Chimeric engulfment receptor molecules

Abstract

The present invention relates to chimeric engulfment receptor molecules, host cells modified to contain phagocytic engulfment molecules, and methods of making and using such receptor molecules and modified cells.

Description

[Technical Field]

[0001] Explanation of Sequence Listing The sequence listing associated with this application is provided in text form in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 200265_401WO_SEQUENCE_LISTING.txt. This text file is 275 KB, was created on September 26, 2017, and has been submitted electronically via EFS-Web. [Background technology]

[0002] background There are two main types of phagocytosis, influenced by the target, cell type, and surrounding environment. Microbial phagocytosis eliminates and degrades disease-causing microorganisms, induces proinflammatory signaling through the secretion of cytokines and chemokines, 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 instances, such as certain persistent infections, an anti-inflammatory response can occur after microbial uptake. Microbial phagocytosis is generally carried out by professional phagocytes of the myeloid lineage, such as immature dendritic cells (DCs) and macrophages, as well as by tissue-resident immune cells.

[0003] In contrast, phagocytosis (e.g., efferocytosis) of damaged, autologous apoptotic cells or cellular debris is generally a non-inflammatory (also referred to as "non-immunogenic") process. Billions of damaged, dying, and unwanted cells undergo apoptosis every day. Unwanted cells include, for example, excess cells produced during development, senescent cells, infected cells (intracellular bacteria or viruses), transformed or malignant cells, and cells irreversibly damaged by cytotoxic agents. Phagocytes provide specific and rapid removal of apoptotic cells without damaging surrounding tissues or inducing a proinflammatory immune response. The steps for the clearance of apoptotic cells include: (1) release of a "find me" signal from the apoptotic cell to recruit phagocytes to the location of the apoptotic cell; (2) binding of the "eat me" signal exposed on the surface of the apoptotic cell to phagocytes via specific receptors; (3) cytoskeletal rearrangement to engulf the apoptotic cell; and (4) digestion of the engulfed apoptotic cell, triggering a specific phagocytic response (e.g., secretion of anti-inflammatory cytokines).

[0004] There is a need for new compositions and methods for treating infectious diseases, inflammatory diseases, immune diseases, and various cancers. The methods and compositions described herein meet this need by promoting the removal of infected, transformed, malignant, apoptotic, damaged, or necrotic cells or particles from the body in the treatment of various cancers, acute and chronic infectious diseases, inflammatory diseases, immune diseases, and selected neurological diseases. Summary of the Invention

[0005] overview Chimeric engulfment receptors are described herein. In certain embodiments, a chimeric engulfment receptor (singular "CER" and plural "CERs") comprises an extracellular domain, a transmembrane domain, and an intracellular engulfment signaling domain. The transmembrane domain is located between and connects the extracellular domain and the engulfment signaling domain. The extracellular domain comprises a binding domain and, optionally, an extracellular spacer domain located between the binding domain and the transmembrane domain. In certain embodiments, a chimeric engulfment receptor described herein is a chimeric protein having (a) an extracellular domain that targets a pro-engulfment marker or target antigen associated with a disease, disorder, condition, or infection, (b) a transmembrane domain, and (c) an engulfment signaling domain. In certain embodiments, the engulfment signaling domain comprises at least one of a homeostatic engulfment domain and a pro-inflammatory engulfment domain. In some embodiments, the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain. In certain embodiments, the chimeric engulfment receptor is a single-chain chimeric protein. The chimeric engulfment receptor can be designed to generate an inflammatory response against a target cell / organ / tissue / region. Although apoptotic cell clearance is generally a non-inflammatory process, inflammation can be beneficial to the host in certain situations, such as the clearance of apoptotic tumor cells to induce an immune response against residual tumor cells.

[0006] In certain embodiments, the extracellular domain of the CER comprises a binding domain specific for an engulfment-promoting marker. In certain such embodiments, the extracellular domain comprises a phosphatidylserine (PtdSer) binding domain. In embodiments of the CER described herein, the PtdSer binding domain can comprise all or a portion of the extracellular domain of T cell immunoglobulin and mucin domain 1 (Tim1), T cell immunoglobulin and mucin domain 4 (Tim4), or T cell immunoglobulin and mucin domain 3 (Tim3). In other embodiments, the PtdSer binding domain can comprise all or a portion of the binding domain from FA58C2, GAS6, protein S, factor VII, factor IX, factor X, or prothrombin PS.

[0007] In further embodiments, the extracellular domain binds to a target antigen. In certain such embodiments, the extracellular domain comprises all or a portion of the extracellular domain of an Fc receptor (FcR), such as, for example, FcGR1, FcGR2A, FcGR2B2, FcGR2C, FcGR3A, FcεR1, and FcαR1. In yet other embodiments, when the extracellular domain binds to a target antigen, the extracellular domain can comprise an antibody or an antigen-binding domain thereof. For example, the extracellular domain can comprise an antibody or antigen-binding domain selected from an intrabody, a peptibody, a nanobody, a single-domain antibody, a SMIP, and a multispecific antibody. In certain such embodiments, the extracellular domain comprises a Fab-binding domain. In yet other such embodiments, the extracellular domain comprises an scFv.

[0008] Upon binding of the extracellular domain of the CER to an engulfment-promoting marker or a targeted antigen, the engulfment signaling domain of the CER stimulates engulfment signaling activity. Thus, upon activation, the engulfment signaling domain contained in the CER transmits an effector functional signal that instructs the host cell to engulf. In certain embodiments, the engulfment signaling domain of the CER comprises a homeostatic engulfment signaling domain. Examples of homeostatic engulfment signaling domains include MRC1, ItgB5, MERTK, Tyro3, ​​and Axl signaling domains. In other embodiments, the engulfment signaling domain comprises a proinflammatory engulfment signaling domain. Examples of proinflammatory engulfment signaling domains include Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, NFAM1, DAP12, and CD79b signaling domains. In yet other embodiments, the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain. In such embodiments, the first engulfment signaling domain and the second engulfment signaling domain can be independently selected from homeostatic and proinflammatory engulfment signaling domains, including those described herein.

[0009] In a further aspect, the present invention relates to cells genetically modified to express a CER. In certain embodiments, the CER confers an engulfment phenotype not exhibited by a single native receptor protein. In other embodiments, the CER of the present invention confers an engulfment phenotype to cells that do not naturally exhibit engulfment activity. In certain embodiments, cells are genetically modified to express a CER that targets an engulfment-promoting marker associated with dead, dying, damaged, infected, or necrotic cells. In other embodiments, cells are genetically modified to express a CER that targets a marker, such as an antibody, that binds to a molecule induced by an infectious microorganism or infectious particle. In such embodiments, the genetically modified cells promote the removal or degradation of the targeted cell or microorganism by binding a marker associated with a target molecule induced by the targeted infectious microorganism or infectious particle through the CER. In other specific embodiments, cells are genetically modified to express a CER that targets an antigenic marker that does not normally cause engulfment. For example, in such embodiments, the extracellular domain of the CER can comprise an antibody or antigen-binding portion thereof, such as a Fab-binding domain or an scFv specific for an antigen marker. In certain such embodiments, the antigen marker can be a surface protein, glycoprotein, or glycolipid characteristic of abnormal cells associated with a disease, disorder, or other undesirable condition. In such embodiments, the genetically modified cell promotes elimination or degradation of the abnormal cells upon binding of the antigen marker by the CER.

[0010] In a further embodiment, the CER-modified cells may be further modified to co-express a small GTPase. The small GTPase can be introduced into the CER-modified cells using a vector encoding both the CER and the small GTPase. Alternatively, the small GTPase can be introduced into the CER-modified cells or cells that can become CER-modified cells using a vector different from the vector used to introduce the CER.

[0011] In yet a further aspect, the present invention relates to methods for treating a subject suffering from a disease, disorder, or undesirable condition. Embodiments of these methods comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more CERs or a cell population genetically modified to express one or more CERs as described herein.

[0012] In another aspect, the present invention provides methods for modifying the engulfment phenotype of a host cell. In certain embodiments, such methods include one or more of the following: generating a cell population that exhibits the engulfment phenotype by introducing and expressing a CER into a host cell that does not naturally exhibit the engulfment phenotype; modifying the engulfment phenotype of a cell population by introducing and expressing a CER in a host cell, where the CER confers an engulfment phenotype specific for an engulfment-promoting marker or antigenic marker that is not naturally targeted by the host cell; and enhancing the engulfment phenotype of a cell population by introducing and expressing a CER in a host cell, where the CER is specific for an engulfment-promoting marker or antigenic marker that is naturally targeted by the host cell, and expression of the CER by the host cell enhances engulfment by the host cell of cells, microorganisms, or particles that exhibit the targeted engulfment-promoting marker or antigenic marker. [Brief explanation of the drawings]

[0013] [Figure 1]Figures 1A to 1D show exemplary schematic diagrams of chimeric engulfment receptors (CERs). Figure 1A shows two exemplary CERs (Tim4 and scFv) with extracellular domains specific for phosphatidylserine and contain a single engulfment signaling domain. Figure 1B shows two exemplary CERs (Tim4 and scFv) with binding domains specific for phosphatidylserine and contain a single engulfment signaling domain, including a first engulfment signaling domain and a second engulfment signaling domain. Incorporation of an accessory domain or a second engulfment signaling domain into a CER can enhance the engulfment response even in the absence of an expressed ligand for the accessory receptor. Figure 1C shows two exemplary CERs with extracellular domains containing Fab or FcR and contain a single engulfment signaling domain. Figure 1D shows two exemplary CERs containing Fab or FcR and contain a single engulfment signaling domain, including a first engulfment signaling domain and a second engulfment signaling domain. “TMD” stands for transmembrane domain. [Figure 2] Figures 2A and 2B show exemplary CER vectors. The CER vector shown in Figure 2A contains a single engulfment signaling domain. The CER vector shown in Figure 2B contains an engulfment signaling domain that includes a first engulfment signaling domain and a second engulfment signaling domain. "ECD" = extracellular domain. [Figure 3] Figures 3A and 3B show a comparison of native lymphocytes and lymphocytes modified with the CERs of the present invention. Figure 3A shows endogenous lymphocytes. Figure 3B shows lymphocytes modified with the CERs of the present invention. [Figure 4] FIG. 4 illustrates an exemplary method of administration of a CER of the present invention. [Figure 5]Figures 5A to 5C show exemplary treatment timelines. Figure 5A shows a therapeutic regimen for treatment with CER-modified cells. Figure 5B shows a therapeutic regimen for CER-modified cells used in combination with non-phagocytic T cell immunotherapy. Figure 5C shows a therapeutic regimen for CER-modified cells used in combination with monoclonal antibodies, conventional chemotherapy, or radiation therapy.

[0014] [Figure 6]Figures 6A to 6F show in vitro engulfment of apoptotic target cells mediated by the Tim4-MERTK chimeric engulfment receptor (CER). Figure 6A shows an exemplary schematic diagram of the Tim4-MERTK CER. ECD = extracellular domain; TMD = transmembrane domain; ESD = engulfment signaling domain. Figure 6B shows a fluorescence-activated cell sorting (FACs) plot of murine Ba / F3 B cells transduced with a pMSCV retroviral vector (GFP Vector) containing a nucleotide sequence encoding the Tim4-MERTK CER of Figure 6A and a nucleotide sequence encoding green fluorescent protein (GFP). Positive Ba / F3 B cell transductants were sorted using flow cytometry by staining for the green fluorescent protein marker and Tim4, demonstrating the presence of the Tim4-MERTK CER on the plasma membrane of Ba / F3 B cells. Figure 6C shows a bar graph of phagocytosis of apoptotic primary thymocytes by Ba / F3 B cells expressing the Tim4-MERTK chimeric engulfment receptor after 2 and 24 hours of incubation, as quantified by FACs. BA / F3 B cells transduced with pMSCV, which contains nucleotide sequences encoding Tim4 and GFP, were used as a negative control. Figure 6D shows a line graph illustrating the correlation between the amount of Tim4-MERTK CER surface expression and the time of target cell incubation with the phagocytosis of apoptotic primary thymocytes. Figure 6E shows images from a fluorescence microscope showing that cells expressing Tim4-MERTK CER engulf apoptotic primary thymocytes stained with pHrodo Red dye. The yellow triangle indicates an apoptotic primary thymocyte inside a phagolysosome. The open square indicates low-intensity staining of unengulfed apoptotic primary thymocytes. FIG. 6F shows FAC and histogram plots of Ba / F3 cells double positive for pHrodo Red and Tim4-MERTK CER expression, indicating phagocytosis in vitro. [Figure 7]Figures 7A-7B show Tim4-MERTK chimeric engulfment receptor (CER)-mediated engulfment of apoptotic target cells. Figure 7A shows time-lapse images of Tim4-MERTK CER-mediated clearance of target apoptotic thymocytes at 12 and 48 h incubation times. More than 95% of the target cells were removed within 4 days. A sheet of apoptotic thymocytes persists in the presence of control Ba / F3 cells expressing Tim4 (lower panel) (white arrows point to thymocytes). Figure 7B shows a line graph quantifying the number of thymocytes present per high-magnification microscopic field in the control sample (Tim4-expressing Ba / F3 cells) and the Tim4-MERTK CER-expressing Ba / F3 cell sample. Figure 7B demonstrates essentially complete removal of apoptotic thymocytes by lymphocytes expressing Tim4-MERTK CER. [Figure 8] Figures 8A-8C show Tim4-MERTK chimeric engulfment receptor-mediated clearance of Raji Burkitt lymphoma cells. Figure 8A shows a FACs plot of Ba / F3 cells double positive for pHrodo Red and Tim4-MERTK CER expression, demonstrating in vitro phagocytosis. Figure 8B shows a bar graph of phagocytosis of Raji Burkitt lymphoma cells by Tim4-MERTK CER-expressing Ba / F3 B cells compared to control Ba / F3 B cells expressing Tim4. Figure 8C shows a fluorescence micrograph of Tim4-MERTK CER-mediated clearance of Raji Burkitt lymphoma cells.

[0015] [Figure 9]Figures 9A-9F show FA58C2-MERTK chimeric engulfment receptor (CER)-mediated in vitro engulfment of apoptotic target cells. Figure 9A shows an exemplary schematic diagram of the FA58C2-MERTK CER. Figure 9B shows a bar graph of phagocytosis of apoptotic primary thymocytes by FA58C2-MERTK CER-expressing Ba / F3 B cells after 2 and 24 hours of incubation, as quantified by FACs. BA / F3 B cells transduced with pMSCV containing nucleotide sequences encoding Tim4 and GFP were used as a negative control. Figure 9C shows a line graph depicting the correlation between the amount of FA58C2-MERTK CER surface expression and the incubation time of target cells and the phagocytosis of apoptotic primary thymocytes. Figure 9D shows images from a fluorescence microscope demonstrating that FA58C2-MERTK CER-expressing cells engulf pHrodo Red-stained apoptotic primary thymocytes. Yellow triangles indicate apoptotic primary thymocytes within phagolysosomes. Figure 9E shows a FACs plot. Figure 9F shows a histogram plot of Ba / F3 cells double positive for pHrodo Red and FA58C2-MERTK CER expression, demonstrating in vitro phagocytosis. [Figure 10]Figures 10A-10E show enhancement of CER-mediated phagocytosis by the small GTPase Rac1. Figure 10A shows a schematic diagram of a bicistronic retroviral expression cassette of FA58C2-MERTK CER and Rac1 or Rab5 with a P2A sequence inserted between them (upper panel) and the resulting coexpression of FA58C2-MERTK CER and GTPase (Rac1) (lower panel). Figure 10B shows a line graph depicting the correlation between the amount of FA58C2-MERTK CER surface expression and the phagocytosis of apoptotic primary thymocytes in Ba / F3 B cells expressing FA58C2-MERTK CER or FA58C2-MERTK CER + Rac1 over a 24-hour incubation period. Figure 10C shows images from a fluorescent microscope showing that FA58C2-MERTK CER + Rac1-expressing cells engulf pHrodo Red-stained apoptotic primary thymocytes. Figure 10D shows a FACs plot. Figure 10E shows a histogram plot of Ba / F3 cells double positive for pHrodo Red and FA58C2-MERTK CER+Rac1 expression, demonstrating in vitro phagocytosis. [Figure 11]Figures 11A-11E show FA58C2-SykCER-mediated in vitro engulfment of target apoptotic cells. Figure 11A shows a schematic diagram of a retroviral expression cassette for FA58C2-SykCER and a bicistronic retroviral expression cassette for FA58C2-SykCER and the small GTPase Rac1 with a P2A sequence inserted between them (upper panel), resulting in coexpression of FA58C2-SykCER and Rac1 (lower panel). Figure 11B shows a bar graph of phagocytosis of apoptotic primary thymocytes by FA58C2-SykCER- or FA58C2-SykCER + Rac1-expressing Ba / F3 B cells after 2 and 24 hours of incubation, as quantified by FACs. BA / F3 B cells transduced with pMSCV, which contains nucleotide sequences encoding Tim4 and green fluorescent protein, were used as a negative control. Figure 11C shows a line graph demonstrating the correlation between the amount of FA58C2-SykCER surface expression and the phagocytosis of apoptotic primary thymocytes upon 24-hour incubation in Ba / F3B cells expressing FA58C2-SykCER or FA58C2-SykCER+Rac1. Phagocytosis is enhanced by the addition of the small GTPase Rac1. Figure 11D shows images from a fluorescent microscope demonstrating that FA58C3-SykCER+Rac1-expressing cells engulf pHrodo Red-stained apoptotic primary thymocytes. Yellow triangles indicate apoptotic primary thymocytes within phagolysosomes. Figure 11E shows a FACs plot of Ba / F3 cells double-positive for pHrodo Red and FA58C2-SykCER+Rac1 expression, demonstrating in vitro phagocytosis.

[0016] [Figure 12]Figures 12A-12D show that coexpression of the small GTPase Rab5 enhances CER-mediated phagocytosis. Figure 12A shows a schematic diagram of a bicistronic retroviral expression cassette of FA58C2-SykCER and the small GTPase Rab5 with a P2A sequence inserted between them (upper panel) and the resulting coexpressed FA58C2-SykCER and Rab5 (lower panel). Figure 12B shows a bar graph of the phagocytosis of apoptotic primary thymocytes by FA58C2-SykCER- or FA58C2-SykCER + Rab5-expressing Ba / F3 B cells after 2 hours of incubation, as quantified by FACs. BA / F3 B cells transduced with pMSCV, which contains nucleotide sequences encoding Tim4 and GFP, were used as a negative control. Figure 12C shows images from a fluorescence microscope demonstrating that FA58C3-SykCER+Rab5-expressing cells engulf apoptotic primary thymocytes stained with pHrodo Red dye. Figure 12D shows FACs plots of Ba / F3 cells double-positive for pHrodo Red and FA58C2-SykCER (left plot), Ba / F3 cells double-positive for FA58C2-SykCER+Rab5 expression (middle plot), or Tim4 control Ba / F3 cells, demonstrating in vitro phagocytosis of FA58C2-SykCER-expressing cells (9%) and increased phagocytosis with the addition of Rab5 (12.5%). [Figure 13]Figures 13A-13H show in vitro engulfment of target B cells via the CD19-MERTK chimeric engulfment receptor (CER). Figure 13A shows an exemplary schematic diagram of a retroviral expression cassette of CD19-MERTK CER (upper panel) and the resulting co-expressed CD19-MERTK CER (lower panel). Figure 13B shows an exemplary schematic diagram of a bicistronic retroviral expression cassette of CD19-MERTK CER and the small GTPase Rac1 with a P2A sequence inserted between them (upper panel) and the resulting co-expressed CD19-MERTK CER and Rac1 (lower panel). Figure 13C shows a bar graph of phagocytosis of Raji Burkitt lymphoma cells by CD19-MERTK CER- or CD19-MERTK CER+Rac1-expressing Ba / F3 B cells after 2 and 24 hours of incubation, as quantified by FACs. Ba / F3 B cells transduced with pMSCV containing nucleotide sequences encoding Tim4 and GFP were used as a negative control. Figure 13D is a line graph showing the correlation between the amount of CD19-MERTK CER surface expression and the phagocytosis of Raji Burkitt lymphoma cells when incubated in Ba / F3 B cells expressing CD19-MERTK CER for 24 hours. Figure 13E shows fluorescence microscopy images demonstrating that CD19-MERTK CER+Rac1-expressing cells engulf pHrodo Red-stained Raji Burkitt lymphoma cells. Yellow triangles indicate apoptotic primary thymocytes within phagolysosomes. Figure 13F shows a FACs plot of Ba / F3 cells double-positive for pHrodo Red and CD19-MERTK CER expression after 2 hours of incubation with Raji Burkitt lymphoma target cells, demonstrating in vitro phagocytosis, and Figure 13G shows a FACs plot of Ba / F3 cells after 24 hours of incubation with Raji Burkitt lymphoma target cells. Figure 13H shows a fluorescent micrograph of CD19-MERTK CER-expressing cells that have engulfed pHrodo Red dye-stained Raji Burkitt lymphoma cells. White arrows indicate engulfment events. [Figure 14]FIG. 14 shows Tables 1 and 2, which show examples of CERs of the present invention. [Figure 15] FIG. 15 shows Table 3, which shows examples of CERs of the present invention. [Figure 16] 16 shows a vector map of a lentiviral vector containing the "CER01" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 71. CER01 contains a Tim4-binding domain, a Tim4 transmembrane domain, and a MERTK signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER01 sequence by a viral T2A sequence. [Figure 17] Figures 17A-D show FACS purification of Ba / F3 mouse cells transduced with CER01. EGFR expression was detected by FACS 48 hours after transduction in untransduced Ba / F3 cells (Figure 17A) and Ba / F3 mouse B cells transduced with CER01-T2A-EGFRt-containing lentivirus (Figure 17B) using biotin-labeled cetuximab (anti-EGFR antibody) followed by streptavidin-R-phycoerythrin (SA-PE) conjugate. CER+EGFRt+-expressing cells (Figure 17C) were selected by FACS and expanded for subsequent assays. Figure 17D shows untransduced Ba / F3 control cells after EGFRt purification.

[0017] [Figure 18] Figures 18A-B show in vitro engulfment of dexamethasone-treated thymocytes by CER01+ Ba / F3 mouse B cells. Figure 18A shows a fluorescent microscopy image of EGFRt+ control-transduced Ba / F3 cells co-cultured with dexamethasone-treated thymocytes; Figure 18B shows a fluorescent microscopy image of CER01-transduced Ba / F3 cells co-cultured with dexamethasone-treated thymocytes (white arrows indicate engulfment events). A magnified image of a portion of Figure 18B is shown on the right. [Figure 19]Figures 19A-B show FACS analysis of CER01+Ba / F3 effector cells (Figure 19A) and quantification of engraftment of dexamethasone-treated thymocytes by CER01+Ba / F3 mouse B cells by measuring the population of cells that double-stained positive for pHrodo Red and CELLTRACE Violet (Figure 19B). [Figure 20] Figures 20A-B show the phagocytosis index for CER01+ cells or EGFRt+ control Ba / F3 cells. Figure 20A shows a table of the percentage of phagocytic cells and hybrid capture values ​​for CER01+ cells or EGFRt+ control Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. Figure 20B shows a graph of the phagocytosis index for CER01+ cells or EGFRt+ control Ba / F3 cells. [Figure 21] Figure 21 shows fluorescence microscopy images of phagocytosis of CT26 colon cancer cells by CER01+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 22] Figures 22A-B: The hybrid capture algorithm was used to detect the fluorescence of pHrodo Red-stained target cells within the CELLTRACE Violet-stained area of ​​CER01+ Ba / F3 cells on the fluorescence image of the phagocytosis assay. Figure 22A shows a histogram plot of the hybrid cell counts extracting the CT26 target cell area from CER01+ Ba / F3 cells. Figure 22B shows the hybrid cell counts for EGFRt+ control Ba / F3 cells. The area ratio represents the overlapping area of ​​CT26 cells within Ba / F3 cells. [Figure 23] Figure 23 shows a scatter plot of the number of hybrid cells extracting the CT26 target cell area from CER01+ Ba / F3 cells or EGFRt+ control Ba / F3 cells. The area ratio represents the overlapping area of ​​CT26 cells within Ba / F3 cells. [Figure 24] Figures 24A-B show the phagocytosis frequency (A) and phagocytosis index (B) of CER01+ Ba / F3 cells or EGFRt+ control Ba / F3 cells co-cultured with CT26 colon cancer cells. [Figure 25]Figure 25 shows fluorescence microscopy images of phagocytosis of A20 lymphoma cells by CER01+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 26] Figure 26A-B: The hybrid capture algorithm was used to detect the fluorescence of pHrodo Red-stained target cells within the CELLTRACE Violet-stained area of ​​CER01+ Ba / F3 cells on the fluorescence image of the phagocytosis assay. Figure 26A shows a histogram plot of the hybrid cell counts extracting the A20 target cell area from CER01+ Ba / F3 cells. Figure 26B shows the hybrid cell counts for EGFRt+ control Ba / F3 cells. The area ratio represents the overlapping area of ​​A20 cells within Ba / F3 cells.

[0018] [Figure 27] Figure 27 shows a scatter plot of the number of hybrid cells extracting the A20 target cell area from CER01+ Ba / F3 cells or EGFRt+ control Ba / F3 cells. The area ratio represents the area of ​​A20 cells within Ba / F3 cells. [Figure 28] FIG. 28 shows a graph of the phagocytic index of CER01+Ba / F3 cells or EGFRt+control Ba / F3 cells co-cultured with A20 cells. [Figure 29] Figure 29 shows a microscopic image of phagocytosis of WR19L T-cell lymphoma cells by CER01+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 30] FIG. 30 shows a graph of the frequency of phagocytosis of WR19L cells by CER01+Ba / F3 cells. [Figure 31] Figures 31A-B show transduction and proliferation of CER01+ human primary B cells. Figure 31A shows FACS analysis of CER01-transduced human primary B cells (right histogram) and control B cells (left histogram) using anti-EGFR antibody followed by anti-Tim4 Kat5-18 antibody. Figure 31B shows purified CER01+ B cells expanded for 24, 48, and 72 hours. [Figure 32]Figures 32A-B show fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated Jurkat cells by CER01+ human primary B cells (Figure 32A) compared to control human primary B cells transduced with truncated EGFR (Figure 32B). White arrows indicate phagocytosis events. [Figure 33] Figure 33 shows phagocytosis of staurosporine-treated, pHrodo Red-stained Jurkat cells by CER01+ human primary B cells analyzed by FACS. Gating was performed on viable CD19+, allophycocyanin (APC)-labeled cells (left plot), and the frequency of double-positive staining events (APC and pHrodo Red) was defined as phagocytosis events (right plot). [Figure 34] FIG. 34 shows a graph of the phagocytosis frequency of staurosporine-treated Jurkat cells co-incubated with CER01+ human primary B cells. [Figure 35] Figure 35 shows fluorescence microscopy images of in vitro phagocytosis of oxaliplatin and fluorouracil-treated Jurkat cells by CER01+ human primary B cells. White arrows indicate phagocytosis events. [Figure 36] Figure 36 shows a vector map of a lentiviral vector containing the "CER08" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 83. CER08 contains a Tim4-binding domain, a Tim4 transmembrane domain, and a Tyro3 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121) separated from the CER08 sequence by a viral T2A sequence. [Figure 37] Figures 37A-B show FACS plots of viable CER08+ modified Ba / F3 cells (Figure 37A) and pHrodo red and CELLTRACE Violet double-stained cell populations showing the frequency of phagocytosis (Figure 37B) in a coculture of dexamethasone-treated, pHrodo Red-stained thymocytes and CELLTRACE Violet-stained CER08+ mouse Ba / F3 cells.

[0019] [Figure 38] Figure 38 shows fluorescence microscopy images of dexamethasone-treated thymocyte phagocytosis by CER08+Ba / F3 cells (bottom photo) compared to EGFRt+Ba / Fe control cells (top photo). White arrows indicate phagocytosis events. A higher magnification of the engulfment event is shown on the right. [Figure 39] Figures 39A-B show the phagocytosis index for CER08+ cells or EGFRt+ control Ba / F3 cells. Figure 39A shows a table of the percentage of phagocytic cells and hybrid capture values ​​for CER08+ cells or EGFRt+ control Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. Figure 39B shows a graph of the phagocytosis index for CER08+ cells or EGFRt+ control Ba / F3 cells. [Figure 40] Figure 40 shows a vector map of a lentiviral vector containing the "CER09" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 84. CER09 contains a Tim4 binding domain, a Tim4 transmembrane domain, and a DAP12 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121) separated from the CER09 sequence by a viral T2A sequence. [Figure 41] Figures 41A-B show FACS plots of viable CER09+ modified Ba / F3 cells (Figure 41A) and pHrodo red and CELLTRACE Violet double-stained cell populations showing the frequency of phagocytosis (Figure 41B) in co-cultures of dexamethasone-treated, pHrodo Red-stained thymocytes with CELLTRACE Violet-stained CER09+ mouse Ba / F3 cells. [Figure 42] Figures 42A-B show fluorescence microscopy images of phagocytosis of dexamethasone-treated thymocytes by CER09+Ba / F3 cells (Figure 42B) compared to EGFRt+Ba / Fe control cells (Figure 42A). White arrows indicate phagocytic events. A magnified image of an engulfment event is shown on the right. [Figure 43]Figures 43A-B show the phagocytosis index for CER09+ cells or EGFRt+ control Ba / F3 cells. Figure 43A shows a table of the percentage of phagocytic cells and hybrid capture values ​​for CER09+ cells or EGFRt+ control Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. Figure 43B shows a graph of the phagocytosis index for CER09+ cells or EGFRt+ control Ba / F3 cells. [Figure 44] Figures 44A-B show fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated CT26 colon carcinoma cells by CER09+ Ba / F3 cells (Figure 44A) and EGFRt+ control Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 45] Figure 45 shows a scatter plot of the number of hybrid cells extracting the CT26 target cell area from CER09+ Ba / F3 cells or EGFRt+ control Ba / F3 cells. The area ratio represents the area of ​​CT26 cells within Ba / F3 cells. [Figure 46] FIG. 46 shows the phagocytosis index for CER09+ cells or EGFRt+ control Ba / F3 cells co-incubated with staurosporine-treated CT26 cells. [Figure 47] Figure 47 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated WR19L lymphoma cells by CER09+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 48] Figure 48 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated A20 lymphoma cells by CER09+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 49] Figures 49A-B show transduction and proliferation of CER09+ human primary B cells. Figure 49A shows FACS analysis of CER09-transduced human primary B cells (right histogram) and control B cells (left histogram) using anti-EGFR antibody followed by anti-Tim4 Kat5-18 antibody. Figure 49B shows purified CER09+ B cells expanded for 24, 48, and 72 hours. [Figure 50]Figure 50 shows phagocytosis of staurosporine-treated pHrodo Red-stained Jurkat cells by CER09+ human primary B cells analyzed by FACS. Gating was performed on viable CD19+, allophycocyanin (APC)-labeled cells (left plot), and the frequency of double-positive staining events (APC and pHrodo Red) was defined as phagocytic events (right plot).

[0020] [Figure 51] FIG. 51 shows a graph of the frequency of phagocytosis of staurosporine-treated Jurkat cells by CER09+ human primary B cells or control EGFRt+ human primary B cells. [Figure 52] Figure 52 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated Jurkat cells by CER09+ human primary B cells (left picture) or EGFRt+ human primary B cells (right picture). White arrows indicate phagocytosis events. [Figure 53] Figure 53 shows a vector map of a lentiviral vector containing a "CER10" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 86. CER10 contains a Tim4-binding domain, a DAP12 transmembrane domain, and a DAP12 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121) with a viral P2A sequence inserted between the CER10 sequence. [Figure 54] Figures 54A-B show FACS analysis of viable CER10+ Ba / F3 effector cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE Violet (Figure 54A) and quantification of engraftment of dexamethasone-treated thymocytes by CER10+ Ba / F3 mouse B cells (Figure 54B). [Figure 55]Figures 55A-B show fluorescence microscopy images of in vitro phagocytosis of dexamethasone-treated thymocytes by CER10+Ba / F3 cells (Figure 55B) or control EGFRt+Ba / F3 cells (Figure 55A). White arrows indicate phagocytosis events. Magnified images of engulfment events are shown on the right. [Figure 56] Figures 56A-B show the phagocytosis index for CER10+ cells or EGFRt+ control Ba / F3 cells. Figure 56A shows a table of the percentage of phagocytic cells and hybrid capture values ​​for CER10+ cells or EGFRt+ control Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. Figure 56B shows a graph of the phagocytosis index for CER10+ cells or EGFRt+ control Ba / F3 cells. [Figure 57] Figure 57 shows a vector map for a lentiviral vector containing a "CER11" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 87. CER11 contains a Tim4-binding domain, a Tim4 transmembrane domain, and an AxI signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER11 sequence by a viral T2A sequence. [Figure 58] Figures 58A-B show FACS analysis of CER11+Ba / F3 effector cells (Figure 58A) and quantification of engraftment of dexamethasone-treated thymocytes by CER11+Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE Violet (Figure 58B). [Figure 59] Figures 59A-B show fluorescence microscopy images of in vitro phagocytosis of dexamethasone-treated thymocytes by CER11+Ba / F3 cells (Figure 59B) or control EGFRt+Ba / F3 cells (Figure 59A). White arrows indicate phagocytosis events. Magnified images of engulfment events are shown on the right. [Figure 60]Figures 60A-B show the phagocytosis index of CER11+ cells or EGFRt+ control Ba / F3 cells. Figure 60A shows a table of the percentage of phagocytosed cells and hybrid capture values ​​of CER11+ cells or EGFRt+ control Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. Figure 60B shows a graph of the phagocytosis index for CER11+ cells or EGFRt+ control Ba / F3 cells. [Figure 61] Figures 61A-B show fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated CT26 colon cancer cells by CER11+Ba / F3 cells (left photograph) or control EGFRt+Ba / F3 cells (right photograph). White arrows indicate phagocytosis events.

[0021] [Figure 62] Figure 62 shows a scatter plot of the number of hybrid cells extracting the CT26 target cell area from CER11+ Ba / F3 cells or EGFRt+ control Ba / F3 cells. The area ratio represents the area of ​​CT26 cells within Ba / F3 cells. [Figure 63] Figure 63 shows fluorescence microscopy images showing in vitro phagocytosis of WR19L cells by CER11+Ba / F3. White arrows indicate phagocytosis events. [Figure 64] Figures 64A-B show FACS analysis of CER11+Ba / F3 effector cells (Figure 64A) and quantification of engulfment of WR19L lymphoma cells by CER11+Ba / F3 murine B cells by measuring the population of cells double-positively stained for pHRodo Red and CELLTRACE Violet (Figure 64B). [Figure 65] Figures 65A-B show fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated A20 lymphoma cells by CER11+Ba / F3 cells (left photograph) or control EGFRt+Ba / F3 cells (right photograph). White arrows indicate phagocytosis events. [Figure 66]FIG. 66 shows the phagocytosis index for CER11+ cells or EGFRt+ control Ba / F3 cells co-incubated with staurosporine-treated A20 cells. [Figure 67] Figure 67 shows fluorescence microscopy images of in vitro phagocytosis of oxaliplatin and fluorouracil-treated Jurkat cells by CER11+ human primary B cells (left photograph) or control EGFRt+ human primary B cells (right photograph). White arrows indicate phagocytosis events. [Figure 68] Figure 68 shows fluorescence microscopy images of in vitro phagocytosis of gemcitabine-treated COLO320HSR colon cancer cells by CER11+ human primary B cells. White arrows indicate phagocytosis events. [Figure 69] Figure 69 shows fluorescence microscopy images of in vitro phagocytosis of paclitaxel-treated A204 rhabdomyosarcoma cells by CER11+ human primary B cells. Arrows indicate phagocytosis events. [Figure 70] Figure 70 shows fluorescence microscopy images of in vitro phagocytosis of paclitaxel or paclitaxel plus gemcitabine-treated H1703 non-small cell lung cancer cells by CER11+ human primary B cells. Arrows indicate phagocytosis events. [Figure 71] Figure 71 shows a vector map of a lentiviral vector containing the "CER12" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 90. CER12 contains a Tim4 binding domain, a Tim4 transmembrane domain, and an FcεRIγ signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER12 sequence by a viral T2A sequence. [Figure 72] Figures 72A-B show FACS analysis of CER12+Ba / F3 effector cells (Figure 72A) and quantification of thymocyte engulfment by CER12+Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE Violet (Figure 72B). [Figure 73]Figures 73A-B show fluorescence microscopy images of in vitro phagocytosis of dexamethasone-treated thymocytes by CER12+Ba / F3 cells (Figure 73B) or control EGFRt+Ba / F3 cells (Figure 73A). White arrows indicate phagocytosis events. Magnified images of engulfment events are shown on the right. [Figure 74] Figures 74A-B show the phagocytosis index for CER12+ cells or EGFRt+ control Ba / F3 cells. Figure 74A shows a table of the percentage of phagocytosed cells and hybrid capture values ​​for CER12+ cells or EGFRt+ control Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. Figure 74B shows a graph of the phagocytosis index for CER12+ cells or EGFRt+ control Ba / F3 cells.

[0022] [Figure 75] Figure 75 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated WR19L lymphoma cells by CER12+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 76] Figure 76 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated A20 lymphoma cells by CER12+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 77] FIG. 77 shows the phagocytosis index for CER12+ cells or EGFRt+ control Ba / F3 cells co-incubated with staurosporine-treated A20 cells. [Figure 78] Figure 78 shows a vector map for a lentiviral vector containing the "CER13" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 91. CER13 contains a Tim4 binding domain, an FcεRIγ transmembrane domain, and an FcεRIγ signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER13 sequence by a viral T2A sequence. [Figure 79]Figures 79A-B show FACS analysis of CER13+ Ba / F3 effector cells (Figure 79A) and quantification of thymocyte engulfment by CER13+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE Violet (Figure 79B). [Figure 80] Figure 80 shows fluorescence microscopy images of in vitro phagocytosis of paclitaxel and gemcitabine-treated Colo320 HSR colon cancer cells by CER13+ human primary B cells. Arrows indicate phagocytosis events. [Figure 81] Figure 81 shows fluorescence microscopy images of in vitro phagocytosis of paclitaxel-treated A204 rhabdomyosarcoma cells by CER13+ human primary B cells. Arrows indicate phagocytosis events. [Figure 82] Figure 82 shows fluorescence microscopy images of in vitro phagocytosis of paclitaxel and gemcitabine-treated Colo320 HSR colon cancer cells by CER13+ human primary B cells. Arrows indicate phagocytosis events. [Figure 83] Figure 83 shows a vector map of a lentiviral vector containing the "CER15" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 79. CER15 contains a Tim4 binding domain, a Tim4 transmembrane domain, and a truncated MyD88 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER15 sequence by a viral T2A sequence. [Figure 84] Figures 84A-B show FACS analysis of CER15+ Ba / F3 effector cells (Figure 84A) and quantification of thymocyte engulfment by CER15+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE Violet (Figure 84B). [Figure 85]Figures 85A-B show fluorescence microscopy images of in vitro phagocytosis of dexamethasone-treated thymocytes by CER15+ Ba / F3 cells (Figure 85B) or control EGFRt+ Ba / F3 cells (Figure 85A). White arrows indicate phagocytosis events. Magnified images of engulfment events are shown on the right.

[0023] [Figure 86] Figures 86A-B show the phagocytosis index for CER15+ cells or EGFRt+ control Ba / F3 cells. Figure 86A shows a table of the percentage of phagocytosed cells and hybrid capture values ​​for CER15+ cells or EGFRt+ control Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. Figure 86B shows a graph of the phagocytosis index for CER15+ cells or EGFRt+ control Ba / F3 cells. [Figure 87] Figure 87 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated CT26 colon cancer cells by CER15+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 88] Figure 88 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated WR19L lymphoma cells by CER15+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 89] Figure 89 shows fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated A20 lymphoma cells by CER15+Ba / F3 cells. White arrows indicate phagocytosis events. [Figure 90] Figures 90A-B show the transduction and expansion of CER15+ human primary B cells. Figure 90A shows FACS analysis of human primary B cells transduced with CER15 (right histogram) and control B cells (left histogram) using an anti-EGFR antibody followed by an anti-Tim4 Kat5-18 antibody. Figure 49B shows purified CER15+ B cells expanded for 24, 48, and 72 hours. [Figure 91]Figure 91 shows phagocytosis of staurosporine-treated, pHrodo Red-stained Jurkat cells by CER15+ human primary B cells analyzed by FACS. Gating was performed on viable CD19+, allophycocyanin (APC)-labeled cells (left plot), and the frequency of double-positive staining events (APC and pHrodo Red) was defined as phagocytic events (right plot). [Figure 92] Figure 92 shows a graph of the frequency of phagocytosis by CER15+ human primary B cells co-incubated with staurosporine-treated Jurkat cells compared to control human primary B cells transduced with truncated EGFR. [Figure 93] Figures 93A-B show fluorescence microscopy images of in vitro phagocytosis of staurosporine-treated Jurkat cells by CER15+ human primary B cells (Figure 93A) compared to control human primary B cells transduced with truncated EGFR (Figure 93B). White arrows indicate phagocytosis events. [Figure 94] Figure 94 shows a vector map of a lentiviral vector containing the "CER16" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 80. CER16 contains a Tim4 binding domain, a Tim4 transmembrane domain, and a MyD88 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER16 sequence by a viral T2A sequence. [Figure 95] Figure 95 shows fluorescence microscopy images of in vitro phagocytosis of Jurkat cells treated with oxaliplatin and fluorouracil by CER16+ human primary B cells. White arrows indicate phagocytosis events. [Figure 96]Figure 96 shows a vector map of a lentiviral vector containing the "CER25" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 93. CER25 contains a Tim4-binding domain, a Tim4 transmembrane domain, and an NFAM1 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER25 sequence by a viral T2A sequence.

[0024] [Figure 97] Figures 97A-B show FACS quantification of engraftment of dexamethasone-treated thymocytes by CER25+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE violet (Figure 97B) compared to control Ba / F3 cells transduced with truncated EGFR (Figure 97A). [Figure 98] Figure 98 shows fluorescence microscopy images of in vitro phagocytosis by CER25+Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. A magnified image of an engulfment event is shown on the right. White arrows indicate phagocytosis events. [Figure 99] Figure 99 shows a graph of the phagocytic index of CER25+ Ba / F3 cells co-cultured with dexamethasone-treated thymocytes compared to Ba / F3 cells transduced with truncated EGFR. [Figure 100] Figure 100 shows a vector map of a lentiviral vector containing the "CER85" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 95. CER85 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a truncated MyD88 signaling domain, and a second engulfment signaling domain that is a BAFFR signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER85 sequence by a viral T2A sequence. [Figure 101]Figures 101A-B show FACS quantification of engraftment of dexamethasone-treated thymocytes by CER85+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE Violet (Figure 101A) compared to control Ba / F3 cells transduced with truncated EGFR (Figure 101B). [Figure 102] Figure 102 shows fluorescence microscopy images of in vitro phagocytosis by CER85+Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. A magnified image of an engulfment event is shown on the right. White arrows indicate phagocytosis events. [Figure 103] FIG. 103 shows a graph of the phagocytic index of CER85+ Ba / F3 cells co-cultured with dexamethasone-treated thymocytes compared to control Ba / F3 cells transduced with truncated EGFR. [Figure 104] Figure 104 shows a vector map of a lentiviral vector containing the "CER86" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 96. CER86 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a truncated MyD88 signaling domain, and a second engulfment signaling domain that is a DAP12 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER86 sequence by a viral T2A sequence. [Figure 105] Figure 105 shows a vector map of a lentiviral vector containing the "CER87" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 130. CER87 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a BAFFR signaling domain, and a second engulfment signaling domain that is a truncated MyD88 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER87 sequence by a viral T2A sequence. [Figure 106] Figures 106A-B show FACS quantification of engraftment of dexamethasone-treated thymocytes by CER87+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE violet (Figure 106A) compared to control Ba / F3 cells transduced with truncated EGFR (Figure 106B).

[0025] [Figure 107] Figure 107 shows fluorescence microscopy images of in vitro phagocytosis by CER87+Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. A magnified image of an engulfment event is shown on the right. White arrows indicate phagocytosis events. [Figure 108] FIG. 108 shows a graph of the phagocytic index of CER87+ Ba / F3 cells co-cultured with dexamethasone-treated thymocytes compared to control Ba / F3 cells transduced with truncated EGFR. [Figure 109] Figure 109 shows a vector map of a lentiviral vector containing a "CER88" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 131. CER88 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a DAP12 signaling domain, and a second engulfment signaling domain that is a truncated MyD88 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER88 sequence by a viral T2A sequence. [Figure 110]Figure 110 shows a vector map of a lentiviral vector containing the "CER89" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 98. CER89 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a truncated MyD88 signaling domain, and a second engulfment signaling domain that is a CD79b signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER89 sequence by a viral T2A sequence. [Figure 111] Figure 111 shows a vector map of a lentiviral vector containing a "CER90" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 100. CER90 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a truncated MyD88 signaling domain, and a second engulfment signaling domain that is an NFAM1 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER90 sequence by a viral T2A sequence. [Figure 112] Figure 112 shows a vector map of a lentiviral vector containing the "CER91" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 105. CER91 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a truncated MyD88 signaling domain, a sequence encoding Rab5a with a viral P2A sequence inserted between the CER sequence, and a sequence encoding a truncated EGFR (SEQ ID NO: 121) separated from the Rab5a sequence by a viral T2A sequence. [Figure 113]Figures 113A-B show FACS quantification of engraftment of dexamethasone-treated thymocytes by CER91+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE violet (Figure 113A) compared to control Ba / F3 cells transduced with truncated EGFR (Figure 113B). [Figure 114] Figure 114 shows fluorescence microscopy images of in vitro phagocytosis by CER91+Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. A magnified image of an engulfment event is shown on the right. White arrows indicate phagocytosis events. [Figure 115] Figure 115 shows a graph of the phagocytic index of CER91+ Ba / F3 cells co-cultured with dexamethasone-treated thymocytes compared to control Ba / F3 cells transduced with truncated EGFR. [Figure 116] Figure 116 shows a vector map of a lentiviral vector containing the "CER92" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 133. CER92 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a MERTK signaling domain, and a second engulfment signaling domain that is a truncated MyD88 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER92 sequence by a viral T2A sequence. [Figure 117] Figures 117A-B show FACS quantification of engraftment of dexamethasone-treated thymocytes by CER92+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE violet (Figure 117A) compared to control Ba / F3 cells transduced with truncated EGFR (Figure 117B).

[0026] [Figure 118]Figure 118 shows fluorescence microscopy images of in vitro phagocytosis by CER92+Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. A magnified image of an engulfment event is shown on the right. White arrows indicate phagocytosis events. [Figure 119] FIG. 119 shows a graph of the phagocytic index of CER92+ Ba / F3 cells co-cultured with dexamethasone-treated thymocytes compared to control Ba / F3 cells transduced with truncated EGFR. [Figure 120] Figure 120 shows a vector map of a lentiviral vector containing a "CER93" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 103. CER93 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a MERTK signaling domain, and a second engulfment signaling domain that is a BAFFR signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER93 sequence by a viral T2A sequence. [Figure 121] Figures 121A-B show FACS quantification of engraftment of dexamethasone-treated thymocytes by CER93+ Ba / F3 mouse B cells by measuring the population of cells double-positively stained for pHrodo Red and CELLTRACE Violet (Figure 121A) compared to control Ba / F3 cells transduced with truncated EGFR (Figure 121B). [Figure 122] Figure 122 shows fluorescence microscopy images of in vitro phagocytosis by CER93+Ba / F3 cells co-cultured with dexamethasone-treated thymocytes. A magnified image of an engulfment event is shown on the right. White arrows indicate phagocytosis events. [Figure 123] FIG. 123 shows a graph of the phagocytic index of CER93+ Ba / F3 cells co-cultured with dexamethasone-treated thymocytes compared to control Ba / F3 cells transduced with truncated EGFR. [Figure 124]Figure 124 shows a vector map of a lentiviral vector containing a "CER94" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 134. CER94 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a MERTK signaling domain, and a second engulfment signaling domain that is a DAP12 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER94 sequence by a viral T2A sequence. [Figure 125] Figure 125 shows a vector map of a lentiviral vector containing the "CER97" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 152. CER97 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is an Axl signaling domain, and a second engulfment signaling domain that is a DAP12 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER97 sequence by a viral T2A sequence. [Figure 126] Figure 126 shows a vector map of a lentiviral vector containing the "CER98" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 153. CER98 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is an Axl signaling domain, and a second engulfment signaling domain that is a CD79b signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER98 sequence by a viral T2A sequence.

[0027] [Figure 127]Figure 127 shows a vector map of a lentiviral vector containing the "CER95" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 101. CER95 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a MERTK signaling domain, and a second engulfment signaling domain that is a CD79b signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER95 sequence by a viral T2A sequence. [Figure 128] Figure 128 shows a vector map of a lentiviral vector containing the "CER96" chimeric engulfment receptor having the amino acid sequence of SEQ ID NO: 102. CER96 contains a Tim4-binding domain, a Tim4 transmembrane domain, a first engulfment signaling domain that is a MERTK signaling domain, and a second engulfment signaling domain that is an NFAM1 signaling domain. The lentiviral vector also contains a sequence encoding a truncated EGFR (SEQ ID NO: 121), separated from the CER96 sequence by a viral T2A sequence. [Figure 129] FIG. 129 shows the phagocytic index of various CER+ Ba / F3 cells co-incubated with dexamethasone-treated thymocytes compared to control Ba / F3 cells transduced with truncated EGFRtt. [Figure 130] FIG. 130 shows the phagocytic index of various CER+ Ba / F3 cells co-incubated with staurosporine-treated CT26 colon cancer cells compared to control Ba / F3 cells transduced with truncated EGFRt. [Figure 131] FIG. 131 shows the phagocytic index of various CER+ Ba / F3 cells co-incubated with staurosporine-treated A20 lymphoma cells compared to control Ba / F3 cells transduced with truncated EGFRt. [Figure 132]Figures 132A-C show the in vivo synergistic effect of CER01 (Tim4MerTk) treatment with low-dose radiation in a mouse model of lymphoma. Figure 132A shows an exemplary schedule of the combination therapy regimen. Figure 132B shows tumor size measurements in untreated mice, mice administered radiation + control T cells, or mice administered radiation + CERO1-modified T cells. [Figure 133] Figures 133A-B show the in vivo synergism of CER01 (Tim4MerTk) treatment with chimeric antigen receptor (CAR) T-cell therapy in a mouse model of lymphoma. Figure 133A shows an exemplary schedule of the combination therapy regimen. Figure 133B shows luciferase images (right image) of tumor size in mice administered anti-CD19 CAR-modified T cells and CER-modified B cells (n=3) or T cells (n=2) 4 days after CER injection, compared with control mice administered anti-CD19 CAR-modified T cells and pMSCV empty retroviral vector-modified T cells (left photograph). [Figure 134] Figure 134 shows an exemplary triple combination treatment schedule including radiation therapy, CER immunotherapy (e.g., targeting phosphatidylserine-expressing cells), followed by TCR or CAR immunotherapy. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description Described herein are chimeric proteins comprising (a) an extracellular domain comprising an extracellular binding domain and optionally an extracellular spacer domain, (b) a transmembrane domain, and (c) an engulfment signaling domain, as well as nucleic acid molecules encoding the chimeric proteins. Additionally provided are cells engineered to express these chimeric proteins, as well as methods and compositions for delivering such engineered cells to a subject in need thereof. The chimeric proteins are referred to herein as "chimeric engulfment receptor(s)" (singular "CER" and plural "CERs"). The chimeric engulfment receptors described herein can confer an engulfment phenotype to a host cell that has been genetically engineered to express the chimeric engulfment receptor. In some such embodiments, expression of a CER described herein confers an engulfment phenotype to a host cell that does not naturally exhibit the engulfment phenotype. In other such embodiments, expression of a CER described herein by a host cell confers an engulfment phenotype specific to an engulfment-promoting marker or antigen marker not naturally targeted by the host cell. In yet other such embodiments, expression of a CER described herein by a host cell confers an engulfment phenotype specific to an engulfment-promoting or antigenic marker that is naturally targeted by the host cell, and expression of the CER by the host cell enhances engulfment by the host cell of cells, microorganisms, or particles that display the targeted engulfment-promoting or antigenic marker.

[0029] In certain embodiments, CER targets the engulfment marker associated with apoptotic cells, dead cells, dying cells, damaged cells, infected cells or necrotic cells.In other embodiments, CER targets antibody-bound cells associated with infectious microorganisms or particles.In still other embodiments, CER targets antigen markers presented by abnormal cells or misfolded proteins associated with disease, disorders or other undesirable pathologies.

[0030] One or more CERs described herein can be transduced and expressed in cells such as T cells, natural killer cells, natural killer T cells, B cells, lymphoid progenitor cells, dendritic cells, Langerhans cells, and myeloid cells. In certain embodiments, in addition to engineering the CER to bind to a specific target molecule (e.g., an enlargement marker or an antigen marker), the enlargement signaling domain of the CER is selected to provide a desired enlargement activity. In one such embodiment, the enlargement signaling domain is selected to induce homeostatic enlargement signaling. In another such embodiment, the enlargement signaling domain is selected to induce proinflammatory enlargement signaling. In yet another embodiment, the enlargement signaling domain comprises a first enlargement signaling domain and a second enlargement signaling domain. The first engulfment signaling domain and the second engulfment signaling domain can both be homeostatic engulfment signaling domains, or both can be pro-inflammatory engulfment signaling domains, or the first engulfment signaling domain can be a homeostatic engulfment signaling domain and the second engulfment signaling domain can be a pro-inflammatory engulfment signaling domain (or vice versa).

[0031] Host cells engineered to express one or more CERs described herein can be used for specific engulfment of target cells or particles expressing a target molecule to which the extracellular domain of a CER binds. In certain embodiments, the target cell or particle can be a tumor cell, cancer cell, microorganism (e.g., bacteria, fungi, virus), protozoan parasite, abnormal cell, or misfolded protein associated with an infection, disease, disorder, or other undesirable condition. In further embodiments, host cells engineered to express one or more CERs described herein can be used as a primary, adjunctive, or combination therapy for treating cancer, infectious diseases (viruses, bacteria, fungi, protozoa), inflammatory diseases, immune diseases (e.g., autoimmune diseases), or neurodegenerative diseases (e.g., Alzheimer's disease) in a subject. The CERs described herein can be designed to confer a particular engulfment phenotype (e.g., homeostatic (non-immunogenic) or pro-inflammatory (immunogenic)) through the selection of a homeostatic or pro-inflammatory engulfment signaling domain, depending on the target molecule and therapeutic indication. Regardless of theory, CERs containing a pro-inflammatory engulfment domain may be useful for improving the cancer microenvironment and enhancing tumor regression.

[0032] definition Before describing the present invention in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms used herein.

[0033] Any concentration range, percentage range, ratio range, or integer range herein should be understood to include any integer value within the recited range, and fractions thereof (e.g., tenths and hundredths of integers, etc.), where appropriate, unless otherwise specified. Additionally, any numerical range recited herein for any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise specified. As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise specified. As used herein, the terms "a" and "an" should be understood to mean "one or more" of the recited components. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof. As used herein, the terms "include," "having," and "comprise" are used interchangeably, and it is intended that the terms and variations thereof be interpreted as open-ended.

[0034] Terms understood by those skilled in the art of antibodies have their respective art-accepted meanings unless clearly defined differently herein. The term "antibody" is used in the broadest sense and encompasses polyclonal and monoclonal antibodies. "Antibody" can refer to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as the antigen-binding portion (or antigen-binding domain) of an intact antibody that has or retains the ability to bind to a target molecule. Antibodies can be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, such as intrabodies, peptibodies, nanobodies, single-domain antibodies, SMIPs, and multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFvs, tandem tri-scFvs, ADAPTIRs). Monoclonal antibodies, or antigen-binding portions thereof, can be non-human, chimeric, humanized, or human, preferably humanized or human. Immunoglobulin structure and function are described, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The "antigen-binding portion" or "antigen-binding domain" of an intact antibody refers to a portion of the intact antibody and is meant to encompass "antibody fragments," which refer to the antigen-determining variable region or complementarity-determining region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, Fab'-SH, F(ab')2, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. "Fab" (antigen-binding fragment) is the portion of an antibody that binds to an antigen and includes the variable region and CH1 of the heavy chain connected to the light chain via an interchain disulfide bond. The antibody can be of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0035] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a specific antigen can be isolated by screening a library of complementary VH or VL domains, respectively, using a VH or VL domain from an antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0036] The terms "complementarity determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to noncontiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0037] The terms "antigen" and "Ag" refer to a molecule that elicits an immune response. The elicited immune response may include antibody production, activation of specific immunologically competent T cells, or both. Macromolecules, including proteins, glycoproteins, and glycolipids, can serve as antigens. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be encoded (i) solely by the full-length nucleotide sequence of a gene, or (ii) by a "gene" at all. Antigens can be generated or synthesized, or they can be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or body fluids.

[0038] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant within an antigen that is specifically bound by a cognate immune binding molecule such as an antibody or fragment thereof (e.g., scFv), a T cell receptor (TCR), a chimeric engulfment receptor, or other binding molecule, domain, or protein. Epitopic determinants generally comprise chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes may be linear or conformational epitopes.

[0039] The term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" may also be manifested by the prevention of hematologic cancers or tumor formation.

[0040] "Autoimmune disease" refers to a disorder resulting from an autoimmune response. An autoimmune disease is the result of an inappropriately excessive response to a self-antigen. An autoimmune response can involve autoreactive B cells, autoreactive T cells, or both, which produce autoantibodies. As used herein, an "autoantibody" is an antibody produced by a subject that binds to an autoantigen that is also produced by the subject.

[0041] "Autologous" means any material derived from the same subject that is later reintroduced.

[0042] "Allogeneic" means a graft derived from a different subject of the same species.

[0043] As used herein, the terms "binding domain," "binding region," and "binding moiety" refer to a molecule, such as a peptide, oligopeptide, polypeptide, or protein, that has the ability to specifically and non-covalently bind, associate, combine, recognize, or bind to a target molecule (e.g., PtdSer, an IgG antibody, an IgE antibody, an IgA antibody, CD138, CD38, CD33, CD123, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, EGFRviii, VEGFR-2, or GD2). Binding domains include any natural, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or other target of interest. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody, or a functional binding domain or antigen-binding portion thereof. Exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g., TNF-α), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to biomolecules.

[0044] A variety of assays are known for identifying binding domains of the invention that specifically bind to a particular target and for determining binding domain affinity, such as Western blot, ELISA, and BIACORE® analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents). As used herein, "specifically binds" refers to a binding domain or fusion protein thereof that specifically binds to a target, such as a target protein, or a protein thereof, that specifically binds to a target protein. 5 M -1 or greater affinity or K a (i.e., the equilibrium binding constant for a particular binding interaction in units of 1 / M) to a target molecule, but does not significantly associate or bind to other molecules or components in the sample.

[0045] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. The abnormal cells may form solid tumors or constitute blood cancers. Cancer cells may spread locally or through the blood and lymphatic systems to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0046] A "disease" is a state of health in a subject in which the subject is unable to maintain homeostasis and, if the disease is not improved, the subject's health continues to deteriorate. In contrast, a "disorder" or "undesirable condition" in a subject is a state in which the subject can maintain homeostasis, but the subject's health is less favorable than if the disorder or undesirable condition were not present. A disorder or undesirable condition left untreated does not necessarily result in a further decline in the subject's health.

[0047] "Microbe" or "microorganism" means any species of bacterium, virus, archaea, or fungus.

[0048] "Particle" means a cell fragment or small object having a diameter of at least 100 nm and at most 6 μm, which is derived from a living cell or organism. The particle may be a virus particle, a small mineral particle, a cell fragment, or a synthetic particle.

[0049] "Encoding" refers to the inherent property of certain polynucleotide sequences, such as DNA, cDNA, and mRNA sequences, to serve as templates for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological products resulting therefrom.

[0050] Thus, a polynucleotide encodes a protein when transcription and translation of mRNA corresponding to that polynucleotide produces the protein in a cell or other biological system. Both the coding and non-coding strands can be referred to as encoding the protein or other product of the polynucleotide.

[0051] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0052] As used herein, the terms "endogenous" or "native" refer to a gene, protein, compound, molecule, or activity that is normally present in a host or host cell.

[0053] As used herein, the term "engulfment" refers to a receptor-mediated process by which endogenous or exogenous cells or particles greater than 100 nm in diameter are internalized by phagocytes or host cells as described herein. Engulfment generally consists of several steps: (1) tethering of the target cell or particle by direct or indirect (via a bridging molecule) binding of an engulfment receptor to an engulfment-promoting or antigenic marker on the target cell or particle; and (2) internalization or engulfment of the entire target cell or particle, or a portion thereof. In certain embodiments, internalization occurs via cytoskeletal rearrangements in the phagocyte or host cell to form a phagosome, a membrane-bound compartment containing the internalized target. Engulfment further includes maturation of the phagosome, in which the phagosome becomes increasingly acidic and fuses with a lysosome (forming a phagolysosome), thereby degrading the engulfed target (e.g., "phagocytosis"). Alternatively, phagosome-lysosome fusion may not be observed in engulfment. In yet another embodiment, the phagosome may regurgitate or release its contents into the extracellular environment before complete degradation. In some embodiments, engulfment refers to phagocytosis. In some embodiments, engulfment includes the tethering, but not internalization, of a target cell or particle by a phagocytic cell of a host cell as described herein. In some embodiments, engulfment includes the tethering and internalization of a portion of a target cell or particle by a phagocytic cell of a host cell as described herein.

[0054] As used herein, the term "phagocytosis" refers to the engulfment process of cells or large particles (≧0.5 μm), which involves target cell or particle tethering, target cell or particle engulfment, and degradation of the internalized target cell or particle. In certain embodiments, phagocytosis involves the formation of a phagosome that encompasses the internalized target cell or particle, and phagosome fusion with a lysosome to form a phagolysosome, where its contents are degraded. In certain embodiments, during phagocytosis, a phagocytic synapse is formed after a CER expressed on a phagocyte or host cell, as described herein, binds to an engulfment marker expressed by the target cell or particle. An actin-rich phagocytic cup is generated at the phagocytic synapse. The phagocytic arms extend around the target cell or particle through cytoskeletal rearrangements. Finally, the target cell or particle is engulfed by forces generated by motor proteins into the phagocyte or host cell. As used herein, "phagocytosis" includes the process of "efferocytosis," which specifically refers to the phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.

[0055] As used herein, the term "engulfment-promoting marker" refers to a moiety (e.g., a protein, lipid, or polysaccharide) displayed on the surface of apoptotic, necrotic, pyroptotic, or infected cells to distinguish them from non-apoptotic, non-necrotic, non-pyroptotic, oncotic, or uninfected cells, respectively. The engulfment-promoting marker can be an intracellular moiety exposed on the surface of apoptotic or necrotic cells, a moiety with altered glycosylation or surface charge on apoptotic or necrotic cells, or a serum moiety associated with apoptotic, necrotic, pyroptotic, or oncotic cells. Examples of engulfment-promoting markers for apoptotic cells include phosphatidylserine (PtdSer), ICAM-3, oxidized low-density lipoprotein, calreticulin, annexin I, complement C1q, and thrombospondin. Necrotic, swollen, and pyroptotic cells also expose PtdSer engulfment-promoting markers on the cell surface. Enulfment receptors can directly or indirectly detect (or bind to) engulfment-promoting markers on target cells (e.g., damaged, infected, apoptotic, necrotic, pyroptotic, or engulfed cells) using soluble bridging molecules as intermediates that bind to the engulfment-promoting markers.

[0056] "Engulfment signaling domain" refers to an intracellular effector domain that, upon binding of a target molecule (e.g., a pro-engulfment marker or antigen marker) targeted by the extracellular domain of a CER expressed by a host cell, activates one or more signaling pathways in the host cell, resulting in engulfment, which, in specific embodiments, includes cytoskeletal rearrangements of the host cell and internalization of the target cell, pathogen, or particle associated with the marker or antigen. In certain embodiments, the engulfment signaling domain activates one or more signaling pathways, resulting in phagocytosis of the target cell, pathogen, or particle. In certain embodiments, the engulfment signaling domain comprises a first engulfment signaling domain. In other specific embodiments, the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain. The first engulfment can be a homeostatic engulfment signaling domain or a pro-inflammatory engulfment signaling domain. In embodiments in which the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain, the first engulfment signaling domain can be a homeostatic engulfment signaling domain or a proinflammatory engulfment signaling domain. Similarly, the second engulfment signaling domain can be selected from a homeostatic engulfment signaling domain and a proinflammatory engulfment signaling domain. In certain embodiments, the CER comprises a first engulfment signaling domain and a second engulfment signaling domain, both of which are homeostatic engulfment signaling domains. In other specific embodiments, the CER comprises a first engulfment signaling domain and a second engulfment signaling domain, both of which are proinflammatory engulfment signaling domains.In yet other embodiments, the CER comprises a first engulfment signaling domain that is a homeostatic engulfment signaling domain and a second engulfment signaling domain that is a proinflammatory engulfment signaling domain, hi yet other embodiments, the CER comprises a first engulfment signaling domain that is a proinflammatory engulfment signaling domain and a second engulfment signaling domain that is a homeostatic engulfment signaling domain.

[0057] The term "homeostatic engulfment signaling domain" refers to an effector domain that (i) stimulates engulfment of a target cell, microorganism, or particle, without (ii) being derived from an endogenous receptor or signaling molecule that typically stimulates an inflammatory or immunogenic response. In certain embodiments, a homeostatic engulfment signaling domain stimulates host cell secretion of anti-inflammatory and / or immunosuppressive cytokines, such as TGF-β and IL-10. In particular embodiments, stimulation of homeostatic engulfment signaling dampens, attenuates, or resolves inflammation in the local tissue environment. A homeostatic engulfment signaling domain may also be referred to as a "non-inflammatory" engulfment signaling domain or a "non-immunogenic" engulfment signaling domain.

[0058] A "proinflammatory engulfment signaling domain" refers to an effector domain derived from an endogenous receptor or signaling molecule that (i) stimulates engulfment of a target cell, pathogen, or particle, and (ii) stimulates one or more of: (a) host cell secretion of inflammatory cytokines, such as TNFα, IL-1, IL-6, IL-12, and IL-23; (b) host cell secretion of inflammatory chemokines, such as CCL5 (RANTES), CXCL9, and CXCL10; (c) upregulation of cell surface costimulatory markers, such as CD80, CD86, HLA-DR, CD40, HVEM, and 4-1BBL; and (d) activation of one or more signaling cascades, such as NF-κB, generally inducing, enhancing, or complementing chemotherapy, antibody-based immunotherapy, or cellular therapy, such as T cell-targeted therapy. In certain embodiments, stimulation of proinflammatory engulfment signaling promotes inflammation in the local tissue environment. A pro-inflammatory engulfment signaling domain may also be referred to as an "immunogenic" engulfment signaling domain or an "inflammatory" engulfment signaling domain.

[0059] As used herein, an "effector domain" is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain upon receiving an appropriate signal. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal upon binding, or it directly binds to a target molecule and elicits a signal from the effector domain. For example, in response to binding of a CER to a target molecule, the effector domain can transmit a signal inside the host cell to induce an effector function, such as engulfment, phagolysosomal maturation, secretion of anti-inflammatory and / or immunosuppressive cytokines, or secretion of inflammatory cytokines and / or chemokines. An effector domain can directly promote a cellular response if it contains one or more signaling domains or motifs. In other embodiments, an effector domain can indirectly promote a cellular response by associating with one or more other proteins that directly promote the cellular response.

[0060] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to a gene, protein, compound, molecule, or activity that is not native to a host cell or subject, or a gene, protein, compound, molecule, or activity that is native to a subject or host cell, but that has been modified or mutated such that the structure, activity, or both differ between the native and mutant molecules. In certain embodiments, a heterologous, non-endogenous, or exogenous molecule (e.g., receptor, ligand) may not be endogenous to a host cell or subject, but instead, nucleic acid encoding such a molecule may be added to a host cell by conjugation, transformation, transfection, electroporation, etc., where the added nucleic acid molecule may be integrated into the host cell genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homolog" refers to a molecule or activity that is found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous molecule or gene encoding the molecule can be homologous to a native host or host cell molecule or gene encoding the molecule, respectively, but can have an altered structure, sequence, expression level, or combination thereof. A non-endogenous molecule can be derived from the same species, a different species, or a combination thereof.

[0061] "Junction amino acids" or "junction amino acid residues" refers to one or more (e.g., about 2 to 20) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide. Junction amino acids can arise from the construct design of a chimeric protein (e.g., can be amino acid residues resulting from the use of restriction enzyme sites during construction of a nucleic acid molecule encoding a fusion protein).

[0062] "Nucleic acid molecules" and "polynucleotides" can be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. Nucleic acid molecules can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-coding (antisense strand). A coding molecule can have a coding sequence identical to a coding sequence known in the art, or can have a different coding sequence that, as a result of redundancy or degeneracy in the genetic code, or by splicing, can encode the same polypeptide.

[0063] The term "overexpressed" or "overexpression" of an antigen refers to an abnormally high level of antigen expression in a cell. Overexpressed antigens or overexpression of antigens are often associated with disease states such as hematological cancers and in cells forming solid tumors within a particular tissue or organ of a subject. Solid tumors or hematological cancers characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0064] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes peptides or proteins comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0065] As used herein, the term "mature polypeptide" or "mature protein" refers to a protein or polypeptide that is secreted or localized in the cell membrane or in a specific cell organelle (e.g., the endoplasmic reticulum, Golgi apparatus, or endosome) and does not include an N-terminal signal peptide.

[0066] A "signal peptide," also known as a "signal sequence," "leader sequence," "leader peptide," "localization signal," or "localization sequence," is a short peptide (usually 15-30 amino acids long) present at the N-terminus of newly synthesized proteins destined for the secretory pathway. Signal peptides generally contain a short sequence of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a cleavage site for a signal peptidase. In eukaryotes, the signal peptide directs the translocation of the newly synthesized protein to the endoplasmic reticulum, where it is cleaved by the signal peptidase to generate the mature protein, which is then progressed to its appropriate destination.

[0067] The "percent identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity (%) = number of identical positions / total number of positions × 100), and also takes into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. The comparison of sequences and determination of percent identity between two or more sequences can be accomplished using mathematical algorithms, such as the BLAST and Gapped BLAST programs, with their default parameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; see also BLASTN (www.ncbi.nlm.nih.gov / BLAST)).

[0068] A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid with similar properties. Examples of conservative substitutions are well known in the art (see, for example, WO97 / 09433, p. 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

[0069] The term "chimeric" refers to a nucleic acid molecule or protein that contains sequences associated or linked together that are not endogenous and are not normally found associated or linked together in nature. For example, a chimeric nucleic acid molecule can contain regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but that are arranged in a manner different from that found in nature.

[0070] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0071] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may include an enhancer sequence and other control elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0072] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0073] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell only when an inducer corresponding to the promoter is substantially present in the cell.

[0074] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded by or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0075] The terms "subject," "patient," and "individual" are used interchangeably herein and are intended to include organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and transgenic species thereof.

[0076] The term "T cell" refers to a cell of the T cell lineage. A "cell of the T cell lineage" refers to a cell that displays at least one phenotypic characteristic of a T cell or its precursor or progenitor, which distinguishes the cell from other lymphoid cells and cells of the erythroid or myeloid lineage. Such phenotypic characteristic may be the expression of one or more proteins specific for T cells (e.g., CD3 + , CD4 + , CD8 + ), or physiological, morphological, functional, or immunological characteristics specific to T cells. For example, cells of the T cell lineage may include expression of precursor or progenitor cells committed to the T cell lineage; CD25 +Immature and inactivated T cells; cells committed to the CD4 or CD8 lineage; CD4 + CD8 + Thymic progenitor cells that are double positive; CD4 + or CD8 + TCRαβ or TCRγκ; or mature and functional or activated T cells. The term "T cells" includes naive T cells (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory T cells (CD45RO-), and + , CD62L + , CD8 + ), effector memory T cells (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), mucosal-associated invariant T cells, natural killer T cells, and tissue-resident T cells.

[0077] The term "B cell" refers to a cell of the B cell lineage. A "cell of the B cell lineage" refers to a cell that displays at least one phenotypic characteristic of a B cell or its precursor or progenitor, which distinguishes the cell from other lymphoid cells and cells of the erythroid or myeloid lineage. Such phenotypic characteristic may be the expression of one or more proteins specific for B cells (e.g., CD19). + , CD72 + , CD24 + , CD20 + ), or physiological, morphological, functional, or immunological characteristics specific to B cells. For example, cells of the B cell lineage can be precursor or progenitor cells committed to the B cell lineage (e.g., prepro-B cells, pro-B cells, and pro-B cells); immature and inactivated B cells, or mature and functional or activated B cells. Thus, "B cells" include naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablast cells, and memory B cells (e.g., CD27 + , IgD - ) are included.

[0078] A "therapeutically effective amount" or "effective amount" of a chimeric protein or cells expressing a chimeric protein of the invention (e.g., CER or cells expressing CER) refers to the amount of protein or cells sufficient to result in amelioration of one or more symptoms of the disease, disorder, or undesired condition being treated. When referring to an individual active ingredient or cells expressing a single active ingredient administered alone, a therapeutically effective amount refers to the effect of that ingredient or cells expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of the active ingredients, or the combined amounts of the supplemental active ingredients in combination with cells expressing the active ingredients, whether administered sequentially or simultaneously, that result in the therapeutic effect.

[0079] "Treating" or "treatment" or "ameliorating" refers to the medical management of a disease, disorder, or undesirable condition in a subject. Generally, an appropriate dose or treatment regimen comprising host cells expressing a CER of the invention is administered in an amount sufficient to provide a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventative benefit includes improved clinical outcome; alleviation or reduction of symptoms associated with the disease, disorder, or undesirable condition; reduced occurrence of symptoms; improved quality of life; a longer disease-free state; a reduction in the severity of the disease, disorder, or undesirable condition; stabilization of the disease state; delay in disease progression; remission; survival; extended survival; or any combination thereof.

[0080] As used herein, the phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct location and orientation relative to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0081] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector can be, for example, a plasmid, cosmid, virus, or phage. The term should be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into a cell. An "expression vector" is a vector that, when present in the appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector.

[0082] In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, gammaretroviral vectors, and lentiviral vectors. "Retrovirus" refers to a virus with an RNA genome. "Gammaretrovirus" refers to the Retroviridae family. Examples of gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "Lentivirus" refers to a genus of retroviruses that can infect dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0083] In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, closed-end linear double-stranded (CELiD) DNA, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). When a non-viral delivery system is used, the delivery vehicle can be a liposome. Lipid formulations can be used to introduce nucleic acids into host cells in vitro, ex vivo, or in vivo. The nucleic acid can be encapsulated inside the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the nucleic acid, comprised in or complexed with a micelle, or otherwise associated with a lipid.

[0084] Further definitions are provided throughout this specification.

[0085] Chimeric Enlargement Receptor (CER) Chimeric engulfment receptors (CERs) are described herein. In certain embodiments, the CER is a chimeric, single-chain protein comprising an extracellular domain and an engulfment signaling domain connected by a transmembrane domain. The extracellular domain comprises an extracellular binding domain and, optionally, an extracellular spacer domain. When expressed in a host cell, the CER confers an engulfment phenotype on modified host cells specific for selected engulfment-promoting or antigenic markers present on or expressed by target cells, pathogens, particles, or other substances (the host cells are "switched" to the engulfment phenotype). In certain embodiments, the CER confers a phagocytic phenotype on modified host cells specific for selected engulfment-promoting or antigenic markers present on or expressed by target cells, pathogens, particles, or other substances. In certain CER embodiments, the chimeric protein comprises, in amino- to carboxyl-terminal order: an extracellular domain having a binding domain specific for a target molecule and an optional extracellular spacer domain; a transmembrane domain; and an enlargement signaling domain (see, e.g., Figures 1A and 1B).

[0086] The components of the CERs described herein can be selected and arranged to provide a desired engulfment phenotype. For example, in certain embodiments, the extracellular domain can include a binding domain specific for (i) an engulfment-promoting marker associated with apoptotic, dead, dying, damaged, or necrotic cells; or (ii) an antigenic marker presented by foreign entities (e.g., pathogens), infected T cells, or aberrant T cells associated with infection, disease, disorder, or other undesirable pathology.

[0087] The enlargement signaling domain can comprise one or more effector domains (also referred to as "signaling" domains) that drive engulfment of a target cell. Signaling by the enlargement signaling domain is triggered by binding of the extracellular domain to a targeted pro-engulfment or antigenic marker. In certain embodiments, the enlargement signaling domain comprises a first enlargement signaling domain. In certain embodiments, the first enlargement signaling domain is selected to initiate a homeostatic enlargement response. In yet other embodiments, the first enlargement signaling domain is selected to initiate a pro-inflammatory enlargement response. In still other embodiments, the enlargement signaling domain comprises a first enlargement signaling domain and a second enlargement signaling domain, wherein the first and second enlargement signaling domains are both homeostatic signaling domains, both pro-inflammatory signaling domains, or either one of each (in either order). The CERs described herein can be designed for application in a variety of therapeutic contexts (e.g., clearance of apoptotic, dead, dying, damaged, infected or necrotic cells, clearance of infectious disease-causing pathogens, and clearance of abnormal cells associated with a disease, disorder, or undesirable condition) to provide engulfment signaling (e.g., homeostatic or pro-inflammatory engulfment signaling) that complements the desired therapeutic outcome.

[0088] Figures 3A and 3B provide a functional comparison of lymphocytes modified with embodiments of the CERs of the present invention with native lymphocytes. Figure 3A shows native lymphocytes, and as shown in the figure, native lymphocytes do not exhibit an engulfment phenotype. However, as shown in Figure 3B, lymphocytes modified to express the CERs described herein exhibit an engulfment phenotype specific to targeted cancer cells, resulting in engulfment (e.g., phagocytosis) and elimination of the targeted cancer cells. Furthermore, as shown in Figure 3B, in certain embodiments, CERs can be designed to polarize the engulfment process. In certain embodiments, the engulfment signaling domains included in the CERs described herein can be selected to drive homeostatic engulfment signaling or proinflammatory engulfment signaling.

[0089] The constituent parts of the fusion proteins of the present invention are further described herein.

[0090] Extracellular domain As described herein, a CER comprises an extracellular domain specific for a target molecule. In certain embodiments, the extracellular domain comprises an extracellular binding domain that specifically binds to a targeted engulfment-promoting marker or antigen. Binding of the target molecule by the binding domain can prevent interaction between the target molecule (e.g., a receptor or ligand) and another molecule, for example, disrupting, reducing, or eliminating a specific function (e.g., signal transduction) of the target molecule. In certain embodiments, binding of the target molecule can induce a specific biological pathway or identify the target molecule or cells expressing the target molecule for elimination.

[0091] A binding domain can be any polypeptide or peptide that specifically binds to a target molecule of interest. Sources of binding domains include receptor binding domains, ligand binding domains, and antibodies or antigen-binding portions, such as antibody variable regions from various species, including human, rodent, avian, or ovine (which can be antibodies, sFv, scFv, Fab, scFv-based grababodies, or soluble VH domains or domain antibodies). Further sources of binding domains include those from camelids (camel, dromedary or llama; Ghahroudi et al., FEBS Lett. 414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; Hamers-Casterman et al., Nature 363:446, 1993 and Nguyen et al., J. Mol. Biol. 275:413, 1998), nurse shark (Roux et al., Proc. Nat'l. Acad. Sci. (USA) 95:11804, 1998), spotted ratfish (Nguyen et al., Immunogen. 54:39, 2002) or lamprey (Herrin et al., Proc. These antibodies include variable regions of antibodies from other species, such as those derived from other antigen-binding domains (Nature, Acad. Sci. (USA) 105:2040, 2008 and Alder et al. Nat. Immunol. 9:319, 2008). These antibodies can form an antigen-binding region using only the heavy chain variable region. That is, these functional antibodies are homodimers of heavy chains only (referred to as "heavy chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008).

[0092] In some embodiments, the extracellular domain binds to an engulfment-promoting marker. In certain such embodiments, the engulfment-promoting marker targeted by the extracellular domain is phosphatidylserine (PtdSer), ICAM-3, oxidized low-density lipoprotein, calreticulin, annexin I, complement C1q, or thrombospondin. In further embodiments, the extracellular domain that binds to an engulfment-promoting marker is derived from an endogenous engulfment receptor or a soluble bridging molecule for the engulfment receptor (e.g., GAS6, protein S, MFG-E8). In some embodiments, the entire extracellular portion (in the case of a transmembrane molecule), the entire bridging molecule, or a truncated portion of the engulfment receptor or bridging molecule is used, provided that the truncated portion retains sufficient binding activity to the engulfment-promoting marker (i.e., is a functional variant). In further embodiments, the extracellular portion of the engulfment receptor or bridging molecule used in the extracellular domain is the entire extracellular portion (in the case of a transmembrane molecule), the entire bridging molecule, or a variant of the extracellular portion of the engulfment receptor or bridging molecule, provided that the variant retains sufficient binding activity to the engulfment-promoting marker (i.e., is a functional variant).

[0093] In certain embodiments, the extracellular domain comprises T cell immunoglobulin and mucin domain 1 (Tim1), T cell immunoglobulin and mucin domain 4 (Tim4), T cell immunoglobulin and mucin domain 3 (Tim3), stabilin-2, RAGE, or an Fc receptor (FcR) extracellular domain. In certain embodiments, the FcR extracellular domain can comprise a binding domain from FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, or FcαR1. In further embodiments, the extracellular domain is selected from the group consisting of Tim1, Tim4, Tim3, stabilin-2, receptor for advanced glycation end products (RAGE), brain-specific angiogenesis inhibitor 1 (BAI1), Milk Fat Globule-EGF factor 8 (MFG-E8) (e.g., the FA58C2 domain that mediates high-affinity binding to PtdSer), growth arrest specific 6 (GAS6), protein S, protein C, factor II, factor VII, factor IX, factor X, β These may include 2-glycoprotein I, α5β3 integrin and other integrins, CR3 complement receptor, CR4 complement receptor, CD14, CD93, annexin V, phosphatidylserine receptor (PSr), prothrombin, or scavenger receptors such as scavenger receptor B (SRB) (e.g., SRB1 (CD36)), scavenger receptor C (SRC) (e.g., LOX-1, SRCL), scavenger receptor D (SRD) (e.g., CD68, macrocyanin), and the PtdSer binding domain from PSOX.

[0094] In certain embodiments, the extracellular domain comprises an FcγRI-binding domain comprising the amino acid sequence of SEQ ID NO: 31 or amino acids 16-292 of SEQ ID NO: 31; a TIM1-binding domain comprising the amino acid sequence of SEQ ID NO: 28 or amino acids 21-290 of SEQ ID NO: 28; a TIM4-binding domain comprising the amino acid sequence of SEQ ID NO: 29 or amino acids 25-314 of SEQ ID NO: 29; a TIM3-binding domain comprising the amino acid sequence of SEQ ID NO: 34 or amino acids 22-202 of SEQ ID NO: 34; an FA58C2-binding domain comprising the amino acid sequence of SEQ ID NO: 30; The amino acid sequence of SEQ ID NO: 33 or a GAS6 binding domain comprising amino acids 31-94 of SEQ ID NO: 32, a BAI1 binding domain comprising the amino acid sequence of SEQ ID NO: 117, or a Protein S binding domain comprising amino acid sequence of SEQ ID NO: 33 or amino acids 25-87 of SEQ ID NO: 33, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the .... In any other embodiment, the extracellular domain comprises or is encoded by a polynucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a polynucleotide sequence encoding the FcγRI-binding domain of SEQ ID NO: 4, a polynucleotide encoding the TIM1-binding domain of SEQ ID NO: 1, a polynucleotide encoding the TIM4-binding domain of SEQ ID NO: 2, a polynucleotide encoding the TIM3-binding domain of SEQ ID NO: 7, a polynucleotide encoding the FA58C2-binding domain of SEQ ID NO: 3, a polynucleotide encoding the GAS6-binding domain of SEQ ID NO: 5, a polynucleotide encoding the BAI1-binding domain of SEQ ID NO: 135, or a polynucleotide sequence encoding the Protein S-binding domain of SEQ ID NO: 6.

[0095] In other embodiments, the extracellular domain is derived from at least one of the following: CD14, which binds to ICAM3; a scavenger receptor extracellular domain, which binds to oxidized LDL; a lectin, which binds to altered sugars; CD36, which binds to thrombospondin; or LRP1 / CD91 or a lectin portion, which binds to calreticulin.

[0096] In yet other embodiments, the extracellular domain comprises an antibody or antigen-binding fragment thereof, such as a single-chain Fv fragment (scFv) comprising VH and VL regions specific for a target molecule of interest. In certain embodiments, the antibody is a chimeric, human, or humanized antibody. In further embodiments, the V H and V LThe region is derived from human or humanized antibody. In certain embodiments, the extracellular domain is an antibody specific to engulfment-promoting marker or its antigen-binding portion.Antibodies specific to phosphatidylserine are known in the art (see U.S. Patent No. 7,247,303; Khogeer et al., 2015, Lupus 24:186-90; Gerber et al., 2015, Am. J. Nucl. Med. Mol. Imaging, 5:493-503, each of which is incorporated herein by reference in its entirety). In certain embodiments, the target molecule of interest is a tumor antigen, such as CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2, and GD2. Exemplary V H and V LThe regions contain anti-CD138, -CD38, -CD33, -CD123, -CD72, -CD79a, -CD79b, -mesothelin, -PSMA, -BCMA, -ROR1, -MUC-16, -L1CAM, -CD22, -CD19, -CD20, -CD23, -CD24, -CD37, -CD30, -CA125, -CD56, -c-Met, -EGFR, -GD-3, -HPV E6, -HPV Included are E7, -MUC-1, -HER2, -folate receptor alpha, -CD97, -CD171, -CD179a, -CD44v6, -WT1, -VEGF-alpha, -VEGFR1, -IL-13Rα1, -IL-13Rα2, -IL-11Rα, -PSA, -FcRH5, -NKG2D ligand, -NY-ESO-1, -TAG-72, -CEA, -ephrinA2, -ephrinB2, -Lewis A antigen, -Lewis Y antigen, -MAGE, -MAGE-A1, -RAGE-1, -folate receptor beta, -EGFRviii, -VEGFR-2, -LGR5, -SSX2, -AKAP-4, -FLT3, -fucosylGM1, -GM3, -o-acetyl-GD2, and -GD2-specific monoclonal antibody segments.

[0097] In further embodiments, the extracellular domain comprises a Fab specific for a target of interest, such as CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand de, NY-ESO-1, TAG-72, CEA, EphrinA2, EphrinB2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP- 4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2, and GD2, and the Fab regions contain anti-CD138, -CD38, -CD33, -CD123, -CD72, -CD79a, -CD79b, -mesothelin, -PSMA, -BCMA, -ROR1, -MUC-16, -L1CAM, -CD22, -CD19, -CD20, -CD23, -CD24, -CD37, -CD30, -CA125, -CD56, -c-Met, -EGFR, -GD-3, and -HPV, respectively. Included are E6, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, WT1, VEGF-alpha, VEGFR1, IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2, and portions of GD2-specific monoclonal antibodies.

[0098] Target molecules that specifically bind to the extracellular domain of a CER of the present invention can be found on or associated with a cell of interest (a "target cell"). Exemplary target cells include cancer cells, cells associated with an autoimmune or inflammatory disease or disorder, and infectious pathogens (e.g., bacteria, viruses, or fungi) or infected cells (e.g., virus-infected cells). Cells of infectious organisms, such as mammalian parasites, are also contemplated as target cells.

[0099] In some embodiments, the extracellular domain optionally includes an extracellular, non-signaling spacer or linker domain. If included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable proper cell-cell contact, binding, and activation. The extracellular spacer domain is generally located between the extracellular binding domain and the transmembrane domain. 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 binding domain size and affinity (see, e.g., Guest et al., J. Immunother. 28:203-11, 2005; PCT Publication WO2014 / 031687). 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. Modified IgG4 hinge regions are described in PCT Publication WO2014 / 031687, the entire contents of which are incorporated herein by reference. In certain embodiments, the extracellular spacer domain comprises a modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 67). Other examples of hinge regions that can be used in the CERs described herein include hinge regions present in the extracellular regions of type I membrane proteins such as CD8a, CD4, CD28, and CD7, which can be wild-type or mutants thereof. In further embodiments, the extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from the following: a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT Publication WO2014 / 031687, the entire contents of which are incorporated herein by reference). In still further 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 a further embodiment, the extracellular spacer domain may be derived from MERTK.

[0100] Engulfment signaling domain The enlargement signaling domain of a CER is an intracellular effector domain that can transmit a functional signal to a cell in response to binding of the extracellular domain of the CER to a target molecule. In certain embodiments, the enlargement signaling domain can include one or more homeostatic enlargement signaling domains, one or more proinflammatory signaling domains, or both a homeostatic signaling domain and a proinflammatory signaling domain.

[0101] In certain embodiments, the engulfment signaling domain is the intracellular signaling domain of an endogenous engulfment receptor. Examples of endogenous engulfment receptors from which the engulfment signaling domain can be derived include Mer tyrosine kinase (MERTK), Tyro3 protein tyrosine kinase, Axl receptor tyrosine kinase, BAI1, mannose receptor type C-1 (MRC1), and Fc receptors (FcR) (e.g., FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, or FcαR1). In other embodiments, the engulfment signaling domain is the intracellular signaling domain of an endogenous kinase or adaptor protein involved in the signal transduction pathway during phagocytosis. Examples of kinases involved in the phagocytosis signal transduction pathway include spleen-associated tyrosine kinase (SYK), the zeta chain of T-cell receptor-associated protein kinase 70 (Zap70), and phosphoinositide 3-kinase (PI3K).

[0102] The enlargement signaling domain can be any portion of the enlargement signaling molecule that retains sufficient signaling activity. In some embodiments, the full-length enlargement signaling molecule or its full-length intracellular component is used. In some embodiments, a truncated portion of the enlargement signaling molecule or a truncated portion of the intracellular component of the enlargement signaling molecule is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the enlargement signaling domain is a variant of the entire enlargement signaling molecule or a truncated portion thereof, provided that the variant retains sufficient signaling activity (i.e., it is a functional variant).

[0103] In certain embodiments, the engulfment signaling domain comprises a homeostatic engulfment signaling domain, such as an MRC1 signaling domain, an ItgB5 signaling domain, a MERTK signaling domain, a Tyro3 signaling domain, an Axl signaling domain, a BAI1 signaling domain, or an ELMO signaling domain. In more particular embodiments, the engulfment signaling domain comprises a homeostatic engulfment signaling domain that comprises or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an MRC1 signaling domain comprising the amino acid sequence of SEQ ID NO:56, an ItgB5 signaling domain comprising the amino acid sequence of SEQ ID NO:114, a MERTK signaling domain comprising the amino acid sequence of SEQ ID NO:69, a Tyro3 signaling domain comprising the amino acid sequence of SEQ ID NO:45, an Axl signaling domain comprising the amino acid sequence of SEQ ID NO:44, a BAI1 signaling domain comprising the amino acid sequence of SEQ ID NO:136, or an ELMO signaling domain comprising the amino acid sequence of SEQ ID NO:120.In other embodiments, the engulfment signaling domain comprises a homeostatic engulfment signaling domain, wherein the homeostatic engulfment signaling domain is encoded by a polynucleotide sequence that comprises or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a polynucleotide encoding the MRC1 signaling domain of SEQ ID NO:55, a polynucleotide encoding the ItgB5 signaling domain of SEQ ID NO:137, a polynucleotide encoding the MERTK signaling domain of SEQ ID NO:138, a polynucleotide encoding the Tyro3 signaling domain of SEQ ID NO:18, a polynucleotide encoding the Axl signaling domain of SEQ ID NO:17, a polynucleotide encoding the BAI1 signaling domain of SEQ ID NO:139, or a polynucleotide encoding the ELMO signaling domain of SEQ ID NO:140.

[0104] In certain embodiments, signaling by the homeostatic engulfment signaling domain results in the expression of at least one of an anti-inflammatory cytokine and an immunosuppressive cytokine. In certain embodiments, at least one of the anti-inflammatory cytokine and the immunosuppressive cytokine is TGF-β, IL-10, or both.

[0105] In certain embodiments, the engulfment signaling domain is, for example, a Traf6 signaling domain, a Syk signaling domain, a MyD88 signaling domain, a truncated MyD88 signaling domain (e.g., a death domain), a proinflammatory engulfment signaling domain, which is a CD79b signaling domain, a CD8+ signaling domain, a CD9+ signaling domain, a CD12 signaling domain, a CD14 signaling domain, a CD16 signaling domain, a CD24 signaling domain, a CD16 signaling domain, a CD18 signaling domain, a CD24 signaling domain, a CD18 ...

[0106] In certain embodiments, the engulfment signaling domain is a Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO: 54, a Syk signaling domain comprising the amino acid sequence of SEQ ID NO: 46, a MyD88 signaling domain comprising the amino acid sequence of SEQ ID NO: 53, a truncated MyD88 signaling domain comprising the amino acid sequence of SEQ ID NO: 78, a Zap70 signaling domain comprising the amino acid sequence of SEQ ID NO: 47, an FcεRIγ signaling domain comprising the amino acid sequence of SEQ ID NO: 88, an FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 48, an FcγR2A signaling domain comprising the amino acid sequence of SEQ ID NO: 49, an FcγR2C signaling domain comprising the amino acid sequence of SEQ ID NO: 50, an FcγR2D signaling domain comprising the amino acid sequence of SEQ ID NO: 51, an FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 52, an FcγR2E signaling domain comprising the amino acid sequence of SEQ ID NO: 53, an FcγR2F signaling domain comprising the amino acid sequence of SEQ ID NO: 54, an FcγR1G signaling domain comprising the amino acid sequence of SEQ ID NO: 55, an FcγR2G signaling domain comprising the amino acid sequence of SEQ ID NO: 56, an FcγR2H signaling domain comprising the amino acid sequence of SEQ ID NO: 57, an FcγR1G signaling domain comprising the amino acid sequence of SEQ ID NO: 58, an FcγR2H signaling domain comprising the amino acid sequence of SEQ ID NO: 59, an FcγR2H signaling domain comprising the amino acid sequence of SEQ ID NO: 60, an FcγR2H signaling domain comprising the amino acid sequence of SEQ ID NO: 61, an FcγR1G signaling domain comprising a proinflammatory engulfment signaling domain that comprises or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a cγR3A signaling domain, a BAFF-R signaling domain comprising the amino acid sequence of SEQ ID NO:94, a DAP12 signaling domain comprising the amino acid sequence of SEQ ID NO:82, an NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO:92, a truncated NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO:132, or a CD79b signaling domain comprising the amino acid sequence of SEQ ID NO:97.

[0107] In other embodiments, the engulfment signaling domain comprises a proinflammatory engulfment signaling domain, wherein the proinflammatory signaling domain is a polynucleotide encoding a Traf6 signaling domain of SEQ ID NO: 27, a polynucleotide encoding a Syk signaling domain of SEQ ID NO: 19, a polynucleotide encoding a MyD88 signaling domain of SEQ ID NO: 26, a polynucleotide encoding a truncated MyD88 signaling domain of SEQ ID NO: 99, a polynucleotide encoding Zap70 of SEQ ID NO: 20, a polynucleotide encoding an FcεRIγ signaling domain of SEQ ID NO: 141, a polynucleotide encoding an FcγR1 signaling domain of SEQ ID NO: 21, a polynucleotide encoding an FcγR2A signaling domain of SEQ ID NO: 22, a polynucleotide encoding a truncated MyD88 signaling domain of SEQ ID NO: 23 a polynucleotide encoding the FcγR2C signaling domain of SEQ ID NO:24, a polynucleotide encoding the FcγR3A signaling domain of SEQ ID NO:124, a polynucleotide encoding the BAFF-R signaling domain of SEQ ID NO:126, a polynucleotide encoding the DAP12 signaling domain of SEQ ID NO:127, a polynucleotide encoding the NFAM1 signaling domain of SEQ ID NO:129, or a polynucleotide encoding the CD79b signaling domain of SEQ ID NO:128.

[0108] In further embodiments, signaling by the proinflammatory engulfment signaling domain results in expression of at least one inflammatory cytokine, inflammatory chemokine, or costimulatory cell surface marker. In still further embodiments, the inflammatory cytokine is TNFα, IL-1, IL-6, IL-12, or IL-23, or any combination thereof, the inflammatory chemokine is CCL5 (RANTES), CXCL9, or CXCL10, or any combination thereof, and the costimulatory cell surface marker is CD80, CD86, HLA-DR, CD40, HVEM, or 4-1BBL, or any combination thereof.

[0109] In still further embodiments, the engulfment signaling domain of a CER can comprise two or more signaling domains. In certain such embodiments, the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain. In embodiments in which the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain, the first engulfment signaling domain can be a homeostatic engulfment signaling domain or a proinflammatory engulfment signaling domain. Similarly, the second engulfment signaling domain can be selected from a homeostatic engulfment signaling domain or a proinflammatory engulfment signaling domain. In certain embodiments, a CER comprises a first engulfment signaling domain and a second engulfment signaling domain, both of which are homeostatic engulfment signaling domains. In certain other embodiments, a CER comprises a first engulfment signaling domain and a second engulfment signaling domain, both of which are proinflammatory engulfment signaling domains. In yet other embodiments, the CER comprises a first engulfment signaling domain that is a homeostatic engulfment signaling domain and a second engulfment signaling domain that is a proinflammatory engulfment signaling domain. In still other embodiments, the CER comprises a first engulfment signaling domain that is a proinflammatory engulfment signaling domain and a second engulfment signaling domain that is a homeostatic engulfment signaling domain. In embodiments in which the first engulfment signaling domain and the second engulfment signaling domain are both homeostatic engulfment signaling domains or both proinflammatory signaling domains, the first and second engulfment signaling domains can be the same or different.In certain embodiments, the domains utilized as the first engulfment signaling domain and the second engulfment signaling domain are selected from one or more of the specific signaling domains described herein, including MRC1, ItgB5, MERTK, ELMO, BAI1, Tyro3, ​​Axl, Traf6, Syk, MyD88, Zap70, PI3K, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, and CD79b.

[0110] In certain embodiments, the presence of the first engulfment signaling domain and the second engulfment signaling domain enhances the engulfment activity of the CER, increases the persistence of CER-modified host cells, increases the proliferation of CER-modified host cells, or a combination thereof. In certain embodiments, the inclusion of the second engulfment signaling domain that is a proinflammatory signaling domain and the first engulfment signaling domain that is a homeostatic engulfment signaling domain enhances the engulfment activity of the CER, increases the persistence of CER-modified host cells, increases the proliferation of CER-modified host cells, or a combination thereof.

[0111] Transmembrane domain The transmembrane domain links the extracellular domain and the engulfment signaling domain and is located between them. The transmembrane domain is a hydrophobic α-helix that crosses the host cell membrane. The transmembrane domain can be fused directly to the binding domain or, if present, to the extracellular spacer domain. In certain embodiments, the transmembrane domain is derived from an integral membrane protein (e.g., a receptor, a cluster of differentiation (CD) molecule, an enzyme, a transporter, a cell adhesion molecule, etc.). The transmembrane domain can naturally be associated with either the extracellular domain or the engulfment signaling domain contained in the CER (e.g., a CER contains a Tim4 binding domain and a Tim4 transmembrane domain). In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules, the transmembrane domain and the engulfment signaling domain are derived from different molecules, or the transmembrane domain, the extracellular domain, and the engulfment signaling domain are all derived from different molecules.

[0112] In certain embodiments, the transmembrane domain is a Tim1 transmembrane domain, a Tim4 transmembrane domain, an FcR transmembrane domain (e.g., FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1 transmembrane domain), a CD8a transmembrane domain, a MERTK transmembrane domain, an AxI transmembrane domain, a Tyro3 transmembrane domain, a BAI1 transmembrane domain, a CD4 transmembrane domain, a CD28 transmembrane domain, an MRC1 transmembrane domain, or a DAP12 transmembrane domain.

[0113] In certain embodiments, the transmembrane domain is a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 35, a Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 37, an FcεRIγ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 89, a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 38, a MERTK transmembrane domain comprising the amino acid sequence of SEQ ID NO: 39, an Axl transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, a Tyro3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 41, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 142, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 143, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 144, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 145, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 146, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 147, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 148, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 149, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 150, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 151, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 152, a BAI1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 68, a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, an MRC1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 118, or a DAP12 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 81, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the BAI1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 68, a CD4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, an MRC1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 118, or a DAP12 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 81.In other embodiments, the transmembrane domain is a polynucleotide sequence encoding a Tim1 transmembrane domain of SEQ ID NO: 8, a polynucleotide sequence encoding a Tim4 transmembrane domain of SEQ ID NO: 9, a polynucleotide sequence encoding an FcεRIγ transmembrane domain of SEQ ID NO: 85, a polynucleotide sequence encoding an FcγRI transmembrane domain of SEQ ID NO: 10, a polynucleotide sequence encoding a CD8a transmembrane domain of SEQ ID NO: 11, a polynucleotide sequence encoding a MERTK transmembrane domain of SEQ ID NO: 12, a polynucleotide sequence encoding an AxI transmembrane domain of SEQ ID NO: 13, a polynucleotide sequence encoding a Tyro3 transmembrane domain of SEQ ID NO: 14. The polynucleotide sequence encoding the CD28 transmembrane domain of SEQ ID NO: 144, the polynucleotide sequence encoding the BAI1 transmembrane domain of SEQ ID NO: 143, the polynucleotide sequence encoding the CD4 transmembrane domain of SEQ ID NO: 15, or the polynucleotide sequence encoding the DAP12 transmembrane domain of SEQ ID NO: 145, is provided by a polynucleotide sequence that comprises or is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the polynucleotide sequence encoding the CD28 transmembrane domain of SEQ ID NO: 144, the polynucleotide sequence encoding the BAI1 transmembrane domain of SEQ ID NO: 143, the polynucleotide sequence encoding the CD4 transmembrane domain of SEQ ID NO: 15, or the polynucleotide sequence encoding the DAP12 transmembrane domain of SEQ ID NO: 145.

[0114] It is understood that the direct linkage of one domain to another of the CERs described herein does not preclude the presence of intervening junction amino acids, which may be natural or non-natural (e.g., resulting from construct design of the chimeric protein).

[0115] CER Example The components of the CERs described herein can be selected and arranged in various combinations to provide a host cell with a desired engulfment phenotype. In addition to inducing engulfment of cells, pathogens, or particles that express or are characterized by molecules targeted by the CER-modified host cell, the CERs described herein can be designed to initiate a homeostatic or proinflammatory engulfment response, depending on the target cell or particle, the disease state, and the desired therapeutic outcome.

[0116] In one aspect, the present invention provides a chimeric engulfment receptor (CER) comprising a single-chain chimeric protein, the single-chain chimeric protein comprising: an extracellular domain comprising a binding domain that binds to phosphatidylserine (PtdSer); an engulfment signaling domain; and a transmembrane domain positioned between and connecting the extracellular domain and the engulfment signaling domain.

[0117] In certain embodiments, the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

[0118] In certain embodiments in which the CER comprises an extracellular domain comprising a binding domain that binds to PtdSer, the engulfment signaling domain is a homeostatic engulfment signaling domain or a proinflammatory engulfment signaling domain. In certain such embodiments, the homeostatic engulfment signaling domain or the proinflammatory engulfment signaling domain can be selected from one or more of those described herein. In other embodiments in which the CER comprises an extracellular domain comprising a binding domain that binds to PtdSer, the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain. The first engulfment signaling domain and the second engulfment signaling domain can both be homeostatic engulfment signaling domains, proinflammatory engulfment signaling domains, or either one of each (in either order). In certain such embodiments, the homeostatic engulfment signaling domain or the pro-inflammatory engulfment signaling domain encompassed by said first signaling domain and said second signaling domain may be selected from one or more of the homeostatic engulfment signaling domains and pro-inflammatory engulfment signaling domains described herein.

[0119] In one embodiment, a CER comprises an extracellular domain comprising a binding domain that binds PtdSer, the extracellular domain comprising a TIM4 PtdSer-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising a MERTK signaling domain (also referred to herein as "CEROl") (see, e.g., Figure 6A). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, a CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 71 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 71).

[0120] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a FA58C2 PtdSer-binding domain and an extracellular spacer domain comprising a modified IgG4 hinge region, a transmembrane domain comprising a CD28 transmembrane domain, and an engulfment signaling domain comprising a MERTK signaling domain (also referred to herein as "CER03") (see, e.g., Figure 9A). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 75 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 75).

[0121] Yet another embodiment, in which a CER comprises an extracellular domain comprising a binding domain that binds PtdSer, comprises an extracellular domain comprising a FA58C2 PtdSer-binding domain and an extracellular spacer domain comprising a modified IgG4 hinge region, a transmembrane domain comprising a CD28 transmembrane domain, and an engulfment signaling domain comprising a SYK signaling domain (also referred to herein as "CER04") (see, e.g., Figure 11A). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, a CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 70 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 70).

[0122] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising a Tyro3 signaling domain (also referred to herein as "CER08"). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 83 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 83).

[0123] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising a DAP12 signaling domain (also referred to herein as "CER09"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 84 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 84).

[0124] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a DAP12 transmembrane domain, and an engulfment signaling domain comprising a DAP12 signaling domain (also referred to herein as "CER10"). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 86 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 86).

[0125] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising an Axl signaling domain (also referred to herein as "CER11"). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 87 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 87).

[0126] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising an FcεRIγ signaling domain (also referred to herein as "CER12"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 90 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 90).

[0127] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising an FcεRIγ transmembrane domain, and an engulfment signaling domain comprising an FcεRIγ signaling domain (also referred to herein as "CER13"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 91 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 91).

[0128] Another embodiment in which a CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising a truncated MyD88 signaling domain that includes the death domain but lacks the TIR domain (also referred to herein as "CER15"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, a CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 79 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 79).

[0129] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising a MyD88 signaling domain (also referred to herein as "CER16"). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 80 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 80).

[0130] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, and an engulfment signaling domain comprising an NFAM1 signaling domain (also referred to herein as "CER25"). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 93 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 93).

[0131] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a truncated MyD88 signaling domain, and a second engulfment signaling domain comprising a BAFF-R signaling domain (also referred to herein as "CER85"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 95 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 95).

[0132] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a truncated MyD88 signaling domain, and a second engulfment signaling domain comprising a DAP12 signaling domain (also referred to herein as "CER86"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 96 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 96).

[0133] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a truncated MyD88 signaling domain, and a second engulfment signaling domain comprising a CD79b signaling domain (also referred to herein as "CER89"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 98 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 98).

[0134] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a truncated MyD88 signaling domain, and a second engulfment signaling domain comprising an NFAM1 signaling domain (also referred to herein as "CER90"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 100 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 100).

[0135] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a MERTK signaling domain, and a second engulfment signaling domain comprising a CD79b signaling domain (also referred to herein as "CER95"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 101 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 101).

[0136] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a MERTK signaling domain, and a second engulfment signaling domain comprising an NFAM1 signaling domain (also referred to herein as "CER96"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 102 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 102).

[0137] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a MERTK signaling domain, and a second engulfment signaling domain comprising a BAFF-R signaling domain (also referred to herein as "CER93"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 103 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 103).

[0138] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a BAFF-R signaling domain, and a second engulfment signaling domain comprising a truncated MyD88 signaling domain (also referred to herein as "CER87"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 130 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 130).

[0139] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a DAP12 signaling domain, and a second engulfment signaling domain comprising a truncated MyD88 signaling domain (also referred to herein as "CER88"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 131 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 131).

[0140] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a MERTK signaling domain, and a second engulfment signaling domain comprising a truncated MyD88 signaling domain (also referred to herein as "CER92"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 133 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 133).

[0141] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a MERTK signaling domain, and a second engulfment signaling domain comprising a DAP12 signaling domain (also referred to herein as "CER94"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 134 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 134).

[0142] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a MERTK signaling domain, and a second engulfment signaling domain comprising an NFAM1 signaling domain (also referred to herein as "CER96"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 102 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 102).

[0143] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a MERTK signaling domain, and a second engulfment signaling domain comprising a truncated NFAM1 signaling domain (also referred to herein as "CER96 with truncated NFAM1"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 116 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 116).

[0144] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising a BAFFR signaling domain, and a second engulfment signaling domain comprising a truncated MyD88 signaling domain (also referred to herein as "CER87"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 130 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 130).

[0145] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising an Axl signaling domain, and a second engulfment signaling domain comprising a DAP12 signaling domain (also referred to herein as "CER97"). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 152. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 152 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 152).

[0146] Another embodiment in which the CER comprises an extracellular domain comprising a binding domain that binds PtdSer comprises an extracellular domain comprising a TIM4-binding domain, a transmembrane domain comprising a TIM4 transmembrane domain, a first engulfment signaling domain comprising an Axl signaling domain, and a second engulfment signaling domain comprising a CD79b signaling domain (also referred to herein as "CER98"). In particular embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 153. In some embodiments, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 153 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 153).

[0147] In another aspect, the present invention provides a CER comprising a single-chain chimeric protein, wherein the single-chain chimeric protein comprises an extracellular domain including a binding domain that binds to a pro-engulfment marker or a target antigen; a pro-inflammatory engulfment signaling domain; and a transmembrane domain located between the extracellular domain and the pro-inflammatory engulfment signaling domain. Such a CER is specifically "polarized" to confer a pro-inflammatory or immunogenic engulfment phenotype upon binding to a target molecule (e.g., a pro-engulfment marker or a target antigen).

[0148] In certain embodiments of a CER comprising a proinflammatory engulfment signaling domain, the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

[0149] In yet another aspect, the present invention provides a CER comprising a single-chain chimeric protein, wherein the single-chain chimeric protein comprises: an extracellular domain comprising a binding domain that binds to an engulfment-promoting marker or a target antigen; an engulfment signaling domain comprising a first engulfment signaling domain and a second engulfment signaling domain; and a transmembrane domain located between and connecting the extracellular domain and a pro-inflammatory engulfment signaling domain, wherein the first engulfment signaling domain and the second engulfment signaling domain are both homeostatic engulfment signaling domains, pro-inflammatory engulfment signaling domains, or either one of them (in either order).

[0150] In any embodiment of a CER comprising an engulfment signaling domain comprising a first engulfment signaling domain and a second engulfment signaling domain, the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

[0151] In yet another aspect, the present invention provides a CER comprising a single-chain chimeric protein, wherein the single-chain chimeric protein comprises: an extracellular domain comprising an scFv that binds to an engulfment-promoting marker or a target antigen; an engulfment signaling domain; and a transmembrane domain located between and connecting the extracellular domain and the engulfment signaling domain, wherein the transmembrane domain and the engulfment signaling domain are each derived from a different molecule.

[0152] In certain embodiments, wherein the CER comprises an extracellular domain comprising an scFv that binds to an engulfment-promoting marker or a target antigen, the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

[0153] Embodiments in which a CER comprises an extracellular domain comprising an scFv that binds to an engulfment-promoting marker or target antigen include an extracellular domain comprising an scFv binding domain specific for CD19 (e.g., FMC63 scFv (SEQ ID NO: 66)) and an extracellular spacer domain comprising a modified IgG4 hinge region; an engulfment signaling domain comprising a MERTK signaling domain; and a transmembrane domain comprising a CD28 transmembrane domain located between and connecting the extracellular domain and the engulfment signaling domain, wherein the extracellular spacer domain is located between the binding domain and the transmembrane domain (also referred to as "CER40") (see, e.g., Figure 13A). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, a CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 64 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 64).

[0154] Embodiments in which a CER comprises an extracellular domain comprising an scFv that binds to an engulfment-promoting marker or target antigen include an extracellular domain comprising an scFv specific for mesothelin (e.g., M912 scFv, amino acids 23-264 of SEQ ID NO: 106, where amino acids 1-22 of SEQ ID NO: 106 are a signal peptide) and an extracellular spacer domain comprising a modified IgG4 hinge region; an engulfment signaling domain comprising a truncated MyD88 signaling domain; and a transmembrane domain comprising a Tim4 transmembrane domain located between and connecting the extracellular domain and the engulfment signaling domain, wherein the extracellular spacer domain is located between the scFv and the transmembrane domain (also referred to as "CER50"). In certain embodiments, such a CER comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, a CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 107 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 107).

[0155] In certain embodiments, after binding of CER expressed on the surface of a host cell to its cognate target molecule, lateral clustering of CER occurs on the host cell surface, resulting in a local increase in CER concentration. Clustering is promoted by the presence of multivalent ligands on the target cell or particle surface.

[0156] In certain embodiments, binding of CER expressed on the surface of a host cell to its cognate target molecule results in dimerization or multimerization of CER, which also binds the intracellular engulfment signaling domain, which then becomes targeted by intracellular kinases.

[0157] In certain embodiments, the CERs of the present invention, when expressed on the surface of a host cell, can tether, internalize, and process (degrade) a target molecule or particle (e.g., phagocytose the target). In other embodiments, the CERs of the present invention can tether and internalize a target molecule or particle (e.g., phagocytose the target). In some embodiments, a target cell or particle within a phagosome can be expelled before or during phagosome maturation. Furthermore, internalization can include internalization of the entire cell or particle bound by the extracellular domain of the CER, or can include internalization of a fragment or portion of the cell or particle bound by the extracellular domain of the CER.

[0158] In certain embodiments, the CERs of the present invention tether target molecules or particles without internalization. Host cells expressing CERs can engulf or tether multiple target cells or particles. Regardless of theory, tethering of target cells or particles by host cells expressing CERs, even without internalization and degradation of the target cells or particles, can result in degradation of the target cells or particles or promote an inflammatory environment, which is desirable in certain therapeutic situations (e.g., cancer).

[0159] Embodiments of CERs according to the present specification are shown in Tables 1-3, Figures 6A, 9A, 10A, 11A, 12A, 13A, 13B, the Sequence Listing and the Examples.

[0160] Host Cells and Nucleic Acids In certain aspects, the present invention provides nucleic acid molecules encoding any one or more of the CERs described herein. A nucleic acid sequence encoding a desired CER can be obtained or produced using standard techniques, such as by screening a library from cells expressing the desired sequence or a portion thereof, by deriving it from a vector known to contain the sequence, or by isolating the sequence or a portion thereof directly from cells or tissues containing the sequence, using recombinant methods known in the art.

[0161] Polynucleotides encoding the CER compositions described herein can be derived from any animal, such as a human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, or pig. In certain embodiments, the polynucleotide encoding the CER is derived from the same animal species as the host cell into which the polynucleotide is inserted.

[0162] Polynucleotides encoding the CER compositions described herein may also contain sequences encoding a signal peptide (also referred to as a leader peptide or signal sequence) at the amino terminus of the CER for targeting the precursor protein to the secretory pathway. The signal peptide may be cleaved from the N-terminus of the extracellular domain, if necessary, during cellular processing and localization of the CER to the cell membrane. A polypeptide from which the signal peptide sequence has been cleaved or removed is also referred to as a mature polypeptide. Examples of signal peptides that can be used in the CERs of the present invention include signal peptides derived from endogenously secreted proteins, including GM-CSF (amino acid sequence of SEQ ID NO: 65) and Tim4 (amino acid sequence of SEQ ID NO: 72). In certain embodiments, the polynucleotide or polypeptide sequence of a CER of the present invention comprises a sequence for a mature polypeptide. For sequences described herein that contain a signal peptide sequence, it will be understood by those skilled in the art that the signal peptide sequence can be replaced with another signal peptide capable of transporting the encoded protein to the extracellular membrane.

[0163] In certain embodiments, nucleic acid molecules encoding the CERs of the present invention are codon-optimized for efficient expression in the expression target host cell.

[0164] Nucleic acid molecules encoding the desired CER can be inserted into appropriate vectors (e.g., viral vectors, non-viral plasmid vectors, and non-viral vectors, such as lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), etc.) for introduction into desired host cells (e.g., T cells, natural killer cells, B cells, lymphocyte progenitor cells, antigen-presenting cells, Langerhans cells, or myeloid cells). Nucleic acid molecules encoding the CERs of the present invention can be cloned into a suitable vector, such as an expression vector, a replication vector, a probe generation vector, or a sequencing vector. In certain embodiments, the nucleic acid sequence encoding the extracellular domain, the nucleic acid sequence encoding the transmembrane domain, and the nucleic acid sequence encoding the engulfment signaling domain are linked together into a single polynucleotide and then inserted into a vector. In other embodiments, the nucleic acid sequence encoding the extracellular domain, the nucleic acid sequence encoding the transmembrane domain, and the nucleic acid sequence encoding the engulfment signaling domain can be inserted separately into vectors such that the resulting amino acid sequence generates a functional CER. A vector encoding a CER is referred to herein as a "CER vector."

[0165] In certain embodiments, the vector contains a nucleic acid molecule encoding one CER. In other embodiments, the vector contains one or more nucleic acid molecules encoding two or more CERs. In one embodiment, two or more nucleic acid molecules, each encoding a CER, can be sequentially cloned into a vector at different multiple cloning sites, allowing each CER to be expressed under the control of a different promoter. In another embodiment, a single nucleic acid molecule encoding multiple CERs can be cloned into a cloning site and expressed from a single promoter, with each CER separated from the other by an IRES or viral 2A peptide sequence, allowing for simultaneous expression of multiple genes from a single open reading frame (e.g., a multicistronic vector). In certain embodiments, the viral 2A peptide is T2A (SEQ ID NO: 147), P2A (SEQ ID NO: 104), E2A (SEQ ID NO: 148), or F2A (SEQ ID NO: 149).

[0166] In some embodiments, vectors are used that allow the long-term integration of transgenes and proliferation into daughter cells.Examples include viral vectors, such as adenovirus, adeno-associated virus, vaccinia virus, herpes virus, cytomegalovirus, poxvirus, or retroviral vectors, such as lentivirus vectors.Lentivirus-derived vectors can be used to achieve long-term gene transfer, and have additional advantages over vectors, including the ability to transduce non-proliferating cells such as hepatocytes and low immunogenicity.

[0167] In certain embodiments, CER vectors can be constructed to optimize spatial and temporal control. For example, CER vectors can contain promoter elements to optimize spatial and temporal control. In some embodiments, CER vectors contain tissue-specific promoters or enhancers that allow specific targeting of CER to organs or pathological microenvironments, such as tumors or infected tissues. An "enhancer" is an additional promoter element that can function cooperatively or independently to activate transcription. In other embodiments, CER vectors contain constitutive promoters. In yet other embodiments, CER vectors contain inducible promoters.

[0168] In further embodiments, the CER vector may contain a homing receptor such as CCR4 or CXCR4 to improve in vivo homing and anti-tumor activity.

[0169] If temporal control is desired, the CER vector may contain an element that allows for inducible elimination of transduced cells. For example, such a vector may contain an inducible suicide gene. The suicide gene may be an apoptosis gene or a gene that confers sensitivity to a substance (e.g., a drug), such as chemically inducible caspase 9 (iCASP9), chemically inducible Fas, or HSV-TK (which confers sensitivity to ganciclovir). In further embodiments, the CER vector may be designed to express known cell surface antigens that allow for depletion of transduced cells upon injection of the relevant antibody. Cell surface antigens and their associated antibodies that can be used for depletion of transduced cells include CD20 and rituximab, RQR8 (a combination of CD34 and CD20 epitopes that allows CD34 selection and anti-CD20 deletion) and rituximab, and EGFR and cetuximab.

[0170] Inducible vector systems, such as the tetracycline (Tet)-On vector system (Heinz et al., Hum. Gene Ther. 2011, 22:166-76), which activates transgene expression using doxycycline, can also be used for inducible CER expression. Inducible CER expression can also be achieved by retention using a selective hook (RUSH) system, which is based on streptavidin immobilized on the membrane of the endoplasmic reticulum through a hook and a streptavidin-binding protein introduced into the CER structure, where adding biotin to the system results in the release of CER from the endoplasmic reticulum (Agaugue et al., 2015, Mol. Ther. 23(Suppl. 1):S88).

[0171] As used herein, the terms "recombinant" or "non-naturally occurring" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic modification or that has been modified by the introduction of an exogenous nucleic acid molecule, where such modification or alteration is introduced by genetic engineering. Genetic modifications include, for example, modifications that introduce an expressible nucleic acid molecule encoding a protein, chimeric protein, or enzyme, or the addition, deletion, substitution, or other functional disruption of other nucleic acid molecules in the genetic material of a cell. Additional modifications include, for example, non-coding regulatory regions, where the modification alters expression of a gene or operon. In certain embodiments, cells, such as T cells obtained from a subject, can be genetically modified into non-natural or recombinant cells (e.g., non-natural or recombinant T cells) by introducing a nucleic acid encoding a CER described herein, such that the cell expresses a CER located on the cell surface.

[0172] Vectors encoding core viruses are referred to herein as "viral vectors." There are numerous available viral vectors suitable for use with the compositions described herein, including those identified for human gene therapy applications (Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, and more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods for using retroviral and lentiviral vectors and packaging mammalian host cells for transduction with viral particles containing chimeric receptor transgenes are known in the art and have been described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0173] In certain embodiments, a viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a target-specific CER. The viral vector may be a retroviral or lentiviral vector. The viral vector may also contain a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selectable markers that can confer drug resistance, or detectable markers such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In certain embodiments, the viral vector further contains a marker for transduction comprising a fluorescent protein (e.g., green, yellow), the extracellular domain of human CD2, or a truncated human EGFR (encoding the amino acid sequence of SEQ ID NO: 121) (huEGFRt; see Wang et al., Blood 118:1255, 2011). When the viral vector genome contains multiple nucleic acid sequences to be expressed in a host cell as separate transcripts, the viral vector may also contain additional sequences between the two (or more) transcripts to enable bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide (eg, T2A, P2A, E2A, F2A), or any combination thereof.

[0174] Figures 2A and 2B provide exemplary CER vectors. The CER vector shown in Figure 2A contains a single engulfment signaling domain. The CER vector shown in Figure 2B contains an engulfment signaling domain that includes a first engulfment signaling domain and a second engulfment signaling domain.

[0175] Other viral vectors can also be used for polynucleotide delivery, including DNA viral vectors, including adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; amplicon vectors, vectors derived from herpes simplex virus (HSV), including replication-deficient herpes simplex virus (HSV) and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).

[0176] Other viral vectors recently developed for gene therapy applications can also be used with the compositions and methods described herein. Such vectors include those derived from baculovirus and alpha-virus (Jolly, D J. 1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (e.g., Sleeping Beauty or other transposon vectors). In some embodiments, the viral or plasmid vector further comprises a genetic marker for transduction (e.g., green fluorescent protein, huEGFRt (encoding the amino acid sequence of SEQ ID NO: 121)).

[0177] In certain embodiments, gene editing methods are used to modify the host cell genome to include the polynucleotide encoding the CER of the present invention.Gene editing or genome editing is a genetic engineering method that uses genetically engineered endonucleases to insert, replace, or remove DNA from the genome of host cells.The nuclease causes specific double-strand breaks at the target locus in the genome.Then, the endogenous DNA repair pathway of host cells repairs the induced breaks by non-homologous end joining (NHEJ) and homologous recombination.Exemplary endonucleases useful for gene editing include zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease systems (such as CRISPR-Cas9), meganucleases, or combinations thereof. Methods for disrupting or knocking out genes or gene expression in immune cells, including B cells and T cells, using gene editing endonucleases are known in the art and are described, for example, in PCT Publications WO 2015 / 066262; WO 2013 / 074916; WO 2014 / 059173; Cheong et al., Nat. Comm. 2016 7:10934; Chu et al., Proc. Natl. Acad. Sci. USA 2016 113:12514-12519 (the methods from each of which are incorporated herein by reference in their entirety).

[0178] In certain embodiments, B cells, lymphoid progenitor cells including common lymphoid progenitors, antigen-presenting cells including dendritic cells, Langerhans cells, myeloid progenitor cells or mature bone marrow cells are modified to contain a non-endogenous nucleic acid molecule encoding a CER of the invention.

[0179] In some embodiments, B cells are generally genetically modified to express one or more CERs. B cells have certain properties that may be advantageous as host cells, including the ability to transport to sites of inflammation (e.g., lymph nodes, tumors), internalize and present antigens, costimulate T cells, be highly proliferative, and self-renew (continue to survive). In certain embodiments, CER-modified B cells can digest engulfed target cells or engulfed target particles into smaller peptides and present them to T cells via MHC molecules. Antigen presentation by CER-modified B cells can contribute to antigen spread in immune responses against non-target antigens. B cells include B cell lineages (e.g., prepro-B cells, pro-B cells, and pre-B cells); immature and inactivated B cells, or progenitors (progenitors or precursors) committed to mature and functional or activated B cells. In certain embodiments, the B cells can be naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablast cells, memory B cells, or any combination thereof. Memory B cells can be distinguished from naive B cells by the expression of CD27, which is not present in naive B cells. In certain embodiments, the B cells can be primary cells or cell lines derived from humans, mice, rats, or other mammals. B cell lines are well known in the art. When obtained from mammals, B cells can be obtained from a number of sources, including blood, bone marrow, spleen, lymph nodes, or other tissues or fluids. In certain embodiments, the B cells are isolated from tumor sites (tumor-infiltrating B cells). The B cell composition can be enriched or purified.

[0180] In certain embodiments, expression of an endogenous gene in a host B cell is inhibited, knocked down, or knocked out. Examples of endogenous genes that can be inhibited, knocked down, or knocked out in B cells include B cell receptor (BCR) genes (e.g., CD79b, IGH, IGκ, IGλ, or any combination thereof), immune checkpoint molecules (e.g., PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, or any combination thereof), or any combination thereof. The expression of the BCR gene, the immune checkpoint molecule gene, or both can be inhibited, knocked down, or knocked out at the gene level, transcription level, or translation level, or a combination thereof. Methods for inhibiting, knocking down, or knocking out the BCR gene, the immune checkpoint molecule gene, or both can be achieved, for example, by RNA interference substances (e.g., siRNA, shRNA, miRNA, etc.) or artificial endonucleases (e.g., CRISPR / Cas nuclease system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, or any combination thereof). In some embodiments, an endogenous gene (e.g., a BCR gene or an immune checkpoint molecule gene) is knocked out by inserting a polynucleotide encoding a CER of the present invention into the locus of the endogenous B cell gene, for example, using an artificial endonuclease.

[0181] In one embodiment, the cells capable of expressing the CERs of the present invention on their cell surface are CD4 + , CD8 + , naive (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory (CD45RO + , CD62L +, CD8 + T cells include effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), virus-specific, mucosal-associated invariant, gamma delta (gd), tissue-resident T cells, and natural killer T cells. In certain embodiments, the T cells can be primary cells or cell lines derived from humans, mice, rats, or other mammals. When obtained from mammals, T cells can be obtained from a number of sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. In certain embodiments, T cells are isolated from tumor sites (tumor-infiltrating T cells). T cell compositions can be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In certain embodiments, T cells lacking endogenous expression of the TCR alpha and beta chains are used. Such T cells may naturally lack endogenous expression of the TCR α and β chains or may be modified to block expression (e.g., T cells from transgenic mice that do not express the TCR α and β chains or cells engineered to inhibit expression of the TCR α and β chains), or to knock out genes for the TCR α chain, the TCR β chain, or both. In certain embodiments, the cells capable of expressing a chimeric protein of the invention on their cell surface are not T cells or cells of a T cell lineage, but rather progenitor cells, stem cells, or cells modified to express cell surface anti-CD3.

[0182] In certain embodiments, the host T cells transfected to express the CERs of the present invention are functional T cells, such as virus-specific T cells, tumor antigen-specific cytotoxic T cells, naive T cells, memory stem T cells, central memory or effector memory T cells, or CD4+ CD25+ regulatory T cells.

[0183] In certain embodiments, expression of endogenous genes in host T cells is inhibited, knocked down, or knocked out. Examples of endogenous genes that can be inhibited, knocked down, or knocked out in T cells include TCR genes (TRA, TRB, or both), HLA genes (HLA class I genes, HLA class II genes, or both), immune checkpoint molecules (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, or any combination thereof), or any combination thereof. The expression of TCR genes, HLA genes, immune checkpoint molecule genes, or any combination thereof can be inhibited, knocked down, or knocked out at the gene level, transcription level, or translation level, or any combination thereof. Methods for inhibiting, knocking down, or knocking out TCR genes, HLA genes, immune checkpoint molecule genes, or any combination thereof can be achieved, for example, by RNA interference substances (e.g., siRNA, shRNA, miRNA, etc.) or artificial endonucleases (e.g., CRISPR / Cas nuclease system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, or any combination thereof). In some embodiments, endogenous genes (e.g., TCR genes, HLA genes, or immune checkpoint molecule genes) are knocked out by inserting a polynucleotide encoding a CER of the present invention into the locus of the endogenous T cell gene, for example, via an artificial endonuclease.

[0184] In certain embodiments, the host cell may be genetically modified to express one type of CER, while in other embodiments, the host cell may express at least two or more different CERs.

[0185] In certain embodiments, the host cell population that has been modified to express one or more CERs can be a population of B cells, a population of T cells, a population of natural killer cells, a population of lymphoid progenitor cells including common lymphoid progenitors, dendritic cells, a population of antigen-presenting cells including Langerhans cells, a population of myeloid progenitor cells, a population of mature myeloid cells, or any combination thereof. In certain embodiments, the host cell population that has been modified to express one or more CERs is a population of B cells, a population of T cells, or both.

[0186] In certain embodiments, each host cell within a host cell population expresses the same CER or set of CERs. In other embodiments, a host cell population comprises a mixture of two or more host cell subpopulations, where each subpopulation expresses a different CER or set of CERs.

[0187] In certain embodiments, host cells genetically engineered to express CER may also be modified to co-express one or more small GTPases. Rho GTPases, a family of small (~21 kDa) signaling G proteins and a subfamily of the Ras superfamily, regulate actin cytoskeleton organization in various cell types and promote pseudopod extension and phagosome closure during phagocytosis (e.g., Castellano et al., 2000, J. Cell Sci. 113:2955-2961). Engulfment requires F-actin recruitment beneath the tethered cell or particle and F-actin rearrangement to allow membrane extension, leading to cell or particle internalization. Rho GTPases include RhoA, Rac1, Rac2, RhoG, and CDC42. Other small GTPases, such as Rap1, are involved in regulating complement-mediated phagocytosis. Coexpression of a small GTPase and a CER can promote internalization and / or phagosome formation of target cells or particles by host cells. In some embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on a vector separate from the CER-containing vector. In other embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on the same CER-containing vector as the multicistronic expression construct. The polynucleotide sequences encoding the CER and small GTPase(s) can be separated from each other by a viral 2A peptide sequence (e.g., T2A (SEQ ID NO: 147), P2A (SEQ ID NO: 104), E2A (SEQ ID NO: 148), F2A (SEQ ID NO: 149)), allowing for multicistronic expression from a single open reading frame. Examples of GTPases that can be coexpressed with a CER include Rac1, Rac2, Rab5 (also known as Rab5a), Rab7, Rap1, RhoA, RhoG, CDC42, or any combination thereof.In certain embodiments, the GTPase comprises or is a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the Rac1 amino acid sequence of SEQ ID NO:76, the Rab5 amino acid sequence of SEQ ID NO:77, the Rab7 amino acid sequence of SEQ ID NO:122, the Rap1 amino acid sequence of SEQ ID NO:123, the RhoA amino acid sequence of SEQ ID NO:124, the CDC42 amino acid sequence of SEQ ID NO:125, or any combination thereof. In certain embodiments of multicistronic expression constructs, an expression construct encoding the Tim4-MyD88t CER and the small GTPase, Rab5a, with a P2A sequence inserted therebetween can comprise the amino acid sequence of SEQ ID NO:105 (CER91). In yet another particular embodiment, the CER mature polypeptide sequence comprises SEQ ID NO:105 without the signal peptide of amino acids 1-22.

[0188] In certain embodiments, when preparing host cells, e.g., B cells or T cells, expressing the CERs described herein, one or more growth factors and cytokines that promote the proliferation of host cells, e.g., B cells or T cells, can be added to the cell culture. The cytokines can be human or non-human. Examples of growth factors and cytokines that can be used to promote T cell proliferation include IL-2, IL-15, etc. Examples of growth factors and cytokines that can be used to promote B cell proliferation include CD40L, IL-2, IL-4, IL-15, IL-21, BAFF, etc.

[0189] In further embodiments, selective gene transfer is used to localize the CER vector to a particular region or organ, hi some embodiments, selective gene transfer is used to localize the CER vector to the liver or lungs of a subject.

[0190] Prior to genetic modification of host cells with a CER vector, a source of host cells (e.g., T cells, B cells, natural killer cells, etc.) is obtained from a subject (e.g., whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue), and the resulting host cells are isolated using methods known in the art. Specific host cell subsets can be collected according to known techniques and enriched or depleted by known techniques such as affinity binding to antibodies, flow cytometry, and / or immunomagnetic selection. After the enrichment and / or depletion steps and introduction of the CER, in vitro propagation of the desired modified host cells can be performed according to known techniques or variations thereof that will be apparent to those skilled in the art.

[0191] In certain embodiments, host cells, including T cells, natural killer cells, B cells, lymphoid progenitor cells, antigen-presenting cells, dendritic cells, Langerhans cells, myeloid progenitor cells, and mature bone marrow cells, comprising a CER according to any of the embodiments described herein have a phagocytic index relative to target cells of about 20 to about 1,500. The "phagocytic index" is a measure of the phagocytic activity of transduced host cells determined by counting the number of target cells ingested per CER-modified host cell during incubation of a suspension of target cells and CER-modified host cells in medium for a period of time. The phagocytosis index can be calculated by multiplying the total number of engulfed target cells / total number of CER-modified cells counted (e.g., phagocytosis frequency) by the average area of ​​target cell staining per CER+Ba / F3 cell × 100 (e.g., hybrid capture), or by multiplying the total number of engulfed particles / total number of CER-modified host cells counted by the total number of CER-modified host cells containing engulfed particles / total number of CER cells counted × 100.In certain embodiments, CER-modified cells are selected from the group consisting of: about 30 to about 1,500; about 40 to about 1,500; about 50 to about 1,500; about 75 to about 1,500; about 100 to about 1,500; about 200 to about 1,500; about 300 to about 1,500; about 400 to about 1,500; about 500 to about 1,500; about 20 to about 1,400; about 30 to about 1,400; about 40 to about 1,400; about 50 to about 1,400; about 100 to about 1,400; about 200 to about 1,400; about 300 to about 1,400; 400 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 100 to about 1,300; about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300; about 500 to about 1,300; about 20 to about 1,200; about 30 to about 1,200; about 40 to about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200; about 300 to about 1,200 ; about 400 to about 1,200; about 500 to about 1,200; about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 200 to about 1,100; about 300 to about 1,100; about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about 30 to about 1,000; about 40 to about 1,000; about 50 to about 1,000; about 100 to about 1,000; about 200 to about 1,000; about 300 to or about 500 to about 1,000; about 20 to about 750; about 30 to about 750; about 40 to about 750; about 50 to about 750; about 100 to about 750; about 200 to about 750; about 300 to about 750; about 400 to about 750; or about 500 to about 750; about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; about 200 to about 500; or about 300 to about 500. In further embodiments, the incubation time is about 2 hours to about 4 hours, about 2 hours, about 3 hours, or about 4 hours.In yet further embodiments, the CER-modified cells exhibit a statistically significantly higher phagocytosis index than cells transduced with a truncated EGFR control. The phagocytosis index can be calculated using methods known in the art and further described in the Examples, including quantification by flow cytometry or fluorescence microscopy.

[0192] The host cells can be derived from animals such as primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. In a preferred embodiment, the animal is a human. The host cells can be obtained from a healthy subject or a subject with a disease associated with the expression of the antigen.

[0193] Uses of CERs and cells engineered to express CERs The present invention provides methods for altering the engulfment phenotype of a host cell. In one aspect, the present invention provides a method for generating a cell population that exhibits an engulfment phenotype, comprising introducing into a host cell population that does not naturally exhibit the engulfment phenotype a nucleic acid molecule encoding at least one CER or a vector containing at least one CER, according to any of the embodiments described herein, and expressing the at least one CER in the host cell population. In a specific embodiment, the engulfment phenotype is phagocytosis.

[0194] In another aspect, the invention provides a method of altering the engulfment phenotype of a cell population, comprising introducing into a host cell population a nucleic acid molecule encoding at least one CER or a vector comprising at least one CER of any of the embodiments described herein, and expressing in the host cell population the at least one CER, wherein the at least one CER confers an engulfment phenotype specific for an engulfment-promoting marker or antigenic marker (target antigen) not naturally targeted by the host cell. In a particular embodiment, the engulfment phenotype is phagocytosis.

[0195] In yet another aspect, the invention provides a method for enhancing the enlargement phenotype of a cell population, comprising introducing into a host cell population a nucleic acid molecule encoding at least one CER or a vector comprising at least one CER of any of the embodiments described herein, and expressing the at least one CER in the host cell population, wherein the at least one CER is specific for an enlargement-promoting marker or antigenic marker (target antigen) that is naturally targeted by the host cells, and wherein expression of the at least one CER by the host cells enhances enlargement of the host cells by cells, pathogens, or particles that display the targeted enlargement-promoting marker or antigenic marker.

[0196] The CERs, nucleic acid molecules encoding CERs, vectors comprising CERs, and host cells expressing CERs of any of the embodiments described herein may also be used in methods of treating a subject suffering from a disease, disorder, or undesirable condition. These method embodiments include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more CERs, nucleic acid molecules encoding one or more CERs, vectors comprising one or more CERs, or a population of host cells genetically modified to express one or more CERs, as described herein.

[0197] Diseases that can be treated with cells expressing the CERs described herein include cancer, infectious diseases (viral, bacterial, fungal, protozoal infections), inflammatory or immune diseases (e.g., autoimmune diseases, inflammatory bowel disease, multiple sclerosis), degenerative diseases (e.g., joint and cartilage), and neurodegenerative diseases (e.g., Alzheimer's disease). Adoptive immunotherapy and gene therapy are promising treatments for various types of cancer (Morgan et al., Science 314:126, 2006; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; June, J. Clin. Invest. 117:1466, 2007) and infectious diseases (Kitchenen et al., PLoS One 4:38208, 2009; Rossi et al., Nat. Biotechnol. 25:1444, 2007; Zhang et al., PLoS Pathog. 6:e1001018, 2010; Luo et al., J. Mol. Med. 89:903, 2010).

[0198] Subjects that can be treated with the compositions and methods described herein include animals such as humans, primates, cattle, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. Subjects can be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects.

[0199] Many cancers, including solid tumors and leukemia, are suitable for the compositions and methods described herein.Exemplary cancer types that can be treated include adenocarcinoma of the breast, prostate, and colon; all types of bronchogenic carcinoma of the lung; bronchogenic carcinoma; melanoma; hepatocellular carcinoma; neuroblastoma; papilloma; adenocarcinoma; choristoma; branchiomas; malignant carcinoid syndrome; carcinoid heart disease; and carcinomas (e.g., Walker's carcinoma, basal cell carcinoma, squamous cell carcinoma, Brown-Pierce carcinoma, ductal carcinoma, Ehrlich tumor, Krebs2, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung carcinoma, oat cell carcinoma, papillary carcinoma, scirrhous carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma, and transitional cell carcinoma). Additional cancer types that may be treated include histiocytosis; malignant histiocytosis; leukemia; Hodgkin's disease; immunoproliferative small intestinal disease; non-Hodgkin's lymphoma; plasmacytoma; multiple myeloma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; germinoma; hamartoma; mesenchymoma; mesonephroma; sarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastic tumor. In addition, the following cancer types are also considered suitable for treatment: adenoma; cholangioma; cholesteatoma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hepatocellular carcinoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; sarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; schwannoma; neuroblastoma; neuroepithelioma; neurofibroma; neurocytoma; paraganglioma; nonchromaffin paraneuromyoma. Cancer types that can be treated include angiokeratoma; angiolymphocytosis with eosinophilia; sclerosing hemangioma; hemangiomatosis; hemangiocytic tumor; hemangioendothelioma; hemangioma; hemangiopericytoma; angiosarcoma; lymphangioma; lymphangioleiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma; phyllodes cystosarcoma; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukemia sarcoma; liposarcoma; lymphangiosarcoma; myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasm; neurofibromatosis; and cervical dysplasia.

[0200] Examples of hyperproliferative diseases suitable for CER therapy are B-cell cancers, including B-cell lymphomas (e.g., various forms of Hodgkin's disease, non-Hodgkin's lymphoma (NHL) or central nervous system lymphoma), leukemias (e.g., lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), hairy cell leukemia, B-cell blastic transformation of chronic myelogenous leukemia), and myelomas (e.g., multiple myeloma). Additional B-cell cancers include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma / leukemia, B-cell proliferation of unspecified grade, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

[0201] Inflammatory and autoimmune diseases include arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, inclusion body myositis, inflammatory myopathy, toxic epidermal necrolysis, systemic scleroderma and sclerosis, CREST syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T-cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis, and lupus myelitis. encephalitis, juvenile-onset diabetes, multiple sclerosis, allergic encephalitis, neuromyelitis optica, rheumatic fever, Sydenham chorea, immune responses related to cytokine- and T-lymphocyte-mediated acute and delayed hypersensitivity reactions, tuberculosis, sarcoidosis, granulomatous diseases including Wegener's granulomatosis and Churg-Strauss syndrome, agranulocytosis, vasculitis (including hypersensitivity vasculitis / vasculitis, ANCA and rheumatoid vasculitis), aplastic anemia, congenital pure red cell aplasia (Diamond Blackfriars). anemia), immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte exudation, central nervous system (CNS) inflammatory diseases, Alzheimer's disease, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behçet's disease, Castleman syndrome, Goodpasture syndrome, Ran's syndrome, Bart-Eaton myasthenic syndrome, Raynaud's syndrome, Jorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), pemphigoid, pemphigus, autoimmune polyendocrinopathy, seronegative spondyloarthropathy, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Henoch-Schönlein purpura,Autoimmune diseases of the testes and ovaries, including autoimmune thrombocytopenia, autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases, including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndrome (or polyendocrinopathy syndrome), type 1 diabetes mellitus (also known as insulin-dependent diabetes mellitus (IDDM)), and Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant), nonspecific interstitial pneumonia (NSIP), and Gila These include: Ang Barre syndrome, large-vessel vasculitis (including polymyalgia rheumatica and large cell (Takayasu) arteritis), medium-sized vasculitis (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Burger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gluten enteropathy), cryoglobinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Wiskott-Aldrich syndrome, and thromboangiitis obliterans. In certain embodiments, in the context of treating inflammatory diseases, it may be preferable to design a CER with a homeostatic (non-inflammatory) engulfment signaling domain.

[0202] Infectious diseases include those associated with infectious agents, including various bacteria (e.g., pathogenic Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Bacillus anthrax, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Helicobacter pylori, Vibrio cholerae, Listeria monocytogenes, Rickettsiae, Chlamydia, etc.), mycobacteria, and parasites (including known members of the protozoan parasite family). Infectious viruses include eukaryotic viruses such as adenoviruses, bunyaviridae, herpesviruses, papovaviridae, papillomaviruses (e.g., HPV), paramyxoviruses, picornaviruses, rhabdoviruses (e.g., rabies), orthomyxoviruses (e.g., influenza), poxviruses (e.g., vaccinia), reoviruses, retroviruses, lentiviruses (e.g., HIV), and flaviviruses (e.g., HCV, HBV). In certain embodiments, compositions comprising the CERs of the present invention are used to treat infections caused by pathogens capable of establishing persistent infections in a subject.

[0203] Neurodegenerative diseases include Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar degeneration, Parkinson's disease, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitin-positive inclusions (FLTD-U), tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (also known as transmissible spongiform encephalopathies, including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), ophthalmoplegia, motor neuron diseases (muscle amyotrophic lateral sclerosis (including Lou Gehrig's disease)), and heterogeneous degenerative diseases of the nervous system (including, but not limited to, Canavan disease, Huntington's disease, neurosteroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes metamorphosis syndrome, Cockayne syndrome, Haarvorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg disease), dementia (including, but not limited to, Pick's disease and spinocerebellar ataxia), cancer (e.g., cancer of the CNS and / or brain tumors, including brain metastases resulting from cancer anywhere in the body). Many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), and prion diseases, share neuropathological manifestations, including proteins such as amyloid beta or tau in Alzheimer's disease; α-synuclein in Parkinson's disease (PD), dementia with Lewy bodies, multiple system atrophy, or Alzheimer's disease; Huntington's disease in Huntington's disease, SOD1 in amyotrophic lateral sclerosis, polyglutamine (polyQ) repeat-containing proteins in Huntington's disease or amyotrophic lateral sclerosis; TDP-43 in amyotrophic lateral sclerosis or FLTD-U; or prion proteins (e.g., PrP) in prion diseases. Sc ) is abnormal accumulation. Thus, in certain embodiments, CER therapy can be designed to target disease-associated proteins to reduce or prevent abnormal protein accumulation, thereby slowing or preventing the progression of neurodegenerative diseases.

[0204] The CERs of the present invention may be expressed in cell-bound form (e.g., target cell populations (mature T cells (e.g., CD8 + or CD4 + The CERs may be administered to a subject for gene therapy of T cells (or other cells of the T cell lineage). Thus, for example, the CERs of the present invention may be administered to a subject expressed on the surface of T cells, natural killer cells, natural killer T cells, B cells, lymphoid progenitor cells, antigen-presenting cells, dendritic cells, Langerhans cells, myeloid progenitor cells, mature myeloid cells (including subsets thereof), or any combination thereof. In certain embodiments, a method of treating a patient comprises administering an effective amount of CER-modified cells (i.e., recombinant cells expressing one or more CERs). In such embodiments, the CER-modified cells are xenogeneic, syngeneic, allogeneic, or autologous cells of a T cell lineage, natural killer cell lineage, natural killer T cell lineage, B cell lineage, lymphoid progenitor cell lineage, dendritic cell lineage, Langerhans cell lineage, myeloid cell lineage, or any combination thereof.

[0205] Pharmaceutical compositions containing CER-modified cells can be administered in a manner appropriate for the disease or condition being treated (or prevented), as determined by those skilled in the pharmaceutical arts. The appropriate dose, duration, and frequency of administration of the composition can be determined by factors such as the patient's condition, size, weight, body surface area, age, sex, type and severity of the disease, the specific therapeutic agent being administered, the specific form of the active ingredient, the time and method of administration, and other drugs being administered concomitantly. The present invention provides pharmaceutical compositions containing CER-modified cells and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextrose, glycerol, and the like, as well as combinations thereof. Other suitable injection vehicles can be any isotonic vehicle formulation, including saline, Normosol® (Abbott), Plasma-Lyte® A (Baxter), 5% dextrose in water, or lactated Ringer's solution.

[0206] A therapeutically effective amount of cells in a pharmaceutical composition is at least one cell (e.g., one CER-modified B cell), or generally 10 2 More than 10 cells, e.g., up to 10 6 cells, up to 10 7 cells, up to 10 8 cells, up to 10 9 cells, up to 10 10 cells, or up to 10 11 In certain embodiments, the cells are about 10 6 from about 10 10 cells / m 2 in the range of about 10 7 from about 10 9 cells / m 2 The number of cells may vary depending on the intended end use of the composition and the cell type contained therein. For example, a composition containing cells modified to contain a CER specific for a particular antigen may contain a cell population containing 5% to about 95% or more of such cells. In certain embodiments, a composition containing CER-modified cells contains a cell population containing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the uses provided herein, the cells are generally administered in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the desired cell density is generally greater than 10 4 cells / ml, generally above 10 7 cells / ml, typically greater than 10 8 The doses are greater than or equal to 10 cells / ml. The cells can be administered as a single injection or in multiple injections over a range of time periods. Repeated injections of CER-modified cells can be separated by days, weeks, months, or even years if there is disease recurrence or disease activity. Clinically meaningful numbers of immune cells can be administered cumulatively at doses of 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 1011 The dose can be divided into multiple injections equal to or greater than 10 cells. A suitable dose for administration of host cells containing a recombinant expression vector described herein is about 10 7 cells / m 2 , about 5x10 7 cells / m 2 , about 10 8 cells / m 2 , about 5x10 8 cells / m 2 , about 10 9 cells / m 2 , about 5x10 9 cells / m 2 , about 10 10 cells / m 2 , about 5x10 10 cells / m 2 , or about 10 11 cells / m 2 In certain embodiments, both a composition of CER-modified B cells and a composition of CER-modified T cells are administered, which administration can be simultaneous, concurrent, or sequential.

[0207] In some embodiments, the compositions described herein are administered intravenously, intraperitoneally, intratumorally, intramedullary, intralymph node, and / or intracerebrospinal fluid. In some embodiments, the chimeric engulfment receptor modifying composition is delivered to the tumor site.

[0208] In some embodiments, CER-modified cells are administered to a subject in conjunction with or in combination with one or more additional therapies. In such embodiments, the one or more additional therapies can be one or more of radiation therapy, genetically engineered cellular immunotherapy (e.g., T cell, dendritic cell, natural killer cell, macrophage, chimeric antigen receptor (CAR) therapy), antibody therapy, immune checkpoint molecule inhibitor therapy, or drug therapy, such as a chemotherapeutic agent, a therapeutic peptide, an antibiotic, an antiviral agent, an antifungal agent, an anti-inflammatory agent, or a small molecule therapeutic agent. In such embodiments, the CER-modified cells can remove apoptotic cells, dead cells, dying cells, damaged cells, infected cells, or necrotic cells that display pro-apoptotic markers induced in the setting of one or more additional therapies. In certain embodiments in which CER-modified cells are administered in combination with one or more additional therapies, the one or more additional therapies can be administered at subtherapeutic doses due to the additive or synergistic effects of the combination with the CER therapy. Combination therapy includes administration of CERs prior to (e.g., 1 to 30 days or more before), concurrently with (the same day as), or after (e.g., 1 to 30 days or more after) the additional therapy. In certain embodiments, the CER-modified cells are administered after administration of one or more additional therapies. In further embodiments, the CER-modified cells are administered 1, 2, 3, 4, 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, or 30 days after administration of one or more additional therapies. In still further embodiments, the CER-modified cells are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of one or more additional therapies. When the one or more additional therapies include multiple administrations, the CER-modified cells can be administered after the first administration of the one or more additional therapies, after the last administration of the one or more additional therapies, or between multiple administrations of the one or more additional therapies.

[0209] An example of a triple therapy (radiation + CER + CAR / or TCR) regimen is shown in Figure 134. After radiation therapy, tumor antigen-specific, CER-modified host cells (e.g., comprising a binding domain that binds to a tumor antigen) described herein are administered to a subject to promote anti-tumor immune responses and recruit immune-activated cells to the tumor microenvironment. In certain embodiments, CER migrates to the local irradiated tumor, rendering the tumor tissue permissive to immune infiltration and destruction (e.g., expression of inflammatory cytokines, activation of effector T cells, activation of dendritic cells, inhibition of regulatory T cells, etc.), thereby sensitizing the tumor microenvironment to subsequent adoptive T cell immunotherapy (e.g., CAR or TCR immunotherapy). In certain embodiments, the CER-modified cells are administered 1, 2, 3, 4, 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, or 30 days after radiation therapy. In further embodiments, the CER-modified cells are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after radiation therapy. In certain embodiments, the CAR or TCR immunotherapy is administered 1, 2, 3, 4, 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, or 30 days after administration of the CER therapy, or within 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of the CER therapy. In certain embodiments, the radiation therapy, the CAR or TCR immunotherapy, or both, are administered at subtherapeutic levels.

[0210] Examples of radiation therapy that may be used in combination with CER therapy include external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy, intensity-modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiation therapy, or any combination thereof.

[0211] Examples of immune checkpoint molecules that can be targeted in combination with CER therapy include PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, or any combination thereof. In certain embodiments, the immune checkpoint molecule inhibitor is an antibody, peptide, RNAi agent, or small molecule. The CTLA-4-specific antibody can be ipilimumab or tremelimumab. The PD-1-specific antibody can be pidilizumab, nivolumab, or pembrolizumab. The PD-L1 specific antibody can be durvalumab, atezolizumab, or avelumab.

[0212] Exemplary chemotherapeutic agents include alkylating agents, platinum-based agents, angiogenesis inhibitors (e.g., VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g., EGF pathway inhibitors), B-Raf inhibitors, MEK inhibitors, mTOR inhibitors, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (e.g., intercalating agents), and DNA repair inhibitors.

[0213] Examples of chemotherapeutic agents that may be considered for use in combination therapy include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxan®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), and cyclosporine. Lumustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (Depocyte®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan®), Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (Daunoxome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Bepcid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrsil®, Efudex®), flutamide (Eurexin®), lexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Campotoster®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Forex®),These include mitoxantrone (Novantron®), Mylotarg, paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, porifeprosan 20 and carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamtin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0214] Examples of alkylating agents include nitrogen mustards (ethyleneimine derivatives, alkylsulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chloretanacil®, Desmethyldopan®, Desmethyldopan®), Hemanthanamine®, Nordopan®, Uracil Nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mastergen®), cyclophosphamide (Cytoxan®, Neosal®, Clafen®, Endoxan®, Procytox®). (R), Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Belsito®), triethylenemelamine (Heimel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Further exemplary alkylating agents include oxaliplatin (Eloxatin®); temozolomide (Temodar®); Temodal®); dactinomycin (also known as actinomycin-D, Cosmegen®); melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); Dacal These include, but are not limited to, vasodilator (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); ifosfamide (Ifex®); prenummustine; procarbazine (Matulane®); mechlorethamine (also known as nitrogen mustard, mustatin, and mechloroethamine hydrochloride, Mustagen®); streptozocin (Zanosar®); thiotepa (also known as thiophosphatamide, TESPA, and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine HCl (Treanda®).

[0215] Examples of platinum-based agents include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin, and triplatin tetranitrate.

[0216] Examples of angiogenesis inhibitors include A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), ABT-627 (atrasentan) (Abbott Laboratories / Xinlay), ABT-869 (Abbott Laboratories), actimid (CC4047, pomalidomide) (Celgene Corporation), AdGVPEDF.11D (GenVec), ADH-1 (exherin) (Adherex Technologies), AEE788 (Novartis), AG-013736 (axitinib) (Pfizer), AG3340 (prinomastat) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP O2) (Alnylam Pharmaceuticals), and AMG. 386 (Amgen), AMG706 (Amgen), apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (ridaforolimus / MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197 (ArQule), ASA404 (Novartis / Antisoma), atiprimod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (bevacizumab) (Genentech), AZD2171 (cediranib / resentin) (AstraZeneca), BAY 57-9352 (teratinib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim Pharmaceuticals), BIBW 2992 (Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-Myers Squibb), BMS-582664 (brivanib) (Bristol-Myers Squibb), BMS-690514 (Bristol-MyersSquibb), calcitriol, CCI-779 (Torisel) (Wyeth), CDP-791 (ImClone Systems), ceflatonin (homoharringtonine / HHT) (ChemGenex Therapeutics), Celebrex (celecoxib) (Pfizer), CEP-7055 (Cephalon / Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Metastat) (Collagenex) Pharmaceuticals), combretastatin (Oxigene), CP-751,871 (figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Daiichi Sankyo), CT-322 (Angiocept) (Adnexus), curcumin, dalteparin (Fragmin) (Pfizer), disulfiram (Antabuse), E7820 (Eisai Co., Ltd.), E7080 (Eisai Co., Ltd.), EMD 121974 (cilengitide) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (ImClone / Bristol-Myers Squibb), EZN-2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals), GC1008 (Genzyme), genistein, GSK1363089 (foretinib) (GlaxoSmithKline), GW786034 (pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-1121B (ramucirumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN241 (IntrogenTherapeutics), Iressa (ZD1839 / gefitinib), LBH589 (Faridak / panobinost) (Novartis), Lucentis (ranibizumab) (Genentech / Novartis), LY317615 (enzastaurin) (Eli Lilly and Company), Macugen (pegaptanib) (Pfizer), MEDI522 (abegulin) (MedImmune), MLN518 (tanutinib) (Millennium), Neovastat (AE941 / Benefin) (Aeterna Zentaris), Nexavar (Bayer / Onyx), NM-3 (Genzyme Corporation), noscapine (Cougar Biotechnology), NPI-2358 (Nereus Pharmaceuticals), OSI-930 (OSI), Palomid 529 (Paloma Pharmaceuticals, Inc.), Panzem Capsules (2ME2) ​​(EntreMed), Panzem NCD (2ME2) ​​(EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen Industries / Medigen Biotechnology), PKC412 (Novartis), Polyphenon E (green tea extract) (Polypheno E International, Inc), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (vatalanib) (Novartis), PXD101 (belinostat) (CuraGen Corporation), RAD001 (everolimus) (Novartis), RAF265 (Novartis), regorafenib (BAY73-4506) (Bayer), Revlimid (Celgene), Retaane (AlconResearch), SN38 (liposomal) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (pasireotide) (Novartis), squalamine (Genaera), suramin, Sutent (Pfizer), Tarceva (Genentech), TB-403 (Thrombogenics), tempostatin (Collard Biopharmaceuticals), tetrathiomolybdic acid (Sigma-Aldrich), TG100801 (TargeGen), thalidomide (Celgene Corporation), tinzaparin sodium, TKI258 (Novartis), TRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (aflibercept) (Regeneron Pharmaceuticals), VEGF Trap Eye (Regeneron Pharmaceuticals), Veglin (VasGene Therapeutics), bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), vorinostat (Merck), and ZSTK474.

[0217] Examples of vascular endothelial growth factor (VEGF) receptor inhibitors include bevacizumab (Avastin®), axitinib (Inrita®); brivanib alaninate (BMS-582664, (S)—((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl) 2-aminopropanoate); sorafenib (Nexavar®); pazopanib (Votrient®); Sunitinib malate (Sutent®); cediranib (AZD2171, CAS288383-20-1); Vargatef (BIBF1120, CAS928326-83-4); foretinib (GSK1363089); telatinib (BAY57-9352, CAS332012-40-5); apatinib (YN968D1, CAS811803-05-1); Imachi nib (Gleevec®); ponatinib (AP24534, CAS943319-70-8); tivozanib (AV951, CAS475108-18-0); regorafenib (BAY73-4506, CAS755037-03-7); vatalanib dihydrochloride (PTK787, CAS212141-51-0); brivanib (BMS-540215, CAS649735-46-6); Nib (Caprelsa® or AZD6474); motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide (described in PCT Publication WO 02 / 066470); dovitinib lactate (TKI258, CAS 852433-84 -2); linfanib (ABT869, CAS796967-16-3); cabozantinib (XL184, CAS849217-68-1); lestaurtinib (CAS111358-88-4); N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS345627-80-7);(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazo These include, but are not limited to, 4-methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); and aflibercept (EYLEA®).

[0218] Examples of EGF pathway inhibitors include tyrphostin 46, EKB-569, erlotinib (Tarceva®), gefitinib (Iressa®), erbitux, nimotuzumab, lapatinib (Tycarb®), cetuximab (anti-EGFR mAb), 188These include, but are not limited to, Re-labeled nimotuzumab (anti-EGFR mAb) and compounds disclosed generally and specifically in WO97 / 02266, EP0564409, WO99 / 03854, EP0520722, EP0566226, EP0787722, EP0837063, U.S. Pat. No. 5,747,498, WO98 / 10767, WO97 / 30034, WO97 / 49688, WO97 / 38983 and WO96 / 33980. Examples of EGFR antibodies include, but are not limited to, cetuximab (Erbitux®); panitumumab (Vectibix®); matuzumab (EMD-72000); trastuzumab (Herceptin®); nimotuzumab (hR3); zalutumumab; TheraCIM h-R3; MDX0447 (CAS339151-96-1); and ch806 (mAb-806, CAS946414-09-1).Examples of epidermal growth factor receptor (EGFR) inhibitors include erlotinib hydrochloride (Tarceva®), gefitinib (Iressa®); N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3"S")-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butanamide, Tovok®); vandetanib (Caprelsa®); lapatinib (Tykerb®); Registered Trademark); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); canertinib hydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS497 839-62-0; Mubritinib (TAK165); Peritinib (EKB569); Afatinib (BIBW2992); Neratinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626); N-(3,4-dichloro-2-furan) and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol (PKI166, CAS 187724-61-4).

[0219] Examples of mTOR inhibitors include rapamycin (Rapamune®) and its analogs and derivatives; SDZ-RAD; temsirolimus (also known as Torisel®, CCI-779); ridaforolimus (formerly known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0] 4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, described in PCT Publication WO 03 / 064383; everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); (5-{ 2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS1013101-36-4); and N 2 -[1,4-dioxo-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-, inner salt (SF1126, CAS936487-67-1).

[0220] Examples of phosphoinositide 3-kinase (PI3K) inhibitors include 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941 and described in PCT Publications WO 09 / 036082 and WO 09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (BEZ 235 or NVP-BEZ 235, described in PCT Publication WO 06 / 122806; 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine (also known as BKM120 or NVP-BKM120, described in PCT Publication WO 2007 / 084786); tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 958852-01-2); (1E,4 S,4aR,5R,6aS,9aR)-5-(acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8); and 8-phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).Examples of protein kinase B (PKB) or AKT inhibitors include 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one (MK-2206, CAS 1032349-93-1); perifosine (KRX0401); 4-dodecyl-N-1,3,4-thiadiazol-2-yl-benzenesulfonamide (PHT-427, CAS 119195 1-57-1;4-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-yl]-2-methyl-3-butyn-2-ol (GSK690693, CAS937174-76-0);8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6 -one (Paromide 529, P529, or SG-00529); trisylvine (6-amino-4-methyl-8-(β-D-ribofuranosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine); (αS)-α-[[[5-(3-methyl-1H-indazol-5-yl)-3-pyridinyl]oxy]methyl]-benzeneethanamine (A674563, CAS 552325-73-2); 4-[(4-chlorophenyl)-

[0037] Examples of compounds that may be used include, but are not limited to, 4-(4-chlorophenyl)-4-[4-(1H-pyrazol-4-yl)phenyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinamine (CCT128930, CAS 885499-61-6); 4-(4-chlorophenyl)-4-[4-(1H-pyrazol-4-yl)phenyl]-piperidine (AT7867, CAS 857531-00-1); and Archexin (RX-0201, CAS 663232-27-7).

[0221] Figures 5A-5C, 93, and 94 illustrate embodiments of regimens utilizing CER-modified cells. As shown in Figure 5A, following leukapheresis, the cells can be treated and activated ex vivo, genetically modified, and expanded for infusion into a subject. Figure 5B shows an example treatment scheme for CER-modified cells used in combination with conventional T cell-based therapy. An initial infusion of engineered T cells induces tumor cell apoptosis, demonstrating an antitumor effect. CER-modified cells are then infused. The CER-modified cells eliminate tumor cells that exhibit pro-engulfment (e.g., PtdSer), which promotes tumor regression while also avoiding the T cell-suppressive tumor microenvironment. Subsequently, changes in the tumor microenvironment resensitize the tumor to T cell therapy, allowing for a second infusion of T cells. Another embodiment of a treatment method is shown in Figure 5C. The treatment scheme shown in Figure 5C utilizes CER-modified cells in combination with monoclonal antibody therapy. Injection of tumor-specific antibodies, such as cetuximab targeting EGFR or rituximab targeting CD20, can induce cell death or induce the targeting moiety that is bound by CER-modified cells.Subsequently, subject is administered CER-modified cells that bind to and remove antibody-bound cells.In such an embodiment, CER extracellular domain can comprise FcR binding domain, PtdSer binding domain or other antigen binding domain.

[0222] In another scenario, CER-modified cells can be used in combination with small molecule inhibitors, such as BTK inhibitors, MEK inhibitors, adenosine pathway inhibitors, A2AR antagonists, IDO1 inhibitors, IMiDs such as lenalidomide, PI3Kδ inhibitors, BRAF inhibitors, or BCR-ABL inhibitors.

[0223] In certain embodiments, the method of the present invention includes a depletion step. The depletion step to remove CER from the subject can be performed after a sufficient time for therapeutic benefit to reduce toxicity to the subject. In such embodiments, the CER vector contains an inducible suicide gene such as iCASP9, inducible Fas, or HSV-TK. Similarly, the CER vector can be designed to express known cell surface antigens such as CD20 or truncated EGFR (SEQ ID NO: 121), which facilitates depletion of transduced cells via infusion of related monoclonal antibodies (mAbs), such as rituximab against CD20 or cetuximab against EGFR. Alemtuzumab, which targets CD52 present on the surface of mature lymphocytes, can also be used to deplete transduced B cells, T cells, or natural killer cells.

[0224] In a further embodiment, cells expressing the CERs of the present invention may be used in diagnostic or imaging methods, including methods used in connection with the indications or conditions identified herein. [Example]

[0225] Example Example 1 Preparation of CER constructs Expression of a natural or synthetic nucleic acid molecule encoding a CER can be achieved by operably linking the nucleic acid molecule encoding the CER protein or a portion thereof to a promoter and incorporating the construct into an expression vector suitable for replication and integration in eukaryotes. The vector contains transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0226] To evaluate the expression of a CER protein or a portion thereof, the expression vector introduced into cells contains a selectable marker gene, such as an antibiotic resistance gene, or a reporter gene, to facilitate identification and selection of expressing cells from a population of cells transfected or infected via a viral vector. The selectable marker is carried on a separate DNA fragment and used in a co-transfection method. The selectable marker gene or reporter gene is adjacent to regulatory sequences to enable expression in the host cells. The expression vector is introduced into the host cells by a retroviral vector. Various assays, including RT-PCR and ELISA, can be performed to confirm the presence of the recombinant DNA sequence in the host cells.

[0227] Evaluation of CER performance To identify and characterize CERs, an in vitro system has been established to reconstitute phagocyte engulfment using retroviral-mediated transduction of candidate CERs. Mouse and human lymphoid cell lines, which normally lack the ability to engulf cells, are transduced with CERs to assess their gain-of-function activity. If the CER is successfully expressed, engulfment occurs in heterologous cells. In addition to their engulfment activity, CERs are assessed for their ability to: (1) polarize cells to release proinflammatory cytokines and chemokines; (2) activate downstream proliferation pathways; and (3) induce target cells into a non-treatment-induced resistance pattern. Multidimensional flow cytometry, cytokine / chemokine arrays, and functional assays (described below) are used to evaluate candidate CERs.

[0228] Example 2 In vitro phagocytosis The murine pro-B cell line Ba / F3 or human Jurkat T cells lack the intrinsic phagocytic capacity to engulf apoptotic or tumor cells in vitro and are used as initial screening cell lines to identify lead CER candidates. After CER retroviral transduction, Ba / F3 or Jurkat T cells are purified, labeled, and immunophenotypically characterized. Phagocytic activity is measured using in vitro coculture assays with defined target cells under various coculture conditions, and engulfment is measured by FAC or luminescence microscopy. Ba / F3 or Jurkat T cells transduced with a CER containing an extracellular PtdSer targeting domain are cocultured with pHrodo-labeled apoptotic cells. This assay allows for the assessment of phagocytosis of apoptotic cells entering the cytoplasmic lysosome. In other cases, Ba / F3 cells or Jurkat T cells transduced with CERs carrying the extracellular domain of an Fc receptor are co-incubated with target cells pre-incubated with an antibody, such as a tumor-specific antibody, to measure the ability of these cells to phagocytose antibody-coated tumor cells. Finally, Ba / F3 cells or Jurkat T cells transduced with CERs that bind to tumor antigens via antibody-binding moieties, such as single-chain variable fragments, are co-cultured with tumor cells to quantify phagocytosis. In some cases, target cells are pre-treated with conventional chemotherapy, radiation therapy, or small molecule therapy prior to co-culture to induce a pro-phagocytic molecular state. Phagocytosis activity is quantified as the percentage of cell tracker-positive cells among labeled Ba / F3 Jurkat transfectants after a 90-minute co-culture.

[0229] Cytokine / chemokine array analysis from conditioned medium In parallel, conditioned medium was collected from cocultures of Ba / F3 cells or Jurkat T cell transfectants and analyzed for the release of proinflammatory cytokines and chemokines. Cytokine / chemokine changes and relative comparisons before and after Ba / F3 Jurkat transduction were quantified to assess gain of function. CER candidates were identified that polarize cells toward an inflammatory state by both (i) downregulating monocyte chemoattractants, such as IL-10 and TGF-β, which are immunosuppressive cytokines involved in the recruitment of immature monocytes and myeloid-derived suppressor cells, and (ii) upregulating proinflammatory cytokines, such as TNFα, IL12p70, IFNα, and IFNγ.

[0230] Multidimensional flow cytometry Ba / F3 and Jurkat transfectants are analyzed in parallel using multidimensional cytometry to characterize activating and inhibitory receptor profiles. Activating profiles may include CD137, CD69, HLA-DR, CD107a, CD123, CD11c, TNF, IFNγ, IL-2, granzymes, perforin, CD25, CD40L, CD80, and CD86, whereas inhibitory profiles may include PD-1, Tim-3, Lag-3, ICOS, and CD172a. Bystander cells in culture are immunophenotypically evaluated for treatment-induced resistance patterns.

[0231] Proliferation assay Primary human T cells transduced with the CER cassette are analyzed for constitutive or non-constitutive growth patterns in the presence or absence of exogenous cytokines or feeder cells.

[0232] Downstream pro-inflammatory signaling pathways To further examine the downstream proinflammatory response, phospho-CYTOF is performed to measure downstream signaling pathways activated by candidate CERs, such as IkBtot, pSTAT1, p38, and JNK.

[0233] Example 3 In vivo analysis To test CER-modified cells in vivo, animal models and ex vivo studies are used. Human primary tumor cells or xenografts are implanted into Nod / SCDγ mice. The proliferation and persistence of CER-modified cells can be quantified from blood and tissue specimens using a droplet PCR (ddPCR) system and primers specific for the CER cassette. To analyze the functional capacity of CER cells ex vivo, tumor tissue and splenocytes are processed and analyzed by FACS and tissue staining for phenotyping after adoptive transfer of CER-modified cells and for demonstration of in vivo phagocytosis. Tumor growth is monitored and quantified in vivo.

[0234] Example 4 Construction of TIM4-MERTK chimeric engulfment receptor (CER) “CER01” The extracellular domain of the phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO: 73), including the signal peptide (encoded by the amino acid sequence of SEQ ID NO: 72) and the transmembrane domain (encoded by the amino acid sequence of SEQ ID NO: 74) (together having the polynucleotide sequence of SEQ ID NO: 57), was fused to the intracellular kinase domain of the tyrosine kinase MERTK (encoded by SEQ ID NO: 58) to generate the chimeric engulfment receptor "CER01" (Tim4-MERTK CER having the amino acid sequence of SEQ ID NO: 71) (FIG. 6A). The MERTK receptor tyrosine kinase transmits the signal for engulfment, and Tim4 has recently been described as a phosphatidylserine-binding receptor (Miyanishi et al., Nature, 2007, 450:435-9; Nishi et al., 2014, Mol. Cell Biol. 34:1512-20). The Tim-4-MERTK chimeric engulfment receptor nucleotide sequence was then inserted into the pMSCV (murine stem cell virus) retroviral vector. Early passage murine Ba / F3 B cells were transduced with the pMSCV Tim4-MERTK retrovirus, which expresses yellow fluorescent protein (YFP) as a transduction marker. Positive Ba / F3 cell transductants were selected by GFP expression using flow cytometry (FAC), expanded in culture, and used for in vitro studies.

[0235] Phagocytic activity towards apoptotic thymocytes in primary culture Primary cultured thymocytes were incubated with 10 μM dexamethasone for 24 hours to induce cell death. Thymocytes were then labeled with 1 μM pHrodo Red dye in PBS for 15 minutes at room temperature, washed twice with RPMI medium containing 10% fetal bovine serum (FBS), and used as target cells for the phagocytosis assay. 50 μl of pHrodo Red-labeled thymocytes (10 6 / mL) was added to 50 μl of sorted Ba / F3 cells (10 5The cells were incubated with 10:1 target cells (10:1 target to effector cell ratio) at 1000 ng / mL. Labeling of target cells with pHrodo Red dye allows visualization of cells that are internalized and translocated into lysosomes due to their increased luminescence in the acidic lysosomal environment (Miksa et al., 2009, Immunol. Methods 342:71-7). Coculture experiments were performed, and Ba / F3 GFP+ cells were serially quantified for phagocytosis by fluorescence microscopy and FAC after 2, 24, 48, and 72 hours of incubation. Ba / F3 cells transduced with a pMSCV vector expressing Tim4 and GFP (a non-engulfment receptor) were used as a negative control.

[0236] Under normal conditions, the Ba / F3 murine B cell line lacks the ability to engulf target cells, and therefore was chosen to establish an assay system for engulfment. Tim4-MERTK CER-mediated engulfment of apoptotic thymocytes was first examined (Figure 6A-F). Expression of Tim4-MERTK CER in the murine Ba / F3 B cell line was confirmed by phosphatidylserine-positive (PtdSer) thymocytes. + ) strongly enhanced phagocytic uptake by thymocytes (Figures 6C-6F). Fluorescence microscopy and FACs observations showed that the amount of phagocytosis correlated with the incubation time with target cells and the amount of Tim4-MERTK CER expression (Figures 6C-6D). After 2 hours of co-incubation, 21.6% of Tim4-MERTK CER-transduced Ba / F3 cells had engulfed target apoptotic thymocytes, compared with 0% in the control group (Figure 6C). The number of phagocytic Ba / F3 cells expressing Tim4-MERTK CER increased to 57.5% after 24 hours of incubation and to 75% after 72 hours of incubation (Figures 6C-6D). Furthermore, Ba / F3 cells that expressed the highest amount of Tim4-MERTK CER exhibited the greatest amount of phagocytosis, approaching 80% in the top quartile of expressing cells (Fig. 6D ), indicating a concentration-dependent effect of Tim4-MERTK CER.

[0237] We next examined the ability of Tim4-MERTK CER to promote the translocation of endocytosed target cells into phagolysosomes. Lysosomes containing hydrolytic enzymes digest endocytosed cells in the low-pH intracellular environment (Arandjelovic et al., 2015, Nat. Immunol. 16:907-17). In this setting, pHrodo Red-labeled target thymocytes exhibited increased fluorescence intensity. Fluorescence microscopy revealed several pHrodo Red-positive cells present inside most Tim4-MERTK CER-expressing Ba / F3 cells (Figure 6E). In complete agreement with this observation, the translocation of target cells into phagolysosomes in Tim4-MERTK CER-expressing Ba / F3 cells was associated with their clearance. By day 4, 97% of target cells were eliminated by phagocytic uptake and lysosomal degradation (Figures 7A-7B). These results suggest that the addition of Tim4-MERTK CER significantly reduced the PtdSer expression. + It has been shown to strongly enhance cell clearance.

[0238] To examine the ability of CER-expressing cells to eliminate tumor cells, we examined Tim4-MERTK CER-mediated engulfment of the Raji human Burkitt B-cell lymphoma cell line (Figures 8A-8B). This study demonstrates that the B-cell receptor (BCR) incorporates PtdSer into membrane microdomains in anti-IgM-activated B cells in the setting of aberrant signaling activity, such as that present in constitutively active Raji lymphoma cells (Dillon et al., 2000, J. Immunol. 164:1322-32). Expression of Tim4-MERTK CER in the murine Ba / F3 B-cell line enhanced phagocytic uptake of Raji cells (Figures 8A-8C), demonstrating a Tim4-MERTK CER-mediated antitumor effect.

[0239] Example 5 Construction of FA58C2-MERTK CER “CER03” The phosphatidylserine-binding motif FA58C2 (amino acid sequence of SEQ ID NO:30) from the macrophage opsonin MFGE8 was linked to a modified IgG4 extracellular spacer domain (amino acid sequence of SEQ ID NO:67), the transmembrane domain of the costimulatory molecule CD28 (amino acid sequence of SEQ ID NO:68), and the cytoplasmic kinase domain of MERTK (amino acid sequence of SEQ ID NO:43) to generate the chimeric engulfment receptor "CER03" (FA58C2-MERTK CER) (polynucleotide sequence of SEQ ID NO:59, amino acid sequence of SEQ ID NO:75) (FIG. 9A). This construct had a signal peptide derived from GM-CSF (encoded by the amino acid sequence of SEQ ID NO:65). The MERTK receptor tyrosine kinase transmits signals for engulfment, and the C-terminal domain of the second FA58C repeat (C2) of MFP-E8 (referred to herein as FA58C2) has been shown to be involved in phosphatidylserine binding (Hanayama et al., 2002, Nature, 417:182-7; Nishi et al., supra). The FA58C2-MERTK CER nucleotide sequence was then inserted into the pMSCV (murine stem cell virus) retroviral vector. Early-passage murine Ba / F3 B cells were transduced with the pMSCV FA58C2-MERTK CER retrovirus, which expresses yellow fluorescent protein (YFP). Positive Ba / F3 cell transductants were selected for GFP expression using flow cytometry (FAC), expanded in culture, and used for in vitro studies.

[0240] Phagocytic activity towards apoptotic thymocytes in primary culture Primary cultured thymocytes were incubated with 10 μM dexamethasone for 24 hours to induce cell death. Thymocytes were then labeled with 1 μM pHrodo Red dye in PBS for 15 minutes at room temperature, washed twice with RPMI medium containing 10% FBS, and used as target cells for the phagocytosis assay. 50 μl of pHrodo Red-labeled thymocytes (10 6 / mL) in 50 μl of FA58C2-MERTK sorted Ba / F3 cells (10 51 / mL) (target cell to effector cell ratio of 10:1). Labeling of target cells with pHrodo Red dye allows visualization of cells that are internalized and translocated into lysosomes due to their increased luminescence in the acidic lysosomal environment (Miksa et al., 2009, supra). Coculture experiments were performed, and Ba / F3 GFP+ cells were serially quantified for phagocytosis by fluorescence microscopy and FAC after 2, 24, 48, and 72 hours of incubation. Ba / F3 cells transduced with pMSCV vector expressing Tim4 and GFP (a non-engulfment receptor) were used as a negative control.

[0241] We first examined FA58C2-MERTK CER-mediated engulfment of apoptotic thymocytes (Figures 9B-9F). Expression of FA58C2-MERTK CER in mouse Ba / F3 B cells was confirmed by the phosphatidylserine-positive (PtdSer) + ) strongly enhanced phagocytic uptake by thymocytes (Figures 9B-9F). Fluorescence microscopy and FACs observations showed that the amount of phagocytosis correlated with the incubation time with target cells and the amount of FA58C2-MERTK CER expression (Figures 9B-9C). After 2 hours of co-incubation, 11% of FA58C2-MERTK CER-transduced Ba / F3 cells were engulfed, compared with 0% in the control group (Figure 9B). The number of phagocytic Ba / F3 cells expressing FA58C2-MERTK CER increased to 48% after 24 hours of incubation (Figures 9B-9F). Furthermore, Ba / F3 cells expressing the highest amount of FA58C2-MERTK CER showed the highest amount of phagocytosis, approaching 80% in the top quartile of expressing cells (Figure 9C), indicating a concentration-dependent effect of FA58C2-MERTK CER.

[0242] Effect of small GTPases on FA58C2-MERTK engulfment We examined the effects of the small GTPases Rac1 and / or Rab5a on engulfment by CER-expressing Ba / F3 cells. Rho and Rab family GTPases regulate the engulfment of apoptotic cells by macrophages and immature dendritic cells. To form a phagocytic cup for engulfing cells, integrin receptors expressed by macrophages activate Rac1, a member of the Rho family of GTPases, to induce actin polymerization (Albert et al., 2000, Nat. Cell Biol. 2:899-905). Rab5, a member of the Rab family of GTPases, regulates the fusion of phagosomes with endosomes and may play a role in lysosomal biogenesis (Duclos et al., 2000, J. Cell Sci. 113:3531-41). A cDNA sequence encoding Rac1 (SEQ ID NO: 60), a cDNA sequence encoding Rab5 (SEQ ID NO: 61), or a cDNA sequence encoding both (SEQ ID NO: 62) was coexpressed with FA58C2-MERTK using bicistronic or tricistronic retroviral expression cassettes (pMSCV FA58C2-MERTK-P2A-Rac1, pMSCV FA58C2-MERTK-P2A-Rab5a, or pMSCV FA58C2-MERTK-P2A-Rac1-T2A-Rab5a) (Figure 10A). As shown in Figures 10B-10E, addition of Rac1 increased FA58C2-MERTK CER-mediated engulfment of targeted apoptotic thymocytes (56% vs. 48%, as shown in Figure 10E vs. Figure 9F). Furthermore, migration of internalized thymocytes into phagolysosomes was observed. Fluorescence microscopy showed that some pHrodo Red-positive cells were present in most FA58C2-MERTK CER / Rac1-expressing Ba / F3 B cells (FIG. 10C).

[0243] Example 6 Construction of FA58C2-SYKCER “CER04” The phosphatidylserine-binding motif FA58C2 from the macrophage opsonin MFGE8 fused to a signal peptide derived from GM-CSF was linked to a modified IgG4 extracellular spacer domain, the transmembrane domain of the costimulatory molecule CD28, and the Syk kinase domain to generate the chimeric engulfment receptor "CER04" (FA58C2-SykCER) (polynucleotide sequence of SEQ ID NO: 63, amino acid sequence of SEQ ID NO: 70, Figure 11A). The clustered Syk tyrosine kinase domain induces phagocytosis in COS cells (Greenberg et al., 1996, Proc. Natl. Acad. Sci. USA 93:1103-7). The FA58C2-SykCER nucleotide sequence was then inserted into the pMSCV (murine stem cell virus) retroviral vector. Early-passage murine Ba / F3 B cells were transduced with the pMSCV FA58C2-Syk retrovirus, which expresses the GFP fluorescent protein. Positive Ba / F3 cell transductants were selected for GFP expression using flow cytometry (FACs), expanded in culture, and used for in vitro studies.

[0244] Phagocytic activity towards apoptotic thymocytes in primary culture Primary thymocytes were induced to undergo apoptosis and labeled with pHrodo Red dye as described in Example 4. Co-culture studies were performed, and Ba / F3 GFP+ cells were serially quantified for phagocytosis by fluorescence microscopy and FACs as described in Example 4. Ba / F3 cells transduced with pMSCV vector expressing Tim4 and GFP (a non-engulfment receptor) were used as a negative control.

[0245] FA58C2-SykCER-mediated engulfment of apoptotic thymocytes was examined (Fig. 11A-11E). Expression of FA58C2-SykCER in the murine Ba / F3 B cell line was observed in phosphatidylserine-positive (PtdSer) B cells. +) strongly enhanced phagocytic uptake by thymocytes (Figures 11B-11E). Fluorescence microscopy and FACs observations showed that the amount of phagocytosis correlated with the incubation time of target cells and the amount of FA58C2-Syk CER expression. After 2 hours of co-incubation, 9.5% of FA58C2-Syk CER-transduced Ba / F3 cells were engulfed, compared with 0% in the control group (Figure 11B). The number of phagocytic Ba / F3 cells expressing FA58C2-Syk CER increased to 48% after 24 hours of incubation (Figures 11B, 11C, and 11E). Furthermore, Ba / F3 cells expressing the greatest amount of FA58C2-Syk CER exhibited the greatest amount of phagocytosis (Figure 11C), indicating a concentration-dependent effect of FA58C2-Syk CER.

[0246] Effect of small GTPases on FA58C2-SYK engulfment The effect of adding the small GTPases Rac1 and / or Rab5a on engulfment in Ba / F3 cells was examined. cDNA sequences encoding Rac1 (SEQ ID NO: 60) and / or Rab5 (SEQ ID NO: 61), or both (SEQ ID NO: 62), were coexpressed with FA58C2-Syk CER using bicistronic or tricistronic retroviral expression cassettes (pMSCV FA58C2-Syk-P2A-Rac1, pMSCV FA58C2-Syk-P2A-Rab5a, and pMSCV FA58C2-Syk-P2A-Rac1-T2A-Rab5a constructs) (Figs. 11A and 12A). As can be seen in Figs. 11B and 11C, the addition of Rac1 either increased FA58C2-Syk CER-mediated engulfment or increased target apoptotic thymocytes. Furthermore, addition of Rab5 also increased phagocytosis (FIGS. 12B-12D).

[0247] Example 7 Construction of CD19-MERTK CER “CER40” An anti-CD19 single-chain variable fragment (scFv) (encoded by the amino acid sequence of SEQ ID NO: 66) derived from the FMC63 mouse IgG2a mouse monoclonal antibody and fused to a GM-CSF-derived signal peptide (encoded by the amino acid sequence of SEQ ID NO: 65) was fused to a modified IgG4 extracellular spacer domain (encoded by the amino acid sequence of SEQ ID NO: 67), the transmembrane domain of the costimulatory molecule CD28 (encoded by the amino acid sequence of SEQ ID NO: 68), and the intracellular kinase domain of MERTK (encoded by the amino acid sequence of SEQ ID NO: 43) to generate the chimeric engulfment receptor "CER40" (CD19-MERTK CER) (having the amino acid sequence of SEQ ID NO: 64) (FIG. 13A) (Kochenderfer et al., 2009, J. Immunother. 32:689-702). To enhance engulfment, a bicistronic retroviral expression construct containing CD19-MERTK CER and Rac1 was constructed (FIG. 13B). The CD19-MERTK CER nucleotide sequence was then inserted into the pMSCV (murine stem cell virus) retroviral vector. Early passage murine Ba / F3 B cells were transduced with the pMSCV CER retrovirus, which expresses green fluorescent protein (GFP). Positive Ba / F3 cell transductants were selected for GFP expression using flow cytometry (FACs), expanded in culture, and used for in vitro studies.

[0248] Phagocytic activity against human lymphoma cell lines CD19 + Raji human Burkitt B-cell lymphoma cells were labeled with 1 μM pHrodo Red dye and used as target cells for the phagocytosis assay described in Example 4. Co-culture studies were performed, and Ba / F3 GFP+ cells were serially quantified for phagocytosis by fluorescence microscopy and FACs as described in Example 4. Ba / F3 cells transduced with pMSCV vector expressing Tim4 and GFP (a non-engulfment receptor) and untransduced Ba / F3 cells were used as negative controls.

[0249] We first investigated CD19-MERTK CER-mediated engulfment of Raji Burkitt B-cell lymphoma cells (Fig. 13C-13F). Expression of CD19-MERTK CER in the murine Ba / F3 B-cell line strongly enhanced the phagocytic uptake of Raji lymphoma cells (Fig. 13C-13F). Observation by fluorescence microscopy and FACs showed that the amount of phagocytosis correlated with the incubation time of target cells and the amount of CD19-MERTK CER expression. After 24 hours of co-incubation, 17% of CD19-MERTK-P2A-Rac1 CER-transduced Ba / F3 cells were engulfed, compared with 0% in the control group (Fig. 13C, 13G). Ba / F3 cells that expressed the highest amount of CD19-MERTK CER showed the greatest amount of phagocytosis (FIG. 13D), indicating a concentration-dependent effect of CD19-MERTK CER.

[0250] The ability of CD19-MERTK CER to promote the translocation of internalized Raji cells into phagolysosomes was examined. Fluorescence microscopy revealed pHrodo Red positivity, indicating that Raji cells were present inside CD19-MERTK CER + Rac1-expressing Ba / F3 cells (Figure 13E). Figure 13H shows engulfment of Raji cells by Ba / F3 cells expressing CD19-MERTK CER (white arrows indicate phagocytic activity). These results demonstrate the ability of CD19-MerTk CER expression to specifically eliminate targets.

[0251] Example 8 Construction of TIM4-MERTK CER “CER01” The Tim-4-MERTK chimeric engulfment receptor nucleotide sequence encoding CER01 having the amino acid sequence of SEQ ID NO: 71, as described in Example 4, was inserted into the pLenti lentiviral vector. Mouse Ba / F3 B cells were cultured in 12-well plates at a density of 0.5 million cells / ml in RMPI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 10 ng / mL mouse IL-3 (Peprotech catalog number 213-13). To transduce Ba / F3 cells, 100 μl of pLenti lentiviral vector expressing Tim4-MERTK (CER01) and truncated EGFR (also known as tEGFR or EGFRt) (see FIG. 16) as a transduction marker and 5 μl of TRANSDUX™ transduction reagent were diluted in 0.5 ml of complete cell growth medium and added to the Ba / F3 cells. The Ba / F3 cells were then centrifuged at 270 x g rpm for 1 hour in a centrifuge prewarmed to 32°C. The Ba / F3 cells were incubated at 37°C for 24 hours. The Ba / F3 cells were then grown in complete cell growth medium for an additional 48 hours. Positive Ba / F3 cell transductants were selected using fluorescence-activated cell sorting (FACs) (Sony Sorter SH800) by staining with a labeled Tim4-specific antibody (Kat5-18, Abcam catalog no. 176486) or a labeled EGFR-specific antibody (cetuximab) (see Figures 17A-B). After sorting, purified transduced Ba / F3 cells containing lentivirus-containing Tim4-MERTK-T2A-truncated EGFR (see Figure 17C) were allowed to rest for 48 hours before being used in the phagocytosis assay. The percentage of cells with positive staining is shown in each histogram.

[0252] Phagocytic activity towards apoptotic thymocytes in primary culture One day before the phagocytosis assay, primary thymocytes were isolated from C3H mice (Charles River Laboratories International, Inc.). Thymocytes were cultured in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in six-well plates. To induce apoptosis and cell surface phosphatidylserine expression, thymocytes were treated with 1 μM dexamethasone for 24 hours. Untreated thymocytes served as a negative control. Thymocytes were harvested from the six-well plates, washed once with sterile 1X PBS, and then stained with 1 ng / μl pH-sensitive pHrodo™ Red dye (ThermoFisher Scientific, catalog number P36600) in PBS for 15 minutes at room temperature. Growth medium was then added to the cells, and they were washed again to remove excess pHrodo Red. pHrodo Red stained thymocytes were plated in flat-bottom 96-well plates at 250,000 cells / well in RMPI 1640 complete medium.

[0253] Ba / F3 CER01+ tEGFR prepared as described above + Cells were washed once with 1X PBS and resuspended in 1 μM CELLTRACE in PBS. (商標) The cells were stained with Violet dye (ThermoFisher Scientific, Cat. No. C34557) for 10 minutes at 37°C. Growth medium was added to the stained transduced Ba / F3 cells, washed once with 1X PBS, and then diluted with excess CELLTRACE (商標) The Violet was removed and cells were plated in RMPI1640 complete medium at approximately 25,000 cells / well in flat-bottom 96-well plates.

[0254] Ba / F3 CER01+tEGFR stained target thymocytes +The cells were cultured at a 10:1 ratio (target cells:effector cells) for 3 hours or overnight (~14 hours) at 37°C. After incubation, the plates were centrifuged, and the medium was replaced with PBS supplemented with 2% fetal bovine serum (pH 9). The 96-well plates were then observed using a KEYENCE BZ-X710 fluorescence microscope with a 20x objective. Duplicate 96-well co-culture plates were also run in parallel for flow cytometry analysis. 7-aminoactinomycin D (7-AAD) dye was used as a cell viability dye, with target thymocytes stained with pHrodo Red and effector cells stained with CELLTRACE Violet. Ba / F3 cells transduced with the pLenti vector expressing a truncated EGFR were used as a negative control. Fluorescence microscopy showed that CER01+ Ba / F3 cells engulfed dexamethasone-treated thymocytes (white arrows indicate engulfment events) (Figure 18B), compared with truncated EGFR-transduced Ba / F3 control cells (Figure 18A). A magnified image of an engulfment event is shown on the right side of Figure 18B.

[0255] The amount of Ba / F3 effector cells measured by FACS is shown in Figure 19A. Phagocytosis was quantified as the population of cells staining double positive for pHrodo Red and CELLTRACE Violet as measured by FACS (see Figure 19B).

[0256] The phagocytosis index was calculated by multiplying the [average of the total number of engulfed target cells / total number of calculated CER-modified cells (e.g., phagocytosis frequency)] by the [average area of ​​target cell staining per CER+ Ba / F3 cell x 100 (e.g., hybrid capture)] compared to EGFRt-transduced Ba / F3 control cells (see Figure 20A-B). .

[0257] Phagocytic activity against mouse cell lines One day before the phagocytosis assay, CT26 mouse colon carcinoma cells were cultured in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 6-well plates and treated with 1 mM staurosporine (STS) for 12 hours to induce apoptosis. Untreated CT26 cells served as a negative control.

[0258] On the day of the phagocytosis assay, CT26 cells were harvested, washed twice with 1X PBS to remove excess staurosporine, and then stained with 1 ng / μl pHrodo Red in PBS for 15 minutes at room temperature. Growth medium was added to the CT26 cells, washed once to remove excess pHrodo Red, and plated at 250,000 cells / well in a flat-bottom 96-well plate in RPMI 1640 complete medium.

[0259] Ba / F3 CER01 prepared as above + EGFR + Cells were washed once with 1X PBS and resuspended in 1 μM CELLTRACE in PBS. (商標) The cells were stained with Violet dye (ThermoFisher Scientific, Cat. No. C34557) for 10 minutes at 37°C. Growth medium was added to the stained, transduced Ba / F3 cells, and the cells were washed once with 1X PBS to remove excess CELLTRACE (商標) The Violet was removed and cells were seeded in the same flat-bottom 96-well plates at approximately 50,000 cells / well in RPMI 1640 complete medium.

[0260] Target CT26 cells were stained with CER01 + tEGFR +The cells were cultured with CT26 cells at a ratio of 5:1 (target cells:effector cells) for 3 hours at 37°C. After incubation, the plates were centrifuged, and the medium was replaced with PBS supplemented with 2% fetal bovine serum (pH 9). The 96-well plates were then observed using a KEYENCE BZ-X710 fluorescence microscope with a 20x objective. Ba / F3 cells transduced with the pLenti vector expressing a truncated EGFR were used as a negative control. Fluorescence micrographs showing in vitro phagocytosis are shown in Figure 21 (white arrows indicate phagocytic events). CT26 cells labeled with pHrodo Red fluoresced within the low-pH compartment of lysosomes when engulfed (pink outline).

[0261] A hybrid capture algorithm that detects pHrodo Red fluorescence within the CELLTRACE Violet stained area is applied to the fluorescence image to determine the area of ​​engulfed target cells / CER. + The area of ​​B cells was quantified. + EGFR + (Figure 22A) or EGFR + Figure 23 shows a histogram plot of the number of hybrid cells representing the CT26 target cell region within Ba / F3 cells transduced with the control (Figure 22B). + EGFR + or EGFR + Scatter plot of hybrid cell number extracting the area of ​​CT26 target cells within control-transduced Ba / F3 cells. Area ratio represents the co-localized area of ​​CT26 cells within Ba / F3 cells. CER01 + EGFR + or EGFR + The frequency of phagocytosis of control-transduced Ba / F3 cells is shown in Figure 24A. + The phagocytic index of Ba / F3 cells is shown in Figure 24B.

[0262] Ba / F3 CER01 + EGFR +Cells were transduced, purified, expanded as above, and analyzed by CELLTRACE (商標) A20 mouse B-cell lymphoma cells were treated with staurosporine and stained with pHrodo Red. The stained CER01 cells were analyzed as described above for the phagocytosis assay using CT26 cells. + tEGFR + Cells were cocultured at a ratio of 5:1 (target cells:effector cells). Ba / F3 cells transduced with the pLenti vector expressing a truncated EGFR were used as a negative control. Phagocytosis events were quantified by fluorescence microscopy (KEYENCE BZ-X710 fluorescence microscope, 20x objective) using the hybrid capture algorithm, as described above for the assay using CT26 cells.

[0263] Fluorescence microscopy images showing in vitro phagocytosis of target TA20 cells are shown in Figure 25 (white arrows indicate phagocytosis events). + EGFR + (Figure 26A) or EGFR + Figure 27 shows a histogram plot of the number of hybrid cells representing the A20 target cell region within Ba / F3 cells transduced with the control (Figure 26B). + EGFR + or EGFR + Scatter plot of hybrid cell number extracting the area of ​​A20 target cells within control-transduced Ba / F3 cells. Area ratio represents the co-localized area of ​​A20 cells within Ba / F3 cells. CER01 + EGFR + or EGFR + The frequency of phagocytosis of control-transduced Ba / F3 cells is shown in Figure 24A. + The phagocytic index of Ba / F3 cells is shown in FIG.

[0264] Ba / F3 CER01 + EGFR +Cells were also cultured with staurosporine-treated WR19L murine T-cell lymphoma cells at a target cell to effector cell ratio of 5:1, as described above in the assay for CT26 cells, and co-incubated for 3 hours. Ba / F3 cells transduced with pLenti vector expressing truncated EGFR were used as a negative control. Phagocytosis of WR19L cells by CER01+ Ba / F3 cells was quantified by fluorescence microscopy as described above. Fluorescence microscopy images showing in vitro phagocytosis are shown in Figure 29 (white arrows indicate phagocytic events). Figure 30 shows the results of CER01 + EGFR + (+ or - staurosporine (STS)) or EGFR + The frequency of phagocytosis of WR19L cells by control-transduced Ba / F3 cells is shown.

[0265] Phagocytic activity of human CER01+ B cells against human cell lines Human primary B cells were transduced with pLenti Tim4-MERTK (CER01) lentivirus expressing a truncated EGFR as a transduction marker as described above for Ba / F3 cells, except that the transduced human B cells were sorted by FACS using an anti-EGFR antibody (cetuximab) labeled and then stained with Kat5-18 antibody (Tim4 specific) (Abcam catalog no. 176486) (see Figure 31A, where the % in the FACS plot on the right indicates the proportion of cells (CER01) expressing the Tim4 binding domain). Purified CER01 + B cells were expanded and imaged at 24, 48 and 72 hours, and are shown in Figure 31B.

[0266] One day before setting up the phagocytosis assay, Jurkat human B lymphocytes were cultured in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 6-well plates and treated with 1 mM staurosporine for 3 hours to induce apoptosis. Jurkat cells were washed twice with 1X PBS to remove excess staurosporine, and then stained with pHrodo Red (1 ng / μl in PBS) for 15 minutes at room temperature. Growth medium was added to the Jurkat cells, which were washed once to remove excess pHrodo Red. Approximately 250,000 cells / well were plated in complete RPMI 1640 medium into flat-bottom 96-well plates.

[0267] Transduced human primary B cells were washed once with 1X PBS and stained with 1 μM CELLTRACE Violet in PBS for 10 minutes at 37°C. Growth medium was added to the human primary B cells, washed once with 1X PBS to remove excess CELLTRACE Violet, and plated at approximately 50,000 cells / well in a 96-well plate in complete RPMI 1640 medium. Human primary B cells and Jurkat cells were co-cultured at a target cell to effector cell ratio of 5:1 for 3 hours at 37°C. After incubation, the co-culture plate was centrifuged, and the medium was replaced with PBS supplemented with 2% fetal bovine serum (pH 9). Phagocytosis events were quantified by fluorescence microscopy (KEYENCE BZ-X710 fluorescence microscope, 20x objective). Fluorescence microscopy images showing in vitro phagocytosis were obtained using CER01. + Shown in Figure 32A for B cells and in Figure 32B for EGFR+ controls (white arrows indicate phagocytic events).

[0268] Duplicate 96-well co-culture plates were also set up in parallel for analysis by flow cytometry using a 10:1 target to effector cell ratio (approximately 300,000 cells / well pHrodo Red-labeled, staurosporine-treated Jurkat cells and approximately 30,000 cells / well CER01). +(The cells were co-cultured with transduced human primary B cells. The co-culture plate was centrifuged at 1200 rpm for 5 minutes, and the medium was replaced with FACS buffer (PBS + 2% fetal bovine serum) containing a 1:50 dilution of allophycocyanin (APC)-labeled CD19 antibody to stain the human primary B cells. The human primary B cells were incubated with the APC-labeled CD19 antibody for 30 minutes at 4°C, washed once, and FACS buffer containing DAPI (4',6-diamidino-2-phenylindole), used as a marker for cell viability, was added to the cell culture plate. During FACS analysis, viable CD19-APC-positive cells were gated (see Figure 33, left FACS plot), and the frequency of CD19-positive and pHrodo Red-positive events (double-positive events), defined as phagocytic events, was assessed (see Figure 33, right FACS plot). FIG. 34 shows the frequency of phagocytosis of Jurkat cells by CER01+EGFR+ or EGFR+ control transduced B cells.

[0269] Human CER01 on chemotherapy-treated human cell lines + B cell phagocytic activity Human primary B cells were transduced with pLentiTim4-MERTK (CER01) lentivirus, which expresses a truncated EGFR as a transduction marker, as described above. One day before setting up the phagocytosis assay, Jurkat human B lymphocytes were cultured in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 6-well plates and treated with oxaliplatin (5 μM) and fluorouracil (5-FU) (10 μM). The next day, target Jurkat cells were harvested, washed twice with 1X PBX, and stained with pHrodo Red (1 ng / mL in PBS) for 15 minutes at room temperature. Growth medium was added to the Jurkat cells, washed once to remove excess pHrodo Red, and plated at approximately 200,000 cells / well in complete RPMI 1640 medium in flat-bottom 96-well plates. Transduced human primary B cells were washed once with 1X PBS and then stained with CELLTRACE Violet (1 mM in PBS) for 10 minutes at 37°C. Growth medium was added to the human primary B cells, which were washed once with 1X PBS to remove excess CELLTRACE Violet. Approximately 50,000 cells were plated in 96-well plates in RPMI complete medium. Human primary B cells and Jurkat cells were co-cultured at a target cell to effector cell ratio of 4:1 for 3 hours at 37°C. The plates were then imaged using a Keyence BZ-X710 microscope with a 20x objective. Figure 35 shows fluorescence microscopy images demonstrating engulfment of chemotherapy-treated Jurkat cells by CER01+ human primary B cells (the image on the right shows a magnified view of the phagocytic event; white arrows indicate phagocytic activity).

[0270] Example 9 Construction of TIM4-TYRO3 CER “CER08” The extracellular domain of the phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO:73), including the signal peptide (amino acid sequence of SEQ ID NO:72) and transmembrane domain (amino acid sequence of SEQ ID NO:74) (together having the polynucleotide sequence of SEQ ID NO:57), was linked to the intracellular signaling domain of Tyro3 (SEQ ID NO:45) to generate the chimeric engulfment receptor "CER08" (Tim4-Tyro3 CER having the amino acid sequence of SEQ ID NO:83). The Tyro3 signaling domain transmits the signal for engulfment, and Tim4 is a phosphatidylserine-binding receptor. The Tim4-Tyro3 (CER08) chimeric engulfment receptor nucleotide sequence was then inserted into the pLenti lentiviral vector with a truncated EGFR as a transduction marker, with a T2A sequence inserted between them (see Figure 36). Murine Ba / F3 B cells were transduced with pLenti vectors expressing Tim4-Tyro3 (CER08) and EGFRt, expanded, sorted by FACs, and used for in vitro studies as described in Example 8.

[0271] Phagocytic activity towards apoptotic thymocytes in primary culture Primary C3H mouse thymocytes were isolated, treated with dexamethasone, and stained with pHrodo Red as described in Example 8. Ba / F3 CER08 + tEGFR + Cells were cultured in a CELLTRACE (商標) Co-culture experiments were performed at a target cell to effector cell ratio of 10:1, and Ba / F3 CER08 + t EGFR + Cells were quantified for their phagocytosis by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with pLenti vector expressing a truncated EGFR were used as a negative control.

[0272] Viable CER08 quantified by FACS +The amount of transduced Ba / F3 cells is shown in Figure 37A. The frequency of phagocytosis was quantified as the population of cells staining double positive for pHrodo Red and CELLTRACE Violet, as detected by FACS (see Figure 37B).

[0273] Fluorescence microscopy showed that CER08 cells were significantly more resistant to tEGFR than tEGFR-transduced Ba / F3 control cells. + Ba / F3 cells were shown to engulf dexamethasone-treated thymocytes (white arrows indicate engulfment events) (see Figure 38). A magnified image of an engulfment event is shown on the right side of Figure 38.

[0274] The phagocytosis index was calculated by multiplying the [average of total number of engulfed target cells / total number of CER-modified cells counted (e.g., phagocytosis frequency)] by the [average area of ​​target cell staining per CER+Ba / F3 cell x 100 (e.g., hybrid capture)] compared to EGFRt-transduced Ba / F3 control cells (see Figures 39A-B).

[0275] Example 10 Construction of TIM4-DAP12CER “CER09” The extracellular domain of the phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO: 73), including the signal peptide (amino acid sequence of SEQ ID NO: 72) and transmembrane domain (amino acid sequence of SEQ ID NO: 74) (together having the polynucleotide sequence of SEQ ID NO: 57), was linked to the intracellular signaling domain of DAP12 (SEQ ID NO: 82) to generate the chimeric engulfment receptor "CER09" (Tim4-DAP12 CER having the amino acid sequence of SEQ ID NO: 84). DAP12 transmits the signal for engulfment, and Tim4 is a phosphatidylserine-binding receptor. The Tim4-DAP12(CER09) chimeric engulfment receptor nucleotide sequence was then inserted into the pLenti lentiviral vector with a truncated EGFR as a transduction marker, with the T2A sequence inserted between them (see Figure 40). Murine Ba / F3 B cells were transduced with pLenti vectors expressing Tim4-DAP12 (CER09) and EGFRt, expanded, sorted by FACs, and used for in vitro studies as described in Example 8.

[0276] Phagocytic activity towards apoptotic thymocytes in primary culture Primary C3H mouse thymocytes were isolated, treated with dexamethasone, and stained with pHrodo Red as described in Example 8. + tEGFR + Cells were cultured in a CELLTRACE (商標) Stained with Violet dye. Ba / F3 CER09 + tEGFR + Co-culture studies of Ba / F3 CER09 cells and primary thymocytes were performed at a target cell to effector cell ratio of 10:1. + EGFR + Cells were quantified for their phagocytosis by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with pLenti vector expressing a truncated EGFR were used as a negative control.

[0277] Viable CER09 quantified by FACS + The amount of transduced Ba / F3 cells is shown in Figure 41 A. The frequency of phagocytosis was quantified as the population of cells staining double positive for pHrodo Red and CELLTRACE Violet, as detected by FACS (see Figure 41 B).

[0278] Fluorescence microscopy showed that CER09 cells were significantly more potent than tEGFR-transduced Ba / F3 control cells. + Ba / F3 cells were shown to engulf dexamethasone-treated thymocytes (white arrows indicate engulfment events) (see Figures 42A-B). A magnified image of an engulfment event is shown on the right side of Figure 42B.

[0279] The phagocytosis index was calculated by multiplying the [average of total number of engulfed target cells / total number of CER-modified cells counted (e.g., phagocytosis frequency)] by the [average area of ​​target cell staining per CER+Ba / F3 cell × 100 (e.g., hybrid capture)] compared to EGFRt-transduced Ba / F3 control cells (see Figures 43A-B).

[0280] Phagocytic activity against mouse cell lines Ba / F3 CER09 + tEGFR + Cells were cultured in a CELLTRACE (商標) CT26 mouse colon carcinoma cells were treated with staurosporine, labeled with pHrodo Red, and Ba / F3 CER09 as described in Example 8. + tEGFR + The cells were co-cultured with target cells to effector cells at a target cell to effector cell ratio of 5:1 for 3 hours. + Phagocytosis of CT26 cells by Ba / F3 cells was quantified by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with pLenti vector expressing truncated EGFR were used as a negative control. CER09+Ba / F3 cells and EGFRt +Fluorescence microscopy images showing in vitro phagocytosis by control cells are shown in Figures 44A-B (white arrows indicate phagocytic events). CT26 cells labeled with pHrodo Red fluoresce within the low pH compartments of lysosomes (pink outline) when engulfed.

[0281] A hybrid capture algorithm that detects pHrodo Red fluorescence within the CELLTRACE Violet stained area is applied to the fluorescence image to identify the area of ​​engulfed target cells / CER. + The area of ​​B cells was quantified. Figure 45 shows the CER09 + tEGFR + or tEGFR + A scatter plot of the number of hybrid cells extracting the area of ​​CT26 target cells within control-transduced Ba / F3 cells is shown. The area ratio indicates the co-localized area of ​​CT26 cells within Ba / F3 cells. The phagocytic index of CER09+ Ba / F3 cells compared to EGFRt-transduced Ba / F3 control cells is shown in Figure 46.

[0282] WR19L mouse lymphoma cells were treated with staurosporine, labeled with pHrodo Red, and CELLTRACE Violet-labeled Ba / F3 CER09 as described in Example 8. + EGFR + The cells were co-cultured with target cells to effector cells at a target cell to effector cell ratio of 5:1 for 3 hours. + Phagocytosis of WR19L cells by Ba / F3 cells was quantified by fluorescence microscopy as described in Example 8. Ba / F3 cells transduced with a pLenti vector expressing a truncated EGFR were used as a negative control. Fluorescence microscopy images showing in vitro phagocytosis of WR19L cells by CER09+ Ba / F3 cells are shown in Figure 47 (white arrows indicate phagocytic events).

[0283] A20 mouse lymphoma cells were treated with staurosporine, labeled with pHrodo Red, and CELLTRACE Violet-labeled Ba / F3 CER09 as described in Example 8. + EGFR +The cells were co-cultured with target cells to effector cells at a target cell to effector cell ratio of 5:1 for 3 hours. + Phagocytosis of A20 cells by Ba / F3 cells was quantified by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with pLenti vector expressing truncated EGFR were used as a negative control. Fluorescence microscopy images showing in vitro phagocytosis of A20 cells by CER09+ Ba / F3 cells are shown in Figure 48 (white arrows indicate phagocytic events).

[0284] Phagocytic activity of human CER09+ B cells against human cell lines As described in Example 8, human primary B cells were transduced with pLenti Tim4-DAP12 (CER09) lentivirus expressing a truncated EGFR as a transduction marker. The transduced human B cells were sorted by FACS using a labeled anti-EGFR antibody (cetuximab) and then stained with Kat5-18 antibody (Tim4 specific) (Abcam catalog no. 176486) (see Figure 49A, where the percentage in the FACS plot on the right indicates the proportion of cells (CER09) expressing the Tim4 binding domain). Purified CER09 + B cells were expanded and imaged at 24, 48 and 72 hours and are shown in Figure 49B.

[0285] In the phagocytosis assay, Jurkat human T lymphocytes were treated with staurosporine, labeled with pHrodo Red, and co-incubated with CER09+ primary B cells at a target cell to effector cell ratio of 5:1 for 3 hours, as described in Example 8. + Phagocytosis of Jurkat cells by human B cells was quantified by fluorescence microscopy and FACs as described in Example 8. The frequency of viable CD19-positive human primary B cells and the frequency of CD19-positive-pHrodo Red-positive events (double-positive events) are shown in Figure 50 (left and right plots, respectively). Figure 51 shows the CER09 + tEGFR + or EGFR +The frequency of phagocytosis of control-transduced B cells is shown.

[0286] CER09 + Fluorescence microscopy images showing in vitro phagocytosis of Jurkat cells by human primary B cells are shown in Figure 52 (left photograph), and phagocytosis of Jurkat cells by tEGFR+ human primary B cell control is shown in Figure 52 (right photograph) (white arrows indicate phagocytosis events).

[0287] Example 11 Construction of TIM4-DAP12-DAP12 CER “CER10” The extracellular domain of the phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO: 73), including its signal peptide (amino acid sequence of SEQ ID NO: 72), was linked to the DAP12 transmembrane domain (SEQ ID NO: 81) and intracellular signaling domain (SEQ ID NO: 82) to generate the chimeric engulfment receptor "CER10" (Tim4-DAP12-DAP12CER having the amino acid sequence of SEQ ID NO: 86). The DAP12 signaling domain transmits the signal for engulfment, and Tim4 is a phosphatidylserine-binding receptor. The Tim4-DAP12-DAP12(CER10) chimeric engulfment receptor nucleotide sequence was then inserted into the pLenti lentiviral vector with a truncated EGFR as a transduction marker, with the P2A sequence (SEQ ID NO: 104) inserted between them (see Figure 53). Murine Ba / F3 B cells were transduced with pLenti vectors expressing Tim4-DAP12-DAP12(CER10) and EGFRt, expanded, sorted by FACs, and used for in vitro studies as described in Example 8.

[0288] Phagocytic activity towards apoptotic thymocytes in primary culture Primary C3H mouse thymocytes were isolated, treated with dexamethasone, and stained with pHrodo Red as described in Example 8. Ba / F3 CER10 + tEGFR + Cells were cultured in a CELLTRACE (商標)Labeled with Violet dye. Ba / F3 CER10 + tEGFR + Ba / F3 CER10 cells and primary thymocytes were co-cultured at a target cell to effector cell ratio of 10:1. + EGFR + Cells were quantified for phagocytosis of target thymocytes by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with pLenti vector expressing a truncated EGFR were used as a negative control.

[0289] Viable CER10 quantified by FACS + The amount of transduced Ba / F3 cells is shown in Figure 54A. The frequency of phagocytosis was quantified as the population of cells staining double positive for pHrodo Red and CELLTRACE Violet, as detected by FACS (see Figure 54B).

[0290] Fluorescence microscopy showed that CER10 cells were significantly more resistant to tEGFR than tEGFR-transduced Ba / F3 control cells. + Ba / F3 cells were shown to engulf dexamethasone-treated thymocytes (white arrows indicate engulfment events) (see Figures 55A-B). A magnified image of an engulfment event is shown in the lower right corner of Figure 55B.

[0291] The phagocytosis index was calculated by multiplying the [average of total number of engulfed target cells / total number of CER-modified cells counted (e.g., phagocytosis frequency)] by the [average area of ​​target cell staining per CER+Ba / F3 cell x 100 (e.g., hybrid capture)] compared to EGFRt-transduced Ba / F3 control cells (see Figures 56A-B).

[0292] Example 12 Construction of TIM4-Axl CER “CER11” The extracellular domain of the phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO: 73), including the signal peptide (amino acid sequence of SEQ ID NO: 72) and transmembrane domain (amino acid sequence of SEQ ID NO: 74), was linked to the Axl intracellular signaling domain (SEQ ID NO: 44) to generate the chimeric engulfment receptor "CER11" (Tim4-Axl CER having the amino acid sequence of SEQ ID NO: 87). The Axl signaling domain transmits the signal for engulfment, and Tim4 is a phosphatidylserine-binding receptor. The Tim4-Axl (CER11) chimeric engulfment receptor nucleotide sequence was then inserted into the pLenti lentiviral vector with a truncated EGFR as a transduction marker, with a T2A sequence inserted between them (see Figure 49). Murine Ba / F3 B cells were transduced with pLenti vectors expressing Tim4-Axl (CER11) and EGFRt, expanded, sorted by FACs, and used for in vitro studies, as described in Example 8.

[0293] Phagocytic activity towards apoptotic thymocytes in primary culture Primary C3H mouse thymocytes were isolated, treated with dexamethasone, and stained with pHrodo Red as described in Example 8. Ba / F3 CER11 + tEGFR + Cells were cultured in a CELLTRACE (商標) Labeled with Violet dye. Ba / F3 CER11 + tEGFR + Ba / F3 CER11 cells and primary thymocytes were co-cultured at a target cell to effector cell ratio of 10:1. + EGFR + Cells were quantified for phagocytosis of target thymocytes by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with pLenti vector expressing a truncated EGFR were used as a negative control.

[0294] Viable CER11 quantified by FACS +The amount of transduced Ba / F3 cells is shown in Figure 58A. The frequency of phagocytosis was quantified as the population of cells staining double positive for pHrodo Red and CELLTRACE Violet, as detected by FACS (see Figure 58B).

[0295] Fluorescence microscopy showed that CER11 cells were significantly more resistant to tEGFR than tEGFR-transduced Ba / F3 control cells. + Ba / F3 cells were shown to engulf dexamethasone-treated thymocytes (white arrows indicate engulfment events) (see Figures 59A-B). A magnified image of an engulfment event is shown in the lower right corner of Figure 59B.

[0296] The phagocytosis index was calculated by multiplying the [average of total number of engulfed target cells / total number of CER-modified cells counted (e.g., phagocytosis frequency)] by the [average area of ​​target cell staining per CER+Ba / F3 cell x 100 (e.g., hybrid capture)] compared to EGFRt-transduced Ba / F3 control cells (see Figures 60A-B).

[0297] Phagocytic activity against mouse cell lines As described in Example 8, Ba / F3 CER11 + tEGFR + Cells, CELLTRACE (商標) CT26 mouse colon carcinoma cells were treated with staurosporine, labeled with pHrodo Red, and Ba / F3 CER11 as described in Example 8. + tEGFR + The target cells were co-cultured with effector cells at a target cell to effector cell ratio of 5:1 for 3 hours. + Phagocytosis of CT26 cells by Ba / F3 cells was quantified by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with the pLenti vector expressing truncated EGFR were used as a negative control. CER11+Ba / F3 cells and EGFRt +Fluorescence microscopy images showing in vitro phagocytosis by control cells are shown in Figures 61A-B (white arrows indicate phagocytic events). CT26 cells labeled with pHrodo Red fluoresce within the low pH compartments of lysosomes (pink outline) when engulfed.

[0298] A hybrid capture algorithm that detects pHrodo Red fluorescence within the CELLTRACE Violet stained area is applied to the fluorescence image to identify the area of ​​engulfed target cells / CER. + The area of ​​B cells was quantified. + tEGFR + or tEGFR + Scatter plot of hybrid cell number extracting the area of ​​CT26 target cells within control-transduced Ba / F3 cells. Area ratio indicates the co-localized area of ​​CT26 cells within Ba / F3 cells.

[0299] WR19L mouse lymphoma cells were treated with staurosporine, labeled with pHrodo Red, and CELLTRACE Violet-labeled Ba / F3 CER11 as described in Example 8. + EGFR + The cells were co-cultured with CER11 cells at a target cell to effector cell ratio of 5:1 for 3 hours. + Phagocytosis of WR19L cells by Ba / F3 cells was quantified by fluorescence microscopy and FACS as described in Example 8. Ba / F3 cells transduced with a pLenti vector expressing truncated EGFR were used as a negative control. Fluorescence microscopy images showing in vitro phagocytosis of WR19L cells by CER11+ Ba / F3 cells are shown in Figure 63 (white arrows indicate phagocytic events). The amount of viable CER11+ transduced Ba / F3 cells quantified by FACS is shown in Figure 64A. The frequency of phagocytosis was quantified as the cell population double-positively stained for pHrodo Red and CELLTRACE Violet, as detected by FACS (see Figure 64B).

[0300] A20 mouse lymphoma cells were treated with staurosporine, labeled with pHrodo Red, and CELLTRACE Violet-labeled Ba / F3 CER11 as described in Example 8. + EGFR + The cells were co-cultured with CER11 cells at a target cell to effector cell ratio of 5:1 for 3 hours. + Phagocytosis of A20 cells by Ba / F3 cells was quantified by fluorescence microscopy and FACs as described in Example 8. Ba / F3 cells transduced with a pLenti vector expressing truncated EGFR were used as a negative control. Fluorescence microscopy images showing in vitro phagocytosis of A20 cells by CER11+ Ba / F3 cells are shown in Figure 65A (white arrows indicate phagocytic events) compared to EGFRt-transduced Ba / F3 controls (Figure 65B). The phagocytic index of CER11+ Ba / F3 cells compared to EGFRt+ control cells was calculated and is shown in Figure 66.

[0301] Human CER11 on chemotherapeutic-treated human cell lines + B cell phagocytic activity Human primary B cells were transduced with pLenti Tim4-Axl (CER11) lentivirus, which expresses a truncated EGFR as a transduction marker, as described in Example 8. One day before setting up the phagocytosis assay, Jurkat human B lymphocyte cells were cultured in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 6-well plates and treated with oxaliplatin (5 μM) and fluorouracil (5-FU) (10 μM). The next day, target Jurkat cells were harvested, washed twice with 1× PBX, and stained with pHrodo Red (1 ng / mL in PBS) for 15 minutes at room temperature. Growth medium was added to the Jurkat cells, washed once to remove excess pHrodo Red, and plated at approximately 200,000 cells / well in a flat-bottom 96-well plate in complete RPMI 1640 medium. Transduced human primary B cells were washed once with 1X PBS and then stained with CELLTRACE Violet (1 mM in PBS) for 10 minutes at 37°C. Growth medium was added to the human primary B cells, which were washed once with 1X PBS to remove excess CELLTRACE Violet. Approximately 50,000 cells were seeded into 96-well plates in RPMI complete medium. Human primary B cells and Jurkat cells were co-cultured at a target cell to effector cell ratio of 4:1 for 3 hours at 37°C. Plates were then imaged using a Keyence BZ-X710 microscope with a 20x objective. Figure 67 shows fluorescence microscopy images demonstrating engulfment of chemotherapy-treated Jurkat cells by CER11+ human primary B cells (the image on the right shows a magnified view of the phagocytic event; white arrows indicate phagocytic activity).

[0302] Human primary B cells were transduced with pLenti Tim4-Axl (CER11) lentivirus, which expresses a truncated EGFR as a transduction marker, as described in Example 8. One day before setting up the phagocytosis assay, Colo320 HSR colon cancer cells were incubated with the phosphatidylserine-inducing chemotherapy drug gemcitabine (10 μM) in serum-free medium for 24 hours. After treatment, floating and adherent target cells were collected, centrifuged, incubated with pHrodo red (1 ng / μL) in PBS at room temperature for 15 minutes, washed, and then plated onto non-adherent 96-well plates. Human CER11+-expressing B cells and Colo320HSR cells were co-cultured at a target cell to effector cell ratio of 4:1 for 3 hours at 37°C. The plates were then imaged using a Keyence BZ-X710 microscope with a 20x objective (see Figure 67; white arrows indicate phagocytosis).

[0303] Human primary B cells were transduced with pLenti Tim4-Axl(CER11) lentivirus, which expresses a truncated EGFR as a transduction marker, as described in Example 8. One day before setting up the phagocytosis assay, A204 rhabdomyosarcoma cells we...

Claims

1. 1. A pharmaceutical composition for use in a method of treating a subject having cancer, comprising: The pharmaceutical composition comprises:

1. A T cell comprising a chimeric engulfment receptor (CER) comprising a single-chain chimeric protein, the single-chain chimeric protein an extracellular domain containing a Tim4-binding domain that binds to phosphatidylserine (PtdSer); an enlargement signaling domain; and A transmembrane domain that is located between and connects the extracellular domain and the enlargement signaling domain T cells, including: a pharmaceutically acceptable excipient.

2. 2. The pharmaceutical composition of claim 1, wherein the binding domain comprises a TIM4 domain comprising the amino acid sequence of SEQ ID NO:29 or amino acids 25-314 of SEQ ID NO:

29.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

4. 4. The pharmaceutical composition of claim 3, wherein the extracellular spacer domain comprises an immunoglobulin hinge region, a hinge region of a type I membrane protein, a stalk region of a type II C-type lectin, an immunoglobulin constant domain, or a fragment thereof.

5. 5. The pharmaceutical composition of claim 4, wherein the extracellular spacer domain comprises an IgG1, IgG2, IgG3, IgG4, IgA, or IgD hinge region.

6. 6. The pharmaceutical composition of claim 5, wherein the extracellular spacer domain comprises a modified IgG4 hinge region comprising the amino acid sequence of SEQ ID NO:

67.

7. 5. The pharmaceutical composition of claim 4, wherein the hinge region of a type I membrane protein is selected from CD8a, CD4, CD28, and CD7.

8. 5. The pharmaceutical composition of claim 4, wherein the stalk region of a type II C-type lectin is selected from CD28, CD69, CD72, CD94, NKG2A, and NKG2D.

9. 5. The pharmaceutical composition of claim 4, wherein the immunoglobulin constant domain is selected from a CH1 domain, a CH2 domain, a CH3 domain, or any combination thereof.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the transmembrane domain comprises a Tim1, Tim4, Tim3, FcR, CD8a, CD28, MERTK, Axl, Tyro3, ​​BAI1, CD4, DAP12 or MRC1 transmembrane domain.

11. 11. The pharmaceutical composition of claim 10, wherein the transmembrane domain comprises a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 35, a Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 37, a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 38, a MERTK transmembrane domain comprising the amino acid sequence of SEQ ID NO: 39, an AxI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, a Tyro3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 41, a CD28 transmembrane domain of SEQ ID NO: 68, a BAI1 transmembrane domain of SEQ ID NO: 142, a CD4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, an FcεRIγ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 89, an MRC1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 118, or a DAP12 transmembrane domain comprising the amino acid sequence of SEQ ID NO:

81.

12. 12. The pharmaceutical composition of claim 10 or 11, wherein the FcR transmembrane domain comprises an FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1 or FcαR1 transmembrane domain.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the engulfment signaling domain comprises an ItgB5, MERTK, Tyro3, ​​Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b engulfment signaling domain.

14. The engulfment signaling domain may be a MERTK signaling domain comprising the amino acid sequence of SEQ ID NO:69, a Tyro3 signaling domain comprising the amino acid sequence of SEQ ID NO:45, an ItgB5 signaling domain comprising the amino acid sequence of SEQ ID NO:114, an MRC1 signaling domain comprising the amino acid sequence of SEQ ID NO:119, a BAI1 signaling domain comprising the amino acid sequence of SEQ ID NO:136, an ELMO signaling domain comprising the amino acid sequence of SEQ ID NO:120, an Axl signaling domain comprising the amino acid sequence of SEQ ID NO:44, a Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO:54, a Syk signaling domain comprising the amino acid sequence of SEQ ID NO:46, a MyD88 signaling domain comprising the amino acid sequence of SEQ ID NO:53, a truncated MyD signaling domain comprising the amino acid sequence of SEQ ID NO:78, or a MyD signaling domain comprising the amino acid sequence of SEQ ID NO:

79.

14. The pharmaceutical composition of claim 13, comprising an FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 88 signaling domain, a Zap70 signaling domain comprising the amino acid sequence of SEQ ID NO: 47, an FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 48, an FcγR2A signaling domain comprising the amino acid sequence of SEQ ID NO: 49, an FcγR2C signaling domain comprising the amino acid sequence of SEQ ID NO: 50, an FcγR3A signaling domain comprising the amino acid sequence of SEQ ID NO: 51, an FcεRIγ signaling domain comprising the amino acid sequence of SEQ ID NO: 88, a BAFF-R signaling domain comprising the amino acid sequence of SEQ ID NO: 94, a DAP12 signaling domain comprising the amino acid sequence of SEQ ID NO: 82, an NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO: 92, or a CD79b signaling domain comprising the amino acid sequence of SEQ ID NO:

97.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the engulfment signaling domain comprises a first engulfment signaling domain and a second engulfment signaling domain.

16. 16. The pharmaceutical composition of claim 15, wherein the first engulfment signaling domain is an ItgB5, MERTK, Tyro3, ​​Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b engulfment signaling domain.

17. The first engulfment signaling domain is selected from the group consisting of a MERTK signaling domain comprising the amino acid sequence of SEQ ID NO:69, a Tyro3 signaling domain comprising the amino acid sequence of SEQ ID NO:45, an ItgB5 signaling domain comprising the amino acid sequence of SEQ ID NO:114, an MRC1 signaling domain comprising the amino acid sequence of SEQ ID NO:119, a BAI1 signaling domain comprising the amino acid sequence of SEQ ID NO:136, an ELMO signaling domain comprising the amino acid sequence of SEQ ID NO:120, an AxI signaling domain comprising the amino acid sequence of SEQ ID NO:44; a Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO:54, a Syk signaling domain comprising the amino acid sequence of SEQ ID NO:46, a MyD88 signaling domain comprising the amino acid sequence of SEQ ID NO:53, a truncated M signaling domain comprising the amino acid sequence of SEQ ID NO:78, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:16, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:17, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:18, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:19, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:20, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:21, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:22, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:23, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:24, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:25, a β-actin signaling domain comprising the amino acid sequence of SEQ ID NO:26, a β- 17. The pharmaceutical composition of claim 16, wherein the signaling domain is a yD88 signaling domain, a Zap70 signaling domain comprising the amino acid sequence of SEQ ID NO:47, an FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO:48, an FcγR2A signaling domain comprising the amino acid sequence of SEQ ID NO:49, an FcγR2C signaling domain comprising the amino acid sequence of SEQ ID NO:50, an FcγR3A signaling domain comprising the amino acid sequence of SEQ ID NO:51, an FcεRIγ signaling domain comprising the amino acid sequence of SEQ ID NO:88, a BAFF-R signaling domain comprising the amino acid sequence of SEQ ID NO:94, a DAP12 signaling domain comprising the amino acid sequence of SEQ ID NO:82, an NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO:92, or a CD79b signaling domain comprising the amino acid sequence of SEQ ID NO:

97.

18. 18. The pharmaceutical composition of any one of claims 15-17, wherein the second engulfment signaling domain is an ItgB5, MERTK, Tyro3, ​​Axl, BAI1, ELMO, MRC1, PI3K, Traf6, Syk, MyD88, Zap70, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, or CD79b signaling domain.

19. The second engulfment signaling domain is selected from the group consisting of a MERTK signaling domain comprising the amino acid sequence of SEQ ID NO:69, a Tyro3 signaling domain comprising the amino acid sequence of SEQ ID NO:45, an ItgB5 signaling domain comprising the amino acid sequence of SEQ ID NO:114, an MRC1 signaling domain comprising the amino acid sequence of SEQ ID NO:119, a BAI1 signaling domain comprising the amino acid sequence of SEQ ID NO:136, an ELMO signaling domain comprising the amino acid sequence of SEQ ID NO:120, an AxI signaling domain comprising the amino acid sequence of SEQ ID NO:44, a Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO:54, a Syk signaling domain comprising the amino acid sequence of SEQ ID NO:46, a MyD88 signaling domain comprising the amino acid sequence of SEQ ID NO:53, a truncated M 19. The pharmaceutical composition of claim 18, wherein the signaling domain is a yD88 signaling domain, a Zap70 signaling domain comprising the amino acid sequence of SEQ ID NO:47, an FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO:48, an FcγR2A signaling domain comprising the amino acid sequence of SEQ ID NO:49, an FcγR2C signaling domain comprising the amino acid sequence of SEQ ID NO:50, an FcγR3A signaling domain comprising the amino acid sequence of SEQ ID NO:51, an FcεRIγ signaling domain comprising the amino acid sequence of SEQ ID NO:88, a BAFF-R signaling domain comprising the amino acid sequence of SEQ ID NO:94, a DAP12 signaling domain comprising the amino acid sequence of SEQ ID NO:82, an NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO:92, or a CD79b signaling domain comprising the amino acid sequence of SEQ ID NO:

97.

20. The T cells are CD4 ＋ T cells, CD8 ＋ The pharmaceutical composition of any one of claims 1 to 19, wherein the T cells are T cells, naive T cells, central memory T cells, effector memory T cells, virus-specific T cells, mucosal-associated invariant T cells, gamma delta (gd) T cells, natural killer T cells, or tissue-resident T cells.

21. The pharmaceutical composition of any one of claims 1-20, wherein the T cells are human T cells.

22. The pharmaceutical composition of any one of claims 1-21, wherein the T cells are autologous T cells.

23. The pharmaceutical composition of any one of claims 1-21, wherein the T cells are allogeneic T cells.

24. A pharmaceutical composition described in any one of claims 1 to 23, used in combination with a second therapeutic agent.

25. 25. The pharmaceutical composition of claim 24, wherein the second therapeutic agent is an antibody, radiation therapy, a chemotherapeutic agent, a cellular immunotherapy, an antibiotic, an antifungal agent, or an antiviral agent.

26. 26. The pharmaceutical composition of claim 24 or 25, wherein the pharmaceutical composition comprising the CER-containing T cells is administered after administration of a second therapeutic agent.

27. 27. The pharmaceutical composition of any one of claims 24-26, wherein the second therapeutic agent is administered in a sub-therapeutic dose.

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