Chimeric engulfment receptor molecules and methods of use

Abstract

To provide novel compositions and methods for treating various cancers, acute and chronic infections, inflammatory diseases, immune diseases and selected nervous diseases by accelerating removal of infected cells, transformed cells, malignant cells, apoptotic cells, damaged cells or necrotic cells from the body.SOLUTION: Provided herein is a chimeric engulfment receptor comprising a single chain chimeric protein, where the single chain chimeric protein comprises: an extracellular domain comprising a binding domain that binds to phosphatidylserine; an engulfment signaling domain comprising toll-like receptor (TLR) signaling domain, Traf6 signaling domain, Traf2 signaling domain or Traf3 signaling domain; and a transmembrane domain positioned between the extracellular domain and engulfment signaling domain to link them.SELECTED DRAWING: None

Description

Technical Field

[0001] Description of Sequence Listing The sequence listing related to this application is provided in text format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 200265_402WO_SEQUENCE_LISTING.txt. This text file is 407 KB, was created on September 19, 2018, and was electronically submitted via EFS-Web.

Background Art

[0002] Background There are two main types of phagocytic cell actions that are affected by the target, cell type, and surrounding environment. Phagocytic cell action against microorganisms eliminates and degrades disease-causing microorganisms, induces inflammatory signaling via the secretion of cytokines and chemokines, and mobilizes immune cells to cause an effective inflammatory response. This type of phagocytic cell action is often referred to as "inflammatory phagocytosis" (or "immunogenic phagocytosis"). However, in some cases, such as certain persistent infections, an anti-inflammatory response can occur after the uptake of microorganisms. Phagocytic cell action against microorganisms is generally carried out by myeloid professional phagocytes such as immature dendritic cells (DCs) and macrophages, as well as tissue-resident immune cells.

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

[0004] There is a need for novel compositions and methods for treating infectious diseases, inflammatory diseases, immune diseases, and various cancers. The methods and compositions described herein meet such needs by promoting the removal of infected cells, transformed cells, malignant cells, apoptotic cells, damaged cells, or necrotic cells 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] Summary This specification describes chimeric engulfment receptors. In certain embodiments, the chimeric engulfment receptor (singular “CER” and plural “CERs”) includes an extracellular domain, a transmembrane domain, and an intracellular engulfment signaling domain. The transmembrane domain is located between and couples the extracellular domain and the engulfment signaling domain. The extracellular domain includes a binding domain and, optionally, an extracellular spacer domain located between the binding domain and the transmembrane domain. In certain embodiments, the chimeric engulfment receptors described herein are chimeric proteins 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 that includes a Toll-like receptor (TLR) signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain. In certain embodiments, the engulfment signaling domain includes at least one of a constitutive engulfment domain and an inflammation-inducing engulfment domain. In one embodiment, the engulfment signaling domain includes 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 elicit an inflammatory response against a target cell / organ / tissue / region. Apoptotic cell clearance is generally a non-inflammatory process, but 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 CER contains a binding domain specific for an engulfment promoting marker. In such specific embodiments, the extracellular domain contains a phosphatidylserine (PtdSer) binding domain. In embodiments of 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 a binding domain derived from FA58C2, GAS6, Protein S, Factor VII, Factor IX, Factor X, or prothrombin PS.

[0007] In a further embodiment, the extracellular domain binds to a target antigen. In such specific embodiments, the extracellular domain can comprise all or a portion of the extracellular domain of an Fc receptor (FcR) such as, for example, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, and FcαR1. In yet another embodiment, 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 intrabodies, peptibodies, nanobodies, single domain antibodies, SMIPs, and multispecific antibodies. In such specific embodiments, the extracellular domain contains a Fab binding domain. In yet another such embodiment, the extracellular domain contains a scFv.

[0008] Binding of the extracellular domain of CER to an engagement promoting marker or a targeted antigen stimulates the engagement signaling domain to stimulate engagement signaling activity. Thus, upon activation, the engagement signaling domain contained in CER transmits an effector functional signal that instructs the host cell to engage. In certain embodiments, the engagement signaling domain comprises a first engagement signaling domain, including a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain; and a second engagement signaling domain. Examples of the second engagement signaling domain include the FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, NFAM1, DAP12, MERTK, CD79b, TLR, Traf2, Traf3, and Traf6 signaling domains.

[0009] In a further aspect, the present invention relates to cells genetically modified to express CER. In certain embodiments, CER results in an engulfment phenotype that is 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 one embodiment, antigen binding by CER induces an engulfment signaling cascade in cells that do not naturally exhibit phagocytic signaling activity. In another embodiment, CER-expressing cells that do not naturally exhibit phagocytic signaling activity and phagocytose target cells can degrade the target cells. In other embodiments, the CER of the present invention further confers to host cells phenotypes such as proliferative activity, expansion activity, activation, cytolytic activity, antigen presentation activity, memory formation, increased persistence, or combinations thereof, that may not be present in host cells that do not express CER. In certain embodiments, the cells are genetically modified to express CER that targets an engulfment-promoting marker associated with dead cells, dying cells, damaged cells, infected cells, or necrotic cells. In other embodiments, the cells are genetically modified to express 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 targeted cells or microorganisms by the binding of CER to the marker associated with the targeted molecule induced by the targeted infectious microorganism or infectious particle. In other specific embodiments, the cells are genetically modified to express CER that targets an antigen marker that normally does not cause engulfment. For example, in such embodiments, the extracellular domain of CER can include an antibody or an antigen-binding portion thereof, such as a Fab binding domain or an scFv specific for the antigen marker. In such specific 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 cells promote the elimination or degradation of abnormal cells upon binding of the antigen marker by CER.In certain embodiments, the cells that are genetically modified to express a CER that targets an antigen marker that does not normally cause engulfment are B cells.

[0010] In yet a further aspect, the present invention relates to methods of treating a subject suffering from a disease, disorder or undesirable condition. Aspects of these methods include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more CERs or a population of cells genetically modified to express one or more CERs according to the description herein.

[0011] 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: introducing and expressing a CER in a host cell that does not naturally exhibit an engulfment phenotype to create a population of cells that exhibit an engulfment phenotype; modifying the engulfment phenotype of a population of cells by introducing and expressing a CER in the host cell, where the CER confers an engulfment phenotype specific to an engulfment promoting marker or antigen marker not naturally targeted by the host cell; and methods for enhancing the engulfment phenotype of a population of cells by introducing and expressing a CER in the host cell, where the CER is specific to an engulfment promoting marker or antigen marker naturally targeted by the host cell and expression of the CER by the host cell enhances the engulfment of cells, microorganisms or particles exhibiting the targeted engulfment promoting marker or antigen marker by the host cell.

Brief Description of the Drawings

[0012] The patent or patent application file contains drawings created in at least one color. Copies of this patent or patent application publication, which include color drawings, are provided by the United States Patent and Trademark Office upon request and payment of the necessary fees.

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Modes for Carrying Out the Invention

[0026] Detailed Description (a) An extracellular domain comprising an extracellular binding domain and optionally an extracellular spacer domain, (b) a transmembrane domain, and (c) an engagement signaling domain comprising a Toll-like receptor (TLR) signaling domain, a Traf6 signaling domain, a Traf2 signaling domain or a Traf3 signaling domain, a chimeric protein comprising the same, and a nucleic acid molecule encoding the chimeric protein are described herein. Further provided are cells modified to express these chimeric proteins, and methods and compositions for delivering such modified cells to a subject in need thereof. The chimeric protein is referred to herein as "chimeric engagement receptor(s)" (singular "CER" and plural "CERs"). The chimeric engagement receptors described herein can confer an engagement phenotype on a host cell that has been genetically modified to express the chimeric engagement receptor. In certain such embodiments, expression of the CERs described herein confers an engagement phenotype on a host cell that does not naturally exhibit the engagement phenotype. In other such embodiments, expression of the CERs described herein by a host cell confers an engagement phenotype specific for an engagement promoting marker or antigen marker that is not naturally targeted by the host cell. In yet other such embodiments, expression of the CERs described herein by a host cell confers an engagement phenotype specific for an engagement promoting marker or antigen marker that is naturally targeted by the host cell, and expression of the CERs by the host cell enhances the engagement by the host cell of cells, microorganisms or particles that exhibit the targeted engagement promoting or antigen marker.

[0027] In certain embodiments, the CER targets an engulfment marker associated with apoptotic cells, dead cells, dying cells, damaged cells, infected cells, or necrotic cells. In other embodiments, the CER targets antibody-bound cells associated with infectious microorganisms or particles. In still other embodiments, the CER targets an antigen marker presented by abnormal cells or misfolded proteins associated with a disease, disorder, or other undesirable medical condition.

[0028] One or more of the 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 particular target molecule (e.g., an engulfment marker or antigen marker), the engulfment signaling domain of the CER is selected to provide a desired engulfment activity. In another embodiment, the CER comprises a first engulfment signaling domain and a second engulfment signaling domain.

[0029] A host cell modified to express one or more CERs described herein can be used for the specific engulfment of target cells or particles that express a target molecule to which the extracellular domain of the CER binds. In certain embodiments, the target cells or particles can be tumor cells, cancer cells, microorganisms (e.g., bacteria, fungi, viruses), protozoan parasites, abnormal cells, or misfolded proteins associated with infection, disease, disorder, or other undesirable pathological conditions. In further embodiments, a host cell genetically modified to express one or more CERs described herein is used as a primary, adjuvant, or combination therapy for treating cancer, infectious diseases (viruses, bacteria, fungi, protozoa), inflammatory diseases, and immune diseases (e.g., autoimmune diseases) in a subject. The CERs described herein confer an inflammatory (immunogenic) phenotype to the host cells expressing the CERs via the TLR, Traf6, Traf2, or Traf3 engulfment signaling domain. In certain embodiments, the CER-modified host cells further exhibit enhanced proliferative activity, activation, memory formation, cytolytic activity, antigen presentation activity, phagocytic signaling activity, lumen degradation, or combinations thereof, which are not present in host cells that do not express the CERs.

[0030] Definitions Before describing the present invention in more detail, it may be helpful to provide definitions of certain terms used herein for its understanding.

[0031] In this specification, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range and, where appropriate, its fractional amounts (e.g., one-tenth and one-hundredth of an integer) unless otherwise specified. Also, any number range recited herein with respect to any physical characteristic, such as a polymer subunit, size, or thickness, should be understood to include any integer within the recited range unless otherwise specified. The term “about” as used herein means ±20% of the indicated range, value, or structure unless otherwise specified. It should be understood that the terms “a” and “an” as used herein 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 of them. The terms “include,” “have,” and “comprise” as used herein are used interchangeably and are intended to be construed as non-limiting and their variations thereof.

[0032] Terms understood by those skilled in the art of antibodies shall have the meanings accepted in the art, respectively, unless otherwise clearly defined herein in a different manner. The term “antibody” is used in the broadest sense and encompasses polyclonal and monoclonal antibodies. An “antibody” can mean an intact antibody that includes at least two heavy (H) chains and two light (L) chains linked to each other 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 immunoglobulins in naturally occurring, recombinantly produced, genetically engineered, or modified forms, such as intrabodies, peptibodies, nanobodies, single-domain antibodies, SMIPs, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetra-bodies, tandem di-scFv, tandem tri-scFv, ADAPTIR). Monoclonal antibodies or their antigen-binding portions can be non-human, chimeric, humanized, or human, preferably humanized or human. Immunoglobulin structures and functions 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 means a part of the intact antibody and includes “antibody fragments” that mean the antigen-determining variable regions or complementarity-determining regions 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, as well as multispecific antibodies formed from antibody fragments. “Fab” (fragment antigen-binding) is a part of an antibody that binds to an antigen and includes the variable region and CH1 of the heavy chain that is bound to the light chain via an interchain disulfide bond. Antibodies can be of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0033] The terms "variable region" or "variable domain" refer to the domain of the antibody heavy or light chain that is involved in binding to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have a similar structure, and each domain contains four conserved framework regions (FRs) 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, an antibody that binds a particular antigen can be isolated by screening a library of complementary VH or VL domains using the 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).

[0034] The terms "complementary determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous sequences of amino acids within the 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).

[0035] The terms "antigen" and "Ag" mean a molecule that elicits an immune response. The elicited immune response may include antibody production, activation of specific immunologically-competent cells, or both. Macromolecules including proteins, glycoproteins and glycolipids can serve as antigens. Antigens can be derived from recombinant DNA or genomic DNA. As contemplated herein, an antigen need not be encoded by (i) only the full-length nucleotide sequence of a gene or (ii) a "gene". An antigen can be generated or synthesized, or an antigen 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.

[0036] 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 homologous immunoconjugate molecule such as an antibody or a fragment thereof (e.g., scFv), a T cell receptor (TCR), a chimeric engager receptor, or other binding molecule, domain or protein. Epitope determinants generally include chemically active surface groups of molecules such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be a linear epitope or a conformational epitope.

[0037] The term "anti-tumor effect" means a biological effect that can be demonstrated by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in mean life span, or an improvement in various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be demonstrated by the prevention of blood cancer or tumor formation.

[0038] "Autoimmune disease" means a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. The autoimmune response may include autoreactive B cells, autoreactive T cells, or both, which produce autoantibodies. As used herein, "autoantibody" is an antibody produced by a subject that binds to an autoantigen also produced by that subject.

[0039] "Autologous" means any material derived from the same subject that is reintroduced later.

[0040] "Allogeneic" means a graft derived from a different subject of the same species.

[0041] As used herein, the terms "binding domain", "binding region" and "binding moiety" refer to molecules such as peptides, oligopeptides, polypeptides, or proteins that have the ability to specifically and non-covalently bind, associate, conjugate, recognize or bind to a related target molecule (e.g., PtdSer, IgG antibody, IgE antibody, IgA antibody, CD138, CD38, CD33, CD123, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, EGFRviii, VEGFR-2 or GD2). A binding domain includes any natural, synthetic, semi-synthetic or recombinantly produced binding partner for a biological molecule or other target of interest. In certain embodiments, the binding domain is an antibody, or an antigen-binding domain such as 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 a biomolecule.

[0042] To identify the binding domains of the present invention that specifically bind to a particular target, and to determine the binding domain affinities, various assays are known, such as Western blot, ELISA, and BIACORE® analysis (see, for example, Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; and also U.S. Patent Nos. 5,283,173, 5,468,614, or their equivalents). As used herein, "specifically binds" means that the binding domain or its fusion protein associates or binds to the target molecule with an affinity of 10 5 M -1 or greater or a K a (i.e., the equilibrium binding constant of a particular binding interaction in units of 1 / M) and does not significantly associate or bind with other molecules or components in the sample.

[0043] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. The abnormal cells can form solid tumors or constitute blood cancers. Cancer cells can spread locally or through the bloodstream and lymphatic system 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 tumors, lymphomas, leukemias, lung cancer, and the like.

[0044] A "disease" is the health state of 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 health state in which the subject can maintain homeostasis, but the subject's health state is less favorable than it would be in the absence of the disorder or undesirable condition. An untreated disorder or undesirable condition does not necessarily result in a further decline in the subject's health state.

[0045] "Pathogen" or "microorganism" means any species of bacterium, virus, archaea, or fungus.

[0046] "Particle" means a fragment of a cell or a small substance with a diameter of at least 100 nm up to a maximum of 6 μm, which is derived from a living cell or organism. The particle can be a virus particle, a small mineral particle, a cell fragment, or a synthetic particle.

[0047] "Encoding" means the unique property of a specific polynucleotide sequence such as a DNA, cDNA, and mRNA sequence that serves as a template for synthesizing other polymers and macromolecules in a biological process, having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological products resulting therefrom.

[0048] Thus, a polynucleotide encodes a protein when transcription and translation of the mRNA corresponding to that polynucleotide produce the protein in a cell or other biological system. Both the coding strand and the non-coding strand can be said to encode a protein or other product of the polynucleotide.

[0049] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" are degenerate to each other and include all nucleotide sequences encoding the same amino acid sequence.

[0050] As used herein, the terms "endogenous" or "native" mean a gene, protein, compound, molecule, or activity that is normally present in a host or host cell.

[0051] As used herein, the term “engulfment” means a receptor-mediated process by which an endogenous or exogenous cell or particle having a diameter greater than 100 nm is internalized by a phagocytic cell or host cell described herein. Engulfment generally consists of the following multiple steps: (1) tethering of a target cell or particle by binding of an engulfment receptor to an engulfment-promoting marker or antigen marker on the target cell or particle, either directly or indirectly (through a bridging molecule); and (2) internalization or uptake of the entire target cell or particle, or a portion thereof. In certain embodiments, internalization occurs via rearrangement of the cytoskeleton of the phagocytic cell or host cell, and a phagosome, a membrane-bound compartment containing the internalization target, may be formed. Engulfment further includes maturation of the phagosome, where the phagosome becomes increasingly acidic and fuses with a lysosome (forming a phagolysosome), whereby the engulfed target is degraded (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 one embodiment, engulfment means phagocytosis. In one embodiment, engulfment includes tethering of a target cell or particle by a phagocytic cell of a host cell described herein, but does not include internalization. In one embodiment, engulfment includes tethering of a target cell or particle by a phagocytic cell of a host cell described herein and internalization of a portion of the target cell or particle.

[0052] As used herein, the term “phagocytosis” refers to the engulfment process of cells or large particles (≧100 nm) in which tethering of target cells or particles, engulfment of target cells or particles, and degradation of internalized target cells or particles occur. In certain embodiments, phagocytosis includes the formation of phagosomes containing internalized target cells or particles, and the formation of phagolysosomes by phagosome fusion with lysosomes, where the contents are degraded. In certain embodiments, during phagocytosis, after CER expressed on the phagocytes or host cells described herein binds to an engulfment marker expressed by the target cells or particles, a phagocytosis synapse is formed. A phagocytic cup rich in actin is generated at the phagocytosis synapse. Phagocytic arms extend around the target cells or particles through rearrangement of the cytoskeleton. And finally, the target cells or particles are taken into the phagocytes or host cells by the force generated by motor proteins. 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.

[0053] As used herein, the term "engulfment-promoting marker" refers to a moiety (e.g., protein, lipid, or polysaccharide) displayed on the surface of apoptotic cells, necrotic cells, pyroptotic cells, or infected cells to distinguish them from non-apoptotic cells, non-necrotic cells, non-pyroptotic cells, oncotic cells, or uninfected cells, respectively. The engulfment-promoting marker can be an intracellular moiety that is surface-exposed on apoptotic or necrotic cells, a moiety on apoptotic or necrotic cells that has undergone glycosylation or a change in surface charge, or a serum moiety that binds to apoptotic cells, necrotic cells, pyroptotic cells, or oncotic cells. Examples of engulfment-promoting markers on apoptotic cells include phosphatidylserine (PtdSer), ICAM-3, oxidized low density lipoprotein, calreticulin, annexin I, complement C1q, and thrombospondin. Necrotic cells, oncotic cells, and pyroptotic cells also expose PtdSer engulfment-promoting markers on the cell surface. Engulfment receptors can detect (or bind to) engulfment-promoting markers on target cells (e.g., damaged cells, infected cells, apoptotic cells, necrotic cells, pyroptotic cells, or oncotic cells) directly or indirectly using soluble bridging molecules as intermediates that bind to the engulfment-promoting markers.

[0054] The term "Toll-like receptor" refers to a member of the conserved immune receptor family of pattern recognition receptors (PRRs) that recognize molecules conserved in pathogens but distinguishable from host molecules (e.g., pathogen-associated molecular patterns (PAMPs)) as well as endogenous molecules released from necrotic or dying cells (danger-associated molecular patterns (DAMPs)). Examples of TLR PAMP ligands include bacterial lipoproteins, bacterial peptidoglycan, double-stranded RNA, lipopolysaccharide, bacterial flagella, single-stranded RNA, and CpG DNA. DAMPs include heat shock proteins and protein fragments from the extracellular matrix. TLRs are type I transmembrane proteins characterized by an extracellular domain containing leucine-rich repeats (LRRs), a juxtamembrane domain containing acidic amino acids between the LRRs and the transmembrane domain, and a cytoplasmic signaling domain containing a conserved region called the Toll / IL-1 receptor (TIR) domain. TLRs are expressed on the membranes of leukocytes, including dendritic cells, macrophages, natural killer cells, and adaptive immune cells (T cells and B cells), as well as non-immune cells (epithelial cells, endothelial cells, and fibroblasts). Ligand binding by TLRs initiates a signaling cascade that leads to the activation of transcription factors such as AP-1, NF-κB, and interferon regulatory factor (IRF), resulting in the production of interferons, pro-inflammatory cytokines, and effector cytokines that induce adaptive immune responses. TLRs can be derived from mammals, such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. TLRs are any one of the 10 TLRs (TLR1-TLR10) identified in humans or any one of the 13 TLRs (TLR1-13) identified in mice. TLRs are on the plasma membrane, except for TLR3, TLR7, TLR8, and TLR9, which are endosomal TLRs.

[0055] "TLR signaling domain" means the cytoplasmic domain of a TLR molecule that includes a TIR domain or a functional fragment thereof. In certain embodiments, the TLR signaling domain can be the signaling domain of any one of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9 or a functional fragment thereof.

[0056] "Engulfment signaling domain" means an intracellular effector domain that activates one or more signaling pathways in a host cell upon binding of a target molecule (e.g., an engulfment promoting marker or an antigen marker) targeted by the extracellular domain of CER expressed by the host cell, resulting in, in a specific embodiment, rearrangement of the host cell's cytoskeleton and engulfment including internalization of target cells, pathogens or particles associated with the marker or antigen. In certain embodiments, the engulfment signaling domain activates one or more signaling pathways, resulting in phagocytosis of target cells, pathogens or particles. In certain embodiments, the engulfment signaling domain includes an engulfment signaling domain that includes a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain or a Traf3 signaling domain. In other specific embodiments, the engulfment signaling domain includes a first engulfment signaling domain and a second engulfment signaling domain. The engulfment signaling domain can include the full-length intracellular component of an engulfment signaling molecule or a functional fragment thereof.

[0057] The "inflammation-induced engulfment signaling domain" refers to an effector domain derived from an endogenous receptor or signaling molecule that stimulates (i) the engulfment of target cells, pathogens, or particles, and (ii) generally induces, enhances, or complements chemotherapy, antibody-based immunotherapy, or cell therapy such as T cell-targeted chemotherapy, including (a) the secretion by host cells of inflammatory cytokines such as TNFα, IL-1, IL-6, IL-12, and IL-23, (b) the secretion by host cells of inflammatory chemokines such as CCL5 (RANTES), CXCL9, and CXCL10, (c) the upregulation of cell surface costimulatory markers such as CD80, CD86, HLA-DR, CD40, HVEM, and 4-1BBL, and (d) the activation of one or more signaling cascades such as NF-κB. In certain embodiments, the stimulation of inflammation-induced engulfment signaling promotes inflammation in the local tissue environment. The inflammation-induced engulfment signaling domain may also be referred to as an "immunogenic" engulfment signaling domain or an "inflammatory" engulfment signaling domain.

[0058] As used herein, an "effector domain" is the intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a cell that expresses the effector domain upon receipt of 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 to induce a signal from the effector domain. For example, in response to the binding of CER to a target molecule, the effector domain may transmit a signal into the interior of the host cell to induce effector functions such as engulfment, phagolysosome maturation, and the secretion of inflammatory cytokines and / or chemokines. The effector domain can directly promote a cellular response if it contains one or more signaling domains or motifs. In other embodiments, the effector domain can indirectly promote a cellular response by associating with one or more other proteins that directly promote the cellular response.

[0059] As used herein, "heterologous" or "non-endogenous" or "exogenous" means a gene, protein, compound, molecule or activity that is not native to the host cell or subject, or a gene, protein, compound, molecule or activity that is native to the subject or host cell but has been modified or mutated such that its structure, activity or both differ between the native molecule and the variant molecule. In certain embodiments, a heterologous, non-endogenous or exogenous molecule (e.g., a receptor, ligand) need not be endogenous to the host cell or subject, but instead a nucleic acid encoding such a molecule can be added to the host cell by conjugation, transformation, transfection, electroporation, etc., where the added nucleic acid molecule can be integrated into the host cell genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The terms "homologous" or "homologue" mean a molecule or activity found in or derived from the host cell, species or strain. For example, a heterologous or exogenous molecule or a gene encoding such a molecule can be homologous to a native host or host cell molecule or a gene encoding such a molecule, respectively, but can have a modified 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.

[0060] "Linker amino acid" or "linker amino acid residue" means one or more (e.g., about 2 to 20) amino acid residues between two adjacent motifs, regions or domains of a polypeptide. Linker amino acids can result from the construction of chimeric protein constructs (e.g., amino acid residues resulting from the use of restriction enzyme sites during the construction of a nucleic acid molecule encoding a fusion protein).

[0061] "Nucleic acid molecule" and "polynucleotide" can be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. The nucleic acid molecule can be double-stranded or single-stranded, and if single-stranded, it can be the coding strand or the non-coding (antisense strand). The coding molecule can have a coding sequence identical to a coding sequence known in the art, or can have different coding sequences that can encode the same polypeptide as a result of the redundancy or degeneracy of the genetic code, or by splicing.

[0062] The term "overexpressed" or "overexpression" of an antigen means an abnormally high level of antigen expression within a cell. Overexpressed antigens or overexpression of antigens are often associated with disease states such as in blood cancers and in cells forming solid tumors in specific tissues or organs of a subject. Solid tumors or blood cancers characterized by overexpression of tumor antigens can be determined by standard assay methods known in the art.

[0063] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and mean a compound containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can contain a protein sequence or peptide sequence. A polypeptide includes a peptide or protein containing two or more amino acids linked to each other by peptide bonds. The terms used herein generally mean in the art both short chains, which are also commonly referred to as peptides, oligopeptides, and oligomers, and long chains, which are generally referred to as proteins (there are many types) in the art. "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.

[0064] As used herein, the term "mature polypeptide" or "mature protein" means a protein or polypeptide that is secreted or localized in the cell membrane or in a specific cell organelle (e.g., endoplasmic reticulum, Golgi apparatus or endosome), and does not contain an N-terminal signal peptide.

[0065] A "signal peptide", also referred to as "signal sequence", "leader sequence", "leader peptide", "localization signal" or "localization sequence", is a short peptide (usually 15 - 30 amino acids in length) present at the N-terminus of a newly synthesized protein destined for the secretory pathway. The signal peptide generally contains 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 signal peptidase. In eukaryotes, the signal peptide facilitates the translocation of the newly synthesized protein into the endoplasmic reticulum where it is cleaved by signal peptidase to generate the mature protein, which then proceeds to its appropriate destination.

[0066] The "percent identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by those sequences (i.e., percent identity (%) = number of identical positions / total number of positions × 100), taking into account also the number of gaps that need to be introduced to optimize the alignment of the two or more sequences and the length of each gap. Comparison of sequences and determination of the 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 (see, e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; see also BLASTN at www.ncbi.nlm.nih.gov / BLAST).

[0067] "Conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid having similar properties. Examples of conservative substitutions are well known in the art (see, e.g., WO97 / 09433, page 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).

[0068] The term "chimeric" means a nucleic acid molecule or protein that includes sequences that are joined or linked together, which are not endogenous and are not normally found joined or linked together in nature. For example, a chimeric nucleic acid molecule can include regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a manner different from that found in nature.

[0069] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of a cell, or introduced synthetic machinery, required to initiate specific transcription of a polynucleotide sequence.

[0070] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for the expression of a gene product operably linked to the promoter / regulatory sequence. In one example, this sequence can be a core promoter sequence, and in other examples, this sequence can include enhancer sequences and other control elements required for the expression of the gene product. The promoter / regulatory sequence can, for example, be one that expresses the gene product in a tissue-specific manner.

[0071] 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 intracellularly under most or all physiological conditions of the cell.

[0072] 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 intracellularly only when an inducer corresponding to the promoter is substantially present intracellularly.

[0073] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes the gene product to be produced intracellularly only when the cell is substantially a cell of the tissue type corresponding to the promoter.

[0074] The terms "subject", "patient" and "individual" are used interchangeably herein and are intended to include organisms (e.g., mammals) capable of eliciting an immune response. Examples of subjects include humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs and their transgenic species.

[0075] The term "T cell" means a cell of the T cell lineage. A "cell of the T cell lineage" means a cell that exhibits at least one phenotypic characteristic of a T cell or its precursor (precursor or progenitor) that distinguishes the cell from other lymphoid cells and erythrocytes or cells of the myeloid lineage. Such phenotypic characteristics can include the expression of one or more proteins specific to T cells (e.g., CD3 + , CD4 + , CD8 + ), or physiological, morphological, functional or immunological characteristics specific to T cells. For example, a cell of the T cell lineage can be a precursor (precursor or progenitor cell) involved in the T cell lineage; CD25 +Immature and inactivated T cells; cells involved in the CD4 or CD8 lineage; CD4 + CD8 + Thymic progenitor cells that are double positive; CD4 + or CD8 + single positive cells; TCRαβ or TCRγκ; or can be mature and functional or activated T cells. The term "T cell" includes naive T cells (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory T cells (CD45RO + , CD62L + , CD8 + ), effector memory T cells (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), mucosa-associated invariant T cells, natural killer T cells, and tissue-resident T cells.

[0076] The term "B cell" means cells of the B cell lineage. "Cells of the T cell lineage" means cells that exhibit at least one phenotypic characteristic of B cells or their precursors (precursor or progenitor) that distinguish the cells from other lymphoid cells and erythrocytes or cells of the myeloid lineage. Such phenotypic characteristics can include the expression of one or more proteins specific to 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 (precursor or progenitor cell) involved in 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 - ).

[0077] The "therapeutically effective amount" or "effective amount" of the chimeric protein of the present invention or a cell expressing the chimeric protein (e.g., CER or a cell expressing CER) means an amount of the protein or cell sufficient to effect an improvement in one or more symptoms of the disease, disorder or unwanted condition being treated. When referring to an individual active ingredient administered alone or a cell expressing a single active ingredient, the therapeutically effective amount means the effect of that ingredient or the cell expressing that ingredient alone. When referring to a combination, the therapeutically effective amount means the combined amount of the active ingredients or the combined amount of the accessory active ingredients in combination with the cells expressing the active ingredients that produce a therapeutic effect, whether administered sequentially or simultaneously.

[0078] "To treat" or "treatment" or "to ameliorate" means the medical management of a subject's disease, disorder or unwanted condition. Generally, an appropriate dosage or treatment regimen comprising a host cell expressing CER of the present invention is administered in an amount sufficient to provide a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefits include improved clinical outcomes; alleviation or reduction of symptoms associated with a disease, disorder or unwanted condition; reduction in the occurrence of symptoms; improvement in quality of life; longer disease-free state; reduction in the degree of a disease, disorder or unwanted condition; stabilization of a disease state; delay in the progression of a disease; remission; survival; extension of life span; or any combination thereof.

[0079] As used herein, the phrases "under transcriptional control" or "operably linked" mean that a promoter is in the correct position and orientation with respect to a polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0080] "Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid. A vector can be, for example, a plasmid, cosmid, virus, or phage. This term should be interpreted to include non-plasmid and non-viral compounds that facilitate the movement of nucleic acids into cells. An "expression vector" is a vector that can direct the expression of a protein encoded by one or more genes carried by the vector when it is in an appropriate environment.

[0081] 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, gamma-retroviral vectors, and lentiviral vectors. A "retrovirus" is a virus having an RNA genome. "Gamma-retrovirus" refers to the Retroviridae family. Examples of gamma-retroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "Lentivirus" refers to the genus of retroviruses that can infect both 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).

[0082] In other aspects, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, closed-ended 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 within the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that associates with both the liposome and the nucleic acid, included in or forming a complex with micelles, or otherwise bound to the lipid.

[0083] Additional definitions are provided throughout this specification.

[0084] Chimeric engager receptor (CER) Chimeric Engulfment Receptor (CER) is described herein. In certain embodiments, CER is a chimeric single-chain protein comprising an extracellular domain and an Engulfment signaling domain linked by a transmembrane domain, the Engulfment signaling domain comprising a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain or a Traf3 signaling domain. The extracellular domain comprises an extracellular binding domain and optionally an extracellular spacer domain. When expressed in a host cell, CER confers an Engulfment phenotype on a modified host cell (the host cell is "switched" to an Engulfment phenotype) that is specific for a selected Engulfment-promoting marker or antigenic marker present on or expressed by a target cell, pathogen, particle or other substance. In certain embodiments of CER, the chimeric protein comprises, in order from amino terminus to carboxy terminus, an extracellular domain having a binding domain specific for a target molecule and optionally an extracellular spacer domain; a transmembrane domain; and an Engulfment signaling domain comprising a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain or a Traf3 signaling domain. In further embodiments, the Engulfment signaling domain comprises a first Engulfment signaling domain and a second Engulfment signaling domain (see, for example, FIGS. 1A and 1B).

[0085] The components of CER described herein can be selected and arranged to provide a desired Engulfment phenotype. For example, in certain embodiments, the extracellular domain can comprise a binding domain specific for (i) an Engulfment-promoting marker associated with apoptotic cells, dead cells, dying cells, damaged cells or necrotic cells; or (ii) an antigen marker presented by a foreign substance (e.g., a pathogen), an infected T cell or an abnormal T cell associated with an infection, disease, disorder or other undesirable condition.

[0086] The engagement signaling domain may include one or more effector domains (also referred to as "signaling" domains) that drive engagement of the target cell. Signaling by the engagement signaling domain is caused by binding of the extracellular domain to a targeted engagement promotion or antigenic marker. In certain embodiments, the engagement signaling domain includes an engagement signaling domain that includes a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain. In certain embodiments, the TLR signaling domain includes a TLR1 signaling domain, a TLR2 signaling domain, a TLR3 signaling domain, a TLR4 signaling domain, a TLR5 signaling domain, a TLR6 signaling domain, a TLR7 signaling domain, a TLR8 signaling domain, or a TLR9 signaling domain. In other embodiments, the engagement signaling domain includes a first engagement signaling domain and a second engagement signaling domain that includes a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain. In certain embodiments, the second engagement signaling domain includes a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TRAF6, TRAF2, TRAF3, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFFR, NFAM1, Dap12, MERTK, or CD79b signaling domain. The CERs described herein can be designed for application in various therapeutic situations (e.g., elimination of apoptotic cells, dead cells, dying cells, damaged cells, infected cells, or necrotic cells, elimination of pathogens causing infections, and elimination of abnormal cells associated with a disease, disorder, or undesirable condition) and provide an engagement signaling (e.g., an inflammation-inducing engagement signaling) that complements the desired therapeutic outcome.

[0087] Figures 2A and 2B provide a functional comparison of lymphocytes modified in accordance with aspects of the CER of the present invention with native lymphocytes. Figure 2A shows endogenous lymphocytes, and as shown in the figure, native lymphocytes do not exhibit an engulfment phenotype. However, as shown in Figure 2B, lymphocytes modified to express the CER described herein exhibit an engulfment phenotype specific for targeted cancer cells, resulting in engulfment (e.g., phagocytosis) and elimination of the targeted cancer cells. In certain embodiments, the engulfment signaling domain included in the CER described herein can drive inflammatory-induced engulfment signaling.

[0088] The components of the fusion proteins of the present invention are further described herein.

[0089] I. Extracellular Domain As described herein, the CER includes an extracellular domain specific for a target molecule. In certain embodiments, the extracellular domain includes an extracellular binding domain that specifically binds to a targeting engulfment promoting marker or antigen. Binding of the target molecule by the binding domain can prevent the interaction between the target molecule (e.g., receptor or ligand) and another molecule, and for example, interfere with, reduce, or eliminate a specific function (e.g., signaling) of the target molecule. In one embodiment, binding of the target molecule can induce a specific biological pathway or can be identified for eliminating the target molecule or the cell expressing the target molecule.

[0090] 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 camelid (from camel, llama, or alpaca; 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. Nat’l. Acad. Sci. (USA) 105:2040, 2008 and Alder et al. Nat. Immunol. 9:319, 2008) and other species-derived antibody variable regions. These antibodies can form antigen-binding regions using only the heavy chain variable region. That is, these functional antibodies are homodimers of only the heavy chain (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).

[0091] In certain embodiments, the extracellular domain binds to an engulfment promoting marker. In such particular 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 the engulfment promoting marker is derived from an endogenous engulfment receptor or a soluble bridging molecule for an engulfment receptor (e.g., GAS6, protein S, MFG-E8). In certain embodiments, the entire extracellular portion (in the case of a transmembrane molecule), the entire bridging molecule, or a cleavage portion of the engulfment receptor or bridging molecule is used, provided that the cleavage 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 for 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).

[0092] In certain embodiments, the extracellular domain comprises the T cell immunoglobulin and mucin domain 1 (Tim1), the T cell immunoglobulin and mucin domain 4 (Tim4), the T cell immunoglobulin and mucin domain 3 (Tim3), stabilin-2, RAGE, or the extracellular domain of an Fc receptor (FcR). In certain embodiments, the extracellular domain of the FcR may comprise a binding domain derived from FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1 or FcαR1. In further embodiments, the extracellular domain comprises Tim1, Tim4, Tim3, stabilin-2, receptor for advanced glycation end products (RAGE), brain-specific angiogenesis inhibitor 1 (BAI1), milk fat globule epidermal growth factor 8 protein (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, beta-2-glycoprotein I, alpha5beta3 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, macrosialin), and a PtdSer-binding domain derived from PSOX.

[0093] In certain embodiments, the extracellular domain comprises the FcγRI binding domain comprising the amino acid sequence of SEQ ID NO: 1 or amino acids 16-292 of SEQ ID NO: 1, the TIM1 binding domain comprising the amino acid sequence of SEQ ID NO: 2 or amino acids 21-290 of SEQ ID NO: 2, the TIM4 binding domain comprising the amino acid sequence of SEQ ID NO: 3 or amino acids 25-314 of SEQ ID NO: 3, the SEQ ID The TIM3 binding domain comprising the amino acid sequence of 4 or amino acids 22-202 of SEQ ID NO: 4, the FA58C2 binding domain comprising the amino acid sequence of SEQ ID NO: 5, the GAS6 binding domain comprising the amino acid sequence of SEQ ID NO: 6 or amino acids 31-94 of SEQ ID NO: 6, the BAI1 binding domain comprising the amino acid sequence of SEQ ID NO: 8, or the protein S binding domain comprising the amino acid sequence of SEQ ID NO: 7 or amino acids 25-87 of SEQ ID NO: 7, and contains 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, or is the said sequence. In any other embodiment, the extracellular domain is encoded by a polynucleotide sequence that contains 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, or is the said sequence, and is the polynucleotide sequence encoding the FcγRI binding domain of SEQ ID NO: 9, the TIM1 binding domain of SEQ ID NO: 10, the TIM4 binding domain of SEQ ID NO: 11, the TIM3 binding domain of SEQ ID NO: 12, the FA58C2 binding domain of SEQ ID NO: 13, the GAS6 binding domain of SEQ ID NO: 14, the BAI1 binding domain of SEQ ID NO: 120, or the protein S binding domain of SEQ ID NO: 15.

[0094] In other embodiments, the extracellular domain is derived from at least one of the following: CD14 that binds to ICAM3; the extracellular domain of a scavenger receptor that binds to oxidized LDL; a lectin that binds to an altered sugar; CD36 that binds to thrombospondin; or LRP1 / CD91 or a lectin moiety that binds to calreticulin.

[0095] In yet other embodiments, the extracellular domain comprises an antibody or an 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 antibody, a human antibody, or a humanized antibody. In further embodiments, the V H and V L regions are from a human or humanized antibody. In certain embodiments, the extracellular domain is an antibody or an antigen-binding portion thereof that is specific for an engulfment-promoting marker. Antibodies specific for 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 these 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 α, 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 β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2, and GD2, and exemplary V H and V LThe field includes, respectively, 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 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, -NY-ESO-1, -TAG-72, -CEA, -ephrin A2, -ephrin B2, -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 segments of GD2-specific monoclonal antibodies.

[0096] In a further aspect, the extracellular domain comprises a Fab specific for a target of interest. In such aspects, targets of interest include 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, NY-ESO-1, TAG-72, CEA, Ephrin A2, Ephrin B2, 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 each comprise a portion of an 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 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, -NY-ESO-1, -TAG-72, -CEA, -Ephrin A2, -Ephrin B2, -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 specific monoclonal antibody.

[0097] Target molecules that specifically bind to the extracellular domain of the CER of the present invention can be found on or associated with the cell of interest (“target cell”). Exemplary target cells include cancer cells, cells associated with autoimmune diseases or disorders or inflammatory diseases or disorders, 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 intended as target cells.

[0098] In certain aspects, the extracellular domain optionally includes an extracellular non-signaling spacer or linker domain. When included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable appropriate cell / cell contact, binding, activation, and proliferation. 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 binding to the target molecule based on the selected target molecule, selected binding epitope, binding domain size, and affinity (see, e.g., Guest et al., J. Immunother. 28:203-11, 2005; Hudecek et al., Clin. Cancer Res. 19:3153-64, 2013; Hudecek et al., Cancer Immunol. Res. 3:125-35, 2015; PCT Publication WO2014 / 031687, the entire contents of which are incorporated herein by reference). In certain aspects, the extracellular spacer domain includes a TLR juxtamembrane domain (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 juxtamembrane domain). In certain aspects, the extracellular spacer domain includes the TLR4 juxtamembrane domain comprising the amino acid sequence of SEQ ID NO: 17. In certain aspects, 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 aspects, the extracellular spacer domain includes a modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 16).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 a further aspect, the extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from: CH1 domain, CH2 domain, CH3 domain, or combinations thereof (see, e.g., PCT Publication WO2014 / 031687, the entire content of which is incorporated herein by reference). In certain aspects, the Fc domain is modified to prevent in vivo interactions with cells expressing FcγR that can result in off-target activation of the CER-modified cells. In yet a further aspect, the extracellular spacer domain can 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 aspect, the extracellular spacer domain can be derived from MERTK.

[0099] II. 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 directly fused to the binding domain or, if present, the extracellular spacer domain. In certain embodiments, the transmembrane domain is derived from an endogenous membrane protein (e.g., a receptor, a differentiation cluster (CD) molecule, an enzyme, a transporter, a cell adhesion molecule, etc.). The transmembrane domain can bind naturally to either the extracellular domain or the engulfment signaling domain included in the CER (e.g., the CER includes a Tim4 binding domain and a Tim4 transmembrane domain). In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules, or 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.

[0100] In certain embodiments, the transmembrane domain is a TLR transmembrane domain (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain), a Tim1 transmembrane domain, a Tim4 transmembrane domain, an FcR transmembrane domain (e.g., an FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, or FcαR1 transmembrane domain), a CD8 transmembrane domain, a MERTK transmembrane domain, an Axl 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.

[0101] In certain embodiments, the transmembrane domain comprises a TLR1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 31, a TLR2 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 32, a TLR3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 33, a TLR4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 34, a TLR5 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 35, a TLR6 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, a TLR7 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 37, a TLR8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 38, a TLR9 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 39, a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 18, a Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 19, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 20, an FcεRIγ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 21, a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 22, an MERTK transmembrane domain comprising the amino acid sequence of SEQ ID NO: 23, an Axl transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24, a Tyro3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 25, a BAI1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 29, a CD28 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 26, a CD4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 27, an MRC1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 30, or a DAP12 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 28, and comprises 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, or is the sequence.

[0102] In other embodiments, the transmembrane domain is the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, the Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 41, the FcεRIγ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 121, the FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, the CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 43, the MERTK transmembrane domain comprising the amino acid sequence of SEQ ID NO: 44, the Axl transmembrane domain comprising the amino acid sequence of SEQ ID NO: 45, the Tyro3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 46, the CD28 transmembrane domain represented by the amino acid sequence of SEQ ID NO: 110, a polynucleotide sequence encoding the BAI1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 113, the CD4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 47, the MRC1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 118, the DAP12 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 111 or the TLR4 transmembrane domain of SEQ ID NO: 112, and comprises 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, or is the sequence, provided by a polynucleotide sequence.

[0103] It is understood that the direct binding of one domain of the CER described herein to another domain does not exclude the presence of intervening junction amino acids. The junction amino acids may be natural or non-natural (e.g., resulting from the design of chimeric protein constructs).

[0104] III. Engulfment signaling domain The engulfment signaling domain of the CER is an intracellular effector domain and can transmit a functional signal to the cell in response to the binding of the extracellular domain of the CER to a target molecule. The CER of the present invention may include one or more constant engulfment signaling domains as described herein.

[0105] In certain embodiments, the engagement signaling domain is the intracellular signaling domain of an endogenous toll-like receptor (TLR) or the intracellular signaling domain of an endogenous signaling protein involved in TLR signaling. In humans, 10 TLRs have been identified. Examples of endogenous TLRs from which the engagement signaling domain can be derived include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. Examples of endogenous signaling proteins involved in TLR signaling that can be used to obtain the engagement signaling domain include Traf6, Traf2, and Traf3.

[0106] In certain embodiments, the first engagement signaling domain comprises a TLR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 48, a TLR2 signaling domain comprising the amino acid sequence of SEQ ID NO: 49, a TLR3 signaling domain comprising the amino acid sequence of SEQ ID NO: 50, a TLR4 signaling domain comprising the amino acid sequence of SEQ ID NO: 51, a TLR5 signaling domain comprising the amino acid sequence of SEQ ID NO: 52, a TLR6 signaling domain comprising the amino acid sequence of SEQ ID NO: 53, a TLR7 signaling domain comprising the amino acid sequence of SEQ ID NO: 54, a TLR8 signaling domain comprising the amino acid sequence of SEQ ID NO: 55, a TLR9 signaling domain comprising the amino acid sequence of SEQ ID NO: 56, a Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO: 57, a truncated Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO: 58, a Traf2 signaling domain comprising the amino acid sequence of SEQ ID NO: 72, or a Traf3 signaling domain comprising the amino acid sequence of SEQ ID NO: 73, and comprises 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, or is, the sequence.

[0107] An engagement signaling domain can be any portion of an engagement signaling molecule that retains sufficient signaling activity. In certain embodiments, the full length of the engagement signaling molecule or its full length intracellular component is used. In certain embodiments, a truncated portion of the engagement signaling molecule or a truncated portion of the intracellular component of the engagement signaling molecule is used, provided that the truncated portion retains sufficient signaling activity. In a further embodiment, the engagement signaling domain is a variant of the whole or a truncated portion of the engagement signaling molecule, provided that the variant retains sufficient signaling activity (i.e., it is a functional variant).

[0108] In certain embodiments, the engagement signaling domain comprises a first engagement signaling domain that includes a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain; and a second engagement signaling domain. The second engagement signaling domain can include an FcR signaling domain (including an FcγR1 signaling domain, an FcγR2A signaling domain, an FcγR2C signaling domain, an FcγR2B2 signaling domain, an FcγR3A signaling domain, an FcγR2C signaling domain, an FcγR3A signaling domain, an FcεR1 signaling domain, and an FcαR1 signaling domain), a B cell-activating factor receptor (BAFF-R) signaling domain, a DAP12 (also referred to as TYRO protein tyrosine kinase-binding protein (TYROBP)) signaling domain, an NFAT-activating protein (NFAM1) signaling domain having an ITAM motif 1, a MERTK signaling domain, a TLR1 signaling domain, a TLR2 signaling domain, a TLR3 signaling domain, a TLR4 signaling domain, a TLR5 signaling domain, a TLR6 signaling domain, a TLR7 signaling domain, a TLR8 signaling domain, a TLR9 signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain, or a CD79b signaling domain.In certain embodiments, the second engagement signaling domain comprises an FcεRIγ signaling domain comprising the amino acid sequence of SEQ ID NO: 62, an FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 63, an FcγR2A signaling domain comprising the amino acid sequence of SEQ ID NO: 64, an FcγR2C signaling domain comprising the amino acid sequence of SEQ ID NO: 65, an FcγR3A signaling domain comprising the amino acid sequence of SEQ ID NO: 66, a BAFF-R signaling domain comprising the amino acid sequence of SEQ ID NO: 67, a DAP12 signaling domain comprising the amino acid sequence of SEQ ID NO: 68, an NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO: 69, a truncated NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO: 70, a CD79b signaling domain comprising the amino acid sequence of SEQ ID NO: 75, a truncated CD79b signaling domain comprising the amino acid sequence of SEQ ID NO: 71, an MERTK signaling domain comprising the amino acid sequence of SEQ ID NO: 59, a TLR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 48, a TLR2 signaling domain comprising the amino acid sequence of SEQ ID NO: 49, a TLR3 signaling domain comprising the amino acid sequence of SEQ ID NO: 50, a TLR4 signaling domain comprising the amino acid sequence of SEQ ID NO: 51, a TLR5 signaling domain comprising the amino acid sequence of SEQ ID NO: 52, a TLR6 signaling domain comprising the amino acid sequence of SEQ ID NO: 53, a TLR7 signaling domain comprising the amino acid sequence of SEQ ID NO: 54, a TLR8 signaling domain comprising the amino acid sequence of SEQ ID NO: 55, a TLR9 signaling domain comprising the amino acid sequence of SEQ ID NO: 56, a Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO: 57, a truncated Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO: 58, a Traf2 signaling domain comprising the amino acid sequence of SEQ ID NO: 72 or a Traf3 signaling domain comprising the amino acid sequence of SEQ ID NO: 73, and comprises 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, or is the sequence.

[0109] In other embodiments, the second engagement signaling domain is a polynucleotide encoding the FcεRIγ signaling domain of SEQ ID NO: 115, a polynucleotide encoding the FcγR1 signaling domain of SEQ ID NO: 77, a polynucleotide encoding the FcγR2A signaling domain of SEQ ID NO: 78, a polynucleotide encoding the FcγR2C signaling domain of SEQ ID NO: 79, a polynucleotide encoding the FcγR3A signaling domain of SEQ ID NO: 80, a polynucleotide encoding the BAFF-R signaling domain of SEQ ID NO: 117, a polynucleotide encoding the DAP12 signaling domain of SEQ ID NO: 116, a polynucleotide encoding the NFAM1 signaling domain of SEQ ID NO: 119, or a polynucleotide encoding the CD79b signaling domain of SEQ ID NO: 118, and comprises 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, or is the sequence, provided by a polynucleotide sequence.

[0110] In certain embodiments, signaling by the engagement signaling domain results in the expression of at least one of an inflammatory cytokine, an inflammatory chemokine, or a costimulatory cell surface marker. In yet 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.

[0111] In certain embodiments, the presence of the second engagement signaling domain enhances the engagement activity of CER, increases the persistence of host cells modified with CER, increases the proliferation of host cells modified with CER, or a combination thereof. In certain embodiments, the inclusion of the second engagement signaling domain and the first engagement signaling domain enhances the engagement activity of CER, enhances the phagocytic signaling of host cells modified with CER, promotes the degradation of luminal contents by host cells modified with CER, increases the activation of host cells modified with CER, increases the persistence of host cells modified with CER, increases the memory formation of host cells modified with CER, increases the proliferation of host cells modified with CER, increases the antigen presentation activity by host cells modified with CER, or a combination thereof.

[0112] In certain embodiments, the presence of a TLR signaling domain with a Traf2, Traf3, or Traf6 signaling domain in CER provides enhanced functions to CER and / or CER-modified host cells. In one embodiment, the first engagement signaling domain includes a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signaling domain, and the second engagement signaling domain includes a Traf6, Traf2, or Traf3 signaling domain. In another embodiment, the first engagement signaling domain includes a Traf6, Traf2, or Traf3 signaling domain, and the second engagement signaling domain includes a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signaling domain. In certain embodiments, a CER comprising both a TLR signaling domain and a Traf2, Traf3, or Traf6 signaling domain exhibits enhanced activation, persistence, memory formation, antigen presentation, or any combination thereof.

[0113] In embodiments where the CER comprises a first engagement signaling domain and a second engagement signaling domain, it is understood that the positions of the engagement signaling domains can be exchanged. For example, in a CER comprising a first engagement signaling domain and a second engagement signaling domain, the second engagement signaling domain may comprise a TLR signaling domain (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9), a Traf2 signaling domain, or a Traf3 signaling domain, and the first engagement signaling domain may comprise an FcR signaling domain (including the FcγR1 signaling domain, the FcγR2A signaling domain, the FcγR2C signaling domain, the FcγR2B2 signaling domain, the FcγR3A signaling domain, the FcγR2C signaling domain, the FcγR3A signaling domain, the FcεR1 signaling domain, and the FcαR1 signaling domain), a BAFF-R signaling domain, a DAP12 signaling domain, an NFAM1 signaling domain, a MERTK signaling domain, a CD79b signaling domain, a TLR signaling domain, a Traf2 signaling domain, a Traf3 signaling domain, or a Traf6 signaling domain.

[0114] IV. Examples of CER The components of the CER described herein can be selected and arranged in various combinations to provide a desired engulfment phenotype to a host cell. In addition to inducing the engulfment of cells, pathogens or particles that express or are characterized by a molecule targeted by a host cell modified with a CER, the CERs described herein can initiate an inflammatory engulfment response, enhance engulfment activity, promote the degradation of luminal contents, enhance cytolytic activity, enhance cell activation, increase cell proliferation, enhance cell memory, increase cell persistence, enhance antigen presentation, or be designed to increase cell proliferation, depending on the target cell or particle, the disease state and the desired therapeutic outcome.

[0115] In one aspect, the present invention provides a chimeric engulfment receptor (CER) comprising a single-chain chimeric protein, said single-chain chimeric protein comprising an extracellular domain comprising a binding domain that binds to phosphatidylserine (PtdSer); an engulfment signaling domain comprising a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain or a Traf3 signaling domain; and a transmembrane domain located between and linking the extracellular domain and the engulfment signaling domain.

[0116] In certain embodiments, the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

[0117] In certain embodiments, the CER further includes a second engagement signaling domain. The second engagement signaling domain can include an FcR signaling domain (including an FcγR1 signaling domain, an FcγR2A signaling domain, an FcγR2C signaling domain, an FcγR2B2 signaling domain, an FcγR3A signaling domain, an FcγR2C signaling domain, an FcγR3A signaling domain, an FcεR1 signaling domain, and an FcαR1 signaling domain), a BAFF-R signaling domain, a DAP12 signaling domain, an NFAM1 signaling domain, a CD79b signaling domain, a MERTK signaling domain, a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain.

[0118] One embodiment of the CER that includes an extracellular domain including a binding domain that binds to PtdSer includes an extracellular domain including a TIM4 PtdSer binding domain, a transmembrane domain including a TIM4 transmembrane domain, and an engagement signaling domain including a TLR4 signaling domain (also referred to herein as "CER05") (see, e.g., FIG. 6). In certain embodiments, such CER includes the amino acid sequence of SEQ ID NO: 81. In one embodiment, the CER mature polypeptide includes the amino acid sequence of SEQ ID NO: 81 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 81).

[0119] Another embodiment of the CER that includes an extracellular domain including a binding domain that binds to PtdSer includes an extracellular domain including a TIM4 PtdSer binding domain, a transmembrane domain including a TIM4 transmembrane domain, and an engagement signaling domain including a TLR4 signaling domain (also referred to herein as "CER06"). In certain embodiments, such CER includes the amino acid sequence of SEQ ID NO: 82. In one embodiment, the CER mature polypeptide includes the amino acid sequence of SEQ ID NO: 82 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 82).

[0120] Another aspect of CER comprising an extracellular domain comprising a binding domain that binds to PtdSer includes an extracellular domain comprising a TIM4 PtdSer binding domain, an extracellular spacer domain comprising a TLR4 membrane-proximal domain, a transmembrane domain comprising a TLR4 transmembrane domain, and an engagement signaling domain comprising a TLR4 signaling domain (also referred to herein as "CER07"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 83. In one aspect, 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).

[0121] Another aspect of CER comprising an extracellular domain comprising a binding domain that binds to PtdSer includes an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, and an engagement signaling domain comprising a TLR3 signaling domain (also referred to herein as "CER17"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 84. In one aspect, 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).

[0122] Another aspect of CER comprising an extracellular domain comprising a binding domain that binds to PtdSer includes an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a TLR3 transmembrane domain, and an engagement signaling domain comprising a TLR3 signaling domain (also referred to herein as "CER18"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 85. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 85 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 85).

[0123] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is an extracellular domain containing a TIM4 PtdSer binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and an engagement signaling domain containing a TLR5 signaling domain (also referred to herein as "CER19"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 86. In one aspect, 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).

[0124] Yet another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is an extracellular domain containing a TIM4 PtdSer binding domain, a transmembrane domain containing a TLR5 transmembrane domain, and an engagement signaling domain containing a TLR5 signaling domain (also referred to herein as "CER20"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 87. In one aspect, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 87 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 87).

[0125] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is an extracellular domain containing a TIM4 PtdSer binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and an engagement signaling domain containing a TLR8 signaling domain (also referred to herein as "CER21"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 88. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 88 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 88).

[0126] Another aspect of the CER comprising an extracellular domain comprising a binding domain that binds to PtdSer comprises an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a TLR8 transmembrane domain, and an engulfment signaling domain comprising a TLR8 signaling domain (also referred to herein as "CER22"). In certain aspects, such a CER comprises the amino acid sequence of SEQ ID NO: 89. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 89 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 89).

[0127] Another aspect of the CER comprising an extracellular domain comprising a binding domain that binds to PtdSer comprises an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, and an engulfment signaling domain comprising a TLR9 signaling domain (also referred to herein as "CER23"). In certain aspects, such a CER comprises the amino acid sequence of SEQ ID NO: 90. In one aspect, 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).

[0128] Another aspect of the CER comprising an extracellular domain comprising a binding domain that binds to PtdSer comprises an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a TLR9 transmembrane domain, and an engulfment signaling domain comprising a TLR9 signaling domain (also referred to herein as "CER24"). In certain aspects, such a CER comprises the amino acid sequence of SEQ ID NO: 91. In one aspect, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 91 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 91).

[0129] Another aspect of CER comprising an extracellular domain comprising a binding domain that binds to PtdSer comprises an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, and an engulfment signaling domain comprising a TLR1 signaling domain (also referred to herein as "CER26"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 92. In one aspect, the CER comprises the amino acid sequence of SEQ ID NO: 92 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 92).

[0130] Another aspect of CER comprising an extracellular domain comprising a binding domain that binds to PtdSer comprises an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, and an engulfment signaling domain comprising a TLR2 signaling domain (also referred to herein as "CER27"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 93. In one aspect, the CER 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 aspect of CER comprising an extracellular domain comprising a binding domain that binds to PtdSer comprises an extracellular domain comprising a TIM4 PtdSer binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, and an engulfment signaling domain comprising a TLR7 signaling domain (also referred to herein as "CER28"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 94. In one aspect, the CER comprises the amino acid sequence of SEQ ID NO: 94 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 94).

[0132] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is a CER (also referred to herein as "CER30") comprising an extracellular domain containing a TIM4 PtdSer binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and an engulfment signaling domain containing a Traf2 signaling domain. In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 96. In one aspect, the CER 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 aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is a CER (also referred to herein as "CER31") comprising an extracellular domain containing a TIM4 PtdSer binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and an engulfment signaling domain containing a Traf3 signaling domain. In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 97. In one aspect, the CER comprises the amino acid sequence of SEQ ID NO: 97 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 97).

[0134] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is a CER (also referred to herein as "CER42") comprising an extracellular domain containing a CD22-specific scFv binding domain, an extracellular spacer domain containing a mutant IgG4 hinge region, a transmembrane domain containing a TLR4 transmembrane domain, and an engulfment signaling domain containing a TLR4 signaling domain. In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 98. In one aspect, the CER comprises the amino acid sequence of SEQ ID NO: 98 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 98).

[0135] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes an extracellular domain containing a TIM4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and an engagement signaling domain containing a truncated Traf6 signaling domain (also referred to herein as "CER29"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 124. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 124 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 124).

[0136] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a TIM4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and an engagement signaling domain comprising a first engagement signaling domain containing a truncated Traf6 signaling domain and a second engagement signaling domain containing an NFAM1 signaling domain (also referred to herein as "CER112"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 128. In one aspect, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 128 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 128).

[0137] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a TIM4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and an engagement signaling domain comprising a first engagement signaling domain containing a truncated Traf6 signaling domain and a second engagement signaling domain containing a DAP12 signaling domain (also referred to herein as "CER110"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 125. In one aspect, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 125 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 125).

[0138] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is an engagement signaling domain comprising an extracellular domain containing a Tim4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, and a first engagement signaling domain containing a truncated Traf6 signaling domain and a second engagement signaling domain containing a BAFFR signaling domain (also referred to herein as "CER113"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 127 or 140. In one aspect, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 127 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 127).

[0139] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer is an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, a first engagement signaling domain containing a truncated Traf6 signaling domain and a second engagement signaling domain containing a CD79b signaling domain (CD79b 185-213) (also referred to herein as "CER111B"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 126. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 126 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 126).

[0140] Another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engulfment signaling domain containing the TLR8 signaling domain, and an engulfment signaling domain containing a second engulfment signaling domain containing the NFAM1 signaling domain (also referred to herein as "CER102"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 130. In one aspect, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 130 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 130).

[0141] Another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engulfment signaling domain containing the TLR8 signaling domain, and an engulfment signaling domain containing a second engulfment signaling domain containing the CD79b (185-229) signaling domain (also referred to herein as "CER103A"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 131. In one aspect, 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).

[0142] Another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR8 signaling domain and a second engagement signaling domain comprising a CD79b(185 - 213) signaling domain (also referred to herein as "CER103B"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 132. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 132 excluding the signal peptide sequence (amino acids 1 - 22 of SEQ ID NO: 132).

[0143] Another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR8 signaling domain and a second engagement signaling domain comprising a DAP12 signaling domain (also referred to herein as "CER104"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 133. In one aspect, 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).

[0144] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR8 signaling domain and a second engagement signaling domain comprising a BAFF-R signaling domain (also referred to herein as "CER105"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 134. In one aspect, 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).

[0145] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a NFAM1 signaling domain and a second engagement signaling domain comprising a TLR8 signaling domain (also referred to herein as "CER106"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 135. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 135 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 135).

[0146] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engagement signaling domain containing the CD79b(185 - 213) signaling domain and a second engagement signaling domain containing the TLR8 signaling domain, and an engagement signaling domain (also referred to herein as "CER107"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 136. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 136 excluding the signal peptide sequence (amino acids 1 - 22 of SEQ ID NO: 136).

[0147] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engagement signaling domain containing the DAP12 signaling domain and a second engagement signaling domain containing the TLR8 signaling domain, and an engagement signaling domain (also referred to herein as "CER108"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 137. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 137 excluding the signal peptide sequence (amino acids 1 - 22 of SEQ ID NO: 137).

[0148] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a BAFF-R signaling domain, and a second engagement signaling domain comprising a TLR8 signaling domain (also referred to herein as "CER109"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 138. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 138 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 138).

[0149] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TRAF6 signaling domain, and a second engagement signaling domain comprising a CD79b(185-229) signaling domain (also referred to herein as "CER111A"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 139. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 139 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 139).

[0150] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engulfment signaling domain containing the TRAF6 signaling domain and a second engulfment signaling domain containing the MERTK signaling domain, and an engulfment signaling domain (also referred to herein as "CER114"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 141. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 141 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 141).

[0151] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engulfment signaling domain containing the MERTK signaling domain and a second engulfment signaling domain containing the TRAF6 signaling domain, and an engulfment signaling domain (also referred to herein as "CER115"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 142. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 142 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 142).

[0152] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TRAF6 signaling domain and a second engagement signaling domain comprising a TLR8 signaling domain (also referred to herein as "CER116"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 143. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 143 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 143).

[0153] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR8 signaling domain and a second engagement signaling domain comprising a TRAF6 signaling domain (also referred to herein as "CER117"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 144. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 144 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 144).

[0154] Another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engagement signaling domain containing the TLR1 signaling domain and a second engagement signaling domain containing the NFAM1 signaling domain (also referred to herein as "CER118"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 145. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 145 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 145).

[0155] Yet another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engagement signaling domain containing the TLR1 signaling domain and a second engagement signaling domain containing the CD79b (185-229) signaling domain (also referred to herein as "CER119A"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 173. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 173 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 173).

[0156] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR1 signaling domain, and a second engagement signaling domain comprising a CD79b(185-213) signaling domain (also referred to herein as "CER119B"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 146. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 146 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 146).

[0157] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR1 signaling domain, and a second engagement signaling domain comprising a DAP12 signaling domain (also referred to herein as "CER120"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 147. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 147 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 147).

[0158] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a Tim4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, a first engagement signaling domain containing a TLR1 signaling domain and a second engagement signaling domain containing a TRAF6 signaling domain (also referred to herein as "CER121"). In certain embodiments, such CER comprises the amino acid sequence of SEQ ID NO: 148. In one embodiment, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 148 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 148).

[0159] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a Tim4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, a first engagement signaling domain containing a TLR2 signaling domain and a second engagement signaling domain containing a DAP12 signaling domain (also referred to herein as "CER122"). In certain embodiments, such CER comprises the amino acid sequence of SEQ ID NO: 149. In one embodiment, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 149 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 149).

[0160] Yet another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR2 signaling domain, and a second engagement signaling domain comprising a TRAF6 signaling domain (also referred to herein as "CER123"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 150. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 150 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 150).

[0161] Another aspect of the CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR2 signaling domain, and a second engagement signaling domain comprising an NFAM1 signaling domain (also referred to herein as "CER124"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 151. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 151 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 151).

[0162] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR2 signaling domain, and a second engagement signaling domain comprising a CD79b (185-229) signaling domain (also referred to herein as "CER125A"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 152. In one aspect, 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).

[0163] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises an engagement signaling domain comprising a Tim4 binding domain, a transmembrane domain comprising a Tim4 transmembrane domain, a first engagement signaling domain comprising a TLR2 signaling domain, and a second engagement signaling domain comprising a CD79b (185-213) signaling domain (also referred to herein as "CER125B"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 153. In one aspect, 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).

[0164] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engagement signaling domain containing the TLR2 signaling domain and a second engagement signaling domain containing the TRAF2 signaling domain (also referred to herein as "CER126"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 174. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 174 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 174).

[0165] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer comprises a Tim4 binding domain, a transmembrane domain containing the Tim4 transmembrane domain, a first engagement signaling domain containing the TRAF2 signaling domain and a second engagement signaling domain containing the TLR2 signaling domain (also referred to herein as "CER127"). In certain aspects, such CER comprises the amino acid sequence of SEQ ID NO: 175. In one aspect, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 175 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 175).

[0166] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a Tim4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, a first engagement signaling domain containing a TRAF2 signaling domain and a second engagement signaling domain containing a TLR8 signaling domain, and an engagement signaling domain (also referred to herein as "CER128"). In certain embodiments, such CER comprises the amino acid sequence of SEQ ID NO: 176. In one embodiment, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 176 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 176).

[0167] Another aspect of CER comprising an extracellular domain containing a binding domain that binds to PtdSer includes a Tim4 binding domain, a transmembrane domain containing a Tim4 transmembrane domain, a first engagement signaling domain containing a TLR8 signaling domain and a second engagement signaling domain containing a TRAF2 signaling domain, and an engagement signaling domain (also referred to herein as "CER129"). In certain embodiments, such CER comprises the amino acid sequence of SEQ ID NO: 177. In one embodiment, the CER mature polypeptide comprises the amino acid sequence of SEQ ID NO: 177 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 177).

[0168] 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 containing a binding domain that binds to an engagement promoting marker or a target antigen; an engagement signaling domain containing a TLR signaling domain, a Traf2 signaling domain or a Traf3 signaling domain; and a transmembrane domain located between the extracellular domain and the engagement signaling domain. Such CER can provide an inflammatory or immunogenic engagement phenotype when bound to a target molecule (e.g., an engagement promoting marker or a target antigen).

[0169] In certain embodiments of the CER comprising an engagement signaling domain comprising a TLR signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain, the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

[0170] 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 engagement promoting marker or a target antigen; an engagement signaling domain comprising a first engagement signaling domain and a second engagement signaling domain comprising a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain; and a transmembrane domain located between and linking the extracellular domain and the engagement signaling domain. In certain embodiments, the second engagement signaling domain is a pro-inflammatory engagement signaling domain comprising an FcR signaling domain (including an FcγR1 signaling domain, an FcγR2A signaling domain, an FcγR2C signaling domain, an FcγR2B2 signaling domain, an FcγR3A signaling domain, an FcγR2C signaling domain, an FcγR3A signaling domain, an FcεR1 signaling domain, and an FcαR1 signaling domain), a BAFF-R signaling domain, a DAP12 signaling domain, an NFAM1 signaling domain, and a CD79b signaling domain.

[0171] In any embodiment of the CER comprising an engagement signaling domain comprising a first engagement signaling domain and a second engagement signaling domain, the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

[0172] 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 comprising a TLR signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain; and a transmembrane domain located between and linking the extracellular domain and the engulfment signaling domain (wherein the transmembrane domain and the engulfment signaling domain are each derived from a different molecule).

[0173] In a particular embodiment of a CER comprising 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.

[0174] An embodiment of a CER comprising an extracellular domain comprising an scFv that binds to an engulfment promoting marker or a target antigen comprises an extracellular domain comprising a CD19-specific scFv binding domain; an extracellular spacer domain comprising a TLR4 membrane-proximal domain; a transmembrane domain comprising a TLR4 transmembrane domain, and an engulfment signaling domain comprising a TLR4 signaling domain (also referred to as “CER43”). In a particular embodiment, such a CER comprises the amino acid sequence of SEQ ID NO: 122. In one embodiment, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 122 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 122).

[0175] Another embodiment of the CER comprising an extracellular domain that binds to an engagement promoting marker or a target antigen and that comprises an scFv is an extracellular domain comprising a CD19-specific scFv binding domain; an extracellular spacer domain comprising a modified IgG4 hinge region; a transmembrane domain comprising a TLR4 transmembrane domain, and an engagement signaling domain comprising a TLR4 signaling domain (also referred to as "CER44"). In certain embodiments, such CER comprises the amino acid sequence of SEQ ID NO: 123. In one embodiment, the CER mature polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 123 excluding the signal peptide sequence (amino acids 1-22 of SEQ ID NO: 123).

[0176] Another embodiment of the CER comprising an extracellular domain that binds to an engagement promoting marker or a target antigen and that comprises an scFv is an extracellular domain comprising a scFv binding domain specific for mesothelin; an extracellular spacer domain comprising a TLR4 juxtamembrane domain; a transmembrane domain comprising a TLR4 transmembrane domain, and an engagement signaling domain comprising a TLR4 signaling domain (also referred to as "CER51").

[0177] Another embodiment of the CER comprising an extracellular domain that binds to an engagement promoting marker or a target antigen and that comprises an scFv is an extracellular domain comprising a scFv binding domain specific for mesothelin; an extracellular spacer domain comprising a modified IgG4 hinge region; a transmembrane domain comprising a TLR4 transmembrane domain, and an engagement signaling domain comprising a TLR4 signaling domain (also referred to as "CER52").

[0178] Another embodiment of the CER comprising an extracellular domain that binds to an engagement promoting marker or a target antigen and that comprises an scFv is an extracellular domain comprising a scFv binding domain specific for LGR5; an extracellular spacer domain comprising a TLR4 juxtamembrane domain; a transmembrane domain comprising a TLR4 transmembrane domain, and an engagement signaling domain comprising a TLR4 signaling domain (also referred to as "CER53").

[0179] Another aspect of the CER comprising an extracellular domain that binds to an engagement promoting marker or target antigen is an extracellular domain comprising an scFv binding domain specific for LGR5; an extracellular spacer domain comprising a modified IgG4 hinge region; a transmembrane domain comprising a TLR4 transmembrane domain, and an engagement signaling domain comprising a TLR4 signaling domain (also referred to as "CER54").

[0180] In certain aspects, after binding of the CER expressed on the surface of a host cell to its cognate target molecule, lateral clustering of the CER occurs on the host cell surface, locally increasing the CER concentration. Clustering is promoted by the presence of multivalent ligands on the target cell or particle surface.

[0181] In certain aspects, after binding of the CER expressed on the surface of a host cell to its cognate target molecule, dimerization or multimerization of the CER occurs, the intracellular engagement signaling domain also binds, and then it becomes a target for intracellular kinases.

[0182] In certain aspects, the CER 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 aspects, the CER of the present invention can tether and internalize a target molecule or particle (e.g., phagocytose the target). In one aspect, the target cell or particle within the phagosome can be expelled before or during phagosome maturation. Further, internalization may include internalizing the entire cell or particle bound by the extracellular domain of the CER, or may include internalizing a fragment or portion of the cell or particle bound by the extracellular domain of the CER.

[0183] In certain embodiments, the CER of the present invention tethers target molecules or particles without internalization. Host cells expressing CER can engulf or tether a number of target cells or particles. Without being bound by theory, tethering of target cells or particles by host cells expressing CER, even without internalization and degradation of the target cells or particles, can lead to degradation of the target cells or particles or promote an inflammatory environment, which is desirable in certain therapeutic situations (e.g., cancer).

[0184] Aspects of the CER according to this specification are shown in FIGS. 6, 15, 18, the Sequence Listing, Table 1, and the Examples.

[0185] [Table 1] [Table 2]

[0186] Nucleic Acids, Vectors, and Host Cells In certain aspects, the present invention provides a nucleic acid molecule encoding any one or more of the CERs described herein. The nucleic acid sequence encoding the desired CER can be obtained or produced using standard techniques, using recombinant methods known in the art, such as screening a library from cells expressing the desired sequence or a portion thereof, deriving it from a vector known to contain the sequence, or isolating the sequence or a portion thereof directly from cells or tissues containing the sequence.

[0187] The polynucleotide encoding the CER composition described herein can be derived from any animal such as human, primate, bovine, equine, ovine, canine, feline, murine, rat, rabbit, guinea pig, or porcine. In certain embodiments, the polynucleotide encoding CER is derived from the same animal species as the host cell into which the polynucleotide is inserted.

[0188] The polynucleotide encoding the CER composition described herein can also include a sequence encoding a signal peptide (also referred to as a leader peptide or signal sequence) at the amino terminus of CER for targeting the precursor protein to the secretory pathway. The signal peptide may, if desired, be cleaved from the N-terminus of the extracellular domain during cell processing and during localization of CER to the cell membrane. The polypeptide with the signal peptide sequence cleaved or removed may also be referred to as the mature polypeptide. Examples of signal peptides that can be used in the CER of the present invention include signal peptides derived from endogenous secreted proteins, including, for example, GM-CSF (amino acid sequence of SEQ ID NO: 99), Tim4 (amino acid sequence of SEQ ID NO: 100 or amino acids 1-24 of SEQ ID NO: 3). In certain embodiments, the polynucleotide or polypeptide sequence of CER of the present invention includes a sequence for the mature polypeptide. For the sequences described herein that include 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 that can transport the encoded protein to the cell outer membrane.

[0189] In certain embodiments, the nucleic acid molecule encoding CER of the present invention is codon-optimized for efficient expression in the target host cell of expression.

[0190] The nucleic acid molecule encoding the desired CER can be inserted into a suitable vector (e.g., viral vector, non-viral plasmid vector and non-viral vector such as lipid-based DNA vector, modified mRNA (modRNA), self-amplifying mRNA, CELiD and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), etc.) for introduction into a host cell of interest (e.g., T cell, natural killer cell, B cell, lymphocyte progenitor cell, antigen-presenting cell, Langerhans cell or bone marrow cell). The nucleic acid molecule encoding the CER of the present invention can be cloned into a suitable vector such as an expression vector, a replication vector, a probe preparation 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 ligated 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 a vector such that the resulting amino acid sequence gives rise to a functional CER. The vector encoding CER is referred to herein as a "CER vector".

[0191] In certain embodiments, the vector comprises a nucleic acid molecule encoding one CER. In other embodiments, the vector comprises 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 the vector at different cloning sites and each CER can be expressed under the control of a different promoter. In another embodiment, a single nucleic acid molecule encoding multiple CERs is cloned into a cloning site and expressed from a single promoter, and each CER is separated from the others by an IRES or viral 2A peptide sequence to allow for co-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: 102, 154, 155 or 156), P2A (SEQ ID NO: 101 or 157), E2A (SEQ ID NO: 103), or F2A (SEQ ID NO: 104).

[0192] In certain embodiments, vectors are utilized that allow for long-term integration of the transgene and propagation to daughter cells. Examples include viral vectors such as adenovirus, adeno-associated virus, vaccinia virus, herpes virus, cytomegalovirus, poxvirus, or retroviral vectors such as lentiviral vectors. Vectors derived from lentivirus can be used to achieve long-term gene transfer and offer the added advantage of vectors that include the ability to transduce non-proliferating cells such as hepatocytes and low immunogenicity.

[0193] In certain embodiments, the CER vector can be constructed to optimize spatial and temporal control. For example, the CER vector can include promoter elements for optimizing spatial and temporal control. In some embodiments, the CER vector includes a tissue-specific promoter or enhancer that enables specific induction of CER into 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, the CER vector includes a constitutive promoter. In still other embodiments, the CER vector includes an inducible promoter.

[0194] In a further embodiment, the CER vector can include a homing receptor such as CCR4 or CXCR4 to improve homing and anti-tumor activity in vivo.

[0195] When temporal control is desired, the CER vector can include an element that enables inducible removal of the transduced cells. For example, such a vector can include an inducible suicide gene. The suicide gene can 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 a further embodiment, the CER vector can be designed to express a known cell surface antigen that enables depletion of the 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 enables CD34 selection and anti-CD20 deletion) and rituximab, and EGFR and cetuximab.

[0196] Inducible vector systems, such as the tetracycline (Tet)-On vector system that activates transgene expression using doxycycline (Heinz et al., Hum. Gene Ther. 2011, 22:166-76), can also be used for inducible CER expression. Inducible CER expression can also be achieved by retention using the selective hook (RUSH) system based on streptavidin immobilized on the endoplasmic reticulum membrane through a hook and a streptavidin-binding protein introduced into the CER construct, where addition of biotin to the system results in release of CER from the endoplasmic reticulum (Agaugue et al., 2015, Mol. Ther. 23(Suppl. 1):S88).

[0197] As used herein, the terms "recombinant" or "non-natural" mean an organism, microorganism, cell, nucleic acid molecule or vector that contains at least one genetic modification or has been modified by the introduction of an exogenous nucleic acid molecule, where such modification or alteration is introduced by genetic manipulation. 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 the cell. Further modifications include, for example, non-coding regulatory regions whose modification changes the expression of a gene or operon. In certain embodiments, cells such as T cells obtained from a subject can be genetically modified to non-natural or recombinant cells (e.g., non-natural or recombinant T cells) by introducing a nucleic acid encoding CER as described herein, whereby the cells express CER located on the cell surface.

[0198] Vectors encoding the core virus 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, for example, vectors derived from Moloney murine leukemia virus (MLV), and more complex retrovirus-derived vectors such as lentivirus-derived vectors. Vectors derived from HIV-1 belong to this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral vectors and lentiviral vectors and methods of packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgenes are known in the art and are reported, 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.

[0199] In certain embodiments, the viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a target-specific CER. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include 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 includes a marker for transduction that includes 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: 105) (huEGFRt; see Wang et al., Blood 118:1255, 2011). If the viral vector genome includes multiple nucleic acid sequences to be expressed in the host cell as separate transcripts, the viral vector can also include additional sequences between two (or more) transcripts that allow for dicistronic or polycistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides (e.g., T2A, P2A, E2A, F2A), or any combination thereof.

[0200] DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; other viral vectors, including amplicon vectors, herpes simplex virus (HSV)-derived vectors including replication-defective HSV and attenuated HSV, can also be used for polynucleotide delivery (Krisky et al., Gene Ther. 5: 1517, 1998).

[0201] Other viral vectors that have recently been developed for gene therapy use can also be used in conjunction 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 certain embodiments, the viral or plasmid vector further includes a gene marker for transduction (e.g., green fluorescent protein, huEGFRt (encoding the amino acid sequence of SEQ ID NO: 105)).

[0202] In certain embodiments, the gene editing method is used to modify the host cell genome to include a polynucleotide encoding the CER of the present invention. Gene editing or genome editing is a method of genetic manipulation that uses a genetically engineered endonuclease to insert, substitute, or remove DNA from the genome of a host cell. The nuclease causes a double-strand break at a target locus within the genome. The host cell's endogenous DNA repair pathways then repair the induced break 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 (e.g., CRISPR-Cas9), meganucleases, or combinations thereof. Methods of 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 Publication Nos. 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 these are incorporated herein by reference in their entirety).

[0203] In certain embodiments, B cells, lymphoid progenitor cells including common lymphoid progenitor cells, antigen-presenting cells including dendritic cells, Langerhans cells, myeloid progenitor cells, or mature myeloid cells are modified to include a non-endogenous nucleic acid molecule encoding the CER of the present invention.

[0204] In certain embodiments, B cells are genetically modified to generally express one or more CERs. B cells have certain properties that can be advantageous as host cells, including the ability to traffic to sites of inflammation (e.g., lymph nodes, tumors), internalize and present antigens, co-stimulate T cells, be highly proliferative, and be able to self-replicate (continue to survive). In certain embodiments, B cells modified with CER can digest engulfed target cells or engulfed target particles into smaller peptides and present them to T cells via MHC molecules. Antigen presentation by B cells modified with CER can contribute to antigen spreading of the immune response to non-target antigens. B cells include B cell lineages (e.g., pre-pro B cells, pro B cells, and pre-B cells); progenitor or precursor cells involved in immature and inactivated B cells or 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 on 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 body fluids. In certain embodiments, the B cells are isolated from a tumor site (tumor-infiltrating B cells). B cell compositions can be concentrated or purified.

[0205] In certain embodiments, the expression of endogenous genes in host B cells 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 of these. The expression of BCR genes, immune checkpoint molecule genes, or both can be inhibited, knocked down, or knocked out at the gene level, transcriptional level, or translational level, or a combination thereof. Methods for inhibiting, knocking down, or knocking out BCR genes, immune checkpoint molecule genes, or both can be achieved, for example, by RNA interfering 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 one embodiment, an endogenous gene (e.g., a BCR gene or an immune checkpoint molecule gene) is knocked out by inserting a polynucleotide encoding the CER of the present invention into the locus of the endogenous B cell gene using an artificial endonuclease or the like.

[0206] In certain embodiments, cells that can express the CER of the present invention on the cell surface are CD4 + , CD8 + , naive (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-) central memory (CD45RO + , CD62L +, CD8 + ) T cells including effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), virus-specific, mucosa-associated invariant, γδ (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 a mammal, the T cells can be obtained from a number of sources including blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. In certain embodiments, the T cells are isolated from a tumor site (tumor-infiltrating T cells). The T cell composition can be concentrated 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 TCRα and β chains are used. Such T cells either naturally lack endogenous expression of the TCRα and β chains or have their expression blocked (e.g., T cells from transgenic mice that do not express the TCRα and β chains or cells engineered to inhibit the expression of the TCRα and β chains), or may be modified to knockout the gene for the TCRα chain, TCRβ chain, or both. In certain embodiments, the cells capable of expressing the chimeric protein of the invention on the cell surface are not T cells or cells of the T cell lineage, but progenitor cells, stem cells, or cells modified to express cell surface anti-CD3.

[0207] In certain embodiments, the CER-modified T cells can digest the engulfed target cells or engulfed target particles into smaller peptides and present them to the T cells via MHC molecules. Antigen presentation by CER-modified T cells can contribute to antigen spreading of the immune response against non-target antigens.

[0208] In certain embodiments, the host T cells transfected to express the CER 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.

[0209] In certain embodiments, the 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 of these. The expression of TCR genes, HLA genes, immune checkpoint molecule genes, or any combination thereof enables inhibition, knockdown, or knockout at the gene level, transcriptional level, or translational level, or any combination of these. 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 interfering 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 certain embodiments, an endogenous gene (e.g., a TCR gene, an HLA gene, or an immune checkpoint molecule gene) is knocked out by inserting a polynucleotide encoding the CER of the present invention into the locus of the endogenous T cell gene, for example, via an artificial endonuclease.

[0210] In certain embodiments, a host cell comprising a CER comprising an extracellular domain comprising a binding domain that binds phosphatidylserine (PtdSer) of any of the embodiments described herein is a T cell, natural killer cell, B cell, lymphoid progenitor cell including a common lymphocyte progenitor cell, antigen-presenting cell including a dendritic cell, Langerhans cell, myeloid progenitor cell, or mature myeloid cell.

[0211] In other embodiments, a host cell comprising a CER comprising an extracellular domain that binds to a binding domain (e.g., scFv) that binds to a target antigen of any of the embodiments described herein is a B cell.

[0212] In yet other embodiments, a host cell comprising a CER comprising an extracellular domain that comprises an engagement promoting marker or a binding domain that binds to a target antigen of any of the embodiments described herein is a cell that does not naturally exhibit an engagement phenotype. In certain embodiments, the host cell is a T cell, natural killer cell, B cell, or lymphoid progenitor cell including a common lymphocyte progenitor cell. In certain embodiments, the host cell is a cell that does not naturally exhibit an engagement phenotype with respect to mammalian cells.

[0213] In certain embodiments, the host cell may be genetically modified to express one type of CER. In other embodiments, the host cell may express at least two or more different CERs.

[0214] In certain embodiments, a population of host cells 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 lymphocyte progenitor cells, a population of antigen-presenting cells including dendritic cells and Langerhans cells, a population of myeloid progenitor cells, a population of mature myeloid cells, or any combination thereof. In certain embodiments, a population of host cells modified to express one or more CERs is a population of B cells, a population of T cells, or both.

[0215] In certain embodiments, each host cell within a host cell population expresses the same CER or set of CERs. In other embodiments, the host cell population comprises a mixture of two or more host cell subpopulations, where each subpopulation expresses a different CER or set of CERs.

[0216] In certain embodiments, when preparing host cells, such as B cells or T cells, that express the CERs described herein, one or more growth factor cytokines that promote the proliferation of the host cells, such as B cells or T cells, can be added to the cell culture. The cytokine can be human or non-human. Examples of growth factor cytokines that can be used to promote T cell proliferation include IL-2, IL-15, and the like. Examples of growth factor cytokines that can be used to promote B cell proliferation include CD40L, IL-2, IL-4, IL-15, IL-21, BAFF, and the like.

[0217] In a further embodiment, selective gene transfer is used to localize the CER vector to a specific region or organ. In one embodiment, selective gene transfer is used to localize the CER vector to the liver or lung of a subject.

[0218] Prior to genetic modification of host cells with the 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, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue), and the host cells obtained therefrom are isolated using methods known in the art. Certain 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 proliferation of the desired modified host cells can be carried out according to known techniques or modifications thereof that will be apparent to those skilled in the art.

[0219] In certain embodiments, host cells comprising T cells, natural killer cells, B cells, lymphoid progenitor cells, antigen presenting cells, dendritic cells, Langerhans cells, myeloid progenitor cells, and mature myeloid cells, comprising CER according to any of the embodiments described herein, have a phagocytic index of from about 20 to about 1,500 against target cells. The "phagocytic index" is a measure of the phagocytic activity of the transduced host cells determined by counting the number of target cells taken up per host cell modified with CER during a defined period of incubation of a suspension of target cells and host cells modified with CER in a medium. The phagocytic index can be calculated by multiplying [total number of engulfed target cells / total number of counted cells modified with CER (e.g., phagocytic frequency)] by [average area of target cell staining per CER+Ba / F3 cell × 100 (e.g., hybrid capture)], or by multiplying [total number of engulfed particles / total number of counted host cells modified with CER] by [total number of host cells modified with CER containing engulfed particles / total number of counted CER cells] × 100.In certain embodiments, the cells modified with CER have a phagocytic index of from about 30 to about 1,500; from about 40 to about 1,500; from about 50 to about 1,500; from about 75 to about 1,500; from about 100 to about 1,500; from about 200 to about 1,500; from about 300 to about 1,500; from about 400 to about 1,500; from about 500 to about 1,500; from about 20 to about 1,400; from about 30 to about 1,400; from about 40 to about 1,400; from about 50 to about 1,400; from about 100 to about 1,400; from about 200 to about 1,400; from about 300 to about 1,400; from about 400 to about 1,400; from about 500 to about 1,400; from about 20 to about 1,300; from about 30 to about 1,300; from about 40 to about 1,300; from about 50 to about 1,300; from about 100 to about 1,300; from about 200 to about 1,300; from about 300 to about 1,300; from about 400 to about 1,300; from about 500 to about 1,300; from about 20 to about 1,200; from about 30 to about 1,200; from about 40 to about 1,200; from about 50 to about 1,200; from about 100 to about 1,200; from about 200 to about 1,200; from about 300 to about 1,200; from about 400 to about 1,200; from about 500 to about 1,200; from about 20 to about 1,100; from about 30 to about 1,100; from about 40 to about 1,100; from about 50 to about 1,100; from about 100 to about 1,100; from about 200 to about 1,100; from about 300 to about 1,100; from about 400 to about 1,100; or from about 500 to about 1,100; from about 20 to about 1,000; from about 30 to about 1,000; from about 40 to about 1,000; from about 50 to about 1,000; from about 100 to about 1,000; from about 200 to about 1,000; from about 300 to about 1,000; from about 400 to about 1,000; or from about 500 to about 1,000; from about 20 to about 750; from about 30 to about 750; from about 40 to about 750; from about 50 to about 750; from about 100 to about 750; from about 200 to about 750; from about 300 to about 750; from about 400 to about 750; or from about 500 to about 750; from about 20 to about 500; from about 30 to about 500; from about 40 to about 500; from about 50 to about 500; from about 100 to about 500; from about 200 to about 500; or from about 300 to about 500. In a further embodiment, the incubation time is from about 2 hours to about 4 hours, about 2 hours, about 3 hours, or about 4 hours.In yet a further aspect, the cells modified with CER show a statistically significantly higher phagocytic index than the cells transduced with a truncated EGFR control. The phagocytic index is known in the art and can be calculated using methods including quantification by flow cytometry or fluorescence microscopy, as further described in the Examples.

[0220] In certain aspects, a host cell modified to express CER, such as one of the aspects described herein, exhibits the following compared to a host cell that does not express CER: cytolytic activity against a target cell, i.e., the ability to lyse a target cell that expresses a target antigen on its surface; enhanced activation (e.g., enhanced cytokine production such as IFNγ); enhanced cell proliferation; enhanced cell expansion; enhanced resistance; enhanced memory formation; antigen-presenting activity; induction of antigen-specific phagocytic signaling or enhancement of antigen-specific phagocytic signaling; degradation of engulfed target cells; or any combination thereof. In certain aspects, the CER-modified host cell can induce antigen spreading through its antigen-presenting activity.

[0221] The host cell can be derived from an animal such as a primate, bovine, equine, ovine, canine, feline, murine, rat, rabbit, guinea pig, or porcine. In a preferred aspect, the animal is human. The host cell can be obtained from a healthy subject or a subject having a disease associated with the expression of an antigen.

[0222] Use of CER and cells modified to express CER The present invention provides a method for altering the engulfment phenotype of a host cell. In one aspect, the present invention provides a method for generating a population of cells that exhibit an engulfment phenotype, comprising introducing into a population of host cells that do not naturally exhibit an engulfment phenotype, a nucleic acid molecule encoding at least one CER according to any of the embodiments described herein or a vector comprising at least one CER, and expressing at least one CER in the population of host cells. In certain embodiments, the engulfment phenotype is phagocytosis. In certain embodiments, the population of host cells expressing at least one CER is capable of antigen-specific phagocytic signaling activity. Induction of the antigen-specific phagocytic signaling cascade can involve activation of CDC42, Rac1, or both. In certain embodiments, the population of host cells expressing at least one CER is capable of degrading engulfed target cells.

[0223] In another aspect, the present invention provides a method for modifying the engulfment phenotype of a population of cells, comprising introducing into a population of host cells a nucleic acid molecule encoding at least one CER according to any of the embodiments described herein or a vector comprising at least one CER, and expressing at least one CER in the population of host cells, wherein the at least one CER confers an engulfment phenotype specific for an engulfment-promoting marker or antigen marker (target antigen) that is not naturally targeted by the host cell. In certain embodiments, the engulfment phenotype is phagocytosis.

[0224] In yet another aspect, the present invention provides a method for enhancing the engulfment phenotype of a cell population, which comprises introducing into a host cell population a nucleic acid molecule encoding at least one CER of any of the embodiments described herein or a vector comprising at least one CER, and expressing at least one CER in the host cell population, wherein the at least one CER is specific for an engulfment promoting marker or an antigen marker (target antigen) that is naturally targeted by the host cell, and expression of the at least one CER by the host cell enhances the engulfment by the host cell of cells, pathogens or particles presenting the targeted engulfment promoting marker or antigen marker.

[0225] In a further aspect of a method for generating a population of host cells exhibiting an engulfment phenotype, a method for altering the engulfment phenotype in a population of cells, or a method for enhancing the engulfment phenotype in a population of cells, expression of at least one CER by the population of host cells enhances the proliferative capacity of the cell population, enhances the activation of the cell population (e.g., enhanced cytokine production such as IFNγ), enhances the proliferation of the host cell population, enhances the resistance of the host cell population, enhances the memory formation of the cell population, confers antigen presenting activity to the population of host cells, enhances the cytolytic activity of the population of host cells, or exhibits any combination thereof. In certain embodiments, a population of CER-modified host cells can induce antigen spreading via antigen presenting activity.

[0226] Any of the CERs, nucleic acid molecules encoding CERs, vectors comprising CERs, and host cells expressing CERs of any of the embodiments described herein can also be used in methods for treating a subject suffering from a disease, disorder or undesirable medical condition. Aspects of these methods 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.

[0227] Diseases that can be treated with cells expressing the CER 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., joints 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, 201).

[0228] Subjects that can be treated by the compositions and methods described herein include animals such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. The subject can be male or female and can be of any suitable age including infant, juvenile, young, adult, and elderly subjects.

[0229] A number of cancers, including solid tumors and leukemias, are suitable for the compositions and methods described herein. Exemplary cancer types that can be treated include adenocarcinomas of the breast, prostate, and colon; all types of bronchogenic carcinomas of the lung; bronchogenic carcinomas; melanomas; hepatocellular carcinomas; neuroblastomas; papillomas; adenomas; choristomas; branchiomas; malignant carcinoid syndrome; carcinoid heart disease; and carcinomas (e.g., Walker carcinoma, basal cell carcinoma, squamous cell carcinoma, Brown-Pearce carcinoma, intraductal carcinoma, Ehrlich tumor, Krebs 2, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung carcinoma, embryonal cell carcinoma, papillary carcinoma, Schiller carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma, and transitional cell carcinoma). Further cancer types that can be treated include histiocytosis; malignant histiocytosis; leukemias; Hodgkin disease; immunoproliferative small intestinal disease; non-Hodgkin 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; germ cell tumor; choristoma; mesenchymal tumor; mesonephroma; sarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastic tumor. Additionally, the following cancer types are also considered suitable for treatment: adenoma; cholangioma; cholesteatoma; cylindroma; 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; epithelioma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neurocytoma; paraganglioma; non-chromaffin paraganglioneuroma. Cancer types that can be treated include angiohemangioma; eosinophilic angiocellular lymphoid hyperplasia; sclerosing hemangioma; hemangiomatosis; glomus tumor; hemangioendothelioma; hemangioma; pericytic hemangioma; angiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma; cystosarcoma phyllodes; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma; lymphangiosarcoma; sarcoma; myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasm; neurofibromatosis; and cervical dysplasia.

[0230] Examples of proliferative diseases suitable for CER therapy include B-cell cancers including B-cell lymphoma (e.g., various forms of Hodgkin's disease, non-Hodgkin lymphoma (NHL), or central nervous system lymphoma), leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, B-cell blast transformation of chronic myelogenous leukemia), and myeloma (e.g., multiple myeloma). Further B-cell cancers include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytic myeloma, solitary plasmacytoma of bone, extramedullary 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 undetermined malignancy, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

[0231] Inflammatory and autoimmune diseases include arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, inclusion body myositis, inflammatory muscle diseases, 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 symptoms, eczema, asthma, symptoms associated with 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, lupus encephalitis, juvenile onset diabetes, multiple sclerosis, allergic encephalitis, neuromyelitis optica, rheumatic fever, Sydenham chorea, immune responses related to acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis and Churg-Strauss syndrome, agranulocytosis, vasculitis (including allergic vasculitis / vasculitis, ANCA and rheumatoid vasculitis), aplastic anemia, congenital erythroblastopenia (Diamond Blackfan 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 associated with leukocyte extravasation, central nervous system (CNS) inflammatory diseases, Alzheimer's disease, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Jorgen syndrome, Stevens-Johnson syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), pemphigoid, pemphigus, autoimmune polyendocrine disorders, 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-Schonlein purpura,Autoimmune diseases of the testis and ovary, including autoimmune thrombocytopenia, autoimmune orchitis and ovaritis; primary hypothyroidism; autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyendocrine syndrome (or, polyendocrine endocrine disorder syndrome), type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM), and Sheehan's syndrome; autoimmune endocrine diseases; autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant), nonspecific interstitial pneumonia (NSIP), Guillain-Barré syndrome, large-vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gluten enteropathy), cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Wiskott-Aldrich syndrome, and thrombangiitis obliterans are included. In certain embodiments, in the context of treating an inflammatory disease, it may be preferable to design a CER having a constitutive (non-inflammatory) engagement signaling domain.,

[0232] Infections include those associated with infectious pathogens and include various bacteria (e.g., pathogenic Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Bacillus anthracis, Clostridium botulinum, Clostridium difficile, Clostridium welchii, Helicobacter pylori, Vibrio cholerae, Listeria, Rickettsia, Chlamydia, etc.), mycobacteria, and parasites (including known parasitic members of protozoa). Infectious viruses include eukaryotic viruses such as adenovirus, bunyavirus, herpesvirus, papovavirus, papillomavirus (e.g., HPV), paramyxovirus, picornavirus, rhabdovirus (e.g., rabies), orthomyxovirus (e.g., influenza), poxvirus (e.g., vaccinia), reovirus, retrovirus, lentivirus (e.g., HIV), flavivirus (e.g., HCV, HBV), etc. In certain embodiments, the composition comprising the CER of the present invention is used to treat an infection caused by a pathogen capable of establishing a persistent infection in a subject.

[0233] Neurodegenerative diseases include, but are not limited to, Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, 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 (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia, also known as transmissible spongiform encephalopathies), ophthalmoplegia, motor neuron diseases (including amyotrophic lateral sclerosis (Lou Gehrig's disease)), and nervous system heterodegenerative diseases (including, but not limited to, Canavan disease, Huntington's disease, neuronal steroid lipoprostinosis, Alexander disease, Tourette syndrome, Menkes variant syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatic lenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg disease), dementia (including, but not limited to, Pick's disease and spinocerebellar ataxia), cancer (e.g., CNS cancer and / or brain tumors, including brain metastases resulting from cancer in any part of 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 hallmarks, including abnormal accumulation of proteins such as amyloid β or tau in Alzheimer's disease; α-synuclein in Parkinson's disease (PD), Lewy body dementia, multiple system atrophy, or Alzheimer's disease; huntingtin in Huntington's disease, SOD1 in amyotrophic lateral sclerosis, polyglutamine (polyQ) repeats in Huntington's disease or amyotrophic lateral sclerosis; TDP-43 in amyotrophic lateral sclerosis or FLTD-U; or prion protein (e.g., PrP Sc ) in prion diseases. Thus, in certain embodiments, CER therapies can be designed to target disease-related proteins to reduce or prevent abnormal protein accumulation and thereby delay or prevent the progression of neurodegenerative diseases.

[0234] The CER of the present invention can be administered to a subject in a cell-binding form (e.g., gene therapy of a target cell population (mature T cells (e.g., CD8 + or CD4 + T cells) or other cells of the T cell lineage). Thus, for example, the CER of the present invention can be administered to a subject that is expressed on the surface of T cells, natural killer cells, natural killer T cells, B cells, lymphocyte 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 cells modified with CER (i.e., recombinant cells expressing one or more CERs). In such embodiments, the cells modified with CER are heterologous cells, syngeneic cells, allogeneic cells or autologous cells of the 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.

[0235] A pharmaceutical composition comprising cells modified with CER can be administered in a manner suitable for the disease or condition to be treated (or prevented) as determined by one of ordinary skill in the pharmaceutical arts. The suitable dosage, suitable period and frequency of administration of the composition can be determined by factors such as the patient's condition, physique, weight, body surface area, age, gender, type and severity of the disease, the specific therapeutic agent administered, the specific form of the active ingredient, the time and method of administration, and other drugs administered simultaneously. The present invention provides a pharmaceutical composition comprising cells modified with CER and a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients include water, physiological saline, dextrose, glycerol, etc., and combinations thereof. Other suitable injection media can be any isotonic media formulation containing physiological saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% aqueous dextrose solution, or lactated Ringer's solution.

[0236] The therapeutically effective amount of cells in the pharmaceutical composition is at least one cell (e.g., a B cell modified with one CER), or generally more than 10 2 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 cells or more. In certain embodiments, the cells are from about 10 6 to about 10 10 cells / m 2 , preferably from about 10 7 to about 10 9 cells / m 2 . The number of cells can 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 can 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 use as provided herein, the cells are generally at a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Thus, the desired cell density generally exceeds 10 4 cells / ml, generally exceeds 10 7 cells / ml, generally exceeds 10 8 cells / ml or more. The cells can be administered as a single injection or as multiple injections over a period of time. Repeated injections of CER-modified cells can be spaced days, weeks, months, or even years apart if disease recurrence or disease activity is present. A clinically significant number of immune cells, cumulatively 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 1011 It can be distributed into multiple injections equal to or exceeding the cells. A suitable dose for administration of host cells containing the 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 the composition of CER-modified B cells and the composition of CER-modified T cells are administered, and the administration can be simultaneous or sequential.

[0237] In one embodiment, the compositions described herein are administered intravenously, intraperitoneally, intratumorally, intramedullary, intranodally and / or into the cerebrospinal fluid. In one embodiment, the chimeric engager receptor-modified composition is delivered to the tumor site.

[0238] In certain embodiments, the CER-modified cells are administered to a subject in combination with, or in conjunction with, one or more additional therapies. In such embodiments, the one or more additional therapies can be radiotherapy, genetically engineered cellular immunotherapy (e.g., T cells, dendritic cells, natural killer cells, macrophages, chimeric antigen receptor (CAR) therapy), antibody therapy, immune checkpoint molecule inhibitor therapy, or drug therapy such as a chemotherapeutic agent, therapeutic peptide, hormonal therapy, antibiotic, antiviral agent, antifungal agent, anti-inflammatory agent, UV therapy, electrical pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy or small molecule therapeutic agent. In such embodiments, the CER-modified cells can eliminate apoptotic cells, dead cells, dying cells, damaged cells, infected cells or necrotic cells that display an apoptosis-promoting marker induced in the setting of one or more additional therapies. In certain embodiments where the CER-modified cells are administered in combination with one or more additional therapies, the one or more additional therapies can be administered at a sub-therapeutic dose for an additive or synergistic effect in combination with the CER therapy. The combination therapy can include administration of CER before (e.g., 1 day to 30 days or more before the additional therapy), simultaneously with (on the same day as) the additional therapy, or after (e.g., 1 day 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 a further embodiment, 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 yet a further embodiment, the CER-modified cells are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of one or more additional therapies. If the one or more additional treatments include multiple administrations, the cells modified with CER can be administered after the first administration of one or more additional therapies, after the final administration of one or more additional therapies, or during multiple administrations of one or more additional therapies.

[0239] An example of a triple combination therapy (radiation + CER + CAR and / or TCR) regimen is shown in FIG. 5. After radiation therapy, the tumor antigen-specific, CER-modified host cells described herein (e.g., including a binding domain that binds to a tumor antigen) are administered to a subject to promote an anti-tumor immune response and mobilize immune-activated cells to the tumor microenvironment. In certain embodiments, CER migrates to the locally irradiated tumor, making 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, within 3 weeks, within 2 weeks, or within 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, within 3 weeks, within 2 weeks, or within 1 week after administration of the CER therapy. In certain embodiments, the radiation therapy, CAR or TCR immunotherapy, or both are administered at sub-therapeutic levels.

[0240] Examples of radiation therapies that can be used in combination with CER therapy include external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiotherapy, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy, intensity-modulated rotational radiotherapy, particle therapy, proton therapy, and Auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiation therapy, or any combination thereof. In certain embodiments, a dose of radiation therapy lower than a typical dose or a dose below a therapeutic dose is used in combination with CER therapy. A dose of radiation therapy at a low dose or below a therapeutic dose may be sufficient to cause lytic membrane damage to cells, but is not necessarily lytic. Lytic membrane damage is sufficient to expose engulfment-promoting markers (e.g., phosphatidylserine) that may be targeted by CER therapy.

[0241] 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, a peptide, an RNAi agent, or a small molecule. An antibody specific for CTLA-4 can be ipilimumab or tremelimumab. An antibody specific for PD-1 can be pidilizumab, nivolumab, or pembrolizumab. An antibody specific for PD-L1 can be durvalumab, atezolizumab, or avelumab.

[0242] Chemotherapeutic agents include non-specific cytotoxic drugs that inhibit mitosis or cell division, as well as molecular target therapies that block the growth and spread of cancer cells by targeting specific molecules involved in tumor growth, progression, and metastasis (e.g., oncogenes). Exemplary non-specific chemotherapeutic agents include alkylating agents, platinum-based drugs, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (e.g., intercalating agents), and DNA repair inhibitors.

[0243] Examples of chemotherapeutic agents considered for use in combination therapy include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoSite®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (VePesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tiazofurin, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (Elspar®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®),Mitoxantrone (Novantrone®), MyloTag, Paclitaxel (Taxol®), Phoenix (Yttrium90 / MX-DTPA), Pentostatin, PolyFeprosan 20 with Carmustine Implant (Gliadel®), Tamoxifen Citrate (Nolvadex®), Teniposide (Vumon®), 6-Thioguanine, Thiotepa, Tirapazamine (Tirapazon®), Topotecan Hydrochloride for Injection (Hycamtin®), Vinblastine (Velban®), Vincristine (Oncovin®), and Vinorelbine (Navelbine®).

[0244] Examples of alkylating agents include nitrogen mustards (ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustards (aminouracil mustard (registered trademark), chloretanasil (registered trademark), desmethyldopan (registered trademark), desmethyldopan (registered trademark)), hemantaneamine (registered trademark), nordopan (registered trademark), uracil nitrogen mustard (registered trademark), uracilrost (registered trademark), uracilmostaza (Uracilmostaza) (registered trademark), uramustin (registered trademark), uramustine (registered trademark)), chloromethine (mustargen (registered trademark)), cyclophosphamide (cytoxan (registered trademark), neosar (registered trademark), clafen (registered trademark), endoxan (registered trademark), prositox (registered trademark), Revimmune (trademark)), ifosfamide (mitoxana (registered trademark)), melphalan (alkeran (registered trademark)), chlorambucil (leukeran (registered trademark)), pipobroman (amedel (registered trademark), bersyt (registered trademark)), triethylenemelamine (heymel (registered trademark), hexalen (registered trademark), hexastat (registered trademark)), triethylenethiophosphoramide, temozolomide (temodal (registered trademark)), thiotepa (thioprexin (registered trademark)), busulfan (Busilvex (registered trademark), myleran (registered trademark)), carmustine (BiCNU (registered trademark)), lomustine (CeeNU (registered trademark)), streptozocin (zanosar (registered trademark)) and dacarbazine (DTIC-Dome (registered trademark)).Additional exemplary alkylating agents include oxaliplatin (Eloxatin (registered trademark)); temozolomide (Temodar, Temodal (registered trademark)); dactinomycin (also known as actinomycin-D, Cosmegen (registered trademark)); melphalan (also known as L-PAM, L-sarcolysin and phenylalanine mustard, Alkeran (registered trademark)); altretamine (also known as hexamethylmelamine (HMM), Hexalen (registered trademark)); carmustine (BiCNU (registered trademark)); bendamustine (Treanda (registered trademark)); busulfan (Busulfex (registered trademark) and Myleran (registered trademark)); carboplatin (Paraplatin (registered trademark)); lomustine (also known as CCNU, CeeNU (registered trademark)); cisplatin (also known as CDDP, Platinol (registered trademark) and Platinol (registered trademark)-AQ); chlorambucil (Leukeran (registered trademark)); cyclophosphamide (Cytoxan (registered trademark) and Neosar (registered trademark)); dacarbazine (DTIC, DIC and also known as imidazole carboxamide, DTIC-Dome (registered trademark)); altretamine (also known as hexamethylmelamine (HMM), Hexalen (registered trademark)); ifosfamide (Ifex (registered trademark)); prednimustine; procarbazine (Matulane (registered trademark)); mechlorethamine (also known as nitrogen mustard, Mustargen and mechlorethamine hydrochloride, Mustargen (registered trademark)); streptozocin (Zanosar (registered trademark)); thiotepa (also known as thiophosphoramide, TESPA and TSPA, Thioplex (registered trademark)); cyclophosphamide (Endoxan (registered trademark), Cytoxan (registered trademark), Neosar (registered trademark), Procytox (registered trademark), Revimmune (registered trademark)); and bendamustine HCl (Treanda (registered trademark)), but are not limited thereto.

[0245] Examples of platinum-based agents include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin, and triplatin tetranitrate.

[0246] 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), 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 / Recentin) (AstraZeneca), BAY 57-9352 (telatinib) (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 Pharaceuticals), Combretastatin (Oxigene), CP-751,871 (Figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Daiichi Sankyo), CT-322 (Angiosept) (Adnexus), Curcumin, Dalteparin (Fragmin) (Pfizer), Disulfiram (Antabuse), E7820 (Eisai Co., Ltd.), E7080 (Eisai Co., Ltd.), EMD 121974 (Sirendide) (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 (Faridac / panobinostat) (Novartis), Lucentis (ranibizumab) (Genentech / Novartis), LY317615 (enzastaurin) (Eli Lilly and Company), Macugen (pegaptanib) (Pfizer), MEDI522 (avelumab) (MedImmune), MLN518 (tandutinib) (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 capsule (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 (batalanib) (Novartis), PXD101 (belinostat) (CuraGen Corporation), RAD001 (everolimus) (Novartis), RAF265 (Novartis), regorafenib (BAY73-4506) (Bayer), lenalidomide (Celgene), Retaane (AlconResearch), SN38 (liposomal) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (pasireotide) (Novartis), squalamine (Genaera), slamine, sustent (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, but are not limited thereto.

[0247] Exemplary molecular targeting inhibitors include angiogenesis inhibitors (e.g., VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g., EGF pathway inhibitors), receptor tyrosine kinase inhibitors, growth factor inhibitors, GTPase inhibitors, serine / threonine kinase inhibitors, transcription factor inhibitors, B-Raf inhibitors, MEK inhibitors, mTOR inhibitors, EGFR inhibitors, ALK inhibitors, ROS1 inhibitors, BCL-2 inhibitors, PI3K inhibitors, VEGFR inhibitors, BCR-ABL inhibitors, MET inhibitors, MYC inhibitors, ABL inhibitors, HER2 inhibitors, BTK inhibitors, H-RAS inhibitors, K-RAS inhibitors and PDGFR inhibitors. In certain embodiments, the use of molecular target therapy involves administering a molecular targeting therapeutic agent specific to the molecular target to a subject identified as having a tumor with the molecular target (e.g., a driver oncogene). In certain embodiments, the molecular target has an activating mutation. In certain embodiments, the use of CER-modified cells in combination with a molecular targeting inhibitor increases the magnitude of the anti-tumor response, the duration of the anti-tumor response, or both. In certain embodiments, a dose lower than the general dose of molecular target therapy or a dose below the therapeutic dose is used in combination with CER-modified cells.

[0248] Examples of vascular endothelial growth factor (VEGF) receptor inhibitors include bevacizumab (Avastin (registered trademark)), axitinib (Inlyta (registered trademark)); alaniinate brivanib (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 (registered trademark)); pazopanib (Votrient (registered trademark)); sunitinib malate (Sutent (registered trademark)); cediranib (AZD2171, CAS288383-20-1); Vargatef (BIBF1120, CAS928326-83-4); foretinib (GSK1363089); teratinib (BAY57-9352, CAS332012-40-5); apatinib (YN968D1, CAS811803-05-1); imatinib (Gleevec (registered trademark)); ponatinib (AP24534, CAS943319-70-8); tivozanib (AV951, CAS475108-18-0); regorafenib (BAY73-4506, CAS755037-03-7); batatinib dihydrochloride (PTK787, CAS212141-51-0); brivanib (BMS-540215, CAS649735-46-6); vandetanib (Caprelsa (registered trademark) or AZD6474); motesanib diphosphate (AMG706, CAS857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide (described in PCT published patent application WO02 / 066470); dovitinib lactate (TKI258, CAS852433-84-2); linifanib (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-quinazolinamine (XL647, CAS 781613-23-8); 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 (registered trademark)) are included, but not limited to these.;

[0249] Examples of EGF pathway inhibitors include tyrphostin 46, EKB-569, erlotinib (Tarceva (registered trademark)), gefitinib (Iressa (registered trademark)), erbitux, nimotuzumab, lapatinib (Tykerb (registered trademark)), cetuximab (anti-EGFRmAb), 188Compounds generally and specifically disclosed in Re-labeled nimotuzumab (anti-EGFR mAb) and WO97 / 02266, EP0564409, WO99 / 03854, EP0520722, EP0566226, EP0787722, EP0837063, U.S. Patent No. 5,747,498, WO98 / 10767, WO97 / 30034, WO97 / 49688, WO97 / 38983 and WO96 / 33980 are included, but not limited thereto. Examples of EGFR antibodies include 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), but not limited thereto.Examples of epidermal growth factor receptor (EGFR) inhibitors include erlotinib hydrochloride (Tarceva (registered trademark)), brigatinib, osimertinib, icotinib, gefitinib (Iressa (registered trademark)); N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3"S")-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butanamide, Tovok (registered trademark)); vandetanib (Caprelsa (registered trademark)); 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, CAS497839-62-0); mobritinib (TAK165); pelitinib (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-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS781613-23-8); and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol (PKI166, CAS187724-61-4), but are not limited thereto.

[0250] Examples of mTOR inhibitors include rapamycin (Rapamune (registered trademark)) and its analogs and derivatives; SDZ-RAD; temsirolimus (Torisel (registered trademark), also known as CCI-779); ridafolimus (formally known as deforolimus, (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 WO03 / 064383); everolimus (Afinitor (registered trademark) or RAD001); rapamycin (AY22989, Sirolimus (registered trademark)); simapimod (CAS164301-51-3); (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-aspartyl-L-serine-, inner salt (SF1126, CAS936487-67-1), among others, but not limited to these.

[0251] 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, described in PCT Publication Nos. WO09 / 036082 and WO09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ 235, described in PCT Publication No. WO06 / 122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine (also known as BKM120 or NVP-BKM120, described in PCT Publication No. WO2007 / 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,4S,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), but are not limited thereto.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 1191951-57-1); 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, CAS 937174-76-0); 8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (palomid 529, P529, or SG-00529); triciribine (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)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidineamine (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), but are not limited thereto.

[0252] In certain embodiments, the tyrosine kinase inhibitor used in combination with the CER-modified cells is an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, alectinib, brigatinib, daratumumab, entrectinib, and lorlatinib.

[0253] Figures 3-5 illustrate embodiments of regimens utilizing CER-modified cells. As shown in FIGS. 3 and 4A, following leukapheresis, cells can be processed and activated ex vivo and subjected to gene modification and expansion for injection into a subject. FIG. 4B shows an example of a treatment scheme for CER-modified cells used in combination with a conventional T cell-based therapy (e.g., CAR or TCR). The first injection of engineered T cells induces tumor cell apoptosis that exhibits an anti-tumor effect. Thereafter, CER-modified cells are injected. The CER-modified cells remove tumor cells that exhibit phagocytosis promotion (e.g., PtdSer), which promotes tumor regression while also avoiding the T cell-suppressive tumor microenvironment. Thereafter, changes in the tumor microenvironment re-sensitize the tumor to T cell therapy and enable a second injection of T cells. Another embodiment of the treatment method is shown in FIG. 4C. The treatment scheme shown in FIG. 4C utilizes CER-modified cells in combination with monoclonal antibody therapy. Injection of a tumor-specific antibody such as cetuximab targeting EGFR or rituximab targeting CD20 can induce cell death or induce a target moiety to be bound by CER-modified cells. Thereafter, the subject is administered CER-modified cells that bind to and remove antibody-bound cells. In such an embodiment, the CER extracellular domain can include an FcR-binding domain, a PtdSer-binding domain, or other antigen-binding domain.

[0254] In another scenario, the CER-modified cells can be used in combination with small molecule inhibitors such as BTK inhibitors, MEK inhibitors, adenosine pathway inhibitor A2AR antagonists, IDO1 inhibitors, IMiDs such as lenalidomide, PI3Kδ inhibitors, BRAF inhibitors, or BCR-ABL inhibitors.

[0255] In certain embodiments, the methods of the invention include a depletion step. The depletion step for removing CER from a subject can be carried out after a sufficient time for a therapeutic benefit, in order to reduce the toxicity to the subject. In such embodiments, the CER vector comprises an inducible suicide gene such as iCASP9, inducible Fas or HSV-TK. Similarly, the CER vector can be designed for the expression of a known cell surface antigen such as CD20 or cleaved EGFR (SEQ ID NO: 105) to promote depletion of transduced cells via infusion of a related monoclonal antibody (mAb), for example 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.

[0256] In a further embodiment, the cells expressing CER of the invention can be used in a diagnostic method or an imaging method, including methods used for the identified indications or medical conditions herein.

Examples

[0257] Example 1 Construction of TIM4-TLR4 CER “CER05” The extracellular domain of phosphatidylserine-binding protein Tim4 (encoded by the amino acid sequence of SEQ ID NO: 106) containing a signal peptide (amino acids 1-22 of SEQ ID NO: 106) and a transmembrane domain (encoded by the amino acid sequence of SEQ ID NO: 108) was fused to the signaling domain of TLR4 (encoded by the amino acid sequence of SEQ ID NO: 51) to generate a chimeric engulfment receptor "CER05" (Tim4-TLR4 CER having the amino acid sequence of SEQ ID NO: 81) (Figure 6A). The TLR4 signaling domain transmits a signal for engulfment, and Tim4 is a phosphatidylserine-binding receptor. Subsequently, the Tim4-TLR4 (CER05) chimeric engulfment receptor nucleotide sequence was inserted into a pLenti lentiviral vector together with a truncated EGFR (EGFRt or tEGFR) (encoded by the amino acid sequence of SEQ ID NO: 105) as a transduction marker separated by a T2A sequence (see Figure 6). Mouse Ba / F3 B cells were cultured at a density of 500,000 cells / ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 10 ng / mL mouse IL-3 (Peprotech catalog number 213-13) in a 12-well plate. Under normal conditions, the Ba / F3 mouse B cell line lacks the ability to phagocytose target cells and was thus selected to establish an assay system for phagocytosis. To transduce Ba / F3 cells, 100 μl of the viral vector expressing Tim4-TLR4 (CER05) and 5 μl of TRANSDUX™ transduction reagent were diluted in 0.5 ml of complete cell growth medium and added to Ba / F3 cells. Subsequently, the Ba / F3 cells were centrifuged at 270 x g rpm for 1 hour in a centrifuge pre-warmed to 32°C. The Ba / F3 cells were incubated at 37°C for 24 hours. The Ba / F3 cells were further grown in complete cell growth medium for 48 hours. Positive Ba / F3 cell transductants were selected using fluorescence-activated cell sorting (FACs) (Sony Sorter SH800) by staining with a labeled EGFR-specific antibody (cetuximab).After sorting, the purified and transduced Ba / F3 cells containing the Tim4-TLR4-T2A-transduction marker containing the viral vector were allowed to stand for 48 hours before being used in the phagocytosis assay.

[0258] Phagocytic activity against primary cultured apoptotic thymocytes One day before the phagocytosis assay, primary cultured thymocytes were isolated from C3H mice (Charles River Laboratories International, Inc.). Thymocytes were cultured in a 6-well plate in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. To induce apoptosis and phosphatidylserine expression on the cell surface, thymocytes were treated with 1 μM dexamethasone for 24 hours. Untreated thymocytes were used as a negative control. Thymocytes were harvested from the 6-well plate, washed once with sterile 1X PBS, and then stained with 1 ng / μl of pH-sensitive pHrodo™ Red dye (ThermoFisher Scientific, catalog number P36600) in PBS for 15 minutes at room temperature. Labeling target cells with the pHrodo Red dye allows visualization of cells that are phagocytosed and transported to lysosomes because the fluorescence increases in the acidic lysosomal environment. Then, growth medium was added to the cells and they were washed once more to remove excess pHrodo Red. The pHrodo Red-stained thymocytes were seeded in a flat-bottom 96-well plate at 250,000 cells / well in RPMI 1640 complete medium.

[0259] Ba / F3 CER01+ tEGFR prepared as described above + The cells were washed once with 1X PBS and stained with 1 μM CELLTRACE (商標) Violet dye (ThermoFisher Scientific, catalog number C34557) at 37 °C for 10 minutes. Growth medium was added to the stained transduced Ba / F3 cells and they were washed once with 1X PBS to remove the excess amount of CELLTRACE (商標)Violet was removed and seeded into a flat-bottom 96-well plate at approximately 25,000 cells / well in RPMI 1640 complete medium.

[0260] Target thymocytes were stained with Ba / F3 CER05+tEGFR + Cells were cultured at 37 °C for 3 hours or overnight (~14 hours) at a ratio of 10:1 (target cells: effector cells). After incubation, the plates were centrifuged and the medium was exchanged with PBS supplemented with 2% fetal bovine serum (pH 9). Subsequently, the 96-well plates were observed using a KEYENCE BZ-X710 fluorescence microscope with a 20x objective lens. Ba / F3 cells transduced with a pLenti vector expressing truncated EGFR were used as a negative control. Fluorescence microscopy showed that CER05+ Ba / F3 cells engulfed dexamethasone-treated thymocytes (white arrows indicate the engulfment event) (see Figure 7).

[0261] The phagocytosis index was calculated by multiplying [total number of engulfed target cells / average of the total number of calculated CER-modified cells (e.g., phagocytosis frequency)] by [average area of target cell staining per CER+ Ba / F3 cell x 100 (e.g., hybrid capture)] compared to control EGFRt+ Ba / F3 cells (see Figure 8).

[0262] Duplicate plates co-culturing Ba / F3 CER05+ cells with dexamethasone-treated thymocytes were cultured in a medium containing IL-3 for 6 hours. 50 nM LysoTracker green, which stains the acidic compartment (e.g., lysosomes) of living cells green, was added 5 minutes before the end of the incubation period. Co-localization of internalized pHrodo red-labeled thymocytes and LysoTracker green vesicles can be visualized by overlaying these two images. Co-localization of red and green fluorescence results in yellow / orange fluorescence in the overlaid image, indicating that pHrodo-labeled target cells are internalized into lysosomes, causing rapid acidification and cell death of the ingested cells (see Figure 9, white arrows indicate co-existence of thymocytes labeled with pHrodo red and LysoTracker green vesicles). Fluorescence microscopy images of co-cultured control Ba / F3 cells transduced with truncated EGFR and dexamethasone-treated thymocytes are shown in Figure 10.

[0263] Phagocytic activity against mouse cell lines One day before the phagocytosis assay, CT26 mouse colon carcinoma cells were cultured in a 6-well plate in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and treated with 1 mM staurosporine (STS) for 12 hours to induce apoptosis. Untreated CT26 cells were used as a negative control.

[0264] 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 seeded at 250,000 cells / well in a flat-bottom 96-well plate in RPMI 1640 complete medium.

[0265] Ba / F3 CER05 prepared as described above + EGFR +Wash the cells once with 1X PBS and stain with 1 μM CELLTRACE (商標) Violet Dye (ThermoFisher Scientific, catalog number C34557) at 37 °C for 10 minutes. Add growth medium to the stained and transduced Ba / F3 cells, wash once with 1X PBS to remove excess CELLTRACE (商標) Violet, and seed at approximately 50,000 cells / well in the same flat-bottom 96-well plate in RPMI 1640 complete medium.

[0266] Seed the target CT26 cells together with the stained CER05 + tEGFR + cells at a ratio of 5:1 (target cells: effector cells) and incubate at 37 °C for 3 hours. After incubation, centrifuge the plate and replace the medium with PBS supplemented with 2% fetal bovine serum (pH 9). Then, observe the 96-well plate using a KEYENCE BZ-X710 fluorescence microscope with a 20x objective lens. Ba / F3 cells transduced with a pLenti vector expressing truncated EGFR were used as a negative control. Fluorescence micrographs showing in vitro phagocytosis are shown in Figure 11 (white arrows indicate phagocytosis events). CT26 cells labeled with pHrodo Red fluoresced within the low pH compartment of lysosomes when engulfed (pink outline).

[0267] Apply a hybrid capture algorithm that detects the fluorescence of pHrodo Red within the CELLTRACE Violet staining region to the fluorescence images to quantify the area of the engulfed target cells / CER + area of B cells or control tEGFR + B cells (Figure 12). Figure 13 shows the CER05 + EGFR + or EGFR + scatter plot of the hybrid cell count that extracts the CT26 target cell region within Ba / F3 cells transduced with the control. The phagocytosis index of CER05 + Ba / F3 cells compared to EGFRt-transduced Ba / F3 control cells is shown in Figure 14.

[0268] Example 2 Construction of TIM4-TLR4 (TLR4 TMD) CER “CER07” The extracellular domain of phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO: 106) containing a signal peptide (amino acids 1-22 of SEQ ID NO: 106) is combined with the transmembrane domain of TLR4 (amino acid sequence of SEQ ID NO: 34) and the intracellular signaling domain of TLR4 (SEQ ID NO: 51) to produce a chimeric engulfment receptor “CER07” (Tim4-Tyro3 CER having the amino acid sequence of SEQ ID NO: 83). The TLR4 signaling domain transmits a signal for engulfment, and Tim4 is a phosphatidylserine-binding receptor. Then, the Tim4-TLR4-TLR4 (CER07) chimeric engulfment receptor nucleotide sequence was inserted into the pLenti lentiviral vector together with truncated EGFR as a transduction marker separated by the T2A sequence (see Figure 15). Mouse Ba / F3 B cells were transduced with the pLenti vector expressing Tim4-TLR4-TLR4 (CER07) and EGFRt, expanded, sorted by FACs, and used for the in vitro tests described in Example 1.

[0269] Phagocytic activity against primary apoptotic thymocytes Primary C3H mouse thymocytes were isolated, treated with dexamethasone, and stained with pHrodo Red as described in Example 1. Ba / F3 CER07+ tEGFR+ cells were labeled with CELLTRACETM Violet dye as described in Example 1. Co-culture tests were performed at a target cell:effector cell ratio of 10:1, and Ba / F3 CER07+ tEGFR+ cells were quantified for phagocytosis by fluorescence microscopy and FAC as described in Example 1. Ba / F3 cells transduced with the pLenti vector expressing truncated EGFR were used as a negative control.

[0270] Fluorescence microscopy showed that CER07+ Ba / F3 cells engulfed dexamethasone-treated thymocytes compared to EGFRt-transduced Ba / F3 control cells.

[0271] The phagocytosis index was calculated by multiplying [total number of target cells engulfed / total number of CER-modified cells counted (e.g., phagocytosis frequency)] by [average area of target cell staining per CER+Ba / F3 cell × 100 (e.g., hybrid capture)] compared to EGFRt-transduced Ba / F3 target cells (see Figure 8).

[0272] Phagocytosis activity against mouse cell lines CER07+ Ba / F3 cells were co-cultured with CT26 mouse colon cancer cells as described in Example 1. Fluorescence microscopy showed that CER07+ Ba / F3 cells engulfed staurosporine-treated CT26 cells (see Figure 16, white arrows indicate phagocytosis). Ba / F3 cells transfected with EGFRt were used as a control.

[0273] A hybrid capture algorithm that detects the fluorescence of pHrodo Red within the CELLTRACE Violet staining region was applied to the fluorescence images to quantify the area of engulfed target cells / area of CER+B cells (see Figure 17). Figure 13 shows a scatter plot of the hybrid cell counts for extracting the CT26 target cell area within Ba / F3 cells transfected with CER07+EGFR+ or EGFR+ control. The area ratio represents the co-localization area of CT26 cells within Ba / F3 cells. The phagocytosis index of CER07+Ba / F3 cells compared to EGFRt-transduced Ba / F3 control cells is shown in Figure 14.

[0274] Example 3 Construction of TIM4-TLR8 (TLR4 TMD and spacer) CER “CER21” and TIM4-TLR5 CER “CER19” The extracellular domain of phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO: 106) containing the signal peptide (amino acids 1-22 of SEQ ID NO: 106) and the Tim4 transmembrane domain (amino acid sequence of SEQ ID NO: 108) were ligated to the intracellular signaling domain of TLR8 (SEQ ID NO: 55) to generate a chimeric engulfment receptor "CER21" (Tim4-TLR8 CER having the amino acid sequence of SEQ ID NO: 88). The TLR8 signaling domain transmits the signal for engulfment and Tim4 is a phosphatidylserine-binding receptor. Subsequently, the Tim4-TLR8 (CER21) chimeric engulfment receptor nucleotide sequence was inserted into a pLenti lentiviral vector together with truncated EGFR as a transduction marker separated by the T2A sequence (see Figure 18). Human primary B cells were transduced with a pLenti vector expressing Tim4-TLR8 (CER21) and EGFRt, expanded, sorted by FACS, and used in the in vitro assays described in Example 1.

[0275] The extracellular domain of phosphatidylserine-binding protein Tim4 (amino acid sequence of SEQ ID NO: 106) containing the signal peptide (amino acids 1-22 of SEQ ID NO: 106) and the Tim4 transmembrane domain (amino acid sequence of SEQ ID NO: 108) were ligated to the intracellular signaling domain of TLR5 (SEQ ID NO: 52) to generate a chimeric engulfment receptor "CER19" (Tim4-TLR5 CER having the amino acid sequence of SEQ ID NO: 86). The TLR5 signaling domain transmits the signal for engulfment and Tim4 is a phosphatidylserine-binding receptor. Subsequently, the Tim4-TLR5 (CER19) chimeric engulfment receptor nucleotide sequence was inserted into a pLenti lentiviral vector together with truncated EGFR as a transduction marker separated by the T2A sequence. Human primary B cells were transduced with a pLenti vector expressing Tim4-TLR8 (CER19) and EGFRt, expanded, sorted by FACS, and used in the in vitro assays as described in Example 1.

[0276] Phagocytic activity of human CER21+ B cells against human cell lines One day before setting up the phagocytosis assay, Jurkat human B lymphocytes were cultured in a 6-well plate in complete RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and treated with 1 mM staurosporine for 3 hours to induce apoptosis. The Jurkat cells were washed twice with 1X PBS, and after removing the excess staurosporine, they were stained with pHrodo Red (1 ng / μl in PBS) at room temperature for 15 minutes. Growth medium was added to the Jurkat cells, and they were washed once to remove the excess pHrodo Red, and then seeded in a flat-bottom 96-well plate at approximately 250,000 cells / well in RPMI 1640 complete medium.

[0277] The transduced CER21+ human primary B cells were washed once with 1X PBS and stained with 1 μM CELLTRACE Violet in PBS at 37°C for 10 minutes. Growth medium was added to the CER21+ human primary B cells, and they were washed once with 1X PBS to remove the excess CELLTRACE Violet, and then seeded in a 96-well plate at approximately 50,000 cells / well in RPMI 1640 complete medium. Human CER21+ primary B cells and Jurkat cells were co-cultured at a target cell to effector cell ratio of 5:1 at 37°C for 3 hours. After incubation, the co-culture plate was centrifuged, and the medium was replaced with PBS supplemented with 2% fetal bovine serum (pH 9). Phagocytic events were quantified by a fluorescence microscope (KEYENCE BZ-X710 fluorescence microscope, 20x objective lens). Fluorescence microscope images showing in vitro phagocytosis are shown in Figure 19A for CER021+ B cells co-cultured with Jurkat cells. Fluorescence microscope images showing in vitro cell lysis of Jurkat cells taken up by CER21+ B cells are shown in Figure 19B. The dashed arrows indicate cell lysis activity.

[0278] Apoptotic cells were quantified from fluorescence images using automated software, and the number of apoptotic cells per high-power view (Figure 20A) and the total fluorescence emission per high-power view (Figure 20B) were calculated using a pH indicator dye. CER21+ B cells co-cultured with staurosporine-treated (sub-therapeutic dose) Jurkat cells showed enhanced cell lethality of the target cells.

[0279] Human CER21 against Chemotherapy-Treated Human Cell Lines + Enhanced Cell Lethality by B Cells Human primary B cells were transduced with pLenti Tim4-TLR8 (CER21) lentivirus expressing truncated EGFR as a transduction marker and stained with CELLTRACE Violet as described in Example 1. One day before setting up the co-culture assay, H1703 non-small cell lung cancer cells were incubated with phosphatidylserine-induced chemotherapy paclitaxel (30 μM) in serum-free medium for 24 hours. Floating and adherent H1703 cells were collected, centrifuged, incubated with pHrodo red (1 ng / μL) in PBS at room temperature for 15 minutes, washed, and seeded into non-adherent 96-well plates. Human CER21+ B cells and H1703 cells were co-cultured at 37°C for 3 hours at a target cell to effector cell ratio of 5:1. B cells transduced with truncated EGFR were used as a control. Next, the plates were imaged using a Keyence BZ-X710 microscope with a 20X objective lens (Figure 21). Apoptotic cells show an increase in red fluorescence as the intracellular pH decreases at the earliest stage of apoptosis (upper row of Figure 21). Adjacent cells not induced by paclitaxel treatment emit only slight fluorescence. Apoptosis measurements in the presence of CER21+ B cells were quantified as the area under the curve of red fluorescence objects for each high-power field and outlined with a blue line using automated software (Figure 21, lower column; Figure 22). White arrows indicate apoptotic events. CER21+ B cells were found to enhance the cell lethality of target cells at sub-therapeutic doses of chemotherapy.

[0280] Enhanced proliferative capacity of CER19+ and CER21+ B cells The proliferative capacity of CER19+ and CER21+ human primary B cells was evaluated by co-culture with paclitaxel (30 μM)-treated Jurkat lymphoma cells. CER-transduced B cells were labeled with CELLTRACE Violet and co-cultured with paclitaxel-treated Jurkat cells at a target cell to effector cell ratio of 5:1 for 5 days at 37°C in the absence of exogenous cytokines. Proliferation of CER19+ or CER21+ B cells was evaluated by flow cytometry by measuring the dilution of CELLTRACE Violet (Figure 23). After 5 days of co-culture in the absence of exogenous cytokines, both CER19+ and CER21+ B cells increased approximately 10-fold. In contrast, cells transduced with the control vector did not show an increase in cell number.

[0281] Enhanced activation state of CER21+ B cells To evaluate the activation state of CER21+ human primary B cells, gene expression profiles were examined from the transduced B cell population. Consistent with previous reports linking activation of the TLR family to the expression of pro-inflammatory IL-1 cytokines (e.g., IL-1B and IL-18) and upregulation of co-stimulatory molecules (e.g., CD80, CD86), CER21 also promoted B cell activation molecules and survival factors (e.g., CD40, CD40L), lymphocyte chemoattractants (RANTES, CXCL10, CXCL11), and expressed molecules involved in lymph node tissue remodeling, such as LTα and TNFα, to promote the emergence of tumor-specific adaptive immune responses (see Figure 24. The bar graph shows the fold change in B cell mRNA levels compared to transduced control B cells).

[0282] Example 4 Construction of FMC63 scFv-TLR4 CER “CER43” and FMC 63 scFv-IgG4-TLR4 CER “CER44” An anti-CD19 single-chain fragment (scFv) (encoded by the amino acid sequence of SEQ ID NO: 109) derived from the FMC63 mouse IgG2a monoclonal antibody and containing the GM-CSF-derived signal peptide (amino acids 1-22 of SEQ ID NO: 109) was fused to the TLR4 membrane-proximal domain (SEQ ID NO: 17), the TLR4 transmembrane domain (amino acid sequence of SEQ ID NO: 34), and the intracellular signaling domain of TLR4 (SEQ ID NO: 51) to generate the chimeric engager receptor "CER43" (FMC63 scFv-TLR4 CER having the amino acid sequence of SEQ ID NO: 122). The TLR4 signaling domain transmits signals for engagement, and the FMC63 scFv binds to CD19. Subsequently, the FMC63 scFv-TLR4 (CER43) chimeric engager receptor nucleotide sequence was inserted into the pLenti lentiviral vector together with the truncated EGFR (EGFRt) as a transduction marker separated by the T2A sequence. Mouse Ba / F3 B cells were transduced with the pLenti vector expressing FMC63 scFv-TLR4 (CER43) and EGFRt, expanded, sorted by FACs, and used in the in vitro assay described in Example 1.

[0283] Derived from the FMC63 mouse IgG2a monoclonal antibody and containing the GM-CSF-derived signal peptide (amino acids 1-22 of SEQ ID NO: 109), an anti-CD19 single-chain fragment (scFv) (encoded by the amino acid sequence of SEQ ID NO: 109) was fused to a modified IgG4 hinge extracellular spacer domain (SEQ ID NO: 16) containing the TLR4 transmembrane domain (amino acid sequence of SEQ ID NO: 34) and the intracellular signaling domain of TLR4 (SEQ ID NO: 51) to generate the chimeric engager receptor "CER44" (FMC63 scFv-IgG4-TLR4 CER having the amino acid sequence of SEQ ID NO: 123). The TLR4 signaling domain transmits a signal for engagement, and the FMC63 scFv binds to CD19. Next, the FMC63 scFv-IgG4-TLR4 (CER44) chimeric engager receptor nucleotide sequence was inserted into the pLenti lentiviral vector together with the truncated EGFR as a transduction marker separated by the T2A sequence. Mouse Ba / F3 B cells were transduced with the pLenti vector expressing FMC63 scFv-IgG4-TLR4 (CER44) and EGFRt, expanded, sorted by FACs, and used in the in vitro assay described in Example 1.

[0284] Phagocytic activity against human lymphoma cell lines Raji human Burkitt lymphoma cells were labeled with 1 μM pHrodo Red dye and used as target cells for the phagocytosis assay. CER43+ or CER44+ modified Ba / F3 cells were stained with CELLTRACE Violet as described in Example 1. A co-culture test using gene-modified CD19-targeted CER43+ or CER44+ Ba / F3 cells and CD19+ Raji cells was performed as described in Example 1. Ba / F3 cells transduced with truncated EGFR were used as a control. CER43+ or CER44+ Ba / F3 cells and Raji cells were co-cultured at a target cell to effector cell ratio of 5:1 for 3 hours at 37°C. Phagocytic events were quantified by a fluorescence microscope (KEYENCE BZ-X710 fluorescence microscope, 20X objective lens). Figure 25 shows the engulfment of Raji cells by CD19-specific CER44-expressing Ba / F3 cells (white arrows indicate engulfment events). The frequency of phagocytosis was quantified as the double-positive staining cell population of pHrodo Red and CELLTRACE Violet detected by FACS. Figure 26 shows a FACS plot of the double-positive cell population of CER43+ Ba / F3 cells (9.10%), CER44+ Ba / F3 cells (6.92%) or control EGFRt+ Ba / F3 cells (4.49%) co-cultured with Raji cells. The frequency of phagocytosis of CER43+, CER44+ or control EGFRt+ Ba / F3 cells co-cultured with Raji cells is also shown in the bar graph of Figure 27. Ba / F3 cells transduced with a lentiviral vector expressing CD19-specific CER43 or CER44 showed enhanced uptake by phagocytosis of Raji lymphoma cells.

[0285] Example 5 Construction of CER, TCR and modified T cells for combination cellular immunotherapy A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 and the transmembrane domain of Tim4 was ligated to the intracellular signaling domain of TLR4 to generate a chimeric engulfment receptor "CER5" encoded by the amino acid sequence of SEQ ID NO: 81. A polynucleotide containing the extracellular domain and the transmembrane domain of phosphatidylserine-binding protein Tim4 was bound to the intracellular signaling domain of TLR3 to produce a chimeric engager receptor "CER17" encoded by the amino acid sequence of SEQ ID NO: 84. A polynucleotide containing the extracellular domain and the transmembrane domain of phosphatidylserine-binding protein Tim4 was bound to the intracellular signaling domain of TLR5 to produce a chimeric engager receptor "CER19" encoded by the amino acid sequence of SEQ ID NO: 86. A polynucleotide containing the extracellular domain and the transmembrane domain of phosphatidylserine-binding protein Tim4 was bound to the intracellular signaling domain of TLR8 to produce a chimeric engager receptor "CER21" encoded by the amino acid sequence of SEQ ID NO: 88. A polynucleotide containing the extracellular domain and the transmembrane domain of phosphatidylserine-binding protein Tim4 was bound to the intracellular signaling domain of TLR9 to produce a chimeric engager receptor "CER23" encoded by the amino acid sequence of SEQ ID NO: 90. A polynucleotide containing the extracellular domain and the transmembrane domain of phosphatidylserine-binding protein Tim4 was bound to the intracellular signaling domain of TLR1 to produce a chimeric engager receptor "CER26" encoded by the amino acid sequence of SEQ ID NO: 92. A polynucleotide containing the extracellular domain and the transmembrane domain of phosphatidylserine-binding protein Tim4 was bound to the intracellular signaling domain of TLR2 to produce a chimeric engager receptor "CER27" encoded by the amino acid sequence of SEQ ID NO: 93. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was linked to the intracellular signaling domain of TLR8 and the truncated intracellular signaling domain of CD79b to produce a chimeric engager receptor “CER103B” encoded by the amino acid sequence of SEQ ID NO: 132. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was linked to the intracellular signaling domain of TLR8 and the intracellular signaling domain of DAP12 to produce a chimeric engager receptor “CER104” encoded by the amino acid sequence of SEQ ID NO: 133. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was linked to the intracellular signaling domain of TLR8 and the intracellular signaling domain of BAFF-R to produce a chimeric engager receptor “CER105” encoded by the amino acid sequence of SEQ ID NO: 134. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was linked to the intracellular signaling domain of NFAM1 and the intracellular signaling domain of TLR8 to produce a chimeric engager receptor “CER106” encoded by the amino acid sequence of SEQ ID NO: 135. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was linked to the intracellular signaling domain of Traf6 and the intracellular signaling domain of TLR8 to produce a chimeric engager receptor “CER116” encoded by the amino acid sequence of SEQ ID NO: 143.

[0286] The polynucleotide encoding the TCRβ chain and the polynucleotide encoding the TCRα of the HPV16 E7-specific TCR (see PCT Publication No. WO2015 / 184228) were fused using a sequence encoding a P2A self-cleaving peptide therebetween. The TCRVα domain contains the amino acid sequence of SEQ ID NO: 162, and the TCRVβ region contains the amino acid sequence of SEQ ID NO: 160. The Cα domain contains cysteine substitutions and LVL substitutions at positions 12, 14, and 15 and contains the amino acid sequence of SEQ ID NO: 163. Cβ also contains a cysteine substitution and contains the amino acid sequence of SEQ ID NO: 161. The encoded HPV16 E7-specific TCR contains the amino acid sequence of SEQ ID NO: 158.

[0287] The selected CER polynucleotide and the HPV16 E7 TCR polynucleotide were each inserted into a pLenti lentiviral vector. Peripheral blood was collected from human donors by venipuncture, and human peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using a lymphocyte separation medium. CD8+ or CD4+ T cells were enriched from PBMCs using a commercially available separation kit and activated with anti-CD3 and anti-CD28 in complete cell growth medium. 50 μl of the viral vector expressing the HPV16 E7 TCR was diluted with 0.5 ml of complete cell growth medium and added to the CD8+ T cells. 50 μl of the viral vector expressing the selected CER was diluted with 0.5 ml of complete cell growth medium and added to the CD4+ T cells. Next, the transduced T cells were centrifuged at 270 x g rpm for 1 hour in a pre-warmed centrifuge at 32°C. The T cells were incubated at 37°C for 24 hours. The T cells were further grown in complete cell growth medium for 72 hours, the beads were removed, and they were grown for 5 days before being used in a functional assay. The transduced CD4 and CD8 T cells were combined at a 1:1 ratio for the functional assay.

[0288] The combination of CD8 T cell-TCR + CD4 T cell-CER shows enhanced antigen-specific cytolytic activity and phagocytic activity Removal of target SCC152 cells via dual HPV16 E7 TCR and CER was detected using cytotoxicity and phagocytosis assays (see Figure 30A). SCC152 cells are HPV+ cells from hypopharyngeal squamous cell carcinoma. The cytotoxic activity of CD8+ T cells transduced with an HPV16 E7-specific TCR was detected using a caspase 3 / 7 apoptosis reagent (IncuCyte®) that binds to an activated caspase 3 / 7 recognition motif and a red reagent that fluoresces upon cleavage. Fluorescent signals were measured using a fluorescence microscope. HPV16 E7 TCR-transduced CD8+ T cells and selected CER-transduced CD4+ T cells were mixed at a 1:1 ratio and co-cultured with HPV16 E7+ head and neck squamous cell carcinoma cells (SCC152) at a 1:1 ratio, and the caspase 3 / 7 apoptosis reagent was added to the co-culture. Cytotoxic activity was measured over time by measuring fluorescence. The control sample was CD8 T cells transduced with HPV16 E7 TCR only. As seen in the graphs of Figures 30B, 31, and 34 and fluorescence micrographs (data not shown), addition of most CER-transduced CD4+ T cells tested to CD8 T cells transduced with HPV16 E7 TCR enhanced cytolytic activity compared to mono-treatment with CD8 T cells transduced with HPV16 E7 TCR.

[0289] Enhanced cytolytic activity of CD4 T cells transduced with CER104 + CD8 T cells transduced with HPV16 E7 TCR was observed when measured using a lactate dehydrogenase (LDH) cytotoxicity assay (see Figure 32). LDH is a cytoplasmic enzyme that is released from cells into the cell culture medium when the cell membrane is damaged. Thus, the presence of LDH in the medium is a marker of cell death. The LDH assay can detect low levels of damage to the cell membrane that cannot be detected by other methods. LDH can be detected using a colorimetric or fluorescent method.

[0290] During co - culture of CD8+ T cells transduced with an HPC16 E7 - specific TCR and CD4 T cells transformed with a selected CER, the elimination of target SCC152 cells was also detected by quantifying the expression of green fluorescent protein over time (0 hours, 24 hours, 48 hours) by SCC152 cells (see Figure 33). By 48 hours, all of the co - cultures of the CD4 T cell / CER + CD8 T cell / HPV16 E7 TCR combination showed enhanced removal of SCC152 cells compared to the control. Time - lapse imaging of the co - culture assay also similarly showed enhanced removal of SCC152 cells by the CD4 T cell / CER + CD8 T cell / HPV16 E7 TCR combination compared to the control (data not shown).

[0291] The cytokine response of the co - culture assay was measured by sampling the cell supernatant using a Mesoscale Multiplex Array Cytokine Plate. The following cytokines were measured: IFNγ, IL - 2, TNFα, IL - 4, IL - 6, IL - 12b, IL - 13, IL - 1b and IL - 10. Enhanced cytokine (e.g., IFNγ, IL - 2) production indicative of an activation profile was induced in co - culture with the CD4 T cell / CER + CD8 T cell / HPV16 E7 TCR combination compared to the control (see Figure 35).

[0292] The phagocytic activity of the CD4 T cell / CER + CD8 T cell / HPV16 E7 TCR combination co - cultured with SCC152 cells was visualized and quantified using a KEYENCE BZ - X710 fluorescence microscope, a 20X objective lens and Hybrid Capture software. Figures 36 - 37 show that CD4+ T cells transduced with various CERs used in co - culture with CD8 T cells / HPV E7 TCR enhanced the engulfment of SCC152 target cells compared to co - culture with control CD8 T cells / HPV16 E7 TCR alone.

[0293] Example 6 Construction of tandem expression cassette A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 and the transmembrane domain of Tim4 was ligated to the intracellular signaling domain of TLR4 to generate a chimeric engager receptor "CER5" encoding the amino acid sequence of SEQ ID NO: 81. A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 and the transmembrane domain of Tim4 was ligated to the intracellular signaling domain of TLR5 to generate a chimeric engager receptor "CER19" encoding the amino acid sequence of SEQ ID NO: 98. A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 was ligated to the transmembrane domain of Tim4 and the intracellular signaling domain of TLR8 to generate a chimeric engager receptor "CER21" encoding the amino acid sequence of SEQ ID NO: 86. A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 and the transmembrane domain of Tim4 was ligated to the intracellular signaling domain of NFAM1 to generate a chimeric engager receptor "CER25" encoding the amino acid sequence of SEQ ID NO: 159. A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 and the transmembrane domain of Tim4 was ligated to the intracellular signaling domain of TLR2 to generate a chimeric engager receptor "CER2" encoding the amino acid sequence of SEQ ID NO: 93. A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 and the transmembrane domain of Tim4 was ligated to the intracellular signaling domain of Traf6 to generate a chimeric engager receptor "CER29" encoding the amino acid sequence of SEQ ID NO: 102. A polynucleotide containing the extracellular domain of phosphatidylserine-binding protein Tim4 and the transmembrane domain of Tim4 was ligated to the intracellular signaling domain of Traf3 to generate a chimeric engager receptor "CER31" encoding the amino acid sequence of SEQ ID NO: 124.

[0294] The polynucleotide encoding the TCRβ chain and the polynucleotide encoding the TCRα of the HPV16 E7-specific TCR (see PCT Publication WO2015 / 184228) were ligated using the sequence of the P2A self-cleaving peptide between them. The TCRVα domain contains the amino acid sequence of SEQ ID NO: 162, and the TCRVβ region contains the amino acid sequence of SEQ ID NO: 160. The Cα domain contains cysteine substitutions and LVL substitutions at positions 12, 14, and 15 and contains the amino acid sequence of SEQ ID NO: 163. Cβ also contains a cysteine substitution and contains the amino acid sequence of SEQ ID NO: 161. The encoded HPV16 E7-specific TCR contains the amino acid sequence of SEQ ID NO: 158. The amino acid sequences of the tandem expression constructs described in this example are provided in Table 2 (see also Figures 38A to 38F).

[0295]

Table 3

Table 4

Table 5

Table 6

Table 7

[0296] The selected CER polynucleotide and the HPV16 E7 TCR polynucleotide were inserted into the same pLenti lentiviral vector having a T2A sequence (encoding the amino acid sequence of SEQ ID NO: 156) therebetween (see FIGS. 1A-1G). Peripheral blood was drawn from a human donor by venipuncture, and human peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using a lymphocyte separation medium. CD8+ T cells were enriched from PBMCs using a commercially available separation kit and activated with anti-CD3 and anti-CD28 in complete cell growth medium. 50 μl of the viral vector expressing the CER-HPV16 E7 TCR combination was diluted in 0.5 ml of complete cell growth medium and added to the CD8+ T cells. The transduced T cells were then centrifuged at 270 x g rpm for 1 hour in a centrifuge pre-warmed to 32°C. The T cells were incubated at 37°C for 24 hours. The T cells were further grown in complete cell growth medium for 72 hours, the beads were removed, and the cells were grown for 5 days before being used in a functional assay.

[0297] CD8 T cells transduced with the CER-TCR tandem expression cassette exhibit antigen-specific cytolysis and phagocytic activity. The cytotoxic activity of tandem expression cassette-transduced CD8+ T cells was detected using a caspase 3 / 7 apoptosis reagent (IncuCyte®) that binds to a red reagent that fluoresces upon cleavage of the activated caspase 3 / 7 recognition motif. Fluorescence signals were measured using a fluorescence microscope. Transduced CD8+ T cells were co-cultured with HPV16 E7+ head and neck squamous cell carcinoma cells (SCC152) at a ratio of 1:1, and the caspase 3 / 7 apoptosis reagent was added to the co-culture. CD8+ T cells containing the CER21-HPV16 E7 TCR tandem expression cassette exhibited cytotoxic activity against SCC152 cells. It was revealed that the cytotoxic response by CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette was exponentially higher by 6 hours than that of CD8+ T cells containing only the HPV16 E7 TCR (see Figure 39). CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette, the CER29-HPV16 E7 TCR tandem expression cassette, or the CER31-HPV16 E7 TCR tandem expression cassette were co-cultured with SCC152 cells at a target:effector cell ratio of 1:1. The caspase 3 / 7 apoptosis reagent was added to the co-culture, and cytotoxic activity was measured over time by measuring fluorescence (see Figure 40). Control samples were CD8 T cells transduced with HPV16 E7 TCR alone or mock-transduced T cells.

[0298] The phagocytic activity of tandem expression cassette-transduced CD8+ T cells was detected by co-culturing tandem expression cassette-transduced CD8+ T cells and SCC152 cells at a ratio of 1:1 for 6 hours. Phagocytic events were visualized and quantified using a KEYENCE BZ-X710 fluorescence microscope, a 20X objective lens, and Hybrid Capture software. CD8+ T cells transduced with the CER21-HPV16 E7 TCR, CER29-HPV16 E7 TCR, or CER31-HPV16 E7 TCR tandem cassette were able to phagocytose SCC152 cells (see Figure 41). The Rac1 inhibitor NSC23766 (50 μM) was also added to the co-culture assay to measure phagocytosis in vitro. Treatment with the Rac1 inhibitor revealed that engulfment of SCC152 cells by CER21-HPV16 E7 TCR-, CER29-HPV16 E7 TCR-, or CER31-HPV16 E7 TCR-transduced T cells occurred in a Rac1-dependent manner (data not shown). CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette engulfed streptavidin-coated latex beads coated with biotinylated phosphatidylserine (data not shown). After approximately 30 minutes of incubation, beads coated with phosphatidylserine were visible within CER21-HPV16 E7 TCR+ T cells.

[0299] The cytokine response of CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette was measured by sampling the cell supernatant during co-culture with SCC152 cells, indicating that CER21-HPV16 E7 TCR+ T cells exhibit antigen-specific effector functions measured by IFNγ response (see Figure 42).

[0300] Example 7 CER Enhancement in Cancer Molecular Targeted Cancer Therapy This example describes an approach for using molecular targeted therapy in combination with CER-expressing cells for the treatment of cancer. In this scenario, a first molecule, for example, a small molecule inhibitor targeting a driver oncogene, induces the expression or membrane exposure of a second molecule recognized by CER-expressing cells. This drug-induced target can be an engulfment-promoting marker (e.g., phosphatidylserine). Upon recognition and binding of the induced second target molecule, CER-expressing cells induce antitumor activity through the activation of the phagocytic signaling cascade. This approach can be utilized to enhance molecular targeted therapy for hematological malignancies and solid tumors.

[0301] CER Enhancement by EGFR Inhibitors Approximately 30 - 40% of non-small cell lung cancer (NSCLC) and approximately 15% of epidermal growth factor (EGFR) activating mutations in Japanese patients. For the treatment of EGFR-mutant NSCLC, EGFR tyrosine kinase inhibitors (EGFR-TKIs) that inhibit the EGFR-induced downstream signaling pathway have been developed. Clinical trials have shown an improvement in the prognosis of EGFR-mutant lung cancer patients with EGFR inhibitors, and the overall survival of advanced NSCLC has been extended from 1 year to 2 - 3 years. EGFR inhibitors can be used for the treatment of other cancers with activating EGFR mutations, including colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, and glioblastoma. Clinical oncology studies have shown that even the most potent targeted therapies, most patients administered drugs designed to interfere with specific genes or proteins ultimately relapse, often with new tumors that no longer respond to treatment.

[0302] CER-modified cells were designed to recognize the engulfment-promoting marker phosphatidylserine and were administered in combination with various EGFR inhibitors, osimertinib, brigatinib, erlotinib, gefitinib, to determine whether CER therapy could enhance EGFR-targeted therapy.

[0303] The HCC159 lung adenocarcinoma cells have EGFR mutations and are sensitive to EGFR inhibition. The HCC159 cells were treated with the EGFR kinase inhibitors osimertinib, brigatinib, erlotinib, or gefitinib at increasing concentrations (50 nM, 250 nM, 500 nM, 1000 nM, 2500 nM, 3700 nM, or 5000 nM of osimertinib, brigatinib, and erlotinib; 50 nM, 250 nM, 500 nM, 1000 nM, 2500 nM, 5000 nM, 10000 nM of brigatinib) for 12 hours and then incubated with the Tim4-IgG1 Fc recombinant fusion protein to evaluate the exposure of the engulfment-promoting marker (phosphatidylserine) to the target cells after EGFR inhibitor treatment. The increase in the concentrations of osimertinib (Figure 43A), brigatinib (Figure 43B), erlotinib (Figure 44A), and gefitinib (Figure 44B) enhanced the surface staining...

Claims

**Claim 1** A chimeric engager receptor (CER) comprising a single-chain chimeric protein, wherein the single-chain chimeric protein comprises an extracellular domain comprising a binding domain that binds phosphatidylserine (PtdSer); an engagement signaling domain comprising a Toll-like receptor (TLR) signaling domain, a Traf6 signaling domain, a Traf2 signaling domain, or a Traf3 signaling domain; and a transmembrane domain located between and connecting the extracellular domain and the engagement signaling domain The chimeric engager receptor (CER) comprising the above. **Claim 2** The chimeric engager receptor (CER) according to claim 1, wherein the binding domain comprises an scFv specific for PtdSer, or a PtdSer binding domain derived from Tim1, Tim4, Tim3, stabilin-2, receptor for advanced glycation end products (RAGE), brain-specific angiogenesis inhibitor 1 (BAI1), milk fat globule-Egf factor 8 protein (MFG-E8), growth arrest-specific factor 6 (GAS6), protein S, protein C, factor II, factor VII, factor IX, factor X, β2 glycoprotein I, α5β3 integrin and other integrins, CR3 complement receptor, CR4 complement receptor, CD14, CD93, annexin V, phosphatidylserine receptor (PSr), prothrombin or scavenger receptor. **Claim 3** The chimeric engager receptor (CER) according to claim 2, wherein the binding domain comprises a Tim1 domain comprising the amino acid sequence of SEQ ID NO: 2, a Tim4 domain comprising the amino acid sequence of SEQ ID NO: 3, a Tim3 domain comprising the amino acid sequence of SEQ ID NO: 4, an FA58C2 domain comprising the amino acid sequence of SEQ ID NO: 5, a GAS6 domain comprising the amino acid sequence of SEQ ID NO: 6, a protein S binding domain comprising the amino acid sequence of SEQ ID NO: 7, or a BAI1 domain comprising the amino acid sequence of SEQ ID NO:

8. **Claim 4** The chimeric engager receptor (CER) according to any one of claims 1 to 3, wherein the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain. **Claim 5** The chimeric engager receptor (CER) according to claim 4, wherein the extracellular spacer domain comprises an immunoglobulin hinge region, the extracellular region of a type I membrane protein, the stalk region of a type II C-type lectin, an immunoglobulin constant domain, the membrane-proximal region of a toll-like receptor, or a fragment thereof.

6. The chimeric engager receptor (CER) according to claim 5, wherein the extracellular spacer domain comprises an IgG1, IgG2, IgG3, IgG4, IgA or IgD hinge region.

7. The chimeric engager receptor (CER) according to claim 6, wherein the extracellular spacer domain comprises a modified IgG4 hinge region comprising the amino acid sequence of SEQ ID NO:

16.

8. The chimeric engager receptor (CER) according to claim 5, wherein the extracellular spacer domain comprises the membrane-proximal region of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9.

9. The chimeric engager receptor (CER) according to claim 8, wherein the extracellular spacer domain comprises the TLR4 membrane-proximal region comprising the amino acid sequence of SEQ ID NO:

17.

10. The chimeric engager receptor (CER) according to any one of claims 1 to 9, wherein the transmembrane domain comprises the transmembrane domain of Tim1, Tim4, Tim3, FcR, CD8a, CD28, MERTK, Axl, MRC1, Tyro3, BA1, CD4, DAP12, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9.

11. The chimeric engager receptor (CER) according to claim 10, wherein the transmembrane domain comprises a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 18, a Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 19, an FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 20, a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 22, a MERTK transmembrane domain comprising the amino acid sequence of SEQ ID NO: 23, an Axl transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24, an MRC1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 30, a Tyro3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 25, a CD28 transmembrane domain of SEQ ID NO: 26, a BA1 transmembrane domain of SEQ ID NO: 29, a CD4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 27, an FcεRIγ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 21, a DAP12 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 28, a TLR1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 31, a TLR2 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 32, a TLR3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 33, a TLR4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 34, a TLR5 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 35, a TLR6 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, a TLR7 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 37, a TLR8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 38, or a TLR9 transmembrane domain comprising the amino acid sequence of SEQ ID NO:

39.

12. The chimeric engager receptor (CER) according to claim 10, wherein the FcR transmembrane domain comprises an FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, or FcαR1 transmembrane domain.

13. The chimeric engager receptor (CER) according to any one of claims 1 to 12, wherein the TLR signaling domain is a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signaling domain.

14. The engagement signal transduction domain comprises a TLR1 signal transduction domain containing the amino acid sequence of SEQ ID NO: 48, a TLR2 signal transduction domain containing the amino acid sequence of SEQ ID NO: 49, a TLR3 signal transduction domain containing the amino acid sequence of SEQ ID NO: 50, a TLR4 signal transduction domain containing the amino acid sequence of SEQ ID NO: 51, a TLR5 signal transduction domain containing the amino acid sequence of SEQ ID NO: 52, a TLR6 signal transduction domain containing the amino acid sequence of SEQ ID NO: 53, a TLR7 signal transduction domain containing the amino acid sequence of SEQ ID NO: 54, a TLR8 signal transduction domain containing the amino acid sequence of SEQ ID NO: 55, a TLR9 signal transduction domain containing the amino acid sequence of SEQ ID NO: 56, a Traf6 signal transduction domain containing the amino acid sequence of SEQ ID NO: 57, a cleaved Traf6 signal transduction domain containing the amino acid sequence of SEQ ID NO: 58, a Traf2 signal transduction domain containing the amino acid sequence of SEQ ID NO: 72 or a Traf3 signal transduction domain containing the amino acid sequence of SEQ ID NO: 73, the chimeric engagement receptor (CER) according to any one of claims 1 to 13.

15. The chimeric engagement receptor (CER) according to any one of claims 1 to 14, wherein signal transduction by the engagement signal transduction domain results in the expression of at least one of an inflammatory cytokine, an inflammatory chemokine or a co-stimulatory cell surface marker.

16. 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 co-stimulatory cell surface marker is CD80, CD86, HLA-DR, CD40, HVEM or 4-1BBL, or any combination thereof, the chimeric engagement receptor (CER) according to claim 15.

17. The engagement signal transduction domain includes a first engagement signal transduction domain and a second engagement signal transduction domain, wherein the first engagement signal transduction domain is a TLR signal transduction domain, a Traf6 signal transduction domain, a Traf2 signal transduction domain or a Traf3 signal transduction domain, the chimeric engagement receptor (CER) according to any one of claims 1 to 12.

18. The chimeric engagement receptor (CER) according to claim 17, wherein the second engagement signal transduction domain is an FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, CD79b, TLR, Traf2, Traf3 or Traf6 signal transduction domain.

19. The second engagement signal transduction domain is an FcγR1 signal transduction domain containing the amino acid sequence of SEQ ID NO: 63, an FcγR2A signal transduction domain containing the amino acid sequence of SEQ ID NO: 64, an FcγR2C signal transduction domain containing the amino acid sequence of SEQ ID NO: 65, an FcγR3A signal transduction domain containing the amino acid sequence of SEQ ID NO: 66, an FcεRIγ signal transduction domain containing the amino acid sequence of SEQ ID NO: 62, a BAFF-R signal transduction domain containing the amino acid sequence of SEQ ID NO: 67, a DAP12 signal transduction domain containing the amino acid sequence of SEQ ID NO: 68, an NFAM1 signal transduction domain containing the amino acid sequence of SEQ ID NO: 69, a cleaved NFAM1 signal transduction domain containing the amino acid sequence of SEQ ID NO: 70, a CD79b signal transduction domain containing the amino acid sequence of SEQ ID NO: 71, a TLR1 signal transduction domain containing the amino acid sequence of SEQ ID NO: 48, a TLR2 signal transduction domain containing the amino acid sequence of SEQ ID NO: 49, a TLR3 signal transduction domain containing the amino acid sequence of SEQ ID NO: 50, a TLR4 signal transduction domain containing the amino acid sequence of SEQ ID NO: 51, a TLR5 signal transduction domain containing the amino acid sequence of SEQ ID NO: 52, a TLR6 signal transduction domain containing the amino acid sequence of SEQ ID NO: 53, a TLR7 signal transduction domain containing the amino acid sequence of SEQ ID NO: 54, a TLR8 signal transduction domain containing the amino acid sequence of SEQ ID NO: 55, a TLR9 signal transduction domain containing the amino acid sequence of SEQ ID NO: 56, a Traf6 signal transduction domain containing the amino acid sequence of SEQ ID NO: 57, a cleaved Traf6 signal transduction domain containing the amino acid sequence of SEQ ID NO: 58, a Traf2 signal transduction domain containing the amino acid sequence of SEQ ID NO: 72 or a Traf3 signal transduction domain containing the amino acid sequence of SEQ ID NO: 73, the chimeric engagement receptor (CER) according to claim 18.

20. The first engagement signal transduction domain is a TLR signal transduction domain, and the second engagement signal transduction domain is a Traf2, Traf3, or Traf6 signal transduction domain, or the first engagement signal transduction domain is a Traf2, Traf3, or Traf6 signal transduction domain and the second engagement signal transduction domain is a TLR signal transduction domain, the chimeric engagement receptor (CER) according to claim 18. **Claim 21** The transmembrane domain and the TLR signal transduction domain are derived from the same TLR, the chimeric engagement receptor (CER) according to any one of claims 1 to 20. **Claim 22** The chimeric engagement receptor (CER) includes an extracellular spacer domain located between the binding domain and the transmembrane domain, and the extracellular spacer domain includes a TLR membrane proximal region and a TLR signal transduction domain derived from the same TLR as the transmembrane domain, the chimeric engagement receptor (CER) according to claim 21. **Claim 23** A nucleic acid molecule encoding at least one of the chimeric engagement receptors (CERs) according to any one of claims 1 to 22. **Claim 24** A nucleic acid molecule encoding two chimeric engagement receptors (CERs), wherein an IRES sequence, a furin cleavage site sequence, or a viral 2A peptide sequence is arranged between the sequences encoding the two CERs, the nucleic acid molecule according to claim 23. **Claim 25** The nucleic acid molecule according to claim 23 or 24, further comprising a sequence encoding a transduction marker, a suicide gene, or both. **Claim 26** The nucleic acid molecule according to claim 25, wherein the transduction marker is a truncated EGFR protein comprising the amino acid sequence of SEQ ID NO:

105. **Claim 27** A vector comprising the nucleic acid molecule according to any one of claims 23 to 26. **Claim 28** The vector according to claim 27, wherein the vector is a multicistronic vector. **Claim 29** The vector according to claim 27 or 28, wherein the vector is a viral vector, a modified mRNA vector, or a transposon-mediated gene transfer vector. **Claim 30** The vector according to claim 29, wherein the viral vector is a retroviral vector or a lentiviral vector. **Claim 31** A host cell comprising a CER according to any one of claims 1 to 22, a nucleic acid according to any one of claims 23 to 26, or a vector according to any one of claims 27 to 30.

32. The host cell according to claim 31, comprising at least two different CERs according to any one of claims 1 to 22.

33. The host cell according to claim 32, wherein each CER is encoded by the same vector.

34. The host cell according to claim 32, wherein each CER is encoded by a different vector.

35. the host cell is CD4 ＋ , CD8 ＋ , naive (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory (CD45RO ＋ , CD62L ＋ , CD8 ＋ ), effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), virus-specific, mucosa-associated invariant, γδ (gd), natural killer cells and tissue-resident T cells including tissue-resident cells, natural killer cells, B cells, lymphocyte progenitor cells including lymphoid common progenitor cells, antigen-presenting cells including dendritic cells, Langerhans cells, myeloid progenitor cells or mature myeloid cells, the host cell according to any one of claims 31 to 34.

36. The host cell according to claim 35, wherein the B cell is a naive B cell, plasma cell, regulatory B cell, marginal zone B cell, follicular B cell, lymphoplasmacytoid cell, plasmablast cell or memory B cell.

37. The host cell according to any one of claims 31 to 36, wherein the host cell is a human cell.

38. The host cell according to any one of claims 31 to 37, wherein the host cell can engulf a target cell expressing PtdSer on its surface.

39. The host cell according to claim 38, wherein the host cell can phagocytose a target cell expressing PtdSer on its surface.

40. The host cell according to any one of claims 31 to 39, wherein the host cell exhibits a phagocytic index of at least 20 against a target cell expressing PtdSer on its surface.

41. The host cell according to any one of claims 31 to 40, wherein the host cell can lyse a target cell expressing PtdSer on its surface.

42. A B cell comprising a chimeric engulfment receptor (CER) comprising a single-chain chimeric protein, wherein the single-chain chimeric protein comprises an extracellular domain comprising a binding domain that binds to a target antigen; an engulfment signaling domain comprising a Toll-like receptor (TLR) signaling domain, a Traf6 signaling domain, a Traf2 signaling domain or a Traf3 signaling domain; and, a transmembrane domain located between and linking the extracellular domain and the engulfment signaling domain comprising a B cell.

43. The B cell according to claim 42, wherein the binding domain is an scFv.

44. The B cell according to claim 42 or 43, wherein the binding domain is specific for a tumor antigen.

45. The B cell according to claim 44, wherein the tumor antigen is 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, NY-ESO-1, TAG-72, CEA, Ephrin A2, Ephrin B2, 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 or GD2.

46. The B cell according to claim 42 or 43, wherein the binding domain is specific for a microbial antigen, an autoimmune disease antigen or a neurodegenerative disease antigen.

47. The B cell according to any one of claims 42 to 46, wherein the extracellular domain further comprises an extracellular spacer domain located between the binding domain and the transmembrane domain.

48. The B cell according to claim 47, wherein the extracellular spacer domain comprises an immunoglobulin hinge region, an extracellular region of a type I membrane protein, a stalk region of a type II-C lectin, an immunoglobulin constant domain, a membrane-proximal region of a toll-like receptor (TLR) or a fragment thereof.

49. The B cell according to claim 48, wherein the extracellular spacer domain comprises an IgG1, IgG2, IgG3, IgG4, IgA or IgD hinge region.

50. The B cell according to claim 49, wherein the extracellular spacer domain comprises a modified IgG4 hinge region comprising the amino acid sequence of SEQ ID NO:

67.

51. The B cell according to claim 48, wherein the extracellular spacer domain comprises a TLR4 membrane-proximal region comprising the amino acid sequence of SEQ ID NO:

17.

52. The B cell according to any one of claims 42 to 51, wherein the transmembrane domain comprises the transmembrane domain of Tim1, Tim4, Tim3, FcR, CD8a, CD28, MERTK, Axl, MRC1, Tyro3, BAIl, CD4, DAP12, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9.

53. The B cell according to claim 52, wherein the transmembrane domain comprises the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 18, the Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 19, the FcγRI transmembrane domain comprising the amino acid sequence of SEQ ID NO: 20, the CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 22, the MERTK transmembrane domain comprising the amino acid sequence of SEQ ID NO: 23, the Axl transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24, the MRC1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 30, the Tyro3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 25, the CD28 transmembrane domain of SEQ ID NO: 26, the BAIl transmembrane domain of SEQ ID NO: 29, the CD4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 27, the FcεRIγ transmembrane domain comprising the amino acid sequence of SEQ ID NO: 21, the DAP12 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 28, the TLR1 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 31, the TLR2 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 32, the TLR3 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 33, the TLR4 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 34, the TLR5 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 35, the TLR6 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, the TLR7 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 37, the TLR8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 38 or the TLR9 transmembrane domain comprising the amino acid sequence of SEQ ID NO:

39.

54. The B cell according to claim 52, wherein the FcR transmembrane domain comprises the transmembrane domain of FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1 or FcαR1.

55. The B cell according to any one of claims 42 to 54, wherein the TLR signaling domain is the TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9 signaling domain.

56. The engagement signal transduction domain comprises a TLR1 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 48, a TLR2 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 49, a TLR3 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 50, a TLR4 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 51, a TLR5 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 52, a TLR6 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 53, a TLR7 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 54, a TLR8 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 55, a TLR9 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 56, a Traf6 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 57, a cleaved Traf6 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 58, a Traf2 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 72 or a Traf3 signal transduction domain comprising the amino acid sequence of SEQ ID NO: 73, the B cell according to any one of claims 42 to 55.

57. The engagement signal transduction domain comprises a first engagement signal transduction domain and a second engagement signal transduction domain, wherein the first engagement signal transduction domain comprises a TLR signal transduction domain, a Traf6 signal transduction domain, a Traf2 signal transduction domain or a Traf3 signal transduction domain, the B cell according to any one of claims 42 to 56.

58. The second engagement signal transduction domain is an FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, CD79b, TLR, Traf2, Traf3 or Traf6 signal transduction domain, the B cell according to claim 57.

59. The second engagement signaling domain is the FcγR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 63, the FcγR2A signaling domain comprising the amino acid sequence of SEQ ID NO: 64, the FcγR2C signaling domain comprising the amino acid sequence of SEQ ID NO: 65, the FcγR3A signaling domain comprising the amino acid sequence of SEQ ID NO: 66, the FcεRIγ signaling domain comprising the amino acid sequence of SEQ ID NO: 62, the BAFF-R signaling domain comprising the amino acid sequence of SEQ ID NO: 67, the DAP12 signaling domain comprising the amino acid sequence of SEQ ID NO: 68, the NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO: 69, the cleaved NFAM1 signaling domain comprising the amino acid sequence of SEQ ID NO: 70, the CD79b signaling domain comprising the amino acid sequence of SEQ ID NO: 71, the TLR1 signaling domain comprising the amino acid sequence of SEQ ID NO: 48, the TLR2 signaling domain comprising the amino acid sequence of SEQ ID NO: 49, the TLR3 signaling domain comprising the amino acid sequence of SEQ ID NO: 50, the TLR4 signaling domain comprising the amino acid sequence of SEQ ID NO: 51, the TLR5 signaling domain comprising the amino acid sequence of SEQ ID NO: 52, the TLR6 signaling domain comprising the amino acid sequence of SEQ ID NO: 53, the TLR7 signaling domain comprising the amino acid sequence of SEQ ID NO: 54, the TLR8 signaling domain comprising the amino acid sequence of SEQ ID NO: 55, the TLR9 signaling domain comprising the amino acid sequence of SEQ ID NO: 56, the Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO: 57, the cleaved Traf6 signaling domain comprising the amino acid sequence of SEQ ID NO: 58, the Traf2 signaling domain comprising the amino acid sequence of SEQ ID NO: 72 or the Traf3 signaling domain comprising the amino acid sequence of SEQ ID NO: 73, the B cell according to claim 58.

60. The B cell according to any one of claims 42 to 59, wherein signaling by the engagement signaling domain results in the expression of at least one of an inflammatory cytokine, an inflammatory chemokine or a costimulatory cell surface marker.

61. The inflammatory cytokine is TNFα, IL-1, IL-6, IL-12 or IL-23; the inflammatory chemokine is CCL5 (RANTES), CXCL9 or CXCL10; and the co-stimulatory cell surface marker is CD80, CD86, HLA-DR, CD40, HVEM or 4-1BBL, or any combination thereof, the B cell according to claim 60.

62. The B cell according to any one of claims 42 to 61, wherein the B cell is a naive B cell, a plasma cell, a regulatory B cell, a marginal zone B cell, a follicular B cell, a lymphoplasmacytoid cell, a plasmablast cell or a memory B cell.

63. The B cell according to any one of claims 42 to 62, wherein the B cell is a human B cell.

64. The B cell according to any one of claims 42 to 63, wherein the B cell is capable of engulfing a target cell expressing a target antigen on its surface.

65. The B cell according to claim 64, wherein the B cell is capable of phagocytosing a target cell expressing a target antigen on its surface.

66. The B cell according to any one of claims 42 to 65, wherein the B cell exhibits at least 20 phagocytic activity indices against a target cell expressing a target antigen on its surface.

67. The B cell according to any one of claims 42 to 66, wherein the B cell is capable of lysing a target cell expressing a target antigen on its surface.

68. A cell population comprising the host cell according to any one of claims 31 to 41 or the B cell according to any one of claims 42 to 67.

69. A pharmaceutical composition comprising the host cell according to any one of claims 31 to 41, the B cell according to any one of claims 42 to 67 or the cell population according to claim 68, and a pharmaceutically acceptable carrier.

70. A method of treating a subject having cancer, the method comprising administering to the subject an effective amount of the host cell according to any one of claims 31 to 41, the B cell according to any one of claims 42 to 67, the cell population according to claim 68, or the pharmaceutical composition according to claim 69.

71. A method of treating a subject having a disease, disorder or undesirable condition associated with overexpression of a tumor antigen, comprising administering to the subject an effective amount of a host cell according to any one of claims 31 to 41, a B cell according to any one of claims 42 to 67, a cell population according to claim 68, or a pharmaceutical composition according to claim 69.

72. A method of treating a subject having an autoimmune disease, disorder or undesirable condition, comprising administering to the subject an effective amount of a host cell according to any one of claims 31 to 41, a B cell according to any one of claims 42 to 67, a cell population according to claim 68, or a pharmaceutical composition according to claim 69.

73. A method of treating or preventing an infectious disease in a subject, comprising administering to the subject an effective amount of a host cell according to any one of claims 31 to 41, a B cell according to any one of claims 42 to 67, a cell population according to claim 68, or a pharmaceutical composition according to claim 69.

74. A method of treating a subject having a neurodegenerative disease, comprising administering to the subject an effective amount of a host cell according to any one of claims 31 to 41, a B cell according to any one of claims 42 to 67, a cell population according to claim 68, or a pharmaceutical composition according to claim 69.

75. The method according to any one of claims 70 to 74, wherein the cell is an autologous cell.

76. The method according to any one of claims 70 to 74, wherein the cell is an allogeneic cell.

77. The method according to any one of claims 70 to 76, further comprising administering a second therapeutic agent to the subject.

78. The method according to claim 77, wherein the second therapeutic agent is an antibody, radiation therapy, chemotherapeutic agent, cellular immunotherapy, antibiotic, antifungal or antiviral agent.

79. The method according to claim 77 or 78, wherein the cell containing CER is administered simultaneously with the second therapeutic agent or after administration of the second therapeutic agent.

80. The method according to any one of claims 77 to 79, wherein the second therapeutic agent is administered at a sub-therapeutic dose.

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