0027]

[0144] In one aspect, provided herein is a pharmaceutical composition, comprising: (a) a recombinant polynucleic acid; Provided are pharmaceutical compositions comprising myeloid cells, such as neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, or macrophage cells, comprising a recombinant polynucleic acid comprising a sequence encoding a CD5-antibody fusion protein (CFP), wherein the CFP comprises (i) an extracellular domain comprising an anti-CD5 binding domain, and (ii) a transmembrane domain operably linked to the extracellular domain; and (b) a pharmaceutically acceptable carrier, wherein the myeloid cells express the CFP and exhibit at least a 1.1-fold increase in phagocytosis of CD5-expressing target cells compared to myeloid cells that do not express the CFP. In some embodiments, the CD5-binding domain is a CD5-binding protein comprising an antigen-binding fragment of an antibody, a Fab fragment, an scFv domain, or an sdAb domain. In some embodiments, the CD5-binding domain comprises (i) a variable heavy chain (V) of SEQ ID NO: 1 or having at least 90% sequence identity to SEQ ID NO: 1. H) sequence; and (ii) sequence SEQ ID NO:2 or a variable light chain (V) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2. L ) sequence of SEQ ID NO: 33 or an scFv having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the HER2 binding domain comprises (i) a variable heavy chain (V) of SEQ ID NO: 8 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. H ) sequence of SEQ ID NO:9, and (ii) a variable light chain (V) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9. L ) sequence. In some embodiments, the CD5 binding domain comprises an scFv comprising SEQ ID NO: 32 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32. In some embodiments, the CFP further comprises an intracellular domain, wherein the intracellular domain comprises one or more intracellular signaling domains, and wherein a wild-type protein comprising the intracellular domain does not comprise an extracellular domain.

[0028]

[0145] In some embodiments, the extracellular domain comprises a hinge domain derived from CD8. and wherein the hinge domain is operably linked to the transmembrane domain and the anti-CD5 binding domain. In some embodiments, the extracellular hinge domain comprises SEQ ID NO:7 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7.

[0029]

[0146] In some embodiments, the CFP is at least as good as or similar to SEQ ID NO:30. In some embodiments, the CFP comprises an extracellular domain fused to a transmembrane domain of SEQ ID NO:31 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:31.

[0030]

[0147] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In embodiments, the transmembrane domain comprises SEQ ID NO: 6 or 29 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6 or 29. In some embodiments, the transmembrane domain comprises SEQ ID NO: 18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises SEQ ID NO: 34 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises SEQ ID NO: 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19.

[0031]

[0148] In some embodiments, the CFP comprises one or more amino acid sequences comprising a phagocytic signaling domain. comprises multiple intracellular signaling domains. In some embodiments, the phagocytosis signaling domain is derived from a receptor other than Megf10, MerTk, FcRα, and Bai1. In some embodiments, the CFP comprises an intracellular signaling domain derived from a receptor other than Megf10, MerTk, FcR, and Bai1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than CD3ζ. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from FcRγ, FcRα, and FcRε. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from CD3ζ. In some embodiments, the CFP comprises an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 20, 27, and 28, or to any one of SEQ ID NOs: 3, 20, 27, and 28. In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the proinflammatory signaling domain comprises a PI3-kinase (PI3K) recruitment domain. In some embodiments, the proinflammatory signaling domain comprises SEQ ID NO:4 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4. In some embodiments, the proinflammatory signaling domain is derived from the intracellular signaling domain of CD40. In some embodiments, the proinflammatory signaling domain comprises SEQ ID NO:5 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5. In some embodiments, the CFP comprises an intracellular signaling domain of SEQ ID NO:21 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:21.In some embodiments, the CFP comprises an intracellular signaling domain of SEQ ID NO:23 or having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:23.

[0032]

[0149] In some embodiments, the CFP is of or at least as good as SEQ ID NO:14. In some embodiments, the CFP comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15 or to SEQ ID NO: 15. In some embodiments, the CFP comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 or to SEQ ID NO: 16. In some embodiments, the CFP comprises SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises SEQ ID NO: 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25.

[0033]

[0150] In some embodiments, the CFP is (a)(i) a sc that specifically binds to CD5. and (ii) an extracellular domain comprising at least a portion of a hinge domain derived from CD8; a hinge domain derived from CD28 or an extracellular domain derived from CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains are selected from the group consisting of (i) an FcR and (ii) a second intracellular signaling domain: (A) comprising a PI3K recruitment domain or (B) derived from CD40. In some embodiments, as an alternative to (c) above, the CFP comprises an intracellular domain comprising at least two intracellular signaling domains, wherein the intracellular domain comprising at least two intracellular signaling domains comprises (i) a first intracellular signaling domain derived from a phagocytic receptor intracellular domain, and (ii) a second intracellular signaling domain derived from a scavenger receptor phagocytic receptor intracellular domain comprising (A) a PI3K recruitment domain or (B) derived from CD40. Exemplary scavenger receptors from which the intracellular signaling domain may be derived can be found in Table 2. In some embodiments, the CFP comprises an intracellular signaling domain derived from the intracellular signaling domain of an innate immune receptor.

[0034]

[0151] In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the recombinant polynucleic acid is a circRNA. In some embodiments, the recombinant polynucleic acid is a viral vector. In some embodiments, the recombinant polynucleic acid is delivered via a viral vector.

[0035]

[0152] In some embodiments, the myeloid cells are CD14+ cells, CD14+ / CD16- cells, CD14+ / CD16+ cells, CD14- / CD16+ cells, CD14- / CD16- cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages or mosaic myeloid cells / macrophages / dendritic cells.

[0036]

[0153] In one aspect, the present disclosure provides a method for treating a cancer, comprising administering to a human subject a pharmaceutical composition comprising administering to said ... Provided is a method of treating cancer in a human subject in need thereof, wherein the pharmaceutical composition comprises: (a) myeloid cells comprising a recombinant polynucleic acid sequence, wherein the polynucleic acid sequence comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising (i) an extracellular domain comprising an anti-CD5 binding domain, and (ii) a transmembrane domain operably linked to the extracellular domain; and (b) a pharmaceutically acceptable carrier, wherein the myeloid cells express the CFP.

[0037]

[0154] In some embodiments, CFP binds to CD5 expressed by target cancer cells of the subject. Upon binding, the killing or phagocytic activity of the myeloid cells is increased by more than 20% compared to myeloid cells that do not express CFP, hi some embodiments, tumor growth is inhibited in the human subject.

[0038]

[0155] In some embodiments, the cancer is a CD5+ cancer. is leukemia, T-cell lymphoma, or B-cell lymphoma.

[0156] In some embodiments, the anti-CD5 binding domain is an antigen-binding fragment of an antibody, scF In some embodiments, the anti-CD5 binding domain is a CD5 binding protein comprising a v domain, a Fab fragment, or an sdAb domain. In some embodiments, the anti-CD5 binding domain is a protein or fragment thereof that binds to CD5, such as a ligand of CD5 (e.g., a natural ligand of CD5).

[0039]

[0157] In some embodiments, the CFP further comprises an intracellular domain, The wild-type protein comprises one or more intracellular signaling domains, wherein the one or more intracellular signaling domains comprise a phagocytic signaling domain, and the wild-type protein comprising the intracellular domain does not comprise an extracellular domain.

[0040]

[0158] In some embodiments, the phagocytosis signaling domain is selected from the group consisting of Megf10, Mer In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Tk, FcRα, or Bai1. It contains an intracellular signaling domain derived from cRε.

[0041]

[0159] In some embodiments, the one or more intracellular signaling domains are pro-inflammatory. In some embodiments, the pro-inflammatory signaling domain further comprises a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3 kinase (PI3K) recruitment domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the extracellular domain comprises at least a portion of a hinge domain derived from CD8, a hinge domain derived from CD28, or an extracellular domain derived from CD68.

[0042]

[0160] In some embodiments, the CFP is (a)(i) a sc that specifically binds to CD5. and (ii) an extracellular domain comprising at least a portion of a hinge domain derived from CD8, a hinge domain derived from CD28, or an extracellular domain derived from CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from FcRγ or FcRε, and (ii) a second intracellular signaling domain, (A) comprising a PI3K recruitment domain or (B) derived from CD40. In some embodiments, the recombinant nucleic acid is an mRNA or a circRNA. In some embodiments, the myeloid cells are CD14+ cells, CD14+ / CD16- cells, CD14+ / CD16+ cells, CD14- / CD16+ cells, CD14- / CD16- cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages, or mosaic myeloid cells / macrophages / dendritic cells.

[0043]

[0161] In some embodiments, the methods include administering to a subject a CD47 agonist, an agent that inhibits Rac, The method further comprises administering an additional therapeutic agent selected from the group consisting of an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4,5-trisphosphate production, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, an agent that promotes lymphocyte segregation in primary and / or secondary lymphoid organs, an agent that increases the concentration of naive T cells and central memory T cells in secondary lymphoid organs, and any combination thereof.

[0044]

[0162] In some embodiments, the myeloid cells are (a) endogenous peptides that dimerize with CFP. and (b) a non-endogenous peptide or protein that dimerizes with CFP, and / or (c) a second recombinant polynucleic acid sequence, wherein the second recombinant polynucleic acid sequence comprises a sequence encoding a peptide or protein that interacts with CFP; wherein the dimerization or interaction enhances phagocytosis by myeloid cells that express CFP compared to myeloid cells that do not express CFP.

[0045]

[0163] In some embodiments, the myeloid cells are capable of (i) effector activity, cross-presentation, and reprogramming. an increase in the absorptive burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4,5-trisphosphate production, trogocytosis by antigen-expressing target cells, resistance to CD47-mediated inhibition of phagocytosis, resistance to LILRB1-mediated inhibition of phagocytosis, or any combination thereof; and / or (ii) an increase in IL-1, IL3, IL-6, IL-10, IL-12, IL-13, IL-23, TNFα, the TNF family of cytokines, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES, interferons, MHC class I proteins, MHC class II proteins, CD40, CD48, CD58, CD80, CD86, CD 112, 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.

[0046]

[0164] In some embodiments, the intracellular signaling domain is a phagocytic or tethering receptor. or the intracellular signaling domain comprises a phagocytic activation domain. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR-alpha, or Bai1. In some embodiments, the intracellular signaling domain is derived from TNFR1, MDA5, CD40, lectin, dectin-1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D , CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fcα receptor I, CR1, CD35, CD3ζ, complement receptor, CR3, CR4, Tim-1, Tim-4, and CD169 (e.g., a phagocytic receptor). In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0047]

[0165] In some embodiments, the intracellular signaling domain is an ITAM domain-containing receptor domain. It comes from the condition.

[0166] As used herein, phagocytic or tethered receptor (PR) fusion proteins (PFPs) and the like The present invention provides a composition comprising a recombinant nucleic acid encoding a CFP, wherein the CFP comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific to an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is derived from a phagocytic receptor other than Megf10, MerTk, FcRα, or Bai1.

[0048]

[0167] In some embodiments, binding of the CFP to an antigen on a target cell increases the killing activity of cells expressing the CFP by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or more than 1000% compared to cells that do not express the CFP. In some embodiments, when CFP is expressed in cells, CFP is functionally integrated into the cell membrane of cells. In some embodiments, when CFP binds to the antigen of target cells, the killing activity of the cells that express CFP increases at least 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 50 times, 75 times or 100 times compared with the cells that do not express CFP.

[0049]

[0168] In some embodiments, the intracellular signaling domain is TNFR1, MDA5 , CD40, lectin, dectin-1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, S The intracellular signaling domain is derived from a receptor such as a phagocytic receptor selected from the group consisting of CARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fcα receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD 169. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain.

[0050]

[0169] As used herein, phagocytic or tethered receptor (PR) fusion proteins (PFPs) and the like are used. A composition comprising a recombinant nucleic acid encoding a CFP, wherein the CFP comprises a PR subunit comprising a transmembrane domain and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific to an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin-1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCAR The present invention provides compositions derived from receptors such as phagocytic receptors selected from the group consisting of A5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fcα receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4 and CD169.

[0051]

[0170] In some embodiments, when CFP binds to an antigen on a target cell, the cell expresses CFP. In some embodiments, the killing activity of cells containing the CFP-expressing antibody 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. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, and Bai1. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain, such as a PI3K recruitment domain derived from CD19. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0052]

[0171] As used herein, phagocytic or tethered receptor (PR) fusion proteins (PFPs) and the like are used. The present invention provides a composition comprising a recombinant nucleic acid encoding a CFP, wherein the CFP comprises a PR subunit comprising a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain, and an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0053]

[0172] As used herein, manipulations that can target target cells, e.g., diseased cells, are The present invention provides compositions of engineered CFPs, such as phagocytic receptor fusion proteins, that can be expressed in cells, such as myeloid cells, such as to generate engineered myeloid cells.

[0054]

[0173] The target cells are, for example, cancer cells. In some embodiments, engineered bone marrow After phagocytosis of cancer cells, cancer cells can present cancer antigens on their cell surface and activate T cells. An "antigen" is a molecule that can stimulate an immune response. Antigens recognized by T cells, helper T lymphocytes (helper T (TH) cells) or cytotoxic T lymphocytes (CTLs) are not recognized as intact proteins, but as small peptides associated with MHC proteins (e.g., class I or class II MHC proteins) on the cell surface. During the course of a natural immune response, class II antigens on antigen-presenting cells (APCs) Antigens recognized in association with MHC molecules are acquired from outside the cell, internalized, and processed into small peptides that associate with class II MHC molecules.

[0055]

[0174] In some embodiments, binding of the CFP to an antigen on a target cell increases the killing activity of cells expressing the CFP by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or more than 1000% compared to cells that do not express the CFP. In some embodiments, when CFP is expressed in cells, CFP is functionally integrated into the cell membrane of cells. In some embodiments, when CFP binds to the antigen of target cells, the killing activity of the cells that express CFP increases at least 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 50 times, 75 times or 100 times compared with the cells that do not express CFP.

[0056]

[0175] In some embodiments, the antigen-expressing target cells are cancer cells. In some embodiments, the antigen-expressing target cells are at least 0.8 microns in diameter.

[0176] In some embodiments, cells expressing CFP exhibit increased phagocytosis of antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit at least a 1.1-fold increase in phagocytosis of antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, or 50-fold increase in phagocytosis of antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased cytokine production compared to cells that do not express CFP. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferon, and combinations thereof. In some embodiments, cells expressing CFP exhibit increased effector activity compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased cross-presentation compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class II proteins compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD80 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD86 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class I proteins compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased expression of TRAIL / TNF family death receptors compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased expression of TRAIL / TNF family death receptors compared to cells that do not express CFP. The cells exhibit increased expression of B7-H2 compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of LIGHT compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of HVEM compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of CD40 compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of TL1A compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of 41BBL compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of OX40L compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of GITRL death receptor compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit increased expression of CD30L compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased expression of TIM4 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TIM1 ligand compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of SLAM compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD48 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD58 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD155 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD112 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of PDL1 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of B7-DC compared to cells not expressing CFP.In some embodiments, cells expressing CFP exhibit an increased respiratory burst compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increased ROS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increased iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increased iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increased extracellular vesicle production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increased trogocytosis by target cells that express an antigen compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increased resistance to CD47-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increased resistance to LILRB1-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased phosphatidylinositol 3,4,5-trisphosphate production.

[0057]

[0177] In some embodiments, the extracellular domain of the CFP comprises an Ig-binding domain. In some embodiments, the extracellular domain comprises an IgA, IgD, IgE, IgG, IgM, FcRγI, FcRγIIA, FcRγIIB, FcRγIIC, FcRγIIIA, FcRγIIIB, FcRn, TRIM21, or FcRL5-binding domain. In some embodiments, the extracellular domain of the CFP comprises an FcR extracellular domain. In some embodiments, the extracellular domain of the CFP comprises an FcRα, FcRβ, FcRε, or FcRγ extracellular domain. In some embodiments, the extracellular domain comprises an FcRα (FCAR) extracellular domain. In some embodiments, the extracellular domain comprises an FcRβ extracellular domain. In some embodiments, the extracellular domain comprises an FCERI extracellular domain. In some embodiments, the extracellular domain comprises an FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, or FCGR3B extracellular domain. In some embodiments, the extracellular domain comprises an integrin domain or an integrin receptor domain. In embodiments, the extracellular domain comprises one or more integrin α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, or β8 domains.

[0058]

[0178] In some embodiments, the CFP further comprises an extracellular domain and an extracellular antigen-binding domain operably linked to the transmembrane domain. In some embodiments, the extracellular domain further comprises the extracellular domain of a receptor, a hinge, a spacer, and / or a linker. In some embodiments, the extracellular domain comprises the extracellular portion of a phagocytic receptor. In some embodiments, the extracellular portion of the CFP is derived from the same receptor from which the intracellular signaling domain is derived. In some embodiments, the extracellular domain comprises the extracellular domain of a scavenger receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises the extracellular domain of an immunoglobulin or an immunoglobulin hinge region. In some embodiments, the extracellular domain comprises a phagocytic domain. In some embodiments, the extracellular domain comprises a structure capable of multimer assembly. In some embodiments, the extracellular domain comprises a multimerization scaffold. In some embodiments, the extracellular domain is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. In some embodiments, the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids in length. In some embodiments, the extracellular antigen-binding domain specifically binds to an antigen on a target cell. In some embodiments, the extracellular antigen-binding domain comprises an antibody domain. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain, an antibody domain, and the antibody domain is a functional antibody fragment, a single-chain variable fragment (scFv), a Fab, a single-domain antibody (sdAb), a nanobody, a V H Domain, V L domain, VNAR domain, V HHIn some embodiments, the extracellular antigen-binding domain comprises an extracellular domain of a ligand, a receptor, or an adaptor. In some embodiments, the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain specific for a single antigen. In some embodiments, the extracellular antigen-binding domain comprises at least two extracellular antigen-binding domains, each of which is specific for a different antigen.

[0059]

[0179] In some embodiments, the antigen is a cancer-associated antigen, a lineage-associated antigen, a pathogenic antigen, or an autoimmune antigen. In some embodiments, the antigen comprises a viral antigen. In some embodiments, the antigen is a T lymphocyte antigen. In some embodiments, the antigen is an extracellular antigen. In some embodiments, the antigen is an intracellular antigen. In some embodiments, the antigen is thymidine kinase (TK1), hypoxanthine-guanine Phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-1, mucin-16 (MUC16), MUC1, epidermal growth factor receptor vIII (EGFRvIII), mesothelin, human epidermal growth factor receptor 2 (HER2), EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B-cell maturation antigen (BCMA), glypican 3 (GPC3), follicle-stimulating hormone receptor, fibroblast activation protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer group 2D (NKG2D) receptor Gand, disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56, CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptors, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, protease, P The antigen is selected from the group consisting of antigens from AP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is an antigen of a protein 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 antigen of an integrin receptor. 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 is capable of binding to two or more different antigens.

[0060]

[0180] In some embodiments, the antigen-binding domain comprises an autoantigen, such as Dsg1 or Dsg3, or a fragment thereof. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain or an antibody domain, and the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.

[0061]

[0181] In some embodiments, the transmembrane domain and the extracellular antigen-binding domain are operably linked through a linker, such as the hinge region of CD8α, IgG1, or IgG4.

[0062]

[0182] In some embodiments, the extracellular domain comprises a multimerization scaffold.

[0183] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises an FcR transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRγ transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRα transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRβ transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRε transmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane domain from a syntaxin, such as syntaxin 3 or syntaxin 4 or syntaxin 5. In some embodiments, when the CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when the CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an exogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the intracellular signaling domain is derived. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the extracellular domain is derived. In some embodiments, the transmembrane domain comprises the transmembrane domain of a phagocytic receptor. In some embodiments, the transmembrane domain and the extracellular domain are derived from the same protein. In some embodiments, the transmembrane domain is derived from the same protein as the intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes a DAP12 recruitment domain. In some embodiments, the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12.

[0063]

[0184] In some embodiments, the transmembrane domain comprises at least 12, 13, 14, 15, It is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acids in length. In some embodiments, the transmembrane domain is up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acids in length.

[0064]

[0185] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, and Bai1. In some embodiments, the intracellular signaling domain is selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin-1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor selected from the group consisting of .5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc-alpha receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a scavenger receptor. In some embodiments, the intracellular domain comprises a CD47 inhibitory domain. In some embodiments, the intracellular domain comprises a Rac inhibitory domain, a Cdc42 inhibitory domain, or a GTPase inhibitory domain. In some embodiments, the Rac inhibitory domain, Cdc42 inhibitory domain, or GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase in the phagocytic cup of PFP-expressing cells. In some embodiments, the intracellular domain comprises an F-actin dissociation activation domain, an ARHGAP12 activation domain, an ARHGAP25 activation domain, or an SH3BP1 activation domain. In some embodiments, the intracellular domain comprises a phosphatase inhibitory domain. In some embodiments, the intracellular domain comprises an ARP2 / 3 inhibitory domain. In some embodiments, the intracellular domain comprises at least one ITAM domain.In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ITAM domains. In some embodiments, the intracellular domain comprises at least one ITAM domain selected from the group consisting of CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and ITAM domains of these amino acid sequences having at least one to no more than 20 modifications thereto. In some embodiments, at least one ITAM domain comprises a Src family kinase phosphorylation site. In some embodiments, at least one ITAM domain comprises a Syk recruitment domain. In some embodiments, the intracellular domain comprises an F-actin depolymerization activation domain. In some embodiments, the intracellular domain lacks enzymatic activity.

[0065]

[0186] In some embodiments, the intracellular domain does not include a domain derived from the CD3 zeta intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the MerTK intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the TLR4 intracellular domain. In some embodiments, the intracellular domain includes a CD47 inhibitory domain. In some embodiments, the intracellular signaling domain includes a PSGL In some embodiments, the intracellular signaling domain comprises a domain that activates an integrin, such as the intracellular region of Rap1-1. In some embodiments, the intracellular signaling domain comprises a domain that activates Rap1 GTPase, such as from EPAC and C3G. In some embodiments, the intracellular signaling domain is derived from paxillin. In some embodiments, the intracellular signaling domain activates focal adhesion kinase. In some embodiments, the intracellular signaling domain is derived from a single phagocytic receptor. In some embodiments, the intracellular signaling domain is derived from a single scavenger receptor. In some embodiments, the intracellular domain comprises a phagocytosis-enhancing domain.

[0066]

[0187] In some embodiments, the intracellular domain comprises a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain. In some embodiments, the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain. In some embodiments, the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, an NLRP family member, NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), a caspase domain, a pro-caspase binding domain, or any combination thereof.

[0067]

[0188] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a connexin (Cx) protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Cx43, Cx46, Cx37, Cx40, Cx33, Cx50, Cx59, Cx62, Cx32, Cx26, Cx31, Cx30.3, Cx31.1, Cx30, Cx25, Cx45, Cx47, Cx31.3, Cx36, Cx31.9, Cx39, Cx40.1, or Cx23. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Cx43.

[0068]

[0189] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a SIGLEC protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-3 (CD33), Siglec-4 (MAG), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-13, Siglec-14, Siglec-15, Siglec-16, or Siglec-17.

[0069]

[0190] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a C-type lectin protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a mannose receptor protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from an asialoglycoprotein receptor protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a macrophage galactose-type lectin (MGL), DC-SIGN (CLEC4L), langerin (CLEC4K), myeloid DAP12-associating lectin (MDL)-1 (CLEC5A), DC-associated C-type lectin 1 (Dectin-1) subfamily proteins, Dectin-1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily proteins, DCIR / CLEC4A, Dectin-2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), myelogenous DAP12-associating lectin (MDL)-1 (CLEC5A ... The signaling domain may include an intracellular signaling domain such as an intracellular signaling domain derived from Bircle (macrophage-induced C-type lectin 4E), a NOD-like receptor protein, a NOD-like receptor MHC class II transactivator (CIITA), IPAF, BIRC1, a RIG-I-like receptor (RLR) protein, RIG-I, MDA5, LGP2, NAIP5 / Bircle, an NLRP protein, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP14, an NLR protein, NOD1 or NOD2, or any combination thereof.

[0070]

[0191] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a cell adhesion molecule. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from an IgCAM, cadherin, integrin, C-type lectin-like domain protein (CTLD), and / or proteoglycan molecule. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a selectin, such as E-selectin, L-selectin, or P-selectin.

[0071]

[0192] In some embodiments, the GFP does not comprise a full-length intracellular signaling domain. In some embodiments, the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. In some embodiments, the intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length.

[0072]

[0193] In some embodiments, the recombinant nucleic acid encodes an FcR α chain extracellular domain, an FcR α chain transmembrane domain, and / or an FcR α chain intracellular domain. In some embodiments, the recombinant nucleic acid encodes an FcR β chain extracellular domain, an FcR β chain transmembrane domain, and / or an FcR β chain intracellular domain. In some embodiments, the FcR α chain or FcR β chain forms a complex with FcRγ when expressed in a cell. In some embodiments, the FcR α chain or FcR β chain forms a complex with endogenous FcRγ when expressed in a cell. In some embodiments, the FcR α chain or FcR β chain is not incorporated into the cell membrane of a cell that does not express FcRγ. In some embodiments, the CFP does not comprise an FcR α chain intracellular signaling domain. In some embodiments, the CFP does not comprise an FcR β chain intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes a TREM extracellular domain, a TREM transmembrane domain, and / or a TREM intracellular domain. In some embodiments, the TREM is TREM1, TREM2, or TREM3.

[0073]

[0194] In some embodiments, the recombinant nucleic acid comprises a sequence encoding a pro-inflammatory polypeptide. In some embodiments, the composition further comprises a pro-inflammatory nucleotide or nucleotides in the recombinant nucleic acid, such as ATP, ADP, UTP, UDP, and / or UDP-glucose.

[0074]

[0195] 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 IL-1, IL3, IL5, IL-6, IL8, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP 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.

[0075]

[0196] In some embodiments, myeloid cells are specifically targeted for delivery. Myeloid cells can be targeted using specific biodegradable polymers, such as PLGA (polylactic-co-glycolic acid) and / or polyvinyl alcohol (PVA). In some embodiments, one or more compounds can be selectively incorporated into such polymeric structures to affect myeloid cell function. In some embodiments, the targeting structure is multilayered, e.g., consisting of one or more PLGA and one or more PVA layers. In some embodiments, the targeting structure is assembled in a layer-by-layer sequence. In some embodiments, the targeting polymeric structure is organized into specifically shaped components, such as labile structures, that can adhere to the myeloid cell surface and deliver one or more components, such as growth factors and cytokines, to maintain myeloid cells in a microenvironment that confers a specific polarization. In some embodiments, the polymeric structures can remain attached to the surface without being phagocytosed by myeloid cells. In some embodiments, the one or more growth factors can be M1-polarizing factors, such as cytokines. In some embodiments, the one or more growth factors can be M2-polarizing factors, such as cytokines. In some embodiments, the one or more growth factors can be macrophage-activating cytokines, such as INFγ. In some embodiments, the polymeric structure is capable of sustained release of the one or more growth factors in an in vivo environment, such as a solid tumor.

[0076]

[0197] In some embodiments, the recombinant nucleic acid comprises a sequence encoding a homeostatic regulator of inflammation. In some embodiments, the homeostatic regulator of inflammation is a sequence in an untranslated region (UTR) of an mRNA. In some embodiments, the sequence in the UTR is a sequence that binds to an RNA-binding protein. In some embodiments, binding of the RNA-binding protein to the sequence in the untranslated region (UTR) inhibits or prevents translation. In some embodiments, the sequence in the UTR comprises the consensus sequence WWWU(AUUUA)UUUW, where W is A or U. In some embodiments, the recombinant nucleic acid is expressed in a bicistronic vector.

[0077]

[0198] In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments, the target cell comprises a cell infected by a pathogen. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a cancer cell that is a lymphocyte. In some embodiments, the target cell is a cancer cell that is an ovarian cancer cell. In some embodiments, the target cell is a cancer cell that is a breast cell. In some embodiments, the target cell is a cancer cell that is a pancreatic cell. In some embodiments, the target cell is a cancer cell that is a glioblastoma cell.

[0078]

[0199] In some embodiments, the recombinant nucleic acid is DNA. In some embodiments, the recombinant nucleic acid is RNA. In some embodiments, the recombinant nucleic acid is mRNA. In some embodiments, the recombinant nucleic acid is unmodified mRNA. In some embodiments, the recombinant nucleic acid is modified mRNA. In some embodiments, the recombinant nucleic acid is circRNA. In some embodiments, the recombinant nucleic acid is tRNA. In some embodiments, the recombinant nucleic acid is microRNA.

[0079]

[0200] Also provided herein are vectors comprising a recombinant nucleic acid sequence encoding a CFP described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the vector further comprises a promoter operably linked to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector is polycistronic. In some embodiments, each of the at least one nucleic acid sequence is operably linked to a separate promoter. In some embodiments, the vector further comprises one or more internal ribosome entry sites (IRES). In some embodiments, the vector further comprises a 5'UTR and / or a 3'UTR adjacent to the at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.

[0080]

[0201] Also provided herein are polypeptides encoded by the recombinant nucleic acids of the compositions described herein.

[0202] Also provided herein are cells comprising the compositions described herein, vectors described herein, or polypeptides described herein. In some embodiments, the cells are phagocytes. In some embodiments, the cells are stem cell-derived cells, myeloid cells, macrophages, dendritic cells, lymphocytes, mast cells, monocytes, neutrophils, microglia, or astrocytes. In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are M1 cells. In some embodiments, the cells are M2 cells. In some embodiments, the cells are M1 macrophage cells. In some embodiments, the cells are M2 macrophage cells. In some embodiments, the cells are M1 myeloid cells. In some embodiments, the cells are M2 myeloid cells.

[0081]

[0203] Also provided herein is a pharmaceutical composition comprising a composition described herein, such as a recombinant nucleic acid described herein, a vector described herein, a polypeptide described herein, or a cell described herein; and a pharmaceutically acceptable excipient.

[0082]

[0204] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes the production of phosphatidylinositol 3,4,5-triphosphate, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, and any combination thereof. In some embodiments, the pharmaceutically acceptable excipient comprises a serum-free medium, a lipid, or a nanoparticle.

[0083]

[0205] Also provided herein are methods for treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is selected from the group consisting of suitable cancers including ovarian cancer, ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is leukemia or lymphoma. In some embodiments, the liquid cancer is T-cell lymphoma. In some embodiments, the disease is a T-cell malignancy.

[0084]

[0206] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent, wherein the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4,5-trisphosphate production, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, and any combination thereof.

[0085]

[0207] In some embodiments, the administering step comprises infusing or injecting. In some embodiments, the administering step comprises administering directly to the solid tumor. In some embodiments, the administering step comprises administering via a circRNA-based delivery procedure, an anon-particle-encapsulated mRNA-based delivery procedure, an mRNA-based In some embodiments, a CD4+ T cell response or a CD8+ T cell response is elicited in the subject.

[0086]

[0208] Also provided herein are methods of preparing a cell, the methods comprising contacting the cell with a composition described herein, a vector described herein, or a polypeptide described herein. In some embodiments, the contacting comprises transducing. In some embodiments, the contacting comprises chemical transfection, electroporation, nucleofection, or viral infection or transduction.

[0087]

[0209] Also provided herein are methods of preparing a pharmaceutical composition, comprising contacting a lipid with a composition described herein or a vector described herein, in some embodiments, the contacting comprises forming lipid nanoparticles.

[0088]

[0210] Also provided herein are methods for preparing a pharmaceutical composition, comprising contacting an antibody with a composition described herein or a vector described herein, in some embodiments, the contacting comprises forming lipid nanoparticles.

[0089] definition

[0211] "Agent" means any cell, small molecule chemical compound, antibody or other It can refer to a fragment, a nucleic acid molecule, or a polypeptide.

[0090]

[0212] An "alteration" or "change" can refer to an increase or decrease. For example, an alteration can be a 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or up to 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90, or 100% increase or decrease. For example, an alteration can be a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, or up to 40-fold, 50-fold, 60-fold, or even up to 70-fold, 75-fold, 80-fold, 90-fold, or 100-fold increase or decrease.

[0091]

[0213] As used herein, "antigen-presenting cells" or "APCs" include professional antigen-presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells). APCs can express major histocompatibility complex (MHC) molecules and present antigens in complexes with MHC on their surface that can be recognized by T cells, inducing T cell activation and immune responses. Professional antigen-presenting cells, particularly dendritic cells, play an important role in stimulating naive T cells. Non-professional antigen-presenting cells, such as fibroblasts, can also contribute to this process. APCs can also process exogenous antigens and cross-present peptide antigens by displaying the processed antigens on class I MHC molecules. Antigens that give rise to proteins recognized in association with class I MHC molecules are generally proteins produced intracellularly, which are processed and associated with class I MHC molecules.

[0092]

[0214] A "biological sample" can refer to any tissue, cell, body fluid, or other substance of biological origin.

[0215] The term "epitope" can refer to any protein determinant, such as a sequence, structure, or amino acid residue, capable of binding to an antibody or binding fragment thereof, a T cell receptor, and / or an antibody-like molecule. Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. A "T cell epitope" can refer to a peptide or peptide-MHC complex recognized by a T cell receptor.

[0093]

[0216] Engineered cells, such as engineered myeloid cells, can refer to cells that have at least one exogenous nucleic acid sequence in the cell, even if the expression is transient. The step of expressing the exogenous nucleic acid can be performed by various methods described elsewhere, including methods known in the art. The present disclosure relates to the preparation and use of engineered cells, such as engineered myeloid cells, for example, engineered phagocytes. The present disclosure particularly relates to engineered cells that contain an exogenous nucleic acid encoding, for example, a chimeric fusion protein (CFP).

[0094]

[0217] The term "immune response" includes, but is not limited to, T cell-mediated, NK cell-mediated, and / or B cell-mediated immune responses. These responses can be influenced by modulation of T cell costimulation and NK cell costimulation. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxic activity. Furthermore, immune responses include immune responses indirectly influenced by NK cell activation, B cell activation, and / or T cell activation, such as antibody production (humoral response), and activation of cytokine-responsive cells, such as macrophages. Immune responses include adaptive immune responses. The adaptive immune system can react to foreign molecular structures, such as antigens of invading organisms. Unlike the innate immune system, the adaptive immune system is highly pathogen-specific. Adaptive immunity can also provide long-lasting protection. Adaptive immune responses include humoral and cell-mediated immune responses. In humoral immune responses, antibodies secreted into body fluids by B cells bind to antigens from pathogens and result in their elimination through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells are activated, which are capable of destroying other cells. For example, when disease-associated proteins are presented by cells, they can be proteolytically fragmented into peptides within the cell. Specific cellular proteins can then attach themselves to antigens or peptides formed in this way, transport them to the cell's surface, and present them to molecular defense mechanisms such as T cells. Cytotoxic T cells can recognize these antigens and kill cells bearing these antigens.

[0095]

[0218] A "ligand" can refer to a molecule capable of binding to or forming a complex with another molecule, such as a receptor. Ligands can include, but are not limited to, proteins, glycoproteins, carbohydrates, lipoproteins, hormones, fatty acids, phospholipids, or any component that binds to a receptor. In some embodiments, a receptor has a specific ligand. In some embodiments, a receptor can exhibit promiscuous binding to ligands, in which case the receptor can bind to several ligands that share at least similarities in conformational configuration, charge distribution, or any other physicochemical characteristics. A ligand can be a biomolecule. A ligand can also be a non-biological substance. For example, a ligand can be a negatively charged particle that is a ligand for the scavenger receptor MARCO. For example, a ligand can be TiO2, which is a ligand for the scavenger receptor SRA1.

[0096]

[0219] The terms "major histocompatibility complex (MHC)," "MHC molecule," or "MHC protein" refer to a protein capable of binding to an antigenic peptide and presenting the antigenic peptide to a T lymphocyte. Such an antigenic peptide can represent a T cell epitope. The HLA complex is also called the HLA complex. Therefore, the terms "human leukocyte antigen (HLA)," "HLA molecule," or "HLA protein" are used interchangeably with the terms "major histocompatibility complex (MHC)," "MHC molecule," and "MHC protein." HLA proteins can be classified as HLA class I or HLA class II. Although the structures of the proteins in the two HLA classes are very similar, they have very different functions. Class I HLA proteins are presented on the surface of almost all cells in the body, including most tumor cells. Class I HLA proteins are usually loaded with antigens originating from endogenous proteins or intracellular pathogens and then presented to naive or cytotoxic T lymphocytes (CTLs). HLA class II proteins are presented by antigen-presenting cells (APCs), including, but not limited to, dendritic cells, B cells, and macrophages. They primarily present peptides processed from external antigen sources, such as the outside of cells, to helper T cells.

[0097]

[0220] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can endocytose material into phagosomes—although B cells more commonly endocytose into endosomes—which fuse with lysosomes, whose acidic enzymes cleave the endocytosed proteins into many different peptides. Autophagy (authophagy) 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.

[0098]

[0221] Presentation of peptides to CD4+ helper T cells by HLA class II molecules can result in an immune response to foreign antigens. Once activated, CD4+ T cells can promote B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells can also secrete cytokines and chemokines that activate and induce the differentiation of other immune cells. HLA class II molecules are typically heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than the class I peptide-binding groove.

[0099]

[0222] HLA alleles are typically expressed in a codominant manner. For example, each person has two alleles of each of the three class I genes (HLA-A, HLA-B, and HLA-C), allowing them to express six different types of class II HLA. At the class II HLA locus, each person inherits a pair of HLA-DP genes (DPAl and DPB1, which encode the α and β chains), HLA-DQ (DQA1 and DQB1, which encode the α and β chains), one gene, HLA-DRα (DRA1), and one or more genes, HLA-DRβ (DRB1 and DRB3, -4, or -5). For example, HLA-DRB1 has nearly 400 known alleles. This means that a heterozygous individual can inherit six or eight functional class II HLA alleles: three or more from each parent. Therefore, HLA genes are highly polymorphic; many different alleles exist in different individuals within a population. Genes that encode HLA proteins have many possible variations, allowing each person's immune system to respond to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is given a specific number. In some embodiments, class I HLA alleles are HLA-A * 02:01, HLA-B * 14:02, HLA-A * 23:01, HLA-E * 01:01 (non-classical). In some embodiments, the class II HLA allele is HLA-DRB* 01:01, HLA-DRB * 01:02, HLA-DRB * 11:01, HLA-DRB * 15:01, and HLA-DRB * It is 07:01.

[0100]

[0223] "Myeloid cells" can broadly refer to cells of the myeloid lineage of hematopoietic stem cells, For example, lymphoid lineages can be excluded. Myeloid cells include, for example, cells of the granulocyte and monocyte lineages. Myeloid cells differentiate from a common ancestor derived from hematopoietic stem cells in the bone marrow. Commitment to the myeloid lineage may be governed by the activation of different transcription factors; therefore, myeloid cells may be characterized as cells with a level of plasticity and the ability to further differentiate into terminal cell types based on extracellular and intracellular stimuli. Myeloid cells can be rapidly mobilized to local tissues via various chemokine receptors on their surface. Myeloid cells respond to various cytokines and chemokines.

[0101]

[0224] Myeloid cells may be cells that arise in the bone marrow from hematopoietic stem cells under the influence of one or more cytokines and chemokines, such as G-CSF, GM-CSF, Flt3L, CCL2, VEGF, and S100A8 / 9. In some embodiments, myeloid cells are progenitor cells. In some embodiments, myeloid cells may be cells that have characteristics of a common myeloid precursor, or a granulocyte precursor, a myeloblast, or a monocyte-dendritic cell precursor, or a combination thereof. Myeloid cells may include granulocytes or monocytes or their precursors. Myeloid cells may include immature granulocytes, immature monocytes, immature macrophages, immature neutrophils, and immature dendritic cells. Myeloid cells may include monocytes or promonocytic cells or monocyte precursors. In some cases, as used herein, myeloid cells may refer to monocytes with an M0 phenotype, an M1 phenotype, or an M2 phenotype. Myeloid cells may include dendritic cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, pre-dendritic cells, or DC precursors. Myeloid cells may include neutrophils, which may be mature neutrophils, neutrophil precursors, or polymorphonuclear cells (PMNs). Myeloid cells may include macrophages, monocyte-derived macrophages, tissue macrophages, and macrophages of M0, M1, or M2 phenotypes. Myeloid cells may include tumor-infiltrating monocytes (TIMs). Myeloid cells may include tumor-associated monocytes (TAMs). Myeloid cells may include myeloid-derived suppressor cells (MDSCs). Myeloid cells may include tissue-resident macrophages. Myeloid cells may include tumor-associated DCs (TADCs). Thus, myeloid cells may express one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD38, CCR5, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, SIGLEC family proteins and CLEC family proteins.In some cases, myeloid cells may be characterized by high or low expression of one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, or a combination thereof.

[0102]

[0225] "Phagocytosis" is used interchangeably with "engulfment" and can refer to the process by which cells engulf particles, such as cancer cells or infected cells. This process can result in an internal compartment (phagosome) containing the particle. This process can be used to ingest and / or remove particles, such as cancer cells or infected cells, from the body. Phagocytic receptors can be involved in the process of phagocytosis. The process of phagocytosis can be closely related to immune responses and antigen presentation. Processing of exogenous antigens is followed by uptake into professional antigen-presenting cells by some type of endocytic event. Phagocytosis can also promote antigen presentation. For example, antigens from phagocytosed cells or pathogens, including cancer antigens, can be processed and presented on the cell surface of APCs.

[0103]

[0226] A "polypeptide" is a glycoprotein, lipoprotein, intracellular protein, or It can refer to molecules containing amino acids linked together via peptide bonds, such as membrane proteins. A polypeptide may comprise one or more subunits of a protein. A polypeptide may be encoded by a recombinant nucleic acid. In some embodiments, a polypeptide may comprise more than one peptide sequence in a single amino acid chain, which may be separated by a spacer, linker, or peptide cleavage sequence. A polypeptide may be a fusion polypeptide. A polypeptide may comprise one or more domains, modules, or moieties.

[0104]

[0227] A "receptor" can refer to a chemical structure composed of a signal-transducing polypeptide, such as a polypeptide that transduces an extracellular signal into a cell. A receptor can contribute to transmitting information in a cell, a cell formation, or an organism. A receptor includes at least one receptor unit and can contain two or more receptor units, each receptor unit including a protein molecule, e.g., a glycoprotein molecule. A receptor can include a structure that binds to a ligand and can form a complex with the ligand. Signaling information can be transmitted by a conformational change of the receptor after binding to the ligand on the surface of the cell.

[0105]

[0228] The term "antibody" refers to the class of proteins commonly known as immunoglobulins, including, but not limited to, IgG1, IgG2, IgG3, and IgG4, IgA (including IgA1 and IgA2), IgD, IgE, IgM, and IgY. The term "antibody" includes, but is not limited to, full-length antibodies, single-chain antibodies, single-domain antibodies (sdAbs), and antigen-binding fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab', and F(ab'), Fd(V). H and C H 1), single chain variable fragments (scFv), single chain antibodies, disulfide-linked variable fragments (dsFv) and V L and / or V H Antibodies may be derived from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, may contain the variable region(s) alone or in combination with one or more of the hinge region, CH1 domain, CH2 domain, and CH3 domain. Also included are any combinations of the variable region and the hinge region, CH1, CH2, and CH3 domain. Antibodies may be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind, for example, an HLA-associated polypeptide or HLA-peptide complex.

[0106]

[0229] The term "recombinant nucleic acid" refers to a nucleic acid prepared, expressed, created, or isolated by recombinant means. Recombinant nucleic acids may contain nucleotide sequences that are not naturally occurring. Recombinant nucleic acids may be synthesized in a laboratory. Recombinant nucleic acids may be prepared by using recombinant DNA techniques, for example, enzymatic modification of DNA, such as restriction enzyme digestion, ligation, and DNA cloning. Recombinant nucleic acids may be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA may be transcribed ex vivo or in vitro to produce messenger RNA (mRNA). Recombinant mRNA may be isolated, purified, and used to transfect cells. Recombinant nucleic acids may encode proteins or polypeptides.

[0107]

[0230] The process of introducing or incorporating nucleic acids into cells can be via transformation, transfection, or transduction. Transformation is the process of uptake of foreign nucleic acids by bacterial cells. This process is adapted for propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into bacterial cells that are competent to take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially introduced in a laboratory setting. Transfection is the introduction of DNA, RNA, or small molecules such as antibodies into eukaryotic cells. Transfection is the transfer of DNA, RNA, or small molecules such as antibodies into bacterial cells. It may also refer to the introduction of a teriophage into a bacterial cell. "Transduction" is often used to describe the introduction of a recombinant viral vector particle into a target cell, while "infection" refers to the natural infection of a human or animal with a wild-type virus.

[0108]

[0231] The term "vector" can refer to a nucleic acid molecule capable of autonomous replication in a host cell, which allows for the cloning of a nucleic acid molecule. As known to those skilled in the art, vectors include, but are not limited to, plasmids, cosmids, phagemids, viral vectors, phage vectors, yeast vectors, mammalian vectors, etc. For example, a vector for exogenous gene transformation may be a plasmid. In certain embodiments, a vector comprises a nucleic acid sequence containing an origin of replication and other elements necessary for the replication and / or maintenance of a nucleic acid sequence in a host cell. In some embodiments, the vectors or plasmids provided herein are expression vectors. Expression vectors are capable of directing the expression of genes and / or nucleic acid sequences to which they are operably linked. In some embodiments, expression vectors or plasmids are in the form of circular double-stranded DNA molecules. A vector or plasmid may or may not be integrated into the genome of a host cell. In some embodiments, the nucleic acid sequence of a plasmid is not integrated into the genome or chromosome of the host cell after introduction. For example, a plasmid may contain elements for transient or stable expression of a nucleic acid sequence in a host cell, such as a gene or open reading frame carried by the plasmid. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that replicates autonomously in host cells. In some embodiments, the nucleic acid sequence of the plasmid is integrated into the genome or chromosome of the host cell when introduced into the host cell. Expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors. The vector can be a DNA or RNA vector. In some embodiments, the vector provided herein is an RNA vector, such as a retroviral vector or a lentiviral vector, that can be integrated into the genome of the host cell (e.g., via reverse transcription) when introduced into the host cell.Other forms of expression vectors known to those of skill in the art which serve equivalent functions, e.g., self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome, can also be used. Exemplary vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked.

[0109]

[0232] The term "spacer" or "linker," when used in reference to a fusion protein, refers to a peptide sequence that connects two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than to link or maintain some minimum distance or other spatial relationship between protein or RNA sequences. In some embodiments, the constituent amino acids of the spacer can be selected to affect certain properties of the molecule, such as the folding, flexibility, net charge, or hydrophobicity of the molecule. Suitable linkers for use in embodiments of the present disclosure are well known to those of skill in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, a linker is used to separate two or more polypeptides, such as two antigenic peptides, by a distance sufficient to ensure that each antigenic peptide folds correctly. Exemplary peptide linker sequences adopt a flexible, extended conformation and do not exhibit the properties required for forming a regular secondary structure. The amino acids of the flexible linker protein region may include Gly, Asn, and Ser, or any permutation of an amino acid sequence containing Gly, Asn, and Ser. Other nearby natural amino acids, such as Thr and Ala, may also be used in the linker sequence.

[0110]

[0233] The terms "treat," "treated," "treating," "t The term "treatment" and the like are meant to refer to reducing, preventing, or ameliorating the associated disorder and / or symptoms (e.g., neoplasm or tumor or infectious agent or autoimmune disease). "Treating" can refer to administering a therapy to a subject after the onset or suspected onset of a disease (e.g., cancer or infection by an infectious agent or autoimmune disease). "Treating" includes the concept of "alleviating" and can refer to reducing the frequency or severity of the occurrence or recurrence of any symptoms or other adverse effects associated with a disease and / or side effects associated with a treatment. The term "treating" also encompasses the concept of "managing" and refers to reducing the severity of a patient's disease or disorder, for example, extending lifespan or increasing the survival rate of a patient with a disease, or delaying its recurrence, for example, extending the period of remission for a patient suffering from a disease. Although not excluded, treating a disorder or condition should not require the complete elimination of the associated disorder, condition, or symptom. As used herein, the terms "prevent," "preventing," "prevention," and their grammatical equivalents may refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who is not experiencing such symptoms at the time administration of an agent or compound begins. In certain embodiments, treating a subject or patient, as described herein, comprises administering a therapeutic composition, such as a drug, metabolite, prophylactic component, nucleic acid, peptide, or protein encoding or otherwise forming a drug, metabolite, or prophylactic component. In some embodiments, treating comprises administering a cell or population of cells to a subject in need thereof. In some embodiments, treating comprises administering to a subject one or more engineered cells described herein, e.g., one or more engineered myeloid cells, such as phagocytes.Treating includes treating a disease, condition, or syndrome, which may be a pathological disease, condition, or syndrome, or a latent disease, condition, or syndrome. In some cases, treating, as used herein, may include administering a therapeutic vaccine. In some embodiments, engineered phagocytes are administered to a patient or subject. In some embodiments, the cells administered to a human subject result in reduced immunogenicity. For example, the engineered phagocytes may not result in or reduce graft-versus-host disease (GVHD) or fratricide. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., have a compatible HLA subtype naturally expressed in the subject). Subject-specific HLA alleles or the subject's HLA genotype may be determined by any method known in the art. In exemplary embodiments, the method includes determining a polymorphism genotype, which may include generating an alignment of reads extracted from sequencing data set to a genetic reference set that includes allelic variants of the polymorphic gene; determining a first posterior probability or posterior probability-derived score for each allelic variant in the alignment; identifying the allelic variant with the highest first posterior probability or posterior probability-derived score as the first allelic variant; identifying one or more overlapping reads aligned with the first allelic variant and one or more other allelic variants; determining a second posterior probability or posterior probability-derived score for the one or more other allelic variants using a weighting factor; identifying the second allelic variant by selecting the allelic variant with the highest second posterior probability or posterior probability-derived score, the first and second allelic variants defining a genotype of the polymorphic gene; and providing an output of the first and second allelic variants.

[0111]

[0234] A "fragment" can refer to a portion of a protein or nucleic acid. In some embodiments, a fragment retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid.

[0112]

[0235] The terms "isolated," "purified," "biologically pure," and their grammatical equivalents refer to a material that is free to varying degrees from the components that normally accompany it as found in its natural state. "Isolate" indicates a degree of separation from its original source or surrounding materials. "Purify" indicates a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that any impurities do not significantly affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the present disclosure is purified when it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or from chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be separately purified.

[0113]

[0236] The term "neoplasia" or "cancer" refers to any disease caused by or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is one non-limiting example of a neoplasia or cancer. The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells with typical characteristics of cancer-causing cells, such as uncontrolled growth, immortality, metastatic potential, rapid proliferation and growth rate, and certain characteristic morphological properties. Cancer cells are often in the form of tumors, but such cells can exist alone in an animal or can be non-tumorigenic cancer cells, such as leukemia cells.

[0114]

[0237] The term "vaccine" is understood to mean a composition for generating immunity for the prevention and / or treatment of disease (e.g., neoplasm / tumor / infectious agent / autoimmune disease). Thus, as used herein, a vaccine is a pharmaceutical comprising a recombinant nucleic acid or cells containing and expressing a recombinant nucleic acid, and is intended for use in humans or animals to generate specific defenses and protectants by vaccination. A "vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent. Aspects of the present disclosure relate to the use of technology in the preparation of phagocyte-based vaccines.

[0115]

[0238] The term "pharmaceutically acceptable" refers to approved or approvable by a federal or state government regulatory agency, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, including humans. A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy the pharmacological activity thereof, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0116]

[0239] Nucleic acid molecules useful in the disclosed methods include, but are not limited to, any nucleic acid molecule that has an activity or encodes a polypeptide. A polynucleotide having substantial identity to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to a pair of nucleic acid molecules forming a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) (See Methods Enzymol. 152:507). For example, stringent salt concentrations may typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In an exemplary embodiment, hybridization can occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization can 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 can occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art. For most applications, post-hybridization washing steps can also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As noted above, wash stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for wash steps are less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash step may include a temperature of at least about 25° C., at least about 42° C., or at least about 68° C. In an exemplary embodiment, the wash step may occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash step may occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash step may occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be readily apparent to one of ordinary skill in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0117]

[0240] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Such 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 identity is typically measured using sequence analysis software (e.g., Sequencing). Identity is measured using the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs (Genetics Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary method for determining the degree of identity is to use the BLAST program, with a probability score between e-3 and em° indicating similar sequences. "Reference" is the basis for comparison. It will be understood that the numbering of specific positions or residues in each sequence depends on the specific protein and the numbering scheme used. Numbering may differ, for example, between precursors of the mature protein and the mature protein itself, and sequence differences between species may affect numbering. One skilled in the art will be able to identify any homologous proteins and their respective residues in their respective encoding nucleic acids by methods well known in the art, for example, by sequence alignment with a reference sequence and determination of homologous residues.

[0118]

[0241] The term "subject" or "patient" refers to an organism, such as an animal (e.g., a human), who is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or non-human primate, a mouse, a cow, a horse, a dog, a sheep, or a cat, or other non-human mammal.

[0119]

[0242] The term "therapeutic effect" refers to some alleviation of one or more symptoms of a disorder (e.g., a neoplasm, a tumor, or an infection by an infectious agent or an autoimmune disease) or its associated pathology. As used herein, a "therapeutically effective amount" refers to an amount of an agent that, upon administration to a cell or subject in single or multiple doses, is effective in extending the survival chances of a patient with such a disorder, reducing, preventing, or delaying one or more signs or symptoms of the disorder beyond that expected in the absence of such treatment, etc. A "therapeutically effective amount" is intended to quantify the amount needed to achieve a therapeutic effect. A physician or veterinarian having ordinary skill can readily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the required pharmaceutical composition.

[0120] Engineered myeloid cells are "targeted" to attack diseased cells

[0243] The present disclosure provides methods for detecting foreign bodies, particles, diseased cells, cell debris, inflammatory signals, and chemoattractants. The present invention also includes compositions and methods for preparing targeted killer myeloid cells by leveraging their innate functional roles in immune defense, ranging from their roles in: egress; activation of endogenous DAMP and PAMP signaling pathways; induction of myelopoiesis, extravasation; chemotaxis; phagocytosis; pinocytosis; mobilization; phagocytosis; scavenging; activation of intracellular oxidative burst and lysis or killing of pathogens; detection, engulfment and killing of diseased or damaged cells; removal of unwanted cells, tissues or cellular debris in vivo; roles in antigen presentation and activation of innate immunity; activation and modulation of immune response cascades; activation of the T cell repertoire; autophagy; inflammatory and non-inflammatory apoptosis; pyroptosis, immunoediting in response to stress and properties related to restoration of tissue homeostasis. In one aspect, the present disclosure provides methods and compositions for enhancing one or more functions of myeloid cells for use in therapeutic applications, where the one or more functions include: detection of foreign bodies, particles, diseased cells, cell debris, inflammatory signals, chemoattractants; activation of endogenous DAMP and PAMP signaling pathways; induction of myelopoiesis; extravasation; chemotaxis; phagocytosis; pinocytosis; recruitment; trogocytosis; phagocytosis; scavenging; activation of intracellular oxidative burst and intracellular lysis or killing of pathogens; detection of diseased or damaged cells; phagocytosis. and killing; removal of unwanted cells, tissues, or cell debris in vivo; a role in antigen presentation and innate immune activation; activation and regulation of immune response cascades; activation of the T cell repertoire; autophagy; inflammatory and non-inflammatory apoptosis; pyroptosis, immunoediting in response to stress, and restoration of tissue homeostasis. In one embodiment, the compositions and methods are also directed to enhancing the targeting and killing functions of specific myeloid cells by genetically modifying these cells. The compositions and methods described herein are also directed to the production of engineered myeloid cells containing at least one genetic modification, which may recognize and induce effector functions against disease cells, such as pathogens, tumors, or cancer cells, such that the engineered myeloid cells are capable of recognizing, targeting, phagocytosis, killing, and / or eliminating pathogens or diseased or cancer cells, and may further activate specific immune response cascades after phagocytosis, killing, and / or elimination of the pathogens or diseased cells.

[0121]

[0244] Myeloid cells appear to be the most abundant cells in tumors (Figure 1B). Myeloid cells can recognize tumor cells over healthy normal cells and amplify the body's immune response to tumor cells. As sentinels of the innate immune response, myeloid cells can identify non-self or abnormal cell types and eliminate them through a process called phagocytosis. This can be therapeutically beneficial in driving myeloid cell-mediated phagocytosis and tumor cell lysis. However, these naturally occurring tumor-infiltrating myeloid cells (TIM) can be influenced by the tumor microenvironment (TME). TIM comprise a heterogeneous population of cells. Many TIM arise from circulating monocytes and granulocytes, which in turn derive from bone marrow-derived hematopoietic stem cells. However, in the presence of persistent stimulation by tumor-derived factors, monocyte and granulocyte precursors can transform their intrinsic pathway of terminal differentiation into mature macrophages, DCs, or granulocytes, becoming tumor-promoting myeloid cell types. Differentiation into pathological or activated immature myeloid cells is favored. These immature myeloid cells include tumor-associated DCs (TADCs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). Instead of this acute myelopoiesis, TAMs may arise from tissue-resident macrophages, which may then be of embryonic or monocytic origin. These tissue-resident macrophages undergo phenotypic and functional changes during carcinogenesis, and proliferation may help maintain TAMs derived from tissue-resident macrophages. The tumor microenvironment may drive tumor-infiltrating myeloid cells to become myeloid-derived suppressor cells and acquire the ability to suppress T cells. Consequently, innovative methods are needed to generate therapeutically effective TAMs that can infiltrate tumors and target tumor cells for phagocytic uptake and killing.

[0122]

[0245] In one aspect, the present disclosure provides engineered myeloid / phagocytic cells capable of targeting specific target cells, such as tumor or pathogenic cells. In some embodiments, the engineered myeloid cells provided herein are effective in infiltrating, targeting, and killing tumor cells. The engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids encoding one or more proteins that assist in targeting phagocytic cells to target cells, such as tumor or cancer cells. In one embodiment, the engineered myeloid cells are capable of readily infiltrating tumors. In one embodiment, the engineered myeloid cells have high specificity for target cells while circulating, with no or negligible cross-reactivity to non-tumor, non-disease cells of interest. In one embodiment, the engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids that will help the cells overcome / evade the effects of the TME and mount a potent anti-tumor response. In one embodiment, the engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids that increase phagocytosis of target cells. In another embodiment, the engineered myeloid / phagocytic cells described herein are engineered to contain recombinant nucleic acids to increase the reduction or elimination of trogocytosis and / or enhance phagocytic lysis of target cells.

[0123]

[0246] Thus, in some embodiments, the compositions described herein include myeloid cells comprising a recombinant nucleic acid encoding a chimeric receptor fusion protein (CFP), e.g., a phagocytic receptor (PR) fusion protein (PFP). The recombinant nucleic acid can include a sequence encoding a PR subunit comprising (i) a transmembrane domain and (ii) an intracellular domain comprising a PR intracellular signaling domain, and an extracellular antigen-binding domain specific for an antigen on a target cell, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked; the PR intracellular signaling domain is derived from a receptor having a signaling domain. The recombinant nucleic acid further encodes one or more polypeptides comprising one or more cell membrane receptors that help phagocytes bind to target cells and enhance their phagocytic activity.

[0124]

[0247] In some embodiments, the myeloid cells described herein comprise one or more recombinant proteins comprising a chimeric receptor, wherein the chimeric receptor is capable of responding to a first phagocytic signal directed toward a target cell, which may be a diseased cell, tumor cell, or pathogen, and a second signal, which is an inflammatory signal that enhances the phagocytic and killing response toward the target initiated by the first signal.

[0125] phagocytes

[0248] As used herein, "improving" or "modifying" or provides methods and compositions for immunotherapy, including "engineering" and targeting to specific targets, which may be specific cell types or classes of cells in a patient or subject. In some embodiments, the subject is a patient with a disease. The terms subject and patient are often used interchangeably in this section. In some embodiments, the phagocytes are derived from a subject with a disease, the disease being, for example, cancer. Autologous cells from the subject may be modified in vitro and administered to the cells, and the modified phagocytes are redesigned to specifically attack and kill the subject's cancer cells.

[0126]

[0249] In some embodiments, the subject has a disease that is not cancer.

[0250] In some embodiments, the subject has a disease that is an infection. In some embodiments, the immunotherapeutic methods and compositions provided herein are for "improving" or "modifying" or "engineering" phagocytes and targeting them to the infection, e.g., infected cells, in the subject.

[0127]

[0251] In some embodiments, the subject has a disease that is a viral, bacterial, fungal, or protozoan infection. In some embodiments, the immunotherapy methods and compositions provided herein are for "improving," "modifying," or "engineering" phagocytes and targeting them to viral, bacterial, fungal, or protozoan-infected cells in an infected subject. In some embodiments, the immunotherapy methods and compositions provided herein are for "improving," "modifying," or "engineering" phagocytes and targeting them to a virus, bacteria, fungus, or any pathogen in the subject, so that the virus, bacteria, fungus, or pathogen in the subject is phagocytosed and / or killed. In some embodiments, the immunotherapy methods and compositions provided herein are for "improving," "modifying," or "engineering" phagocytes and targeting them to a viral antigen, bacterial antigen, fungal antigen, or pathogen antigen in the subject, so that there is at least one improved immune response in the subject against the virus, bacteria, fungus, or pathogen in the subject.

[0128]

[0252] In some embodiments, the myeloid cells, such as phagocytes, are allogeneic. In some embodiments, the methods and compositions for immunotherapy provided herein are derived from an allogeneic source. The method includes obtaining myeloid cells, such as phagocytes, derived from an allogeneic source. The myeloid cells, such as phagocytes, can then be modified or engineered and introduced into a diseased subject so that the modified or engineered cells from the allogeneic source can attack, phagocytose, and / or directly or indirectly kill diseased cells in the subject, or improve at least one immune response of the subject to the disease. In some embodiments, the allogeneic source is a human. In some embodiments, the allogeneic source is a healthy human.

[0129]

[0253] Phagocytes are natural sentinels of the immune system, forming the body's first line of defense. They engulf pathogens, pathogen-infected cells, foreign bodies, or cancer cells, eliminating them from the body. Most potential pathogens are quickly neutralized by this system before they can cause serious infections, for example. This can involve receptor-mediated uptake via the clathrin-coated pit system, pinocytosis, and especially macropinocytosis, as a result of membrane ruffling and phagocytosis. Thus, phagocytes can be activated by a variety of non-self (and self) elements and exhibit a level of plasticity in their "target" recognition.

[0130]

[0254] The mononuclear phagocyte system (MPS), composed of monocytes, macrophages, and dendritic cells, is essential for maintaining tissue homeostasis and determining the balance of immune responses through its role in antigen presentation. The MPS is a cell lineage that arises from bone marrow progenitor cells and gives rise to blood monocytes, tissue macrophages, and dendritic cells. Thus, the process of generating macrophages from the MPS begins with BM promonocytes that undergo a process of differentiation into monocytes poised to enter the systemic circulation. After a short period of circulation (<48 hours), these newly formed monocytes rapidly infiltrate peripheral tissues, where most of them differentiate into macrophages or dendritic cells (DCs). Antimicrobial phagocytosis eliminates and degrades disease-causing microorganisms, induces proinflammatory signaling through cytokine and chemokine secretion, and recruits immune cells to initiate an effective inflammatory response. This type of phagocytosis is often referred to as "inflammatory phagocytosis" (or "immunogenic phagocytosis"). However, in some cases, such as certain persistent infections, an anti-inflammatory response follows microbial uptake. Antimicrobial phagocytosis is generally carried out by professional phagocytes of the myeloid lineage, such as immature dendritic cells (DCs) and macrophages, as well as tissue-resident immune cells. Phagocytosis of damaged, apoptotic cells or cells is typically a non-inflammatory (also called "non-immunogenic") process. Transformed or malignant cells (autologous cells) and cells undergo phagocytosis, and apoptotic cells are rapidly removed without causing damage to surrounding tissues or inducing a pro-inflammatory immune response. This type of apoptotic cell clearance is non-inflammatory and involves the release of a "find me" signal from the apoptotic cell to recruit phagocytes to its location; an "eat me" signal exposed on the apoptotic cell's surface that is bound by phagocytes via specific receptors; cytoskeletal rearrangements that engulf the apoptotic cell; and subsequent digestion of the ingested apoptotic cell, which attracts specific phagocytic receptors (e.g., secretion of anti-inflammatory cytokines).

[0131]

[0255] Phagocytosis, the cellular uptake of microparticles, e.g., particles >0.5 μm within the plasma membrane envelope, is closely related to and partially overlaps with fluid-phase macropinocytosis and receptor-mediated endocytosis of soluble ligands. Variants associated with the uptake of apoptotic cells, also known as efferocytosis, and those of necrotic cells result from infection and inflammation (necroptosis and pyroptosis). The uptake of exogenous particles (heterophagy) shares characteristics with autophagy, an endogenous process for the segregation and lysosomal disposal of damaged organelles. There is a spectrum of uptake mechanisms, depending on particle size, diversity of receptor-ligand interactions, and cytoskeletal involvement. Once internalized, phagosomes can selectively fuse with transient lysosomes or products of the endoplasmic reticulum (ER) and Golgi complex to form secondary phagolysosomes (Russell, DG (2011). Immunol. Rev. 240, 252-268). This route is dynamic They undergo fusion and division with intracellular and secretory vesicles, macrophages, DCs, osteoclasts, and eosinophils. Antimicrobial phagocytosis eliminates and degrades disease-causing microorganisms, induces proinflammatory signaling through cytokine and chemokine secretion, and recruits immune cells to initiate an effective inflammatory response. This type of phagocytosis is often referred to as "inflammatory phagocytosis" (or "immunogenic phagocytosis"). However, in some cases, such as certain persistent infections, an anti-inflammatory response follows microbial uptake. Antimicrobial phagocytosis is generally carried out by professional phagocytes of the myeloid lineage, such as immature dendritic cells (DCs) and macrophages, as well as tissue-resident immune cells. In contrast, phagocytosis (e.g., efferocytosis) of damaged, autologous, apoptotic cells or cell debris is typically a non-inflammatory (also called "non-immunogenic") process. Billions of damaged, dying, and unwanted cells undergo apoptosis every day. Unwanted cells include, for example, excess cells generated during development, senescent cells, infected cells (intracellular bacteria or viruses), transformed or malignant cells, and cells irreversibly damaged by cytotoxic agents.

[0132]

[0256] The bone marrow is a source of circulating neutrophils and monocytes that replace selected tissue-resident macrophages during inflammation and infection, expanding the tissue myeloid population. After phagocytosis, newly recruited monocytes and tissue macrophages secrete their products by generating them from pre-existing phospholipids and arachidonic acid in the cell membrane and by releasing radicals generated by activation of the respiratory burst or induction of inducible nitric oxide synthesis. Apart from the synthesis of low-molecular-weight products (arachidonic acid metabolites, superoxide anion, and nitric oxide) generated as described above, macrophage phagocytosis-induced secretion is primarily achieved by de novo synthesis of RNA and a change in pH, resulting in gradual acidification.

[0133]

[0257] In some embodiments, the phagocytes provided herein are monocytes or cells of the monocyte lineage.

[0258] In some embodiments, myeloid cells are phagocytic macrophages that are MARCO+SignR1+ and are found in the outer marginal zone, where they rapidly eliminate encapsulated bacteria. Similar CD169+ F4 / 80 macrophage strains are present in the subcapsular sinus of lymph nodes and have been implicated in viral infections. Endothelial macrophages, including Kupffer cells in the liver, are known to eliminate microorganisms and antigenic ligands from the blood and lymph nodes, providing sinusoidal immune functions comparable to but distinct from mucosal immunity. Despite expressing typical macrophage markers, not all tissue macrophages are constitutively phagocytic. In the marginal zone of rodent spleens, metallophilic macrophages lacking F4 / 80 strongly express CD169 and sialic acid-binding immunoglobulin (Ig)-like lectin 1 (SIGLEC1 [sialoadhesin]) but are weakly phagocytic. Non-professional phagocytes include epithelial cells and fibroblasts. Fibroblasts are "working-class phagocytes" that eliminate apoptotic debris by using integrins other than CD11b-CD18 through adhesion molecules ICAM and vitronectin receptors. Astrocytes have also been reported to phagocytose apoptotic corpses, although they do not efficiently degrade them. Cell membrane receptors associated with phagocytosis can be opsonins, primarily FcRs (activating or inhibiting), a conserved domain of IgG antibodies, and complement receptors, such as CR3 of iC3b accumulated by the classical pathway of complement activation (IgM or IgG) or the alternative lectin pathway. CR3 can also mediate recognition in the absence of opsonins, possibly by accumulating macrophage-derived complement. Antimicrobial phagocytosis is generally carried out by professional phagocytes of the myeloid lineage, such as immature dendritic cells (DCs) and macrophages, and by tissue-resident immune cells.

[0134]

[0259] In some embodiments, due to the instant cell engineering programs disclosed herein, the cells used to engineer for immunotherapeutic use are strongly phagocytic.

[0260] In some embodiments, for the instant cell engineering programs disclosed herein, the cells used to engineer for use in immunotherapy are obtained from whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, or spleen tissue.

[0135]

[0261] In some embodiments, the cells used to engineer for use in immunotherapy are obtained from peripheral blood.

[0262] Within liver MPS, various structural and functional distinctions have been characterized as both stimulatory and inhibitory with a view to generating cells for cancer immunotherapy.

[0136] [Table 1]

[0137]

[0263] In some embodiments, the myeloid cells engineered for use in immunotherapy of the immediate application comprise myeloid cells selected from the group consisting of macrophages, dendritic cells, adipocytes, monocytes, neutrophils, microglia, and astrocytes.

[0138]

[0264] In some embodiments, the myeloid cells engineered for use in immunotherapy are phagocytes, hi some embodiments, the phagocytes are monocytes.

[0265] In some embodiments, the myeloid cells engineered for use in immunotherapy of the immediate application are monocytes, monocyte-derived macrophages, and / or dendritic cells.

[0139]

[0266] In some embodiments, the myeloid cells engineered for use in immunotherapy of the immediate application are monocytes or macrophages.

[0267] In some embodiments, the myeloid cells are obtained from peripheral blood.

[0140]

[0268] In some embodiments, the myeloid cells are labeled with the selectable marker CD14 + CD16 lowIn some embodiments, the myeloid cells are selected via elutriation.

[0141]

[0269] In some embodiments, the myeloid cells are isolated from a leukapheresis column in a subject, hi some embodiments, the subject is the same subject to which the pharmaceutical composition comprising the engineered cells is administered.

[0142]

[0270] In some embodiments, the subject is not the same subject that is administered the pharmaceutical composition comprising the engineered cells.

[0271] In some embodiments, leukapheresis is performed on the same subject once a week to recover more myeloid cells. In some embodiments, leukapheresis is performed on the same subject more than once over an 8-10 day period to recover more myeloid cells. In some embodiments, leukapheresis is performed on the same subject more than twice over a one month period to recover more myeloid cells.

[0143]

[0272] In some embodiments, myeloid cells are isolated from a leukapheresis sample or a peripheral blood sample. In some embodiments, the myeloid cells are progenitor cells. In some embodiments, the myeloid cells are monocyte progenitor cells. In some embodiments, the myeloid cells described herein do not differentiate into end-stage cells or exhibit end-stage cell phenotypes, such as tissue macrophages. In some embodiments, the myeloid cells comprise CD14+ cells. In some embodiments, the myeloid cells do not express CD16. In some embodiments, the myeloid cells express low amounts of CD16. In some embodiments, myeloid cells are preselected for manipulation from a biological sample, such as peripheral blood or an apheresis sample, by selection of CD14+ cells. In some embodiments, selection is performed without contacting or binding with the selected myeloid cells. In some embodiments, myeloid cells are selected prior to manipulation from a biological sample by sorting, e.g., flow cytometry-based cell sorter (FACS). In some embodiments, myeloid cells expressing CD16 are captured with an antibody, and the remaining myeloid cells are collected and used for manipulation. In some embodiments, to obtain myeloid cells, one or more other cell surface molecules, such as CD3, CD8, CD11c, CD40, or CD206, were targeted in addition to CD16 for capture in a negative selection process.

[0144]

[0273] In one aspect, the present disclosure provides a myeloid cell comprising at least one exogenous recombinant nucleic acid encoding a fusion protein. The fusion protein may be a chimeric protein comprising at least one transmembrane domain and an extracellular domain comprising a region capable of binding to a target cell. For example, the chimeric protein may bind to a target, such as a target antigen, antigenic peptide, or ligand of the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the target is a cancer antigen. In some embodiments, the chimeric protein is expressed in the myeloid cell and activates the myeloid cell, overcomes TME-induced inhibitory signals, and acts as an activated pro-inflammatory myeloid cell. In one embodiment, the chimeric protein expressed in the myeloid cell is capable of responding to a second signal other than the target (first signal), where the second signal is a pro-inflammatory signal and an activating signal. In some embodiments, the chimeric protein expressed in the myeloid cell is capable of responding to multiple signals other than the target or first signal. The chimeric protein may be capable of responding to one, two, three, four, five, or more signals in addition to the target or first signal.

[0145]

[0274] In another embodiment, the chimeric protein expressed in myeloid cells specifically binds to a target. In some embodiments, the target is a cancer antigen. Expression of the chimeric protein contributes to targeting specificity to myeloid cells.

[0146]

[0275] In one embodiment, chimeric proteins expressed in myeloid cells can be multiplexed, e.g., with multiple domains for activation and processing of more than one signal or signal type. In some embodiments, activation of multiple domains simultaneously results in an increased effector response to myeloid cells. Myeloid cell effector responses include, for example, enhanced phagocytosis, pro-inflammatory activation, and target cell killing. In some embodiments, chimeric proteins expressed in myeloid cells and capable of multiplexing can bind to more than one ligand, such as a target antigen and a helper molecule. In some embodiments, chimeric proteins can bind to multiple target antigens on cancer cells. In some embodiments, chimeric proteins capable of multiplexing can bind to multiple target antigens on multiple cells. In some embodiments, chimeric proteins can bind to macrophage-monocyte inhibitory targets on cancer cells and generate a stimulatory signal upon contact using a pro-inflammatory domain fused to the intracellular tail, a process referred to as "signal switching." For example, the extracellular domain of the chimeric protein may contain a CD47-binding domain, whereas the chimeric fusion protein lacks the transmembrane and / or intracellular domain of the native CD47 receptor but contains a PI3K recruitment domain in the intracellular region, thereby converting a macrophage-monocyte inhibitory signal from contact with tumor cells into a pro-inflammatory phagocytosis-enhancing signal.

[0147]

[0276] In some embodiments, the chimeric protein is capable of binding multiple units of the expressed chimeric protein, e.g., multimerizing. Multimerization includes the formation of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers. In some embodiments, multimerization can occur through the association of transmembrane regions, extracellular regions, or intracellular regions, or a combination thereof. For example, a chimeric protein containing the collagen domain of the phagocytic receptor MARCO may form a trimer for its effective function. In some embodiments, the chimeric protein is capable of associating with other molecules, e.g., another receptor. For example, the chimeric protein may contain an Fc-alpha transmembrane domain that dimerizes with an Fcγ TM domain, where Fcγ may be an endogenous receptor.

[0148]

[0277] In some embodiments, the chimeric protein that can be multiplexed comprises multiple intracellular domains that can be activated by more than one signal and then activate multiple intracellular signaling molecules.For example, the chimeric protein can comprise a phagocytic receptor domain and a pro-inflammatory domain.For example, the chimeric protein can comprise an FcR signaling domain and an additional phosphorylation domain that recruits procaspase.

[0149] Phagocytosis receptor (PR) subunit of the PFP fusion protein

[0278] Herein, CFP, a phagocytic receptor (PR) fusion protein (PFP), is Recombinant nucleic acids encoding PFPs are provided. PFPs may comprise PR subunits comprising a transmembrane (TM) domain and an intracellular domain (IDC) comprising a PR intracellular signaling domain. In some embodiments, when a recombinant nucleic acid encoding PFP is expressed in a cell, the PFP is functionally integrated into the cell membrane of the cell. In some embodiments, the recombinant nucleic acid comprises a transmembrane domain that is specifically integrated into the membrane of a phagocyte, e.g., a myeloid cell such as a macrophage. Encode the

[0150]

[0279] In some embodiments, suitable PR is selected after screening a library of transmembrane proteins. The PR subunit is fused with a cancer cell-binding antibody at the extracellular domain. In some embodiments, the PR may be fused with one or more additional domains at the intracellular end.

[0151] Intracellular domain of CFP fusion protein

[0280] In some embodiments, the CFP subunit comprises the TM domain of a phagocytic receptor. .

[0152]

[0281] In some embodiments, the CFP subunit comprises the ICD domain of a phagocytic receptor.

[0282] In some embodiments, the phagocytic receptor is a scavenger receptor. While many scavenger receptors cooperate in the detection and uptake of materials, not all receptors exclusively engage in phagocytosis-induced engulfment. The association of specific phagocytic and scavenger receptors can have dramatic effects on downstream immune responses. For example, induction of the A-type scavenger receptor MARCO by 500 nm negatively charged nanoparticles is associated with an anti-inflammatory and tolerogenic immune response. On the other hand, positively charged particles are engulfed by a subset of phagocytic receptors that activate pro-inflammatory pathways such as NLRP3 and / or fibrotic responses. Furthermore, certain scavenger receptor pathways, such as the scavenger receptor expressed by endothelial cells (SREC-1), have been shown to play a role in antigen cross-presentation. Therefore, identifying and understanding potential receptors that can be utilized to enhance macrophage activity and clinical efficacy is a critical step in the CFP development platform.

[0153]

[0283] Non-opsonic receptors naturally and variably expressed by professional phagocytes include lectin-like recognition molecules such as CD169 and CD33, as well as related receptors for sialylated residues. Additionally, phagocytes express Dectin-1 (a receptor for fungal β-glucans with well-defined signaling capabilities), related C-type lectins (e.g., MICL, Dectin-2, Mincle, and DNGR-1), and a group of scavenger receptors. SR-A, MARCO, and CD36 differ in domain structure and have overlapping yet distinct recognition of apoptotic and microbial ligands. CD36-related family members reveal that apoprotein ligands bind to the receptor helix bundle, while their extracellular surface domains form channels that translocate lipids, such as cholesterol, across the membrane bilayer.

[0154] [Table 2-1]

[0155] [Table 2-2]

[0156] [Table 3-1]

[0157] [Table 3-2]

[0158]

[0284] In some embodiments, the recombinant nucleic acid encodes a chimeric antigen receptor for phagocytosis (CAR-P). In some embodiments, the recombinant nucleic acid encodes a phagocytic receptor (PR) fusion protein.

[0159]

[0285] In some embodiments, the ICD of the CFP encoded by the recombinant nucleic acid comprises a domain from a protein selected from the group consisting of TNFR1, CD40, MDA5, lectin, Dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor.

[0160]

[0286] In some embodiments, the ICD is selected from the group consisting of lectin, dectin-1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO (collagenous macrophage receptor, also known as SRA6, SCARA2), CD36 (thrombosponge receptor, also known as scavenger receptor class B, member 3), CD163 (scavenger receptor, cysteine-rich type 1), MSR1, SCARA3, COLEC12 (also known as C-type lectin, SCARA4) , or scavenger receptor with collectin 12), SCARA5, SCARB1, SCARB2, CD68 (SCARD, microsialin), OLR1 (oxidized low-density lipoprotein receptor 1, Lox-1, or C-type lectin domain family 8 member A), SCARF1, SCARF2, SRCRB4D, SSC5D, and CD169 (also known as sialoadhesin receptor, SIGLEC1).

[0161]

[0287] In some embodiments, the recombinant nucleic acid encodes, for example, the intracellular domain of human MARCO. The PR subunit may include an intracellular domain having the 44 amino acid ICD of human MARCO, which has the amino acid sequence: MRNKKILKEDELLSETQQAAFHQIAMEPFEINVPKPKRRNGVNF. In some embodiments, the PR subunit includes a variant that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of MARCO. In some embodiments, the PR includes the transmembrane region of human MARCO.

[0162]

[0288] In some embodiments, the recombinant nucleic acid encodes the intracellular domain of human SRA1. The CFP comprises an intracellular domain having the 50 amino acid ICD of human SRA1, which has the amino acid sequence: MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFK. In some embodiments, the PR subunit comprises a variant that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of human SRA1. The intracellular region of SRA contains phosphorylation sites.

[0163]

[0289] In some embodiments, the CFP comprises the transmembrane region of human SRA1.

[0290] In some embodiments, the recombinant nucleic acid comprises a sequence encoding the intracellular domain of CD36. In some embodiments, the recombinant nucleic acid comprises a sequence encoding the TM domain of CD36. Naturally occurring full-length CD36 has two TM domains and two short intracellular domains, and the extracellular domain of CD36 binds to oxidized LDL. Both intracellular domains contain acylated fatty acid cysteine ​​pairs. It lacks known signaling domains (e.g., kinase, phosphatase, G-protein binding, or scaffolding domains). The N-terminal cytoplasmic domain is extremely short (5-7 amino acid residues) and closely associates with the inner leaflet of the plasma membrane. The carboxy-terminal domain contains 13 amino acids containing a CXCX5K motif, which is homologous to regions of the intracellular domains of CD4 and CD8 known to interact with signaling molecules. The intracellular domain of CD36 is capable of assembling signaling complexes that activate lyn kinase, MAP kinase, and focal adhesion kinase (FAK) and inactivating Src homology 2-containing phosphotyrosine phosphatase (SHP-2). Members of the guanine nucleotide exchange factors (GEFs) have been identified as potential key signaling intermediates.

[0164]

[0291] In some embodiments, the recombinant nucleic acid encodes, for example, the intracellular domain of human SCARA3. The CFP may comprise the intracellular domain of human SCARA3 having the 56 amino acid ICD, which has the amino acid sequence: MKVRSAGGDGDALCVTEEDLAGDDEDMPTFPCTQKGRPGPRCSRCQKNLSLHTSVR. In some embodiments, the CFP comprises a variant that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to the intracellular domain of human SCARA3. In some embodiments, the CFP comprises the TM domain of SCARA3.

[0165]

[0292] In some embodiments, the TM domain of a PR is about 20-30 amino acids in length. In some embodiments, the TM domain comprises multiple transmembrane spans. In some embodiments, the TM domain comprises about 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or more amino acids in length. In some embodiments, the TM domain of an SR is about 20-30 amino acids in length.

[0166]

[0293] Scavenger receptors can occur as homo- or heterodimers. For example, MARCO occurs as a homotrimer. In some embodiments, the scavenger receptor is a monomer. In some embodiments, the scavenger receptor is a homodimer. In some embodiments, the scavenger receptor is a heterodimer. In some embodiments, the scavenger receptor is a homotrimer. In some embodiments, the scavenger receptor is a heterotrimer. In some embodiments, the scavenger receptor is a homotetramer. In some embodiments, the scavenger receptor is a heterotetramer. In some embodiments, the scavenger receptor is a multimer comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more subunits, which may be the same or different.

[0167]

[0294] In some embodiments, the TM domain or ICD domain of the PSP is not derived from FcR, Megf10, Bai1 or MerTK. In some embodiments, the ICD of the RP does not comprise the CD3 zeta intracellular domain.

[0168]

[0295] In some embodiments, the intracellular and transmembrane domains are derived from FcRβ.

[0296] In one embodiment, the recombinant nucleic acid encodes a chimeric antigen receptor with enhanced phagocytosis (CAR-P), which comprises: (a) an extracellular domain comprising an extracellular antigen-binding domain specific for an antigen of a target cell; (b) a transmembrane domain; and (c) a recombinant PSR intracellular signaling domain. A phagocytic scavenger receptor (PSR) fusion protein (PFP) containing a recombinant PSR intracellular signaling domain comprises a first portion derived from a phagocytic receptor and a second portion derived from a non-phagocytic receptor.

[0169]

[0297] In some embodiments, the second moiety is not a PI3K recruitment domain.

[0298] The second portion derived from a non-phagocytic receptor may include an intracellular signaling domain that enhances phagocytosis and / or the inflammatory potential of engineered myeloid cells, such as phagocytes, that express the recombinant nucleic acid. In some embodiments, the second portion derived from a non-phagocytic receptor includes more than one intracellular domain (ICD). In some embodiments, the second portion derived from a non-phagocytic receptor includes a second ICD. In some embodiments, the second portion derived from a non-phagocytic receptor includes a second and a third ICD. In some embodiments, the second portion derived from a non-phagocytic receptor includes a second, a third, and a fourth ICD, and the second portion is encoded by a recombinant nucleic acid. In some embodiments, the intracellular portion includes two, three, four, five, six, seven, or more ICDs. Respective second portions including a second, a third, or a fourth ICD from a non-phagocytic receptor are described below.

[0170] Chimeric antigen receptors enhance intracellular signaling and inflammatory activation

[0299] In one embodiment, the recombinant nucleic acid encodes a second intracellular domain in addition to the phagocytic ICD. This domain confers potent pro-inflammatory immune activation, such as in the case of myeloid cells such as macrophages involved in infection defense. A second intracellular domain (ICD) is fused to the cytoplasmic end of the first phagocytic ICD. The second intracellular domain provides the second signal required to trigger inflammasomes and pro-inflammatory signals. Nod-like receptors (NLRs) are a subset of receptors activated in the innate immune response. They oligomerize to form multiprotein complexes that serve as platforms for recruiting pro-inflammatory caspases and inducing their cleavage and activation. This often leads to the direct activation of ROS, resulting in the severe cell death known as pyroptosis. There are four inflammasome complexes: NLRP1m, NLRP3, IPAF, and AIM2.

[0171]

[0300] The tumor microenvironment (TME) constitutes an immunosuppressive environment. The effects of IL-10, glucocorticoid hormones, apoptotic cells, and immune complexes can interfere with innate immune cell function. Immune cells, including phagocytes, adopt a tolerogenic phenotype. In myeloid cells, such as macrophages, this phenotype, commonly known as the M2 phenotype, differs from the M1 phenotype in that the cells are capable of killing pathogens. Myeloid cells, such as macrophages, exposed to LPS or IFNγ, for example, can polarize to the M1 phenotype, whereas myeloid cells, such as macrophages, exposed to IL-4 or IL-13, can polarize to the M2 phenotype. LPS or IFNγ interacts with Toll-like receptor 4 (TLR4) on the surface of myeloid cells such as macrophages, inducing the Trif and MyD88 pathways and inducing activation of transcription factors IRF3, AP-1, and NFKB, thereby activating genes such as TNF, interferon, CXCL10, NOS2, and IL-12. Similarly, IL-4 and IL-13 bind to IL-4R, activate the Jak / Stat6 pathway, and regulate the expression of genes associated with anti-inflammatory responses (M2 responses), such as CCL17, ARG1, IRF4, IL-10, and SOCS3. The expression of CD14, CD80, and D206, but low expression of CD163, is indicative of the polarization of myeloid cells such as macrophages toward an M1 phenotype.

[0172]

[0301] In some embodiments, the recombinant nucleic acid encodes one or more additional intracellular domains comprising a cytoplasmic domain for an inflammatory response. In some embodiments, expression of a recombinant nucleic acid encoding a phagocytic receptor (PR) fusion protein (PFP) comprising a cytoplasmic domain for an inflammatory response in engineered myeloid cells, such as macrophages, confers a strong pro-inflammatory response resembling an M1 phenotype.

[0173]

[0302] In some embodiments, the cytoplasmic domain for an inflammatory response comprises the intracellular signaling domain of TLR3, TLR4, TLR9, MYD88, TRIF, RIG-1, MDA5, CD40, an IFN receptor, NLRP-1, NLRP-2, NLRP-3, NLRP-4, NLRP-5, NLRP-6, NLRP-7, NLRP-8, NLRP-9, NLRP-10, NLRP-11, NLRP-12, NLRP-13, NLRP-14, NOD1, NOD2, Pyrin, AIM2, NLRC4 and / or CD40.

[0174]

[0303] In some embodiments, the phagocytic scavenger receptor (PR) fusion protein (PFP) comprises a pro-inflammatory cytoplasmic domain for activation of the IL-1 signaling cascade.

[0175]

[0304] In some embodiments, the cytoplasmic portion of the chimeric receptor (e.g., phagocytic receptor (PR) fusion protein (PFP)) comprises a cytoplasmic domain from a Toll-like receptor, such as the intracellular signaling domain of Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), Toll-like receptor 8 (TLR8), or Toll-like receptor 9 (TLR9).

[0176]

[0305] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from interleukin-1 receptor-associated kinase (IRAK1).

[0306] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from the differentiation primary response protein (MYD88).

[0177]

[0307] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from myelin and lymphocyte protein (MAL).

[0308] In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region derived from the retinoic acid-inducible gene (RIG-1).

[0178]

[0309] In some embodiments, the cytoplasmic portion of the CFP comprises the cytoplasmic domain of any one of MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, or IRAK1.

[0179]

[0310] In some embodiments, the recombinant CFP intracellular signaling domain comprises a first portion derived from a phagocyte receptor and a second portion derived from a non-phagocytic receptor, wherein the second portion derived from the non-phagocytic receptor comprises a phosphorylation site. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for autophosphorylation. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for phosphorylation by a Src family kinase. In some embodiments, the phosphorylation site comprises an amino acid sequence capable of binding to the SH2 domain of a kinase upon phosphorylation. In some embodiments, a receptor tyrosine kinase domain is fused to the cytoplasmic end of PFP in addition to the first cytoplasmic portion.

[0180]

[0311] In some embodiments, the phosphorylation is tyrosine phosphorylation.

[0312] In some embodiments, the second intracellular domain is an immunoreceptor tyrosine-based activation motif (ITAM), which is present in mammalian α and β immunoglobulin proteins, TCRγ receptors, FCRγ receptor subunits, CD3 chain receptors, and NFAT activating molecules.

[0181]

[0313] In some embodiments, the PFP intracellular domain comprises one ITAM motif. In some embodiments, the PFP intracellular domain comprises more than one ITAM motif. In some embodiments, the PFP intracellular domain comprises two or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises three or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises four or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises five or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises six or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises seven or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises eight or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises nine or more ITAM motifs. In some embodiments, the PFP intracellular domain comprises ten or more ITAM motifs.

[0182]

[0314] In some embodiments, one or more domains of the first phagocytic ICD comprise a mutation.

[0315] In some embodiments, one or more domains of the second ICD comprise mutations that enhance a kinase binding domain, create a phosphorylation site, create an SH2 docking site, or a combination thereof.

[0183] Co-expression of inflammatory genes

[0316] In one embodiment, the recombinant nucleic acid comprises a pro-inflammatory polypeptide that is co-expressed with PFP in the engineered cells. The pro-inflammatory gene comprises a coding sequence of a gene. In some embodiments, the pro-inflammatory gene is a cytokine. Examples include, but are not limited to, TNF-α, IL-1α, IL-1, IL-6, CSF, GMCSF, or IL-12 or an interferon.

[0184]

[0317] The recombinant nucleic acid encoding the pro-inflammatory gene can be monocistronic, wherein the two coding sequences, (a) the PSP and (b) the pro-inflammatory gene, are cleaved post-transcriptionally or post-translationally for independent expression.

[0185]

[0318] In some embodiments, the two coding sequences include a self-cleaving domain, for example, encoding a P2A sequence.

[0319] In some embodiments, the two coding regions are separated by an IRES site.

[0186]

[0320] In some embodiments, the two coding sequences are encoded by a bicistronic genetic element. The coding regions for (a) PSP and (b) the pro-inflammatory gene can be unidirectional, each under separate regulatory control. In some embodiments, both coding regions are bidirectional, driving in opposite directions. Each coding sequence is under separate regulatory control.

[0187]

[0321] Co-expression of pro-inflammatory genes is designed to provide a strong inflammatory stimulus for myeloid cells such as macrophages, activating the surrounding tissues in inflammation. Integrin Activation Domain

[0322] Cell-cell and cell-substratum adhesion are mediated by integrative responses to diverse protein ligands. Although adhesive interactions are mediated by the binding of integrin extracellular domains, cellular regulation of these interactions and their translation into dynamic cellular responses, such as cell spreading or migration, requires the integrin cytoplasmic tails. These short tails bind intracellular ligands that connect the receptor to signaling pathways and cytoskeletal networks (Calderwood DA, 2004, Integrin Activation, Journal of Cell Science 117, 657-666, incorporated herein in its entirety). Integrins are heterodimeric adhesion receptors formed by the non-covalent association of α and β subunits. Each subunit is a type I transmembrane glycoprotein with a relatively large extracellular domain and, except for the β4 subunit, a short cytoplasmic tail. Individual integrin family members have the ability to recognize multiple ligands. Integrins bind to many extracellular groups. Integrins can bind to matrix proteins (bone matrix proteins, collagen, fibronectin, fibrinogen, laminin, thrombospondin, vitronectin, and von Willebrand factor), representing their primary function in cell adhesion to the extracellular matrix. Many "counter-receptors" are ligands, representing the role of integrins in mediating cell-cell interactions. Integrins undergo conformational changes that increase ligand affinity.

[0188]

[0323] The integrin β2 subfamily consists of four distinct receptors: α M β2 (CD11b / CD18, Mac-1, CR3, Mo-1), α L β2 (CD11a / CD18, LFA-1), α X β2 (CD11c / CD18), and α D β2 (CD11d / CD18). These leukocyte integrins are involved in virtually all aspects of leukocyte function, including immune responses, adhesion to and transmigration from the endothelium, phagocytosis of pathogens, and leukocyte activation.

[0189]

[0324] All β2 integrin α subunits contain an inserted region of approximately 200 amino acids, called the I or A domain. Highly conserved I domains are found in several other integrin α subunits and in other proteins, such as certain coagulation and complement proteins. I domains mediate protein-protein interactions, and in integrins, they are integrally responsible for binding protein ligands. While the I domain governs the ligand-binding function of these integrins, other regions of the α subunit influence ligand recognition. For example, α M In β2, mAb (OKM1) recognizes an epitope outside the I domain, whereas in α M The α subunit inhibits ligand binding; L The EF-hand regions of β2 and α2β1, as well as I-domain integrins in their α subunits, contribute to ligand recognition. MThe α subunit, and possibly other α subunits, contain lectin-like domains involved in binding of non-protein ligands, the occupancy of which may regulate the function of the I domain.

[0190]

[0325] When integrins lack enzymatic activity, signaling is instead induced by the assembly of signaling complexes on the cytoplasmic surface of the cell membrane. The formation of these complexes is achieved in two ways: first, by receptor clustering, which increases the avidity of molecular interactions and thereby increases the on-rate for effector molecule binding; and second, by inducing conformational changes in the receptor that create or expose effector-binding sites. Within the ECM, integrins have the ability to bind fibronectin, laminin, collagen, tenascin, vitronectin, and thrombospondin. Clusters of integrin / ECM interactions form focal adhesions, concentrating cytoskeletal components and intracellular signaling molecules. The cytoplasmic tails of integrins serve as binding sites for α-actinin and talin, which then recruit vinculin, a protein involved in binding F-actin to membranes. Talin is activated by kinases such as protein kinase C (PKCα).

[0191]

[0326] Integrins are activated by selectins. Leukocytes express L-selectin, activated platelets express P-selectin, and activated endothelial cells express E- and P-selectin. P-selectin-mediated adhesion allows chemokine- or platelet-activating factor-induced activation of β2 integrins and stabilizes adhesion. It also promotes the release of chemokines from adherent leukocytes. The cytoplasmic domain of P-selectin glycoprotein ligand 1 forms a constitutive complex with Nef-related factor 1. After P-selectin binding, Src kinase phosphorylates Nef-related factor 1, recruiting the phosphoinositide-3-OH kinase p85-p110δ heterodimer, leading to leukocyte integrin activation. E-selectin ligand transduces signals that also affect β2 integrin function. Selectins induce activation of Src family kinases. SFKs activated by selectin binding are immunoreceptor tyrosine-based activators of the cytoplasmic domains of DAP12 and FcRγ. CD44 phosphorylates the ITAM (Integrins-Transcriptional Amino Acid Motif) of integrins. In some respects, CD44 is sufficient to transduce signals from E-selectin. CD44 induces inside-out signaling of integrins. The final common step in integrin activation is the binding of talin to the cytoplasmic tail of the β subunit. Kindlin, another group of cytoplasmic adaptors, binds to a different region of the integrin β tail. Kindlin increases the clustering of talin-activated integrins. Kindlin is responsive to selectin signaling, but is found predominantly in hematopoietic stem cells such as neutrophils. Selectin signaling and signaling upon integrin activation by chemokine components share components including SFK, Syk, and SLP-76.

[0192]

[0327] In some embodiments, the intracellular domain of the recombinant CFP comprises an integrin activation domain. The integrin activation domain comprises the intracellular domain of a selectin, such as P-selectin, L-selectin, or E-selectin.

[0193]

[0328] In some embodiments, the intracellular domain of the recombinant CFP comprises the integrin activation domain of laminin.

[0329] In some embodiments, the intracellular domain of the recombinant CFP comprises an integrin activation domain for activation of talin.

[0194]

[0330] In some embodiments, the intracellular domain of the recombinant CFP comprises an integrin activation domain fused to the cytoplasmic end of a phagocytic receptor ICD domain. Chimeric receptors for enhancing antigen cross-presentation

[0331] In some embodiments, the recombinant nucleic acid can enable cross-presentation of antigens These domains encode antigens. Generally, MHC class I molecules present self- or pathogen-derived antigens synthesized within cells, whereas exogenous antigens derived from endocytic uptake are loaded onto MHC class II molecules for presentation to CD4+ T cells. MHC I is limited to the presentation of endogenous antigens, and peptides are generated by proteosomes. However, in some cases, DCs can process exogenous antigens into the MHC-I pathway for presentation to CD8+ T cells. This is called antigen cross-presentation. Soluble or exogenous antigen components may be degraded by lysosomal proteases in the vacuole and cross-presented by DCs instead of through the endocytic pathway. In some cases, chaperones such as heat shock protein 90 (Hsp90) have been shown to aid in antigen cross-presentation by specific APCs. HSP-peptide complexes are known to be internalized by a different set of receptors compared to free polypeptides. These receptors are from the scavenger receptor family and include LOX-1, SREC-I / SCARF-I, and FEEL1 / stabilin-1. Both SREC-I and LOX-1 have been shown to mediate cross-presentation of molecular chaperone-bound antigens, leading to activation of CD8+ T lymphocytes.

[0195]

[0332] SREC-1 (scavenger receptor expressed by endothelial cells) does not show significant homology to other types of scavenger receptors but has a unique domain structure. It contains 10 repeats of an EGF-like cysteine-rich motif in its extracellular domain. Recently, the structure of SREC-1 has been shown to be similar to that of a transmembrane protein with 16 EGF-like repeats encoded by the Caenorhabditis elegans gene ced-I, which functions as a cell surface phagocytic receptor that recognizes apoptotic cells.

[0196]

[0333] Cross-presentation of cancer antigens via the class I MHC pathway results in enhanced CD8+ T cell responses, which are associated with cytotoxicity and therefore beneficial for tumor regression. In embodiments, the intracellular domain of PFP comprises a SREC1 intracellular domain. In some embodiments, the intracellular domain of PFP comprises a SRECII intracellular domain.

[0197]

[0334] In some embodiments, the CFP comprises an intracellular domain that includes a PSR intracellular signaling domain from SREC1 or SRECII.

[0335] In some embodiments, the CFP comprises (i) a transmembrane domain and (ii) an intracellular domain comprising a CFP intracellular signaling domain derived from SREC1 or SRECII.

[0198]

[0336] In some embodiments, the CFP comprises (i) a transmembrane domain, (ii) an intracellular domain comprising an intracellular signaling domain, and (iii) an extracellular domain derived from SREC1 or SRECII.

[0199] Transmembrane domain of PFP fusion protein

[0337] In some embodiments, the TM encoded by the recombinant nucleic acid is a scavenger. In some embodiments, the TM can be a TM domain of, or derived from, any one or more of: lectin, dectin-1, mannose receptor (CD206), SRA1, MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, SRCRB4D, SSC5D, and CD169.

[0200]

[0338] In some embodiments, the TM domain is about 20-30 amino acids in length. The TM domain of an SR is about 20-30 amino acids in length.

[0339] In some embodiments, the TM domain or ICD domain of the CFP is not derived from Megf10, Bai1, or MerTK. In some embodiments, the ICD of the CFP does not include the CD3ζ intracellular domain.

[0201]

[0340] In some embodiments, the TM is derived from the same phagocytic receptor as the ICD.

[0341] In some embodiments, the TM region is derived from a cell membrane protein. The TM may be selected from an Fc receptor (FcR). In some embodiments, nucleic acid sequences encoding domains from specific FcRs are used for cell-specific expression of recombinant constructs. An FCR-alpha region containing the TM domain may be used for specific expression of the construct in myeloid cells such as macrophages. FcRα recombinant proteins may be expressed in adipocytes.

[0202]

[0342] In some embodiments, the PFP comprises a TM of FcRβ.

[0343] In some embodiments, PFP comprises both an FcRβ and an ICD domain, hi some embodiments, PFP comprises both an FcRα and an ICD domain.

[0203]

[0344] In some embodiments, the TM domain is derived from CD8.

[0345] In some embodiments, the TM is derived from CD2.

[0346] In some embodiments, the TM is derived from FcRα.

[0204] Extracellular domain of PFP fusion protein

[0347] In some embodiments, the extracellular domain of the PFP fusion proteins provided herein The extracellular domain comprises an antigen-binding domain that binds to one or more targets. The binding target can be an antigen or a ligand. For example, the binding target can be an antigen on a target cell. In some embodiments, the target-binding domain is specific for the target. In some embodiments, the extracellular domain is an antibody, or an intrabody, peptibody, nanobody, or single-domain antibody. The antibody may comprise an antigen-binding domain selected from a SMIP, a SMIP, and a multispecific antibody.

[0205]

[0348] In some embodiments, the antibody fragment comprises a portion of an intact antibody, e.g., the antigen-binding region or variable region of the intact antibody. In a further aspect of the invention, the anti-HIV antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, diabodies, linear antibodies, multispecific antibodies formed from antibody fragments and scFv fragments, as well as other fragments described below. In another embodiment, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, or other antibody class or isotype described herein. (See, e.g., Hudson et al., Nat. Med. 9:129-134 (2003); Pluckthin, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); WO 93 / 01161; and U.S. Pat. Nos. 5,571,894, 5,869,046, 6,248,516, and 5,587,458.) A full-length, intact, or whole antibody is an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0206]

[0349] Fv is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment comprises a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops from each of the H and L chains) that contribute antigen-binding amino acid residues and confer antigen-binding specificity to the antibody. However, even a single variable region (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0207]

[0350] Single-chain Fvs (sFvs or scFvs) are VFs linked in a single polypeptide chain. H Antibody domains and V L sFv polypeptides are antibody fragments containing the V H Domains and V L It may further comprise a polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding (see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.) sFvs can be used in chimeric antigen receptors (CARs).

[0208]

[0351] Diabodies are V domains that are engineered to achieve interchain, rather than intrachain, pairing of V domains, resulting in bivalent fragments. H Domains and V L A bispecific diabody is a small antibody fragment prepared by constructing an sFv fragment with a short linker (approximately 5-10 residues) between the V domains of two antibodies. H Domains and V LIt is a heterodimer of two crossover sFv fragments in which the domains are present on different polypeptide chains (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0209]

[0352] Domain antibodies (dAbs) that can be produced in a fully human form range from about 11 kDa to about 15 kDa. dAbs are the smallest known antigen-binding fragments of antibodies, ranging from 0.1 kDa to 0.5 kDa. H and V L ) are robust variable regions. They are highly expressed in microbial cell culture and exhibit favorable biophysical properties, including, for example, but not limited to, solubility and temperature stability, making them well suited for selection and affinity maturation by in vitro selection systems such as phage display. dAbs are biologically active as monomers, and their small size and inherent stability allow them to be formatted into larger molecules to generate drugs with extended serum half-lives or other pharmacological activities. (See, e.g., WO9425591 and US20030130496.)

[0353] Fv and sFv are the only species with intact binding sites devoid of constant regions. Therefore, they are suitable for reducing nonspecific binding when used in vivo. sFv fusion proteins can be constructed to fuse an effector protein to either the amino or carboxy terminus of an sFv. Antibody fragments can also be "linear antibodies." (See, e.g., U.S. Pat. No. 5,641,870.) Such linear antibody fragments can be monospecific or bispecific.

[0210]

[0354] In some embodiments, the extracellular domain comprises a Fab binding domain, hi yet other such embodiments, the extracellular domain comprises an scFv.

[0355] In some embodiments, the chimeric antigen receptor is an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a V H Domain, V L Domains, Single Domain Antibodies (sdAbs), VNAR Domains, and V HH In some embodiments, the antibody comprises an extracellular antigen-binding domain derived from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a V domain, a bispecific antibody, a diabody, or a functional fragment of any of these. H Domain, V L Domains, Single Domain Antibodies (sdAbs), VNAR Domains, and V HH The domain, bispecific antibody, diabody, or functional fragment of any of these specifically binds to one or more antigens.

[0211]

[0356] In some embodiments, the antigen is a cancer antigen and the target cell is a target cancer cell. In some embodiments, the antigen on the target cell is CD3, CD4, CD5, CD7, CD19, CCR2, CCR4, CD30, CD37, TCRB1 / 2, TCRαβ, TCRαδ, CD22, HER2 (ERBB2 / neu), mesothelin, PSCA, CD123, CD30, CD171, CD138, CS-1, CLECL1, CD33, CD79b, EGFRvIII, GD2, GD3, BCMA, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3 (CD276), KIT (CD117), CD213A2, IL-1 Selected from the group consisting of IRa, PRSS21, VEGFR2, CD24, MUC-16, PDGFR-β, SSEA-4, CD20, MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, FAP, EphA2, GM3, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CD97, CD179a, ALK, and IGLL1.

[0212]

[0357] In some embodiments, the target antigen is an autoimmune antigen. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a B cell that produces autoantibodies. In some embodiments, the target antigen is Dsg1 or Dsg3.

[0213]

[0358] Various cancer antigen targets can be selected from cancer antigens known to those skilled in the art.Depending on the cancer and cell type involved, cancer antigens are mutated natural proteins.Antigen binding domains are screened for specificity to mutated / cancer antigens, not natural antigens.

[0214]

[0359] In some embodiments, for example, the cancer antigens to target cancer cells are mutant / cancer antigens: MUC16, CCAT2, CTAG1A, CTAG1B, MAGEA1, MAGEA 2, MAGEA3, MAGEA4, MAGEA6, PRAME, PCA3, MAGEC1, MAGEC2, MAGED2, AFP, MAGEA8, MAGE9, MAGEA11, MAGEA12, IL13RA2, PLAC1, SDCCAG8, LSP1, CT45A1, CT45A2, CT45A3, CT45A5, CT45A6, CT45A8, CT45A10, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A8, CT47A9, CT47A10, CT47A11, CT47A12, CT47B1, SAGE1, and CT55.

[0215]

[0360] In some embodiments, for example, the cancer antigen of the target cancer cells may be one or more of the following mutant / cancer antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30, CXCR4, CD45, CD56, and the cancer is a T-cell lymphoma.

[0216]

[0361] In some embodiments, for example, the cancer antigen of the target cancer cells may be one or more of the following mutated / cancer antigens: IDH1, ATRX, PRL3, or ETBR, and the cancer is glioblastoma.

[0217]

[0362] In some embodiments, for example, the cancer antigen of the target cancer cells may be one or more of the following mutant / cancer antigens: CA125, β-hCG, urinary gonadotropin fragment, AFP, CEA, SCC, inhibin, or extradiol, and the cancer is ovarian cancer.

[0218]

[0363] In some embodiments, the cancer antigen of the target cancer cell can be CD5.

[0364] In some embodiments, the cancer antigen of the target cancer cells can be HER2.

[0365] In some embodiments, the cancer antigen of the target cancer cells can be EGFR variant III.

[0219]

[0366] In some embodiments, the cancer antigen of the target cancer cell can be CD19.

[0367] In some embodiments, the SR subunit region comprises the extracellular domain (ECD) of a scavenger receptor. In some embodiments, the ECD of the scavenger receptor comprises an ECD domain of an SR, which includes an ICD domain and a TM domain. In some embodiments, the target antigen is an SR ligand on a cancer cell, for example, any one of the ligand components in Table 2 or Table 3. In some embodiments, the SR-ECD contributes to the binding of phagocytes to target cells, and is then activated to activate phagocytosis of the target cells.

[0220]

[0368] In some embodiments, the CFP comprises the ECD of a scavenger receptor or a portion thereof. In some embodiments, the CFP comprises the ICD of a scavenger receptor or a portion thereof. In some embodiments, the CFP comprises the TM domain of a scavenger receptor. In some embodiments, the ECD encoded by the recombinant nucleic acid comprises a domain selected from the group consisting of lectin, Dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169. The extracellular domains of most scavenger receptors comprise scavenger receptors with broad binding specificities that can be used to distinguish between self and non-self in nonspecific, antibody-independent recognition of foreign substances. The type I and type II class A scavenger receptors (SR-AI1 and SR-AII) are trimeric membrane glycoproteins with a small NH2-terminal intracellular domain and an extracellular portion containing a short spacer domain, an α-helical coiled-coil domain, and a triple-helical collagen domain. Type I receptors contain an additional cysteine-rich COOH-terminal (SRCR) domain. These receptors are present on myeloid cells, such as macrophages, in various tissues throughout the body and exhibit very broad ligand-binding specificity. They bind a wide variety of polyanions, including chemically modified proteins, such as modified LDL, and are involved in cholesterol deposition during atherogenesis. They may also play a role in macrophage-associated host defense and cell adhesion processes in inflammatory conditions.

[0221]

[0369] In some embodiments, the SR ECD is designed to bind to pro-apoptotic cells. In some embodiments, the scavenger receptor ECD comprises a binding domain for a cell surface molecule of a cancer cell or an infected cell.

[0222]

[0370] In some embodiments, the extracellular domain of the PR subunit is linked by a linker to a target cell binding domain, e.g., an antibody or portion thereof, specific for a cancer antigen.

[0223]

[0371] In some embodiments, the extracellular antigen-binding domain comprises one antigen-binding domain. In some embodiments, the extracellular antigen-binding domain comprises more than one binding domain. In some embodiments, the binding domain is an scFv. Figure 2 shows a schematic diagram of an embodiment in which PFP targets a single target (left) or multiple targets (right) on cancer cells. One or more scFvs are fused to a recombinant PR at the extracellular domain. In some embodiments, the scFv fraction and the extracellular domain of the PR are linked via a linker.

[0224]

[0372] In some embodiments, the ECD antigen binding domain is capable of binding to an intracellular antigen. In some embodiments, the intracellular antigen is a cancer antigen.

[0373] In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 1000 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 500 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 450 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 400 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 350 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 250 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 200 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 100 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity in the range of 200 nM to 1000 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity in the range of 300 nM to 1.5 nM. In some embodiments, the antigen binding domain binds to the target ligand with an affinity of >200 nM, >300 nM, or >500 nM.

[0225]

[0374] In some embodiments, the extracellular antigen-binding domain binds to a target ligand, where the target ligand is a T cell, and its binding characteristics are such that the target T cell is not triggered to activate T cell-mediated lysis of the engineered cell. In some embodiments, binding of the TCR to the ligand on the engineered cell is avoided, bypassed, or inhibited.

[0226] Linker

[0375] The linker may be any of the polypeptide or peptide domains of the present disclosure. The PFP fusion proteins described herein can include one or more linkers. For example, one or more domains of the PFP fusion protein can be used to and subunits may be directly fused to another domain or subunit, or may be linked to another domain or subunit via a linker. In some embodiments, an extracellular antigen-binding domain comprising an antibody specific for an antigen on a target cell, a portion of an antibody capable of specifically binding to an antigen on a target cell, or an scFv specific for an antigen on a target cell is linked to a TM domain or other extracellular domain by a linker. In some embodiments where more than one scFv is present in the extracellular antigen-binding domain, the more than one scFv are linked to each other by a linker.

[0227]

[0376] In some embodiments, the linker is a short peptide sequence.

[0377] The linker can be a single, covalent bond, or a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or amino acid-based. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.).

[0228]

[0378] In some embodiments, the linker is a single amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, a group, a polymer, or a chemical moiety. In some embodiments, the linker is about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length. In some embodiments, the linker is a stretch of glycine residues and one or more serine residues. Other preferred amino acids for short peptide linkers include, but are not limited to, threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), alanine (Ala), arginine (Arg), phenylalanine (Phe), and glutamic acid (Glu). Among these, Pro, Thr, and Gln are frequently used amino acids in natural linkers. Pro is a unique amino acid with a cyclic side chain that induces highly restricted conformations. Pro-rich sequences have been used as interdomain linkers, including the linker between the lipoyl and E3-binding domains of pyruvate dehydrogenase (GA2PA3PAKQEA3PAPA2KAEAPA3PA2KA). For the purposes of this disclosure, empirical linkers can be flexible linkers, rigid linkers, and cleavable linkers. Sequences such as (G4S)x (where x is multiple copies of the moiety, designated 1, 2, 3, 4, etc.) comprise flexible linker sequences. Other flexible sequences used herein include several repeats of glycine, such as (Gly)6 or (Gly)8. On the other hand, rigid linkers can be used, for example, the linker (EAAAK)x (where x is an integer, 1, 2, 3, 4, etc.) produces a rigid linker.A variety of linker lengths and interdomain or intersubunit flexibility can be used in the fusion proteins provided herein to achieve optimal length, ranging from highly flexible linkers of the form (GGGS)n, (GGGGS)n, and (G)n, to more rigid linkers of the form (EAAAK)n, (SGGS)n, SGSETPGTSESATPES (see, e.g., Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014;32(6):577-82; the entire contents of which are incorporated herein by reference), and (XP)n. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, where n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGGGGSG. In some embodiments, the linker comprises the amino acid sequence GSGS.

[0229]

[0379] In some embodiments, the linker is flexible. In some embodiments, the linker comprises a hinge region. When included, such a spacer or linker domain can position the binding domain away from the host cell surface, further enabling proper cell-to-cell contact, binding, and activation. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, the selected binding epitope, and the size and affinity of the binding domain. In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In some embodiments, the linker or spacer used herein comprises an IgG4 hinge region having the sequence: ESKYGPPCPPCP. In some embodiments, the hinge region comprises a hinge or spacer comprising a sequence present in the extracellular region of a type 1 membrane protein, such as CD8a, CD4, CD28, and CD7, which may be wild-type or a variant thereof. In some embodiments, the extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof. In some embodiments, the spacer or linker may be further modified by post-translational modification, such as glycosylation.

[0230]

[0380] In some embodiments, the extracellular spacer domain may comprise the stalk region of a type II C lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D. In yet further embodiments, the extracellular spacer domain may be derived from the scavenger receptor MERTK.

[0231]

[0381] In some embodiments, the linker comprises at least 2 or at least 3 amino acids. In some embodiments, the linker comprises 4 amino acids. In some embodiments, the linker comprises 5 amino acids. In some embodiments, the linker comprises 6 amino acids. In some embodiments, the linker comprises 7 amino acids. In some embodiments, the linker comprises 8 amino acids. In some embodiments, the linker comprises 9 amino acids. In some embodiments, the linker comprises 8 amino acids. In some embodiments, the linker comprises 10 amino acids. In some embodiments, the linker comprises more than 10 amino acids. In some embodiments, the linker comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, there are 12 or more amino acids in the linker. In some embodiments, there are 14 or more amino acids in the linker. In some embodiments, there are 15 or more amino acids in the linker.

[0232] Other fusion proteins for enhancing phagocytosis

[0382] In one aspect of the present disclosure, myeloid cells are primarily inhibited by blocking inhibitory signals. Recombinant nucleic acids are prepared that encode one or more chimeric receptors that enhance phagocytosis in cells, such as macrophages. Particularly in tumor environments, myeloid cells, such as macrophages, face phagocytosis-suppressing or inhibitory signals, such as CD47-mediated antiphagocytic activity, against target cells, such as cancer cells. Chimeric receptors are generated that block CD47 signaling when expressed in phagocytes.

[0233]

[0383] In some embodiments, other CAR fusion proteins can be designed for expression in phagocytes that can enhance phagocytosis. In one embodiment, the chimeric antigen receptor (CAR) described herein Compositions are provided that include a recombinant nucleic acid encoding a (CAR) fusion protein (CFP), wherein the CFP comprises (a) a subunit comprising (i) an extracellular domain and (ii) a transmembrane domain, and (b) an extracellular antigen-binding domain specific to CD47 on a target cell, wherein the extracellular domain and the extracellular antigen-binding domain of the subunit are operably linked; and the subunit does not contain a functional intracellular domain of an endogenous receptor that binds to CD47 or an intracellular domain that activates phosphatase. In some embodiments, the extracellular antigen-binding domain is derived from signal-regulatory protein alpha (SIRPα). In some embodiments, the extracellular antigen-binding domain is derived from signal-regulatory protein alpha (SIRPβ). In some embodiments, the transmembrane domain is derived from SIRPα. In some embodiments, the transmembrane domain is derived from SIRPβ.

[0234]

[0384] In some embodiments, additional CAR fusion protein (CFP) can be co-transfected with the above-mentioned recombinant PFP. In some embodiments, the scavenger receptor intracellular domain comprises a second intracellular domain that comprises a signal transduction domain that activates phagocytosis; or a pro-inflammatory domain at the cytoplasmic end, and these are operably linked. The signal transduction domain that activates phagocytosis is derived from a receptor selected from the group consisting of the receptors listed in Table 2.

[0235]

[0385] In some embodiments, the intracellular domain having the phagocytosis signaling domain comprises a domain having one or more immunoreceptor tyrosine-based activation motif (ITAM) motifs. ITAMs are conserved sequences present in the cytoplasmic tails of several receptors of the immune system, such as T cell receptors, immunoglobulins (Ig), and FcRs. These are located at a defined interval (YXXL / IX 6-8ITAMs share...

Claims

1. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; the antigen is not a CD19 or CD22 antigen; when the CFP binds to the antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 20% compared to cells not expressing the CFP; and the killing or phagocytic activity is measured by flow cytometry.

2. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and when the CFP binds to an antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 10-fold compared to cells not expressing the CFP, wherein the killing or phagocytic activity is calculated as phagocytosis per 100 cells expressing the CFP.

3. 3. The composition of claim 1 or 2, wherein the intracellular signaling domain is derived from a phagocytic or tethering receptor, or wherein the intracellular signaling domain comprises a phagocytic activation domain.

4. The composition of any one of claims 1 to 3, wherein the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR, or Bai1.

5. 5. The composition of any one of claims 1 to 4, wherein the intracellular signaling domain is derived from a receptor selected from the group consisting of the receptors listed in Table 2.

6. The composition of any one of claims 1 to 5, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain.

7. The composition of claim 6 , wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

8. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; The composition, wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, and Bai1.

9. The composition of claim 8, wherein when the CFP binds to an antigen on the target cell, the killing activity of cells expressing the CFP increases by at least 20% compared to cells not expressing the CFP; the killing or phagocytic activity of cells expressing the CFP increases by at least 20% compared to cells not expressing the CFP; and the killing or phagocytic activity is measured by flow cytometry.

10. The intracellular signaling domain is selected from the group consisting of lectin, dectin 1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, and CD209.

10. The composition of claim 8 or 9, wherein the composition is derived from a protein selected from the group consisting of: 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, TNFR1, MDA5, CD40, and CD169.

11. The composition of any one of claims 8 to 10, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain.

12. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is selected from the group consisting of 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 SRCRB4. D, SSC5D, 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, TNFR1, MDA5, CD40, and CD169.

13. The composition of claim 12, wherein when the CFP binds to an antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 55% compared to cells that do not express the CFP.

14. The composition of claim 12 or 13, wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, or Bai1.

15. The composition of any one of claims 12 to 14, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain.

16. 16. The composition of claim 15, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

17. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

18. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a signaling domain derived from the TFNR1 receptor.

19. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a signaling domain derived from the CD40 receptor.

20. 1. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (a) (i) a transmembrane domain, and (ii) an intracellular domain containing an intracellular signaling domain a phagocytic or tethering receptor (PR) subunit, comprising: (b) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain comprises a signaling domain derived from MDA5.

21. The composition of any one of claims 17 to 20, wherein when the CFP binds to an antigen on the target cell, the killing or phagocytic activity of cells expressing the CFP is increased by at least 20% compared to cells not expressing the CFP, and the killing or phagocytic activity is measured by flow cytometry.

22. The composition of any one of claims 17 to 21, wherein the intracellular signaling domain is derived from a phagocytic receptor.

23. 23. The composition of any one of claims 17 to 22, wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR, or Bai1.

24. The intracellular signaling domain is selected from the group consisting of lectin, dectin 1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, and RAG.

24. The composition of any one of claims 17 to 23, wherein the phagocytic receptor is derived from a phagocytic receptor selected from the group consisting of E, 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, TNFR1, MDA5, CD40, and CD169.

25. The composition of any one of claims 1 to 15, wherein the intracellular signaling domain comprises a PI3K recruitment domain.

26. 2. The composition of any one of the preceding claims, wherein the intracellular signaling domain comprises an FcRγ intracellular domain.

27. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in N to C order, an FcRγ intracellular domain and a CD40 receptor intracellular signaling domain.

28. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in C to N order, an FcRγ intracellular domain and a CD40 receptor intracellular signaling domain.

29. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in N to C order, an FcRγ intracellular domain and a TNFR1 receptor intracellular signaling domain.

30. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in C to N order, an FcRγ intracellular domain and a TNFR1 receptor intracellular signaling domain.

31. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in N to C order, an FcRγ intracellular domain and an MDA5 signaling domain.

32. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in C to N order, an FcRγ intracellular domain and an MDA5 signaling domain.

33. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in N to C order, an FcRγ intracellular domain and a PI3K recruitment domain.

34. 27. The composition of claim 26, wherein the intracellular signaling domain comprises, in C to N order, an FcRγ intracellular domain and a PI3K recruitment domain.

35. 2. The composition of claim 1, wherein the CFP is functionally integrated into the cell membrane of a cell when expressed in the cell.

36. The cells expressing the CFP express the antigen more effectively than the cells not expressing the CFP.

10. The composition of any one of the preceding claims, which exhibits increased phagocytosis of target cells expressing it.

37. The composition of claim 36, wherein cells expressing the CFP exhibit at least a 1.1-fold increase in phagocytosis of target cells expressing the antigen compared to cells not expressing the CFP, wherein phagocytosis is measured by flow cytometry.

38. 10. The composition of claim 1, wherein cells expressing the CFP exhibit at least a 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold increase in phagocytosis of target cells expressing the antigen compared to cells not expressing the CFP, wherein phagocytosis is measured by flow cytometry.

39. 10. The composition of any one of the preceding claims, wherein the target cells expressing the antigen are cancer cells.

40. 10. The composition of any one of the preceding claims, wherein the target cells expressing the antigen are at least 0.8 microns in diameter.

41. 10. The composition of any one of the preceding claims, wherein the intracellular signaling domain is derived from a scavenger receptor.

42. 2. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased cytokine production compared to cells not expressing the CFP.

43. 43. The composition of claim 42, wherein the cytokine is selected from the group consisting of IL-1, IL3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferon, and combinations thereof.

44. 2. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased effector activity compared to cells not expressing the CFP.

45. 10. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased cross-presentation compared to cells not expressing said CFP.

46. 2. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of MHC class II proteins compared to cells not expressing the CFP.

47. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of CD80 compared to cells not expressing the CFP.

48. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of CD86 compared to cells not expressing said CFP.

49. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of MHC class I proteins compared to cells not expressing the CFP.

50. 2. The method of claim 1, wherein cells expressing the CFP exhibit increased expression of TRAIL / TNF family death receptors compared to cells not expressing the CFP. composition.

51. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of B7-H2 compared to cells not expressing said CFP.

52. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of LIGHT compared to cells not expressing said CFP.

53. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of HVEM compared to cells not expressing the CFP.

54. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of CD40 compared to cells not expressing said CFP.

55. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of TL1A compared to cells not expressing said CFP.

56. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of 41BBL compared to cells not expressing the CFP.

57. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of OX40L compared to cells not expressing the CFP.

58. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of the GITRL death receptor compared to cells not expressing said CFP.

59. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of CD30L compared to cells not expressing the CFP.

60. 2. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of TIM4 compared to cells not expressing the CFP.

61. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of TIM1 ligand compared to cells not expressing the CFP.

62. 2. The composition of claim 1, wherein cells expressing the CFP exhibit increased expression of SLAM compared to cells not expressing the CFP.

63. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of CD48 compared to cells not expressing said CFP.

64. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of CD58 compared to cells not expressing said CFP.

65. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of CD155 compared to cells not expressing the CFP.

66. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased expression of CD112 compared to cells not expressing the CFP.

67. 2. The composition of claim 1, wherein cells expressing the CFP exhibit increased expression of PDL1 compared to cells not expressing the CFP.

68. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased expression of B7-DC compared to cells not expressing said CFP.

69. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit an increased respiratory burst compared to cells not expressing said CFP.

70. 2. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased ROS production compared to cells not expressing the CFP.

71. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased iNOS production compared to cells not expressing said CFP.

72. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased iNOS production compared to cells not expressing said CFP.

73. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased extracellular vesicle production compared to cells not expressing the CFP.

74. 2. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased trogocytosis by target cells expressing the antigen compared to cells not expressing the CFP.

75. 2. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased resistance to CD47-mediated inhibition of phagocytosis compared to cells not expressing said CFP.

76. The composition of any one of the preceding claims, wherein cells expressing the CFP exhibit increased resistance to LILRB1-mediated inhibition of phagocytosis compared to cells not expressing the CFP.

77. The composition of any one of the preceding claims, wherein the intracellular domain comprises a Rac inhibitory domain, a Cdc42 inhibitory domain, or a GTPase inhibitory domain.

78. 65. The composition of claim 64, wherein the Rac inhibitory domain, the Cdc42 inhibitory domain, or the GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase in the phagocytic cup of a cell expressing the CFP.

79. The composition of any one of the preceding claims, wherein the intracellular domain comprises an F-actin dissociation activation domain, an ARHGAP12 activation domain, an ARHGAP25 activation domain, or an SH3BP1 activation domain.

80. The composition of any one of the preceding claims, wherein cells expressing said CFP exhibit increased phosphatidylinositol 3,4,5-triphosphate production.

81. The composition of any one of the preceding claims, wherein the extracellular domain comprises an Ig-binding domain.

82. 10. The composition of any one of the preceding claims, wherein the extracellular domain comprises an IgA, IgD, IgE, IgG, IgM, FcRγI, FcRγIIA, FcRγIIB, FcRγIIC, FcRγIIIA, FcRγIIIB, FcRn, TRIM21, or FcRL5 binding domain.

83. The composition of any one of the preceding claims, wherein the extracellular domain comprises an FcR extracellular domain.

84. The composition of any one of the preceding claims, wherein the extracellular domain comprises an FcR-alpha, GcRβ, FcRε or FcRγ extracellular domain.

85. The composition of any one of the preceding claims, wherein the extracellular domain comprises an FcRα (FCAR) extracellular domain.

86. The composition of any one of the preceding claims, wherein the extracellular domain comprises an FcRβ extracellular domain.

87. The composition of any one of the preceding claims, wherein the extracellular domain comprises the FcRε (FCER1A) extracellular domain.

88. 3. The composition of any one of the preceding claims, wherein the extracellular domain comprises an FcRγ (FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, FCGR3B) extracellular domain.

89. The composition of any one of the preceding claims, wherein the extracellular domain comprises an integrin domain.

90. 3. The composition of any one of the preceding claims, wherein the extracellular domain comprises one or more integrin α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, or β8 domains.

91. The composition of any one of the preceding claims, wherein the intracellular domain comprises a CD47 inhibitory domain.

92. The composition of any one of the preceding claims, wherein the PR subunit further comprises an extracellular domain and an extracellular antigen-binding domain operably linked to the transmembrane domain.

93. 93. The composition of claim 92, wherein the extracellular domain further comprises an extracellular domain of a receptor, a hinge, a spacer, or a linker.

94. 94. The composition of claim 93, wherein the extracellular domain comprises the extracellular portion of a PR.

95. 95. The composition of claim 94, wherein the extracellular portion of the PR is derived from the same PR as the PR intracellular signaling domain.

96. 96. The composition of any one of claims 92 to 95, wherein the extracellular domain comprises the extracellular domain of a scavenger receptor or an immunoglobulin domain.

97. The immunoglobulin domain is an extracellular domain of an immunoglobulin or an immunoglobulin 97. The composition of claim 96, comprising a hinge region.

98. The composition of any one of claims 92 to 97, wherein the extracellular domain comprises a phagocytic phagocytosis marker.

99. The composition of any one of claims 92 to 98, wherein the extracellular domain comprises a structure capable of multimeric assembly.

100. 99. The composition of any one of claims 92 to 98, wherein the extracellular domain comprises a multimerization scaffold.

101. 10. The composition of any one of the preceding claims, wherein the extracellular domain is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 amino acids in length.

102. 10. The composition of any one of the preceding claims, wherein the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids in length.

103. 10. The composition of claim 1, wherein the extracellular antigen-binding domain specifically binds to an antigen on a target cell.

104. The composition of any one of the preceding claims, wherein the extracellular antigen-binding domain comprises an antibody domain.

105. The extracellular antigen-binding domain comprises a receptor domain, an antibody domain, and the antibody domain is a functional antibody fragment, a single chain variable fragment (scFv), a Fab, a single domain antibody (sdAb), a nanobody, a V Ｈ Domain, V Ｌ domain, VNAR domain, V ＨＨ 10. The composition of any one of the preceding claims, comprising a domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof.

106. The composition of any one of the preceding claims, wherein the extracellular antigen-binding domain comprises the extracellular domain of a ligand, a receptor, or an adaptor.

107. 10. The composition of claim 1, wherein the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain specific for a single antigen.

108. 10. The composition of claim 1, wherein the extracellular antigen-binding domain comprises at least two extracellular antigen-binding domains, each of the at least two extracellular antigen-binding domains being specific for a different antigen.

109. The composition according to any one of the preceding claims, wherein the antigen is a cancer antigen, a pathogenic antigen, or an autoimmune antigen.

110. The composition of any one of the preceding claims, wherein the antigen comprises a viral antigen.

111. 10. The composition of any one of the preceding claims, wherein the antigen is a T lymphocyte antigen.

112. The composition of any one of the preceding claims, wherein the antigen is an extracellular antigen.

113. The composition of any one of the preceding claims, wherein the antigen is an intracellular antigen.

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

115. 10. The composition of any one of the preceding claims, wherein the antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CXCR4, CD8, CD30, CD45, CD56 and cutaneous lymphocyte-associated antigen (CLA).

116. 115. The composition of claim 114, wherein the antigen is a CD5 antigen.

117. 115. The composition of claim 114, wherein the antigen is a HER2 antigen.

118. The composition of any one of the preceding claims, wherein the antigen is an ovarian cancer antigen or a T lymphoma antigen.

119. The composition of any one of the preceding claims, wherein the antigen is an integrin receptor.

120. 3. The composition of any one of the preceding claims, wherein the antigen is an integrin receptor 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.

121. 10. The composition of any one of the preceding claims, wherein the antigen comprises two or more antigens.

122. The composition of any one of the preceding claims, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked through a linker.

123. 10. The composition of any one of the preceding claims, wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked through a linker, such as the hinge region of CD8α, IgG1 or IgG4.

124. 10. The composition of any one of the preceding claims, wherein the extracellular domain comprises a multimerization scaffold.

125. The composition of any one of the preceding claims, wherein the transmembrane domain comprises an FcR transmembrane domain.

126. The composition of any one of the preceding claims, wherein the transmembrane domain comprises an FcR-ε with no more than 20, 10, or 5 modified transmembrane domains.

127. 10. The composition of claim 1, wherein the transmembrane domain comprises a transmembrane domain from a syntaxin, such as syntaxin 3 or syntaxin 4 or syntaxin 5.

128. 10. The composition of any one of the preceding claims, wherein the transmembrane domain comprises a CD8 transmembrane domain.

129. The composition of any one of claims 1 to 127, wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD2 transmembrane domain.

130. The composition of any one of claims 1 to 127, wherein the transmembrane domain comprises a CD68 transmembrane domain.

131. 2. The composition of claim 1, wherein when the CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor.

132. 2. The composition of claim 1, wherein the transmembrane domain of the CFP oligomerizes with the transmembrane domain of an exogenous receptor when the CFP is expressed in a cell.

133. 2. The composition of claim 1, wherein when the CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor.

134. 2. The composition of claim 1, wherein the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor when the CFP is expressed in a cell.

135. 10. The composition of any one of the preceding claims, wherein the transmembrane domain is derived from a protein different from the protein from which the intracellular signaling domain is derived.

136. 10. The composition of claim 1, wherein the transmembrane domain is derived from a protein different from the protein from which the extracellular domain is derived.

137. 10. The composition of claim 1, wherein the transmembrane domain comprises the transmembrane domain of a phagocytic receptor.

138. The composition of any one of the preceding claims, wherein the transmembrane domain and the extracellular domain are derived from the same protein.

139. 10. The composition of any one of the preceding claims, wherein the transmembrane domain is derived from the same protein as the intracellular signaling domain.

140. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid encodes a DAP12 recruitment domain.

141. 10. The composition of claim 1, wherein the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12.

142. 2. The composition of any one of the preceding claims, wherein the transmembrane domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acids in length.

143. 10. The composition of any one of the preceding claims, wherein the transmembrane domain is at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acids in length.

144. 10. The composition of claim 1, wherein the intracellular domain comprises a phosphatase inhibitor domain.

145. The composition of any one of the preceding claims, wherein the intracellular domain comprises an ARP2 / 3 inhibitory domain.

146. The composition of any one of the preceding claims, wherein the intracellular domain comprises at least one ITAM domain.

147. The composition of any one of the preceding claims, wherein the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains.

148. The composition of any one of the preceding claims, wherein the intracellular domain further comprises at least one ITAM domain.

149. 10. The composition of any one of the preceding claims, wherein the intracellular domain further comprises at least one ITAM domain selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one to no more than 20 modifications thereto.

150. 150. The composition of claim 149, wherein the at least one ITAM domain comprises a Src family kinase phosphorylation site.

151. 150. The composition of claim 149, wherein said at least one ITAM domain comprises a Syk recruitment domain.

152. The composition of any one of the preceding claims, wherein the intracellular domain comprises an F-actin depolymerization activating domain.

153. 10. The composition of any one of the preceding claims, wherein the intracellular domain lacks enzymatic activity.

154. the intracellular domain does not contain a domain derived from the CD3 zeta intracellular domain; A composition according to any one of the preceding claims.

155. The composition of any one of the preceding claims, wherein the intracellular domain comprises a CD47 inhibitory domain.

156. 10. The composition of any one of the preceding claims, wherein the intracellular signaling domain comprises a domain that activates an integrin, such as the intracellular region of PSGL-1.

157. the intracellular signaling domain is derived from Rap1, such as from EPAC and C3G 10. A composition according to any one of the preceding claims, comprising a GTPase-activating domain.

158. 10. The composition of any one of the preceding claims, wherein the intracellular signaling domain is derived from paxillin.

159. The composition of any one of the preceding claims, wherein the intracellular signaling domain activates focal adhesion kinase.

160. The composition of any one of the preceding claims, wherein the intracellular signaling domain is derived from a single phagocytic receptor.

161. 10. The composition of any one of the preceding claims, wherein the intracellular signaling domain is derived from a single scavenger receptor.

162. 10. The composition of any one of the preceding claims, wherein the intracellular domain further comprises a phagocytosis enhancing domain.

163. 10. The composition of any one of the preceding claims, wherein the intracellular domain comprises a pro-inflammatory signaling domain.

164. 164. The composition of claim 163, wherein the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain.

165. The composition of claim 163 or 164, wherein the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain.

166. 166. The composition of any one of claims 163-165, wherein the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-I, MYD88, MAL, IRAKl, MDA-5, an IFN receptor, an NLRP family member, NLRPl-14, NODl, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank-binding kinase (TNK), a caspase domain, or a pro-caspase binding domain, or any combination thereof.

167. The composition of any one of the preceding claims, wherein the CFP does not include a full-length intracellular signaling domain.

168. 10. The composition of any one of the preceding claims, wherein the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length.

169. 10. The composition of any one of the preceding claims, wherein the intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length.

170. The composition of any one of the preceding claims, wherein the recombinant nucleic acid encodes the FcRα chain extracellular domain, the FcRα chain transmembrane domain and / or the FcRα chain intracellular domain.

171. The composition of any one of the preceding claims, wherein the recombinant nucleic acid encodes an FcR β chain extracellular domain, an FcR β chain transmembrane domain and / or an FcR β chain intracellular domain.

172. The composition of claim 170 or 171, wherein the FcRα chain or the FcRβ chain forms a complex with FcRγ when expressed in a cell.

173. The composition of claim 172, wherein the FcRα chain or FcRβ chain forms a complex with endogenous FcRγ when expressed in a cell.

174. The composition of any one of claims 170 to 173, wherein the FcRα chain or the FcRβ chain is not incorporated into the cell membrane of a cell that does not express FcRγ.

175. The composition of any one of claims 170 to 174, wherein the CFP does not contain an FcRα chain intracellular signaling domain.

176. The composition of any one of claims 170 to 175, wherein the CFP does not comprise an FcR β chain intracellular signaling domain.

177. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid encodes a TREM extracellular domain, a TREM transmembrane domain and / or a TREM intracellular domain.

178. The composition of claim 177, wherein the TREM is TREM1, TREM2 or TREM3.

179. A composition comprising a recombinant nucleic acid encoding a phagocytic or anchoring receptor (PR) fusion protein (CFP), wherein the CFP comprises, from the N-terminus to the C-terminus: (a) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; (b) (i) a CD8 transmembrane domain, and (ii) an intracellular domain comprising an Fcγ intracellular signaling domain and a CD40 intracellular signaling domain; and a PR subunit comprising wherein the transmembrane domain and the extracellular domain are operably linked by a linker.

180. A composition comprising a recombinant nucleic acid encoding a phagocytic or anchoring receptor (PR) fusion protein (CFP), wherein the CFP comprises, from the N-terminus to the C-terminus: (a) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; (b) (i) a CD8 transmembrane domain, and (ii) an intracellular domain comprising an Fcγ intracellular signaling domain and a TNFR1 intracellular signaling domain; and a PR subunit comprising wherein the transmembrane domain and the extracellular domain are operably linked by a linker.

181. A composition comprising a recombinant nucleic acid encoding a phagocytic or anchoring receptor (PR) fusion protein (CFP), wherein the CFP comprises, from the N-terminus to the C-terminus: (a) an extracellular domain comprising an antigen-binding domain specific for an antigen on a target cell; (b) (i) a CD8 transmembrane domain, and (ii) an intracellular domain comprising an Fcγ intracellular signaling domain and an MDA5 signaling domain; and a PR subunit comprising wherein the transmembrane domain and the extracellular domain are operably linked by a linker.

182. The composition of any one of claims 179 to 181, wherein the antigen is a CD5 antigen.

183. The composition of any one of claims 179 to 181, wherein the antigen is a HER2 antigen.

184. The composition of any one of claims 179-183, wherein the CFP further comprises a signal peptide.

185. The composition of claim 184, wherein the signal peptide is a GMCSF signal peptide.

186. The composition of claim 184, wherein the CFP comprises the amino acid sequence of SEQ ID NO:

24.

187. The composition of claim 184, wherein the CFP comprises the amino acid sequence of SEQ ID NO:

25.

188. The composition of claim 184, wherein the CFP comprises the amino acid sequence of SEQ ID NO:

26.

189. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid comprises a pro-inflammatory nucleotide or polynucleotide sequence encoding a pro-inflammatory polypeptide.

190. 10. The composition of any one of the preceding claims, wherein the composition further comprises a pro-inflammatory polypeptide.

191. 191. The composition of claim 189 or 190, wherein the pro-inflammatory polypeptide is a chemokine or cytokine.

192. 192. The composition of claim 191, wherein 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.

193. 192. The composition of claim 191, wherein 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. thing.

194. 190. The composition of claim 189, wherein the nucleotide is selected from ATP, ADP, UTP, UDP, and / or UDP-glucose.

195. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid comprises a sequence encoding a homeostatic regulator of inflammation.

196. The composition of claim 195, wherein the homeostatic regulator of inflammation is a sequence in the untranslated region (UTR) of an mRNA.

197. 197. The composition of claim 196, wherein the sequence of the UTR is a sequence that binds to an RNA-binding protein.

198. 198. The composition of claim 196 or 197, wherein translation is inhibited or prevented when the RNA binding protein binds to a sequence in the untranslated region (UTR).

199. 199. The composition of claim 197 or 198, wherein the sequence of the UTR comprises a consensus sequence of WWWU(AUUUA)UUUW, where W is A or U.

200. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid is expressed in a bicistronic vector.

201. 10. The composition of any one of the preceding claims, wherein the target cell is a mammalian cell.

202. 10. The composition of any one of the preceding claims, wherein the target cells are human cells.

203. 10. The composition of any one of the preceding claims, wherein the target cells comprise cells infected by a pathogen.

204. 10. The composition of any one of the preceding claims, wherein the target cells are cancer cells.

205. 10. The composition of any one of the preceding claims, wherein the target cells are lymphocyte cancer cells.

206. 10. The composition of any one of the preceding claims, wherein the target cells are cancer cells that are ovarian cancer cells.

207. 10. The composition of any one of the preceding claims, wherein the target cells are cancer cells that are ovarian pancreatic cells.

208. 10. The composition of any one of the preceding claims, wherein the target cells are cancer cells that are glioblastoma cells.

209. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid is DNA.

210. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid is RNA.

211. The composition of any one of the preceding claims, wherein the recombinant nucleic acid is mRNA.

212. 10. The composition of any one of the preceding claims, wherein the recombinant nucleic acid is a circRNA.

213. The composition of any one of the preceding claims, wherein the recombinant nucleic acid is a tRNA.

214. The composition of any one of the preceding claims, wherein the recombinant nucleic acid is a microRNA.

215. A vector comprising a recombinant nucleic acid of the composition of any one of claims 1 to 214.

216. The vector of claim 215, which is a viral vector.

217. The vector of claim 216, wherein the viral vector is a retroviral vector or a lentiviral vector.

218. 218. The vector of any one of claims 215 to 217, further comprising a promoter operably linked to at least one nucleic acid sequence encoding one or more polypeptides.

219. The vector of any one of claims 215 to 218, which is polycistronic.

220. 220. The vector of claim 218 or 219, wherein each of the at least one nucleic acid sequence is operably linked to a separate promoter.

221. 221. The vector of any one of claims 215 to 220, further comprising one or more internal ribosome entry sites (IRES).

222. 222. The vector of any one of claims 215 to 221, further comprising 5'UTR and / or 3'UTR flanking at least one nucleic acid sequence encoding one or more polypeptides.

223. The vector of any one of claims 215 to 222, further comprising one or more regulatory regions.

224. 215. A polypeptide encoded by a recombinant nucleic acid of the composition of any one of claims 1 to 214.

225. A cell comprising a composition according to any one of claims 1 to 214, a vector according to any one of claims 216 to 223 or a polypeptide according to claim 224.

226. The cell of claim 225, which is an immature myeloid cell.

227. The cell of claim 225, which is a non-polarized or undifferentiated myeloid cell.

228. CD14+ / CD16 ｌｏｗ The cell of claim 225, which is a cell.

229. CD14+ / CD16 － cells, CD14 － / CD16 ＋ The cell of claim 225, which is a cell.

230. The cell of claim 225, which is a phagocyte.

231. 226. The cell of claim 225, which is a stem cell-derived cell, a myeloid cell, a macrophage, a dendritic cell, a lymphocyte, a mast cell, a monocyte, a neutrophil, a microglia, an eosinophil, a basophil, a myeloid progenitor cell, a mosaic phenotype cell, or an astrocyte.

232. The cell of claim 225, which is an M1 macrophage cell.

233. The cell described in claim 225, which is an M2 macrophage cell.

234. The cell of any one of claims 225 to 233, which is an autologous cell.

235. The cell of any one of claims 225 to 233, which is an allogeneic cell.

236. 224. A population of modified cells, wherein a plurality of the populations of modified cells comprises a composition of any one of claims 1-214, a vector of any one of claims 216-223, or a polypeptide of claim 224.

237. 237. The population of modified cells of claim 236, wherein the plurality comprises at least 80% of the population of modified cells.

238. The population of modified cells of claim 237, wherein the population of cells is not concentrated.

239. The population of cells may be CD14+ / CD16- cells, CD14- / CD16+ cells or CD14+ / CD16 ｌｏｗ 239. The population of modified cells of claim 237 or 238, which are cells.

240. 240. The population of modified cells of claim 237 or 239, wherein the population of cells is phagocytes.

241. (a) a composition according to any one of claims 1 to 214, a vector according to any one of claims 216 to 223 or a polypeptide according to claim 224, a cell according to any one of claims 225 to 235 or a population of cells according to any one of claims 236 to 240, and (b) a pharmaceutically acceptable excipient 10. A pharmaceutical composition comprising:

242. 242. The pharmaceutical composition of claim 241, further comprising an additional therapeutic agent.

243. The pharmaceutical composition of claim 241 or 242, wherein the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to CFP, an agent that promotes PI3K activity, an agent that promotes the production of phosphatidylinositol 3,4,5-trisphosphate, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, and any combination thereof.

244. 244. The pharmaceutical composition of any one of claims 241-243, wherein the pharmaceutically acceptable excipient comprises serum-free medium, lipids, or nanoparticles.

245. 245. A method of treating a disease in a subject in need thereof, comprising administering to said subject a pharmaceutical composition according to any one of claims 241 to 244.

246. 246. The method of claim 245, wherein the disease is cancer.

247. The method of claim 246, wherein the cancer is a solid cancer.

248. The method of claim 247, wherein the solid cancer is selected from the group consisting of ovarian cancer, suitable cancers including ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer.

249. The method of claim 246, wherein the cancer is a liquid cancer.

250. The method of claim 249, wherein the liquid cancer is leukemia or lymphoma.

251. The method of claim 249, wherein the liquid cancer is T-cell lymphoma.

252. 246. The method of claim 245, wherein the disease is a T-cell malignancy.

253. 253. The method of any one of claims 245 to 252, further comprising administering to the subject an additional therapeutic agent.

254. The method of claim 253, wherein the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin dissociation, an agent that promotes PI3K recruitment to CFP, an agent that promotes PI3K activity, an agent that promotes the production of phosphatidylinositol 3,4,5-trisphosphate, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, and any combination thereof.

255. 255. The method of any one of claims 245 to 254, wherein the administering step comprises infusing or injecting.

256. 256. The method of any one of claims 245-255, wherein the administering step comprises administering directly to the solid tumor.

257. 257. The method of any one of claims 245-256, wherein said administering comprises administering a circRNA, mRNA, a viral vector, a particle, a nanoparticle, a liposome, an exosome, or a cell.

258. 258. The method of any one of claims 245 to 257, wherein a CD4+ T cell response or a CD8+ T cell response is elicited in the subject.

259. 224. A method for preparing a cell, the method comprising contacting the cell with a composition of any one of claims 1 to 214, a vector of any one of claims 216 to 223, or a polypeptide of claim 224.

260. 260. The method of claim 259, wherein the contacting step comprises transducing.

261. 261. The method of claim 260, wherein the transducing step comprises chemical transfection, electroporation, nucleofection, or viral infection.

262. A method for preparing a pharmaceutical composition, comprising administering a lipid to a composition according to any one of claims 1 to 214, a vector according to any one of claims 216 to 223 or a pharmaceutical composition according to claim 224. The method comprises contacting a mammalian cell with the polypeptide described above.

263. 263. The method of claim 262, wherein the contacting step includes forming lipid nanoparticles.

264. A method for preparing a pharmaceutical composition, the method comprising the step of contacting an antibody with a composition according to any one of claims 1 to 214 or a vector according to any one of claims 216 to 223.

265. 265. The method of claim 264, wherein the contacting step includes forming lipid nanoparticles.

Images