Chimeric TIM receptor and its use

Abstract

The present disclosure relates to chimeric Tim receptors, host cells modified to contain chimeric Tim receptor molecules, and methods of making and using such receptor molecules and modified cells.

Description

Technical Field

[0001] Declaration Regarding Array List The array list associated with this application is provided in text format instead of a paper copy and is hereby incorporated herein by reference. The name of the text file containing the array list is 200265_414WO_ST25.txt. The text file is 416 KB, was created on August 13, 2021, and was electronically submitted via EFS-Web.

Background Art

[0002] Upon exposure to an antigen, naive antigen-specific T cells undergo activation that promotes their development into functional effector T cells capable of clonal expansion, differentiation, and killing cells expressing cognate antigens (e.g., tumor cells). After antigen clearance, most of the effector T cells undergo apoptosis, and a subset of the surviving effector T cells differentiates into memory T cells that can provide long-term protection against antigen re-exposure. However, prolonged antigen exposure can cause T cell exhaustion and enable tumor cell persistence. T cell exhaustion refers to a dysfunctional state acquired by T cells undergoing persistent TCR stimulation, characterized by upregulated expression of immune checkpoint molecules (e.g., PD-1, CTLA-4, Tim-3), reduced effector function, poor proliferation, and metabolic impairment. Engineered T cells expressing chimeric antigen receptors (CARs) can also develop exhaustion.

Brief Description of the Drawings

[0003] [Figure 1]Figures 1A-1C: In vitro co-culture systems for evaluating T cell antigen-presenting function show that the addition of T cells containing a chimeric Tim4 receptor with a TLR intracellular signaling domain attached to the CD3ζ signaling domain (and optionally a CD28 signaling domain) enhances their ability to function as antigen-presenting cells (APCs). Figure 1A: Schematic diagram of co-culture of pCTX247 and pCTX1107 chimeric Tim4 receptors and pCTX1107 T cells pulsed with E7 peptide, and E7-specific T cells for evaluation of their antigen-presenting ability. Figure 1B: E7-specific proliferation response was measured by CT Violet dye dilution after 6 days in the presence of autologous CER-T (TLR-containing or non-TLR-containing CER) pulsed with E7 peptide. Figure 1C: Addition of TLR-2 ICD (pCTX1107) induced a proliferation response in E7-specific TCRs, while non-TLR-containing CERs were relatively less irritating. All data were collected by FACS. Cell tracing showed E7 TCR-T cells using anti-mouse TCRb. E7 TCR-T cells were labeled with CT Violet at the time of co-culture. [Figure 2] Figure 2: T cells containing a chimeric Tim4 receptor with a TLR intracellular signaling domain attached to the CD3ζ signaling domain (and optionally a CD28 signaling domain) are potent stimulants of the autologous E7-specific T cell response. CER-T cells were pulsed with the E7 peptide and tested for their ability to induce the autologous E7-specific T cell response. CD25, a T cell activation marker, was evaluated against E7 TCR-T cells 24 hours after CER-T cells were pulsed with the E7 peptide. CER-T pCTX1107 contains the TLR-2 ICD and is a potent stimulant of E7-specific activation. [Figure 3]Figure 3: T cells containing a chimeric Tim4 receptor with a TLR intracellular signaling domain attached to the CD3ζ signaling domain (and optionally a CD28 signaling domain) are potent stimulants of autologous E7-specific T cell responses. CER-T cells were pulsed with E7 peptides and tested for their ability to induce autologous E7-specific T cell responses. CD69, an early marker of T cell activation, was evaluated against E7 TCR-T cells 24 hours after CER-T cells were pulsed with the E7 peptide. CER-T pCTX1107 contains a TLR-2 ICD and is a potent stimulant of E7-specific activation. [Figure 4] Figure 4: Robust cell surface chimeric Tim4 receptor expression and detection using anti-Tim4 antibody. Chimeric Tim4 receptor cell surface staining was evaluated using anti-Tim4 antibody (9F4) on day 5 post-transduction. pCTX1107 contains the TLR-2 intracellular sequence. The lentiviral cassette contains the p2A fragment and the subsequent cleaved EGFRt polypeptide. [Figure 5]Figures 5A-5C: pCTX131 CER-T cells enhance the efficacy of CD1928z CAR-T cells. Figure 5A: Dynamics of Ptd-Ser induction in JeKo-1 MCL cells in response to CD1928z CAR-T cells. JeKo-1 MCL cells were co-cultured in an increasing effector:target ratio, and Ptd-Ser induction was evaluated over time. The dynamic curve represents the percentage of viable JeKo-1 targets that bind to rTim-4, a Ptd-Ser binding protein. Figure 5B (upper): JeKo-1 cells were co-cultured for 48 hours in a 1:1 T cell:JeKo-1 ratio with pCTX184(1928z)+pCTX131, pCTX184+CTX156 control T cells, or pCTX184 cells alone. Samples treated with CTX184+CER131 showed substantially fewer tumor cells in culture after approximately 2 days compared to samples treated with CTX184 alone or with CTX184+CTX156 control T cells. All data were collected via FACS. Figure 5B bottom: Representative flow plot for enumeration of residual JeKo-1 cells after 48 hours of co-culture. Figure 5B top: Raw flow data from Figure 5B bottom used to calculate the bar graph of residual JeKo-1 cells. Figure 5C: JeKo-1 cells were co-cultured for 48 hours with pCTX184(1928z)+pCTX131, pCTX184+CTX156 control T cells, or pCTX184 cells alone at a T cell:JeKo-1 ratio of 0.5:1. Samples treated with CTX184+CER131 showed increased IFN-γ secretion compared to samples treated with CTX184 alone or with CTX184+CTX156 control T cells. [Figure 6]Figures 6A-6B: pCTX131 chimeric Tim4 receptor-T cells enhance the potency of CD1928z CAR-T cells. Evaluating the cytotoxic response of a mixture of CD1928z CAR-T (also known as pCTX184, CAR184) + pCTX131 chimeric Tim4 receptor-T cells. Figure 6A: pCTX131 chimeric Tim4 receptor-T cells were combined with CD1928z CAR-T cells (pCTX184) in various ratios, and JekO-1 cell counts were quantified over time. Figure 6B: Caspase 3 / 7 response. All data were collected via incucyte. [Figure 7] Figures 7A-7B: TLR-2 containing pCTX133 and a chimeric Tim4 receptor enhances the efficacy of niraparib in an ovarian cancer model. Figure 7A: Flow cytometry measurement of surface PtdSer. Kuramochi cells were treated with 1.56 or 25 μM niraparib, or with an equivalent volume of DMSO (control). After 48 hours, samples were trypsinized and stained with Tim4-Fc and subsequent fluorescently labeled secondary antibodies against Tim4-Fc. Figure 7B: Kuramochi cells pretreated with 1.56 μM niraparib for approximately 20 hours were co-cultured with pCTX133 and untransduced CD4 T cells from donor 32 at a T cell:Kuramochi ratio of 2:1 and a final niraparib concentration of 1.56 μM. Samples treated with niraparib + pCTX133 showed significantly fewer tumor cells in culture after approximately 3 days compared to samples treated with niraparib alone or niraparib + untransduced T cells. All data were collected via IncuCyte. [Figure 8]Figures 8A-8B: Combination of chimeric Tim4 receptor-T cells + BTK inhibitor (ibrutinib) for hematological malignancies. Figure 8A: Ibrutinib induces phosphatidylserine expression on target cells. Figure 8B: Synergistic chimeric Tim4 receptor-T cell-mediated cell death in combination with a BTK inhibitor small molecule. CTX136(Tim4-CD28-CD3z) T cells co-cultured in 3:1, 2:1, and 1:1 E:T in the presence of ibrutinib showed a substantial increase in cell death compared to empty vector transducers or ibrutinib treatment alone. [Figure 9] Figures 9A and 9B show the transfection of Jurkat cells with various chimeric Tim4 receptor constructs: pCTX1183, pCTX1161, pCTX1189, pCTX1184, pCTX1163, pCTX1162, pCTX1190, pCTX1186, pCTX1187, pCTX1164, pCTX1185, and pCTX1165. Figure 9B shows the results standardized against untransfected cells. [Figure 10] Figure 10 is a bar graph showing that activation of HPV E7 TCR T cells, mediated by antigen presentation by chimeric Tim4-T cells, is blocked by anti-HLA-I antibodies. [Modes for carrying out the invention]

[0004] In one embodiment, the present disclosure provides a chimeric T cell immunoglobulin mucin protein (Tim) receptor, also referred to as a chimeric phagocytic receptor (CER). The chimeric Tim receptor of the present disclosure confers phagocytic and / or cytotoxic activity to chimeric Tim receptor-modified host cells (e.g., T cells), the cytotoxic activity being induced upon binding of the chimeric Tim receptor to its target antigen, phosphatidylserine. In some embodiments, the chimeric Tim receptor confers phagocytosis, cytotoxicity, and enhanced antigen capture, antigen processing, and antigen presentation activity to modified host cells (e.g., T cells).

[0005] In some embodiments, the chimeric Tim receptor described herein comprises a single-chain chimeric protein comprising (a)(i) a Tim4 IgV domain and a Tim1 mucin domain; or (ii) an extracellular domain comprising a binding domain comprising a Tim1 IgV domain and a Tim4 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain and an appropriate secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0006] In some embodiments, the chimeric Tim receptor described herein comprises a single-chain chimeric protein comprising: (a) an extracellular domain comprising a binding domain comprising a Tim1 IgV domain and a Tim1 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain and an optional secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0007] In some embodiments, the chimeric Tim receptor described herein comprises a single-chain chimeric protein comprising: (a) an extracellular domain comprising (i) a Tim1 IgV domain and a Tim1 mucin domain; (ii) a Tim4 IgV domain and a Tim4 mucin domain; (iii) a Tim1 IgV domain and a Tim4 mucin domain; or (iv) a binding domain comprising a Tim4 IgV domain and a Tim1 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain selected from a Tim1 signaling domain or a Tim4 signaling domain and an appropriate secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0008] In some embodiments, the chimeric Tim receptor described herein comprises a single-chain chimeric protein comprising: (a)(i) an extracellular domain comprising a binding domain comprising a Tim4 IgV domain and a Tim4 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain selected from a CD28 signaling domain, a CD3ζ signaling domain, and a 4-1BB signaling domain, and a secondary intracellular signaling domain selected from a TLR signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0009] In some embodiments, the chimeric Tim receptor described herein comprises a single-chain chimeric protein comprising (a)(i) an extracellular domain comprising a binding domain comprising a Tim4 IgV domain and a Tim4 mucin domain; (b) a primary intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling domain; a secondary intracellular signaling domain comprising a co-stimulatory signaling domain, a Tim1 signaling domain, or a Tim4 signaling domain; and an intracellular signaling domain comprising a tertiary intracellular signaling domain comprising a TLR signaling domain.

[0010] In some embodiments, the extracellular domain of the chimeric Tim receptor described herein may include an extracellular spacer domain located between and connecting the binding domain and the transmembrane domain.

[0011] In some embodiments, chimeric Tim receptors may also be able to co-stimulate T cells via signaling pathways different from the “classical” T cell co-stimulatory pathway (e.g., CD28). For example, in addition to binding to phosphatidylserine, Tim4 is also a ligand for Tim1 expressed on the surface of activated T cells. Tim1 can also bind to phosphatidylserine. Tim4-induced Tim1 signaling has been found to co-stimulate T cell proliferation and survival (Hartt Meyers et al., 2005, Nat. Immunol. 6:455). Thus, in certain embodiments, cytotoxic chimeric Tim receptors may reduce or inhibit T cell exhaustion or restore exhausted T cells by providing a co-stimulatory signal via at least one signaling pathway. In certain embodiments, cytotoxic chimeric Tim receptors provide a co-stimulatory signal via at least two different signaling pathways (e.g., via a selected co-stimulatory signaling domain and Tim1 in the cytotoxic chimeric Tim receptor).

[0012] In some embodiments, when expressed in host cells, the chimeric Tim receptor of this disclosure also confers phagocytic activity to the host cells. For example, in certain such embodiments, binding of the chimeric Tim receptor expressed in host cells to a phosphatidylserine target can induce both cytolytic and phagocytic responses by the host cells. In certain embodiments of modified host cells described herein, prior to modification using the chimeric Tim receptor, the host cells do not naturally exhibit a phagocytic phenotype.

[0013] In another embodiment, administration of host cells modified using the chimeric Tim receptor of this disclosure can be used, for example, in a method for removing target cells that harbor surface-exposed phosphatidylserine for the treatment of cancer. In normal healthy cells, phosphatidylserine is located in the inner leaflet of the plasma membrane. However, certain cellular events such as injury, apoptosis, necrosis, and stress activate "scramblases" that rapidly expose phosphatidylserine to the cell surface, and these scramblases can bind to receptors such as Tim4 or Tim1. Endogenous tumor-specific T effector cells can induce exposure of phosphatidylserine on the outer membrane of targeted tumor cells during cell lysis. Furthermore, certain cancer therapies (e.g., chemotherapy, radiotherapy, CAR-T cells, etc.) can induce exposure of phosphatidylserine on targeted tumor cells or cells in the tumor microenvironment by inducing apoptosis, cellular stress, cell damage, etc. Engineered host cells expressing the chimeric Tim receptor disclosed herein can clear damaged tumor cells, stressed tumor cells, apoptotic tumor cells, or necrotic tumor cells that retain surface-exposed phosphatidylserine by inducing apoptosis in tumor cells that retain surface-exposed phosphatidylserine. In certain embodiments, host cells expressing the chimeric Tim receptor disclosed herein clear damaged tumor cells, stressed tumor cells, apoptotic tumor cells, or necrotic tumor cells that retain surface-exposed phosphatidylserine by inducing apoptosis and by phagocytosis. Engineered host cells containing the chimeric Tim receptor described herein may be administered to a subject alone or in combination with one or more additional therapeutic agents, such as CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, oncolytic viruses, electropulse therapy, etc.

[0014] In another embodiment, host cells modified with the chimeric Tim receptor of this disclosure may be used in a method to enhance effector responses (e.g., tumor-specific immune responses). In an embodiment, host cells modified with the chimeric Tim receptor of this disclosure may be used in a method to enhance antitumor efficacy (e.g., tumor transport, growth and persistence). Embodiments of the chimeric Tim receptor of this disclosure can co-stimulate T cells via at least one costimulatory signaling pathway upon phosphatidylserine binding. In a particular embodiment, the chimeric Tim receptor described herein provides costimulatory signals via at least two different signaling pathways. In a particular embodiment, the enhanced effector response may be enhanced T cell proliferation, cytokine production, cytotoxic activity, persistence, or any combination thereof. Host cells expressing the chimeric Tim receptor described herein may be administered to a subject alone or in combination with one or more additional therapeutic agents, such as CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, oncolytic viruses, electropulse therapy, etc.

[0015] In another embodiment, host cells modified with the chimeric Tim receptor of this disclosure may be used in methods for inhibiting or reducing immune cell exhaustion. In certain embodiments, immune cell exhaustion refers to T cell exhaustion, NK cell exhaustion, or both. Tumor cells can often provide continuous antigenic stimulation to immune cells in the absence of a costimulatory ligand that can lead to immune cell exhaustion (e.g., reduced proliferative capacity, reduced effector function, and upregulation of immunosuppressive molecules). Cancer therapies, such as chemotherapy, radiotherapy, and CAR-T cell therapy, can also provide prolonged antigenic stimulation in the absence of costimulatory signals, or when the intensity or duration of costimulatory signals is limited. The chimeric Tim receptor of this disclosure can co-stimulate immune cells via at least one costimulatory signaling domain upon binding of phosphatidylserine. In certain embodiments, the chimeric Tim receptor provides costimulatory signals via at least two different signaling pathways. Host cells expressing chimeric Tim receptors may be administered to a target alone or in combination with one or more additional therapeutic agents, such as CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, oncolytic viruses, and electropulse therapy.

[0016] In some embodiments, host cells (e.g., T cells) modified with the chimeric Tim receptor of this disclosure exhibit enhanced antigen capture, antigen processing, and antigen presentation activity. Ligand binding to the phagocytic receptor moiety of the chimeric Tim receptor mediates a cascade of events including T cell activation, signaling, cytolytic function, cytokine and chemokine production, partial phagocytosis of target cells, and a downstream transcriptional program resulting in the presentation of target cell antigens. Expression of the chimeric Tim receptor in non-phagocytic or attenuated phagocytic immune cells, e.g., mature polyclonal T cells, can enable and enhance antigen-specific capture through phagocytosis of target cell fragments. In some embodiments, the added functionality of chimeric Tim receptor-mediated antigen capture supports enhanced presentation of non-targeting antigens while inducing direct cytolytic activity against primed tumor cell targets.

[0017] For combination therapeutic compositions and methods comprising a chimeric Tim receptor and a cellular immunotherapy, e.g., CAR or TCR, as described herein, the chimeric Tim receptor and the cellular immunotherapy agent (e.g., CAR or TCR) may be expressed on separate engineered cells or on the same engineered cells to generate bispecific, multifunctional engineered cells. The chimeric Tim receptor and the cellular immunotherapy agent expressed on the same engineered cells may be expressed from separate vectors or on the same vector as the multicistronic construct.

[0018] In another embodiment, host cells modified with the chimeric Tim receptor of this disclosure may be used to enhance the effects of therapeutic agents that induce cellular stress, injury, necrosis, or apoptosis. For example, certain therapeutic agents, such as chemotherapy, specific inhibitors of driver mutations associated with cancer (targeting therapies, e.g., BRAF inhibitors, EGRF inhibitors, ALK / ROS1 kinase inhibitors, BTK inhibitors), radiotherapy, UV phototherapy, electropulsed therapy, adoptive cell immunotherapy (e.g., CAR-T cells, TCRs), and oncolytic virus therapy, can induce cellular injury or cell death in tumor cells or diseased cells. Cells expressing the chimeric Tim receptor described herein can bind to phosphatidylserine moieties exposed on the outer leaflets of injured or dying cells resulting from any one or more of such therapeutic agents, and can induce cytolysis or both cytolysis and phagocytosis of targeted cells.

[0019] Before providing further details of this disclosure, it may be helpful to provide definitions of certain terms used herein to aid in understanding this disclosure.

[0020] In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the listed range, and, where appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer), unless otherwise indicated. Similarly, any numerical range listed herein relating to any physical feature, e.g., polymer subunits, size, or thickness, should be understood to include any integer within the listed range, unless otherwise indicated. Where used herein, the term “approximately” means ±20% of the indicated range, value, or structure, unless otherwise indicated. Where used herein, the terms “a” and “an” should be understood to refer to “one or more” of the listed components. The use of options [e.g., “or”] should be understood to mean one of the options, both, or any combination thereof. Where used herein, the terms “include,” “have,” and “comprise” are used synonymously, and their variations are intended to be interpreted as non-limiting.

[0021] Terms understood by those skilled in the art in the field of antibody technology shall be given the meanings obtained in the art unless explicitly defined otherwise herein. The term “antibody” is used in its broadest sense and includes polyclonal and monoclonal antibodies. “Antibody” may mean an intact antibody comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, and an antigen-binding portion (or antigen-binding domain) of an intact antibody having or retaining the ability to bind a target molecule. Antibodies may be natural, recombinant, genetically engineered or modified forms of immunoglobulins, e.g., intrabodies, peptidebodies, nanobodies, single-domain antibodies, SMIPs, and multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv, ADAPTIR). The monoclonal antibody or its antigen-binding moiety may be a non-human, chimeric, humanized, or human monoclonal antibody or its antigen-binding moiety, preferably a humanized or human monoclonal antibody or its antigen-binding moiety. Immunoglobulin structure and function are outlined, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The “antigen-binding moiety” or “antigen-binding domain” of an intact antibody means that it encompasses an “antibody fragment,” which represents a portion of the intact antibody and refers to the antigenicity-determining variable region or complementarity-determining region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, Fab'-SH, F(ab')2, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. "Fab (fragment antigen binding)" is a part of an antibody that binds to an antigen and includes a variable region and a heavy chain CH1 linked to the light chain via an interchain disulfide bond.The antibody may be an antibody of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0022] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs [see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)]. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using the VH or VL domain from an antibody that binds to the antigen, and libraries of complementary VL or VH domains may be screened, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0023] The terms "complementarity-determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to discontinuous 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).

[0024] 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, combine, recognize, or combine with a target molecule (e.g., phosphatidylserine). A binding domain includes any natural, synthetic, semi-synthetic, or recombinant-produced binding partner for the biomolecule of interest or other target. In some embodiments, a binding domain is an antigen-binding domain, such as an antibody or its functional binding domain or antigen-binding moiety. Exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor extradomains (e.g., Tim4), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to biomolecules.

[0025] A "T cell receptor" (TCR) generally refers to a molecule found on the surface of T cells (also called T lymphocytes) that is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In most T cells, the TCR consists of a disulfide-bonded heterodimer of highly variable α and β chains (also known as TCRα and TCRβ, respectively). In a small subset of T cells, the TCR consists of a heterodimer of γ and δ chains (also known as TCRγ and TCRδ, respectively). Each chain of the TCR is a member of the immunoglobulin superfamily and has one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus (Janeway et al., Immunobiology: The Immune System in Health and Disease, 3). rd(See Ed., Current Biology Publications, p. 4:33, 1997). The TCRs of this disclosure may be from a variety of animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. The TCRs may be cell-binding (i.e., having transmembrane regions or domains) or soluble. TCRs include recombinant production, genetic engineering, fusion or modification forms of TCRs, including, for example, scTCRs, soluble TCRs, and TCR fusion constructs [TRuC®; see U.S. Patent Application Publication No. 2017 / 0166622].

[0026] The terms "variable regions" or "variable domains" of the TCR α chain (Vα) and β chain (Vβ), or Vγ and Vδ of the γδ TCR, refer to the TCR's involvement in antigen binding. α and V β They generally have a similar structure, and each variable domain contains four conserved FRs and three CDRs. α The domain is encoded by two separate DNA segments: a variable gene segment (V gene) and a zygote gene segment (J gene); V β The domain is encoded by three separate DNA segments: the variable gene segment (V gene), the diversity gene segment (D gene), and the juxtaposition gene segment (J gene). α or V β The domains may be sufficient to confer antigen-binding specificity. "Major histocompatibility complex (MHC) molecules" refer to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers consisting of a transmembrane α chain (having three α domains) and non-covalently associated β2-microglobulin. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which transmembrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, and on the cell surface, the peptide:MHC complex binds to CD8 +It is recognized by T cells. MHC class II molecules deliver peptides of endocytic origin to the cell surface, where they are recognized by CD4 + It is recognized by T cells. MHC molecules can be MHC molecules from a variety of animal species, including humans, mice, rats, or other mammals.

[0027] A "chimeric antigen receptor" (CAR) refers to a chimeric protein containing two or more distinct domains that, when expressed on the cell surface, can function as a receptor. CARs generally consist of an extracellular domain containing a binding domain that binds to a target antigen, an optional extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain [e.g., a T cell activation motif containing an immunoreceptor tyrosine-based activation motif (ITAM), and optionally, an intracellular co-stimulatory domain]. In certain embodiments, the intracellular signaling domain of the CAR has an ITAM-containing T cell activation domain (e.g., CD3ζ) and an intracellular co-stimulatory domain (e.g., CD28). In certain embodiments, the CAR is synthesized as a single polypeptide chain or encoded by a nucleic acid molecule as a single-chain polypeptide.

[0028] To identify the binding domains of the present disclosure that specifically bind to a particular target and to determine binding domain affinity, various assays are known, such as Western blot, ELISA, and BIAcore® analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; and 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 has an affinity or K 5 M -1 equal to or greater than that to the target molecule, or K aThis refers to association or coalescence based on a specific bond interaction equilibrium association constant (i.e., a unit of 1 / M), where the molecules do not significantly associate or coalesce with any other molecules or components in the sample.

[0029] The terms “antigen” and “Ag” refer to molecules that can induce an immune response. The induced immune response may include antibody production, activation of specific immunocompetent cells, or both. Macromolecules, including proteins, glycoproteins, and glycolipids, can act as antigens. Antigens may be recombinant or derived from genomic DNA. As intended herein, antigens do not necessarily have to be encoded by (i) the full-length nucleotide sequence of a gene alone, or by (ii) a “gene.” Antigens may be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids.

[0030] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant within an antigen to which a geneic immunobinding molecule, such as an antibody or fragment thereof (e.g., scFv), a T cell receptor (TCR), a chimeric Tim receptor, or other binding molecule, domain, or protein specifically binds. Epitope determinants generally consist of a group of chemically active surfaces of a molecule, such as amino acids or sugar side chains, and may have specific three-dimensional structural and specific charge characteristics. Epitopes can be linear or conformational epitopes.

[0031] As used herein, the term “Tim4” (T cell immunoglobulin and mucin domain-containing protein 4), also known as “TimD4,” refers to a phosphatidylserine receptor typically expressed on antigen-presenting cells such as macrophages and dendritic cells. Tim4 mediates phagocytosis of apoptotic, necrotic, injured, wounded, or stressed cells by presenting phosphatidylserine (PtdSer) on the outer surface (outer) leaflet of the cell membrane. Tim4 can also bind to Tim1 expressed on the surface of T cells and induce proliferation and survival. In certain embodiments, Tim4 refers to human Tim4. An exemplary human Tim4 protein contains the amino acid sequence of SEQ ID NO: 1.

[0032] As used herein, the term “Tim4-binding domain” refers to the N-terminal immunoglobulin fold domain of Tim4, which possesses a metal ion-dependent pocket that selectively binds to PtdSer. An exemplary human Tim4-binding domain comprises the amino acid sequence of SEQ ID NO: 2, and an exemplary mouse Tim4-binding domain comprises the amino acid sequence of SEQ ID NO: 24.

[0033] The Tim4 binding domain comprises a variable immunoglobulin (IgV)-like domain (hereinafter referred to herein as the "IgV domain") and a mucin-like domain (hereinafter referred to herein as the "mucin domain"). An exemplary human Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO: 34, and an exemplary human Tim4 mucin domain comprises the amino acid sequence of SEQ ID NO: 35. In certain embodiments, the Tim4 binding domain does not contain a signal peptide. An exemplary human Tim4 signal peptide has the amino acid sequence of SEQ ID NO: 11. An exemplary mouse Tim4 signal peptide has the amino acid sequence of SEQ ID NO: 25.

[0034] As used herein, the term "Tim1" (T cell immunoglobulin and mucin domain-containing protein 1) refers to a phosphatidylserine receptor expressed on the surface of T cells. Tim1 as described above can also bind to Tim4 expressed on the surface of antigen-presenting cells. In certain embodiments, Tim1 refers to human Tim1. An exemplary human Tim1 protein contains the amino acid sequence of SEQ ID NO: 36.

[0035] As used herein, the term “Tim1-binding domain” refers to the N-terminal immunoglobulin fold domain of Tim1 that selectively binds to PtdSer. An exemplary human Tim1-binding domain includes the amino acid sequence of SEQ ID NO: 37.

[0036] The Tim1 binding domain includes an IgV domain and a mucin domain. An exemplary human Tim1 IgV domain comprises the amino acid sequence of SEQ ID NO: 38, and an exemplary human Tim1 mucin domain comprises the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the Tim1 binding domain does not include a signal peptide. An exemplary human Tim1 signal peptide has the amino acid sequence of SEQ ID NO: 40.

[0037] As used herein, “effector domain” is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote an intracellular biological or physiological response that expresses the effector domain upon receiving a suitable signal. In certain embodiments, the effector domain is a portion of a protein or protein complex that receives a signal upon binding, or it directly binds to a target molecule that elicits a signal from the effector domain. The effector domain may directly promote a cellular response if it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, the effector domain indirectly promotes a cellular response by associating with one or more other proteins that directly promote the cellular response.

[0038] As used herein, “costimulatory signaling domain” refers to an intracellular signaling domain of a costimulatory molecule, or a functional portion thereof, that, when activated in conjunction with a major or classical (e.g., ITAM-driven) activation signal (e.g., provided by the CD3ζ intracellular signaling domain), promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. Co-stimulatory signaling domains include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, or any combination thereof.

[0039] As used herein, “immune receptor tyrosine-based activation motif (ITAM) activation domain” refers to an intracellular signaling domain or functional portion thereof that is naturally or endogenously present on an immune cell receptor or cell surface marker and contains at least one immune receptor tyrosine-based activation motif (ITAM). ITAM is YXXL / IX 6-8 - Refers to the conserved motif YXXL / I. In certain embodiments, the ITAM signaling domain contains one, two, three, four or more ITAMs. The ITAM signaling domain can initiate T cell activation signaling after antigen binding or ligand engagement. The ITAM signaling domain includes, for example, the intracellular signaling domains CD3γ, CD3δ, CD3ε, CD3ζ, CD79a, and CD66d.

[0040] A "junctional amino acid" or "junctional amino acid residue" refers to one or more (e.g., about 2 to 20) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide. Junctional amino acids can arise from the construct design of chimeric proteins (e.g., amino acid residues resulting from the use of restriction enzyme sites during the construction of nucleic acid molecules encoding chimeric proteins).

[0041] "Nucleic acid molecules" and "polynucleotides" can be in the form of RNA, or DNA, including cDNA, genomic DNA, and synthetic DNA. Nucleic acid molecules may consist of native nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of native nucleotides (e.g., α-enantiomers of native nucleotides), or a combination of both. Modified nucleotides may have modifications or substitutions of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such bonds. Analogues of phosphodiester bonds include phosphorothioates, phosphorodioates, phosphoroselenoates, phosphorodiodiselenoates, phosphoranilothioates, phosphoranilidates, phosphoramidates, etc. Nucleic acid molecules may be double-stranded or single-stranded, and if single-stranded, they may be coding strands or non-coding (antisense strands). The coding molecule may have the same coding sequence as a coding sequence known in the art, or it may have a different coding sequence that can code for the same polypeptide as a result of duplication or degeneracy of the gene code, or by splicing.

[0042] "Code" refers to the inherent property of a particular polynucleotide sequence, such as DNA, cDNA, and mRNA sequences, to act as a template for the synthesis of other polymers and macromolecules having either a specific nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a specific amino acid sequence in biological processes, and the biological properties that result therefrom. Thus, a polynucleotide codes for a protein if the transcription and translation of the mRNA corresponding to that polynucleotide in a cell or other biological system produces a protein. Both the coding and non-coding strands may be referred to as coding for a protein or other product of the polynucleotide. Unless otherwise specified, "nucleotide sequences that code for an amino acid sequence" include all nucleotide sequences that are degenerate of each other and code for the same amino acid sequence.

[0043] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds consisting of 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 constitute a protein sequence or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and long chains, generally called proteins in the art, of which there are many types. Polypeptides include, among others, bioactive fragments, substantially homopolymers, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0044] As used herein, the terms “mature polypeptide” or “mature protein” refer to proteins or polypeptides that are secreted or localized to the cell membrane or to certain organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes) and that do not contain an N-terminal signal peptide.

[0045] A "signal peptide," also known as a "signal sequence," "leader sequence," "leader peptide," "localization signal," or "localization sequence," is a short peptide (typically 15-30 amino acids in length) located at the N-terminus of a newly synthesized protein destined for secretory pathways. A signal peptide typically contains a short range 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 the signal peptidase. In eukaryotes, the signal peptide induces the translocation of the newly synthesized protein to the endoplasmic reticulum, where it is cleaved by the signal peptidase to create a mature protein, which then proceeds to its appropriate destination.

[0046] The term "chimera" refers to any nucleic acid molecule or protein that contains conjugated or linked sequences that are not endogenous and are not typically found conjugated or linked together in nature. For example, a chimeric nucleic acid molecule may contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that found in nature.

[0047] As used herein, the terms “endogenous” or “native” refer to genes, proteins, compounds, molecules, or activities that are normally present in a host or host cell, including naturally occurring variants of genes, proteins, compounds, molecules, or activities.

[0048] As used herein, “homologous” or “same species” means a molecule or activity that is ancestrally related to a second gene or activity from a host cell, for example, from the same host cell, from a different host cell, from a different organism, from a different lineage, from a different species. For example, a heterologous molecule, or a heterologous gene encoding a molecule, may be homologous to the native host cell molecule or gene encoding the molecule, and may have altered structure, sequence, expression level, or any combination thereof.

[0049] As used herein, “heterogeneous” nucleic acid molecule, construct, or sequence means a nucleic acid molecule or portion of a nucleic acid molecule that is not native to the host cell but may be homogeneous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of heterogeneous nucleic acid molecules, constructs, or sequences may be from a different genus or species. In some embodiments, heterogeneous nucleic acid molecules are not native. In certain embodiments, heterogeneous nucleic acid molecules are added to a host cell or host genome (i.e., not endogenous or native) by means of, for example, conjugation, transformation, transfection, transduction, electroporation, or otherwise, and the added molecule may be integrated into the host cell genome, exist as extrachromosomal gene material (e.g., as plasmid or other forms of self-replicating vectors), or exist in multiple copies. In addition, “heterogeneous” means non-native enzymes, proteins, or other activities encoded by non-endogenous nucleic acid molecules introduced into the host cell, even if the host cell encodes a homogeneous protein or activity.

[0050] As used herein, the terms “manipulated,” “recombinant,” “modified,” or “non-natural” refer to organisms, microorganisms, cells, nucleic acid molecules, or vectors modified by the introduction of heterologous nucleic acid molecules, or to cells or microorganisms genetically engineered by human intervention, i.e., modified by the introduction of heterologous nucleic acid molecules, or to cells or microorganisms in which the expression of endogenous nucleic acid molecules or genes is regulated, deregulated, or altered to be constitutive, and such alteration or modification can be introduced by genetic engineering. Human-produced genetic modifications may include, for example, modifications that introduce nucleic acid molecules encoding one or more proteins, chimeric receptors, or enzymes (which may include expression regulatory elements such as promoters); or additions, deletions, substitutions of other nucleic acid molecules; or other functional disruptions of the cellular genetic material or additions to the cellular genetic material. Exemplary modifications include modifications in coding regions or functional fragments thereof, heterologous or homologous polypeptides from the reference molecule or parent molecule. Additional exemplary modifications include, for example, modifications in non-coding regulatory regions in which the modification alters the expression of a gene or operon.

[0051] As used herein, the term “transgene” refers to a gene or polynucleotide encoding a target protein (e.g., a chimeric Tim receptor) that is desired to be expressed in a host cell and has been translocated to the cell by genetic engineering techniques. Transgenes may encode proteins for therapeutic purposes, as well as proteins that are reporters, tags, markers, suicide proteins, etc. Transgenes may be transgenes from natural sources, modified natural genes, recombinant or synthetic molecules. In certain embodiments, the transgene is a component of a vector.

[0052] The term "overexpressed" antigen or "overexpression" of an antigen refers to abnormally high levels of an antigen in cells. Overexpressed antigens or overexpression of an antigen are often associated with disease conditions, such as hematological malignancies and disease conditions in cells that form solid tumors within specific tissues or organs of interest. Solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0053] The "identity percentage" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by those sequences, taking into account 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 (i.e., identity % = number of identical positions / total number of positions x 100). The comparison of sequences between two or more sequences and the determination of the identity percentage can be achieved using mathematical algorithms such as the BLAST and Gapped BLAST programs with their default parameters (e.g., Altschul et al., J. Mol. Biol. 215:403, 1990; also see BLASTN at www.ncbi.nlm.nih.gov / BLAST).

[0054] A "conservative substitution" is recognized in the art as the substitution of one amino acid with another amino acid having similar properties. Exemplary conservative substitutions are well known in the art [see, for example, WO97 / 09433, published March 13, 1997, page 10; 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].

[0055] The term "promoter," as used herein, is defined as a DNA sequence that is recognized by or introduced into a cellular synthetic mechanism and is required to initiate the specific transcription of a polynucleotide sequence.

[0056] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence required for the expression of a gene product operably ligated to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, it may also include an enhancer sequence and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, be a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0057] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that codes for or identifies a gene product, results in the gene product being produced within the cell under most or all physiological conditions.

[0058] An "inducible" promoter is a nucleotide sequence that, when operably ligated to a polynucleotide encoding or identifying a gene product, results in the gene product being produced within the cell only if a corresponding inducer is present within the cell.

[0059] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that codes for a gene or is identified by a gene, results in the gene product being produced within the cell only if the cell is effectively a cell of the tissue type corresponding to the promoter.

[0060] When used herein, the phrases “transcriptionally controlled” or “operatably linked” mean that the promoter is present in the correct location and orientation with respect to the polynucleotide in order to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0061] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. Vectors can be, for example, plasmids, cosmids, viruses, or phages. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells. An "expression vector" is a vector that, when present in the appropriate environment, can direct the expression of proteins encoded by one or more genes contained within the vector.

[0062] In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma-retrovirus vectors, and lentivirus vectors. A “retrovirus” is a virus that has an RNA genome. A “gamma-retrovirus” refers to a genus of the retroviridae family. Examples of gamma-retroviruses include mouse stem cell viruses, mouse leukemia viruses, feline leukemia viruses, feline sarcoma viruses, and avian reticuloendotheliosis viruses. A “lentivirus” refers to a genus of retroviruses that can infect dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus) (including HIV1 and HIV2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0063] In other embodiments, the vector is a nonviral vector. Examples of nonviral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplified mRNA, closed-ended linear duplex (CELiD) DNA, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). When a nonviral delivery system is used, the delivery medium may be a liposome. Lipid formulations may be used to introduce nucleic acids into host cells in vitro, ex vivo, or in vivo. The nucleic acid may be encapsulated within a liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule that associates with both the liposome and the nucleic acid, contained in a micelle or complexed with a micelle, or otherwise associated with lipids.

[0064] As used herein, the term “phagocytosis” refers to a receptor-mediated process in which an endogenous or exogenous cell or particle with a diameter greater than 100 nm is internally transported by a phagocyte or host cell of the present disclosure. Phagocytosis typically consists of several steps: (1) tethering of the target cell or particle via direct or indirect (via cross-linking molecules) binding of a phagocytic receptor to a prophagocytic marker or antigenic marker on the target cell or particle; and (2) internal transport or phagocytosis of the whole or whole or part thereof of the target cell or particle. In certain embodiments, internal transport may occur via cytoskeletal rearrangement of the phagocyte or host cell, forming a phagosome, which is a membrane-bound compartment containing the internally transported target. Phagocytosis may further involve the maturation of the phagosome, which becomes increasingly acidic and fuses with a lysosome (forming a phagolysosome), in which case the phagocyted target is degraded (e.g., “phagocytosis”). Alternatively, phagosome-lysosome fusion may not be observed in phagocytosis. In another embodiment, phagosomes may regurgitate or excrete their contents into the extracellular environment before complete degradation. In some embodiments, phagocytosis refers to phagocytosis. In some embodiments, phagocytosis includes the tethering of a target cell or particle by phagocytic cells of the host cell of the disclosure, but does not include internal migration. In some embodiments, phagocytosis includes the tethering of a target cell or particle by phagocytic cells of the host cell of the disclosure, and internal migration of portions of the target cell or particle.

[0065] As used herein, the term “phagocytosis” refers to the process of phagocytosis of a cell or large particle (0.5 μm or larger) in which the target cell or particle is tethered, engulfed, and the internalized target cell or particle is degraded. In certain embodiments, phagocytosis includes the formation of a phagosome containing the internalized target cell or particle, and phagosome fusion with a lysosome to form a phagolysosome, whose contents are degraded. In certain embodiments, during phagocytosis, a phagocytic synapse is formed after binding of a chimeric Tim receptor expressed on the host cell of this disclosure to phosphatidylserine expressed by the target cell or particle; an actin-rich phagocytic cup is generated at the phagocytic synapse; a phagocytic arm extends around the target cell or particle through cytoskeletal rearrangement; and finally, the target cell or particle is drawn into the phagocytic cell or host cell through a force generated by a motor protein. 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.

[0066] The term “immune system cells” or “immune cells” refers to any cell of the immune system derived from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells give rise to two main lineages: myeloid progenitor cells (which give rise to bone marrow cells, e.g., monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphocyte progenitor cells (which give rise to lymphocyte cells, e.g., T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8-2 negative T cells, γδ T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells are sometimes also called “antigen-presenting cells” or “APCs (antigen presenting cells),” and these are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of APCs, which are complexed with peptides, interact with the TCR on the surface of T cells.

[0067] The term "T cell" refers to cells of the T cell lineage. "Cells of the T cell lineage" refers to cells that exhibit at least one phenotypic feature of T cells or their precursors or progenitors that distinguishes them from other lymphocytes and erythrocyte or myeloid lineages. Such phenotypic features include one or more T cell-specific proteins (e.g., CD3). + CD4 + CD8 + This may include the expression of ) or T cell-specific physiological, morphological, functional, or immunological features. For example, cells of a T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25 + Immature and inactivated T cells; cells committed to the CD4 or CD8 lineage; CD4 + CD8 + Two-positive thymic progenitor cells; single-positive CD4 + Or CD8 + ;TCRαβ or TCRγδ; or mature and functional or activated T cells. The term "T cell" refers to naive T cells (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory T cells (CD45RO + CD62L + CD8 + This includes effector memory T cells (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), mucosa-associated invariant T (MAIT) cells, Tregs, natural killer T cells, and tissue-resident T cells.

[0068] The term "B cell" refers to cells of the B cell lineage. "Cells of the B cell lineage" refers to cells that exhibit at least one phenotypic feature of B cells or their precursors or progenitors that distinguishes them from other lymphocyte and erythrocyte or myeloid lineages. Such phenotypic features include one or more proteins specific to B cells (e.g., CD19). + CD72+, CD24+, CD20 +) may include the expression of or physiological, morphological, functional, or immunological features specific to B cells. For example, cells of a B cell lineage may be progenitor or precursor cells committed to the B cell lineage (e.g., prepro B cells, pro B cells, and pre-B cells); immature and inactivated B cells; or mature and functional or activated B cells. Thus, “B cells” can include naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic cells, plasmablasts, and memory B cells (e.g., CD27). + IgD - ) includes.

[0069] The term “cytotoxic activity” for cells expressing an immune receptor (e.g., TCR) or a chimeric Tim receptor as described herein (e.g., T cells or NK cells) on their surface is also called “cytolytic activity” and means that the cell induces target cells to undergo apoptosis in response to antigen-specific signaling (e.g., via TCR, chimeric Tim receptor). In some embodiments, cytotoxic cells may induce apoptosis in target cells via the release of cytotoxins from granules, e.g., perforin, granzyme, and granulosin. Perforin inserts into the target cell membrane, forming a pore that allows water and salt to rapidly enter the target cell. Granzyme is a serine protease that induces apoptosis in target cells. Granulosin is a pro-inflammatory molecule that can also form a pore in the target cell membrane. In some embodiments, cytotoxic cells may induce apoptosis in target cells via the interaction of a Fas ligand, which is upregulated on T cells after antigen-specific signaling, with a Fas molecule expressed on the target cell. Fas is an apoptosis signaling receptor molecule found on the surface of several different cells.

[0070] The term “exhaustion” in relation to immune cells refers to a state of immune cell dysfunction defined by poor effector function (e.g., reduced cytokine production, reduced cytotoxic activity), reduced proliferative capacity, increased expression of immune checkpoint molecules, and transcriptional states different from those of functional effector or memory cells. In certain embodiments, exhausted immune cells become unresponsive to the presence of their target antigen. Immune cell exhaustion can result from chronic exposure to a target antigen (e.g., as can result from chronic infection) or when it enters an immunosuppressive environment (e.g., a tumor microenvironment). In certain embodiments, immune cell exhaustion refers to T cell exhaustion, NK cell exhaustion, or both. In certain embodiments, exhausted T cells exhibit (a) increased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof; (b) decreased production of IFN-γ, IL-2, TNF-α, or any combination thereof; or both (a) and (b). In certain embodiments, exhausted NK cells exhibit (a) increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) decreased production of IFN-γ, TNF-α, or both; or both (a) and (b).

[0071] A “disease” is a health condition in which the subject is unable to maintain homeostasis, and if the disease does not improve, the subject’s health continues to deteriorate. In contrast, a “disability” or “undesirable condition” in a subject is a health condition in which the subject is able to maintain homeostasis, but the subject’s health condition is worse than it would be in the absence of the disability or undesirable condition. If left untreated, a disability or undesirable condition does not necessarily lead to a further deterioration of the subject’s health condition.

[0072] When used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. These abnormal cells may form solid tumors or constitute hematological malignancies. Cancer cells may 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 cancer, lymphoma, leukemia, and lung cancer.

[0073] The terms “subject,” “patient,” and “individual” are used interchangeably herein and are intended to include living organisms (e.g., mammals) from which an immune response can be induced. Examples of subjects include humans, primates, cattle, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0074] "Adoptive cell immunotherapy" refers to the administration of disease antigen-specific immune cells (e.g., T cells), whether natural or genetically modified. Adoptive cell immunotherapy can be autologous (immune cells are from the recipient), allogeneic (immune cells are from the same species of donor), or syngeneic (immune cells are from a donor genetically identical to the recipient).

[0075] "Autologous" refers to any material (e.g., organ, tissue, or cell graft) that originates from the same subject into which the material is later reintroduced.

[0076] "Homogenous grafts" refers to grafts originating from different subjects of the same species.

[0077] In this disclosure, “therapeutic effective dose” or “effective dose” of a chimeric protein or cells expressing a chimeric protein (e.g., chimeric Tim receptor or cells expressing a chimeric Tim receptor) means a sufficient amount of protein or cells to produce improvement in one or more symptoms of the disease, disorder or undesirable condition being treated. When referring to individual active ingredients or cells expressing a single active ingredient administered alone, the therapeutic effective dose refers to the effect of that ingredient or cells expressing that ingredient alone. When referring to a combination, the therapeutic effective dose refers to the amount of the active ingredient or the combined associated active ingredient, in combination with cells expressing the active ingredient, that produces a therapeutic effect, whether administered sequentially or concurrently.

[0078] "To treat," "to cure," or "to improve" refers to the medical management of the disease, disorder, or undesirable condition in question. Generally, an appropriate dose or therapeutic regimen containing host cells expressing the chimeric protein of this disclosure is administered in an amount sufficient to induce a therapeutic or preventive benefit. Therapeutic or preventive / preventive benefits include: improved clinical outcomes; reduced or mitigated symptoms associated with the disease, disorder, or undesirable condition; reduced incidence of symptoms; improved quality of life; longer disease-free status; reduced severity of the disease, disorder, or undesirable condition; stabilization of the disease state; delayed disease progression; remission; survival; extended survival; or any combination thereof.

[0079] The term "antitumor effect" refers to a biological effect that can be demonstrated by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer. Furthermore, an "antitumor effect" can be demonstrated by the prevention of hematological malignancies or tumor formation.

[0080] "Autoimmune disease" refers to a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to autoantigens. Autoimmune responses may include autoreactive B cells that produce autoantibodies, autoreactive T cells, or both. As used herein, "autoantibody" is an antibody produced by a subject that also binds to autoantigens produced by that subject.

[0081] Additional definitions are provided throughout this disclosure.

[0082] Chimeric Tim receptor In some embodiments, the Disclosure provides a chimeric Tim receptor comprising a single-chain chimeric protein comprising (a)(i) a Tim4 IgV domain and a Tim1 mucin domain; or (ii) an extracellular domain comprising a binding domain comprising a Tim1 IgV domain and a Tim4 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain and an optional secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0083] In some embodiments, the disclosure provides a chimeric Tim receptor comprising a single-chain chimeric protein comprising: (a) an extracellular domain comprising a binding domain comprising a Tim1 IgV domain and a Tim1 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain and an optional secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0084] In some embodiments, the Disclosure provides a chimeric Tim receptor comprising a single-chain chimeric protein comprising: (a)(i) a Tim1 IgV domain and a Tim1 mucin domain; (ii) a Tim4 IgV domain and a Tim4 mucin domain; (iii) a Tim1 IgV domain and a Tim4 mucin domain; or (iv) an extracellular domain comprising a binding domain comprising a Tim4 IgV domain and a Tim1 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain selected from a Tim1 signaling domain or a Tim4 signaling domain and an appropriate secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0085] In some embodiments, the disclosure provides a chimeric Tim receptor comprising a single-chain chimeric protein comprising: (a)(i) an extracellular domain comprising a binding domain comprising a Tim4 IgV domain and a Tim4 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain selected from a CD28 signaling domain, a CD3ζ signaling domain, and a 4-1BB signaling domain, and a secondary intracellular signaling domain selected from a TLR signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

[0086] In some embodiments, the disclosure provides a chimeric Tim receptor comprising a single-chain chimeric protein comprising (a)(i) an extracellular domain comprising a binding domain comprising a Tim4 IgV domain and a Tim4 mucin domain; (b) a primary intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling domain; a secondary intracellular signaling domain comprising a co-stimulatory signaling domain, a Tim1 signaling domain, or a Tim4 signaling domain; and an intracellular signaling domain comprising a tertiary intracellular signaling domain comprising a TLR signaling domain.

[0087] Additional chimeric Tim receptors are provided in this disclosure.

[0088] In certain embodiments, the extracellular domain of the chimeric Tim receptor described herein may include an extracellular spacer domain located between and connecting the binding domain and the transmembrane domain.

[0089] When expressed in host cells, the chimeric Tim receptors of this disclosure may confer a phosphatidylserine-specific cytotoxic phenotype to modified host cells (e.g., host cells become cytotoxic to stressed, damaged, wounded, apoptotic, or necrotic cells that express phosphatidylserine on their surface). In some embodiments, the chimeric Tim receptor induces apoptosis in targeting cells via the release of granzymes, perforins, granulosins, or any combination thereof. In some embodiments, cells expressing the chimeric Tim receptors described herein exhibit a phagocytic phenotype specific to phosphatidylserine-presenting cells. In some embodiments, cells expressing the chimeric Tim receptors described herein, such as T cells, exhibit enhanced antigen-presenting activity.

[0090] The intracellular signaling domain may contain one or more effector domains that can transmit functional signals to cells in response to the binding of the extracellular domain of the chimeric Tim receptor to phosphatidylserine. Signaling by the intracellular signaling domain is induced by the binding of the extracellular domain to phosphatidylserine. The signals transmitted by the intracellular signaling domain promote effector functions in chimeric Tim receptor-containing cells. Examples of effector functions include cytotoxic activity, cytokine secretion, proliferation, anti-apoptotic signaling, persistence, expansion, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen presentation, or any combination thereof.

[0091] In certain embodiments, the intracellular signaling domain includes a first intracellular signaling domain. In embodiments, the intracellular signaling domain includes a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a first intracellular signaling domain, a second intracellular signaling domain and a third intracellular signaling domain. The chimeric Tim receptor described herein can be used in a variety of therapeutic methods in which the clearance of apoptotic cells, necrotic cells, damaged cells, or stressed cells is beneficial, while providing co-stimulation that enhances the cellular immune response, reduces immune cell exhaustion, or both.

[0092] The component portions of the fusion protein of this disclosure are described in further detail herein.

[0093] Extracellular domain As described herein, the chimeric Tim receptor comprises an extracellular domain containing a Tim-binding domain. The Tim-binding domain is a phospholipid with a negatively charged head group and confers specificity to phosphatidylserine (PtdSer), a component of the cell membrane. In healthy cells, phosphatidylserine is predominantly found in the inner leaflet of the cell membrane. However, when cells are stressed, damaged, or undergo apoptosis or necrosis, phosphatidylserine is exposed on the outer leaflet of the cell membrane. Therefore, phosphatidylserine can be used as a marker to distinguish stressed cells, damaged cells, apoptotic cells, necrotic cells, pyroptotic cells, or swollen cells. Binding of phosphatidylserine by the Tim-binding domain can block interactions between phosphatidylserine and other molecules and may interfere with, reduce, or eliminate certain functions of phosphatidylserine, such as signal transduction. In some embodiments, binding of phosphatidylserine may induce a specific biological pathway for removal, or identify the phosphatidylserine molecule or cells expressing phosphatidylserine.

[0094] Suitable Tim-binding domains for use in the chimeric Tim receptors of this disclosure may be any polypeptide or peptide derived from Tim1 and / or Tim4 molecules that specifically bind to phosphatidylserine. In embodiments, the Tim-binding domain includes an IgV domain derived from Tim1 or Tim4, and a mucin domain derived from Tim1 or Tim4. For example, the Tim-binding domain may include a Tim1 IgV domain and a Tim1 mucin domain. In another example, the Tim-binding domain may include a Tim1 IgV domain and a Tim4 mucin domain. In yet another example, the Tim-binding domain may include a Tim4 IgV domain and a Tim1 mucin domain. In yet another example, the Tim-binding domain may include a Tim4 IgV domain and a Tim4 mucin domain.

[0095] Phosphatidylserine binding is generally regulated by the IgV domain. The core phosphatidylserine binding domain is a four-amino acid sequence in the IgV domain (e.g., amino acids 95-98 of SEQ ID NO: 34 or amino acids 92-95 of SEQ ID NO: 38). The Tim4 binding domain binds minimally to cells with low phosphatidylserine densities. The Tim1 binding domain binds more strongly to lower phosphatidylserine densities, resulting in a lower threshold for response. An overview of Tim1 and Tim4 binding to phosphatidylserine is provided in Table 1. By combining the Tim1 IgV domain with the Tim4 mucin domain, or the Tim4 IgV domain with the Tim1 mucin domain, the binding affinity of the binding domain to phosphatidylserine can be modulated. Furthermore, such combinations in the Tim binding domain also provide a combination of Tim4's sensitivity to phosphatidylserine and Tim1's stability in protein expression.

[0096] Table 1. [Table 1]

[0097] In addition, the RGD domain in the IgV domain (e.g., amino acids 68-70 of SEQ ID NO: 34) can regulate integrin binding as a co-receptor for phagocytosis.

[0098] In some embodiments, the Tim-binding domain is obtained from or derived from human Tim1 and / or Tim4. An exemplary human Tim1 molecule is provided in Uniprot.Ref.Q96D42 (SEQ ID NO: 36). An exemplary human Tim1-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 43. In some embodiments, the Tim1-binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with respect to SEQ ID NO: 37 or SEQ ID NO: 43. In a particular embodiment, the Tim1 binding domain includes an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 43.

[0099] An exemplary human Tim4 molecule is provided in Uniprot.Ref.Q96H15 (SEQ ID NO: 1). An exemplary human Tim4-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 42, or SEQ ID NO: 119. An exemplary mouse Tim4-binding domain comprises or consists of the amino acid sequence of amino acids 23-279 of SEQ ID NO: 24 or SEQ ID NO: 24. In certain embodiments, the Tim4-binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with respect to amino acids 23-279 of SEQ ID NO: 2, SEQ ID NO: 42, SEQ ID NO: 119, or SEQ ID NO: 24 or SEQ ID NO: 24. In a particular embodiment, the Tim4 binding domain includes an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of amino acids 23-279 of SEQ ID NO: 2, SEQ ID NO: 42, SEQ ID NO: 119, or SEQ ID NO: 24.

[0100] In some embodiments, the Tim-binding domain includes a Tim1-derived IgV domain. An exemplary human Tim1 IgV domain is provided in SEQ ID NO: 38. In some embodiments, the Tim1 IgV domain is a modified Tim1 IgV domain including an R66G substitution in SEQ ID NO: 38. The R66G substitution (e.g., amino acids 68-70 in SEQ ID NO: 34) gives an RGD domain in the Tim1 IgV domain that can regulate integrin binding as a coreceptor for phagocytosis. In certain embodiments, the modified Tim1 IgV domain includes the amino acid sequence of SEQ ID NO: 41. In some embodiments, this modified Tim1 domain can increase phagocytic activity while preserving Tim1 sensitivity. In some embodiments, the Tim1 IgV domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with respect to SEQ ID NO: 38, SEQ ID NO: 38 with R66G substitution, or SEQ ID NO: 41. In certain embodiments, Tim1 IgV comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) with respect to the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 38 with R66G substitution, or SEQ ID NO: 41.

[0101] In some embodiments, the Tim-binding domain includes a Tim4-derived IgV domain. An exemplary human Tim4 IgV domain is provided in SEQ ID NO: 34. In some embodiments, the Tim4 IgV domain includes or comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with respect to SEQ ID NO: 34. In certain embodiments, the Tim4 IgV domain includes an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) with respect to the amino acid sequence of SEQ ID NO: 34.

[0102] In some embodiments, the Tim-binding domain includes a mucin domain derived from Tim1. An exemplary human Tim1 mucin domain is provided in SEQ ID NO: 39. In certain embodiments, the Tim1 mucin domain includes or comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with respect to SEQ ID NO: 39. In certain embodiments, the Tim1 mucin domain includes an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) with respect to the amino acid sequence of SEQ ID NO: 39.

[0103] In other embodiments, the Tim-binding domain includes a mucin domain derived from Tim4. An exemplary human Tim4 mucin domain is provided in SEQ ID NO: 35. In certain embodiments, the Tim4 mucin domain includes or comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with respect to SEQ ID NO: 35. In certain embodiments, the Tim4 mucin domain includes an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) with respect to the amino acid sequence of SEQ ID NO: 35.

[0104] In some embodiments, the Tim-binding domain includes a Tim1 IgV domain and a Tim1 mucin domain. In some embodiments, the Tim1 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 38, and the Tim1 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the Tim1 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 38 having an R66G substitution, and the Tim1 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the Tim1 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 41, and the Tim1 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, both the Tim1 IgV domain and the Tim1 mucin domain include or comprise the amino acid sequence shown in SEQ ID NO: 37 or SEQ ID NO: 43.

[0105] In some embodiments, the Tim-binding domain includes a Tim4 IgV domain and a Tim4 mucin domain. In some embodiments, the Tim4 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 34, and the Tim4 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, both the Tim4 IgV domain and the Tim4 mucin domain include or comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 42.

[0106] In some embodiments, the Tim-binding domain includes a Tim1 IgV domain and a Tim4 mucin domain. In some embodiments, the Tim1 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 38, and the Tim4 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the Tim1 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 38 having an R66G substitution, and the Tim4 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the Tim1 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 41, and the Tim4 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the Tim1 IgV domain further includes the Tim1 signal sequence of SEQ ID NO: 40.

[0107] In some embodiments, the Tim-binding domain includes a Tim4 IgV domain and a Tim1 mucin domain. In some embodiments, the Tim4 IgV domain includes or comprises the amino acid sequence shown in SEQ ID NO: 34, and the Tim1 mucin domain includes or comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the Tim4 IgV domain further includes the Tim4 signal sequence of SEQ ID NO: 11.

[0108] In some embodiments, the extracellular domain may include an extracellular non-signaling spacer or linker domain. If included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable appropriate cell / cell contact, binding, and activation. When included in the chimeric receptors described herein, the extracellular spacer domain is generally located between the extracellular binding domain and the transmembrane domain of the chimeric Tim receptor. The length of the extracellular spacer may vary to optimize target molecule binding 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; PCT Publication WO2014 / 031687). In some embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. The modified IgG4 hinge region is described in PCT Publication WO2014 / 031687, which is incorporated herein by reference in its entirety. In some embodiments, the extracellular spacer domain includes a modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 3). Other examples of hinge regions that may be used in the chimeric Tim receptor described herein include hinge regions from the extracellular regions of type 1 membrane proteins, which may be wild-type or variants, such as CD8a, CD4, CD28, and CD7. In some embodiments, the extracellular spacer domain includes a CD28 hinge region having the amino acid sequence SEQ ID NO: 32. In some embodiments, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, for example, PCT Publication WO2014 / 031687, the spacer being incorporated herein by reference in its entirety).In some embodiments, the extracellular spacer domain may include the stalk region of type II C lectin (the extracellular domain located between the type C lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.

[0109] In some embodiments, the extracellular domain comprises an amino acid sequence derived from any mammalian species, including humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, their transgenic species, or any combination thereof. In certain embodiments, the extracellular domain is mouse, human, or chimera.

[0110] Intracellular signal transduction domains The intracellular signaling domain of the chimeric Tim receptor described herein is an intracellular effector domain that can transmit functional signals to cells in response to the binding of the extracellular domain of the chimeric Tim receptor to phosphatidylserine. The signals transmitted by the intracellular signaling domain promote effector functions in chimeric Tim receptor-containing cells. Examples of effector functions include cytotoxic activity, cytokine secretion, proliferation, anti-apoptotic signaling, persistence, expansion, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen capture, antigen processing, antigen presentation, or any combination thereof.

[0111] The intracellular signaling domain includes a primary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain, a secondary intracellular signaling domain, and a tertiary intracellular signaling domain. The primary, secondary, and / or tertiary intracellular signaling domains can independently be any part of a signaling molecule that retains sufficient signaling activity. In some embodiments, a full-length signaling molecule or a full-length intracellular component of a signaling molecule is used. In some embodiments, a cleaved portion of a signaling molecule or an intracellular component of a signaling molecule is used, provided that the cleaved portion retains sufficient signaling activity. In some embodiments, the signaling domain is the entire part of a signaling molecule or a variant of a cleaved portion, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

[0112] In some embodiments, the primary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a 4-1BB signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

[0113] In some embodiments, the secondary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a 4-1BB signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

[0114] In some embodiments, the tertiary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a 4-1BB signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

[0115] In some embodiments, the primary intracellular signaling domain includes an immune receptor tyrosine-based activation motif (ITAM)-containing signaling domain; the secondary intracellular signaling domain includes a co-stimulatory signaling domain, a Tim1 signaling domain, or a Tim4 signaling domain; and the tertiary intracellular signaling domain includes a TLR signaling domain. The ITAM-containing signaling domain is generally YXXL / IX. 6-8It contains at least one (1, 2, 3, 4 or more) ITAMs that refer to the conserved motif -YXXL / I. The ITAM-containing signaling domains can initiate T cell activation signaling after antigen binding or ligand engagement. ITAM signaling domains include, for example, intracellular signaling domains for CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278(ICOS), DAP12, FcRγ, and CD66d. Co-stimulatory signaling domains, when activated in conjunction with major or classical (e.g., ITAM-driven) activation signals, promote or enhance T cell responses, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or combinations thereof. Co-stimulatory signaling domains for use in chimeric Tim receptors include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, ZAP70, or any combination thereof. In some embodiments, the co-stimulatory signaling domain includes the OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137) signaling domain. The TLR signaling domain may be the TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signaling domain. In some embodiments, the TLR signaling domain is the TLR2 signaling domain or the TLR8 signaling domain.

[0116] As used herein, the designation of primary, secondary, and tertiary intracellular signaling domains includes, but is not limited to, the arrangement of a primary intracellular signaling domain at the N-terminus, a secondary intracellular signaling domain in the center, and a tertiary intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Therefore, the designation of a primary intracellular signaling domain does not limit the use of a selected intracellular signaling domain at the N-terminus of the intracellular portion of the chimeric Tim receptor. The designation of a secondary intracellular signaling domain does not limit the use of a selected intracellular signaling domain at the center (or at the C-terminus, for chimeric Tim receptors having only two intracellular signaling domains). The designation of a tertiary intracellular signaling domain does not limit the use of a selected intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Therefore, different arrangements of primary, secondary, and / or tertiary intracellular signaling domains within the intracellular portion of the chimeric Tim receptor are contemplated.

[0117] An exemplary Tim1 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 44. An exemplary Tim4 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 124, or SEQ ID NO: 125. An exemplary TRAF2 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 48. An exemplary TRAF6 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 46. An exemplary CD28 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 4 or 26. An exemplary DAP12 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 9. An exemplary CD3ζ signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 27 or 5. An exemplary 4-1BB signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 100. An exemplary TLR2 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 122. An exemplary TLR8 signaling domain includes or comprises the amino acid sequence of SEQ ID NO: 47.

[0118] In some embodiments, the Tim1 signaling domain includes the amino acid sequence shown in SEQ ID NO: 44. In some embodiments, the Tim4 signaling domain includes the amino acid sequence shown in SEQ ID NO: 45, SEQ ID NO: 124, or SEQ ID NO: 125. In some embodiments, the TRAF2 signaling domain includes the amino acid sequence shown in SEQ ID NO: 48. In some embodiments, the TRAF6 signaling domain includes the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the CD28 signaling domain includes the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the CD28 signaling domain includes the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the DAP12 signaling domain includes the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the CD3ζ signaling domain includes the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the CD3ζ signaling domain includes the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the 4-1BB signaling domain includes the amino acid sequence of SEQ ID NO: 100. In some embodiments, the TLR2 signaling domain includes the amino acid sequence of SEQ ID NO: 122. In some embodiments, the TLR8 signaling domain includes the amino acid sequence shown in SEQ ID NO: 47.

[0119] In some embodiments, the primary and / or secondary signaling domains include or consist of amino acid sequences having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with any one of SEQ ID NOs: 4, 5, 9, 26, 27, 44-48, 100, 122, 124, and 125. In some embodiments, the primary and / or secondary signaling domains include an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to any one of the amino acid sequences of SEQ ID NOs: 4, 5, 9, 26, 27, 44-48, 100, 122, 124, and 125. In some embodiments, the primary and secondary signaling domains are the same or different.

[0120] In some embodiments, the primary, secondary, and / or tertiary intracellular signaling domains include or consist of amino acid sequences having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with any one of sequence numbers 4, 5, 7, 9, 26, 27, 44-48, 122, and 124. In some embodiments, the primary, secondary, and / or tertiary intracellular signaling domains include an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to any one of the amino acid sequences of SEQ ID NOs: 4, 5, 9, 26, 27, 44-48, 100, 122, 124, and 125. In some embodiments, the primary, secondary, and tertiary intracellular signaling domains are the same. In some embodiments, two or three of the primary, secondary, and tertiary intracellular signaling domains are different.

[0121] In some embodiments, the intracellular signaling domain includes the Tim1 intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the Tim4 intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the CD3ζ intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the CD28 intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the 4-1BB intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the TRAF6 intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the TRAF2 intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the TLR2 intracellular signaling domain. In some embodiments, the intracellular signaling domain includes the TLR8 intracellular signaling domain.

[0122] In some embodiments, the intracellular signaling domain includes a Tim1 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a Tim4 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a TLR8 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain and a DAP12 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a CD3ζ secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain, and a CD3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a CD3ζ secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a TLR2 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD3ζ primary intracellular signaling domain and a TLR2 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a TLR8 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain.In some embodiments, the intracellular signaling domain includes a CD3ζ primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a TRAF6 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD3ζ primary intracellular signaling domain and a TRAF6 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a CD3ζ secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain, and a CD3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain, a CD3ζ secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD3ζ primary intracellular signaling domain and a TLR2 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a TLR2 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a CD3ζ primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a TLR8 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain.In some embodiments, the intracellular signaling domain includes a CD3ζ primary intracellular signaling domain and a TRAF6 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a TRAF6 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a Tim4 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain and a CD3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a Tim4 primary intracellular signaling domain, a CD3ζ secondary intracellular signaling domain and a TLR2 tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a Tim4 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain and a CD3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain includes a Tim4 primary intracellular signaling domain, a CD3ζ secondary intracellular signaling domain and a TLR8 tertiary intracellular signaling domain.

[0123] In some embodiments, the intracellular signaling domain includes a Tim1 primary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 44 and a CD3ζ secondary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 27 or 5. In some embodiments, the intracellular signaling domain includes a Tim4 primary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 45, 124, or 125 and a CD3ζ secondary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 27 or 5. In some embodiments, the intracellular signaling domain includes a TLR8 primary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 47 and a CD3ζ secondary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 27 or 5. In some embodiments, the intracellular signaling domain includes a CD28 primary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 4 or 26 and a DAP12 secondary intracellular signaling domain containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the intracellular signaling domain includes a combination of primary, secondary, and optionally tertiary intracellular signaling domains as shown in Table 8.

[0124] Intracellular signaling domains may originate from mammalian species, including humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0125] transmembrane domain The transmembrane domain of the chimeric Tim receptor connects and is located between the extracellular domain and the intracellular signaling domain. The transmembrane domain is a hydrophobic alpha-helix that traverses the host cell membrane. The transmembrane domain may be fused directly to the binding domain or, if present, to the extracellular spacer domain. In certain embodiments, the transmembrane domain is derived from an endogenous membrane protein [e.g., receptors, surface antigen classification (CD) molecules, enzymes, transporters, cell adhesion molecules, etc.]. In one embodiment, the transmembrane domain is selected from the same molecule from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule from which the intracellular signaling domain is derived. For example, the chimeric Tim receptor may include a Tim4 binding domain and a Tim4 transmembrane domain. In another example, the chimeric Tim receptor may include a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain may originate from different molecules; the transmembrane domain and the intracellular signaling domain may originate from different molecules; or the transmembrane domain, extracellular domain, and intracellular signaling domain may all originate from different molecules. Examples of transmembrane domains that may be used in the chimeric Tim receptor of this disclosure include transmembrane domains derived from Tim1, Tim4, and CD28. An exemplary Tim1 transmembrane domain may include or consist of the amino acid sequence of SEQ ID NO: 8. An exemplary Tim4 transmembrane domain may include or consist of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 23, or SEQ ID NO: 121. An exemplary CD28 transmembrane domain may include or consist of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 120. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with any one of SEQ ID NOs: 6-8, 23, 120, and 121.In a particular embodiment, the transmembrane domain includes an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletions, additions, substitutions) to any one of the amino acid sequences among SEQ ID NOs: 6-8, 23, 120, and 121.

[0126] The transmembrane domain may originate from any mammalian species, including humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0127] In certain embodiments, the chimeric Tim receptor is encoded by a polynucleotide sequence derived from any mammalian species, including humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, their transgenic species, or any combination thereof. In certain embodiments, the chimeric Tim receptor is mouse, chimeric, human, or humanized.

[0128] The direct fusion of one domain of a chimeric Tim receptor to another domain, as described herein, is understood not to exclude the presence of intervening junctional amino acids. These junctional amino acids may be native or non-native (e.g., arising from the construct design of the chimeric protein). For example, junctional amino acids may arise from restriction enzyme sites used for the fusion of one domain to another or for the cloning of polynucleotides encoding a chimeric Tim receptor into a vector.

[0129] Exemplary chimeric Tim receptor The component portions of the chimeric Tim receptor disclosed herein can be selected and arranged in various combinations to provide desired specificity and effector phenotypes for host cells.

[0130] Exemplary chimeric Tim receptors of this disclosure are described in Table 2.

[0131] Table 2. [Table 2]

[0132] Further exemplary chimeric Tim receptors are described in Table 3.

[0133] Table 3. [Table 3]

[0134] In some embodiments, the chimeric Tim receptor of this disclosure includes the constructs shown in Table 2. In some embodiments, the chimeric Tim receptor of this disclosure includes the constructs shown in Table 3.

[0135] In some embodiments, the chimeric Tim receptor of construct 1 or construct 1' contains amino acids 21-456 of SEQ ID NO: 49. In certain embodiments, the chimeric Tim receptor of construct 1 or construct 1' contains the amino acid sequence of SEQ ID NO: 49.

[0136] In some embodiments, the chimeric Tim receptor of construct 2 or construct 2' contains amino acids 21-471 of SEQ ID NO: 50. In certain embodiments, the chimeric Tim receptor of construct 2 or construct 2' contains the amino acid sequence of SEQ ID NO: 50.

[0137] In some embodiments, the chimeric Tim receptor of construct 3 or construct 3' contains amino acids 21-363 of SEQ ID NO: 51. In certain embodiments, the chimeric Tim receptor of construct 3 or construct 3' contains the amino acid sequence of SEQ ID NO: 51.

[0138] In some embodiments, the chimeric Tim receptor of construct 4 or construct 4' contains amino acids 21-590 of SEQ ID NO: 52. In certain embodiments, the chimeric Tim receptor of construct 4 or construct 4' contains the amino acid sequence of SEQ ID NO: 52.

[0139] In some embodiments, the chimeric Tim receptor of construct 5 or construct 5' contains amino acids 21-596 of SEQ ID NO: 53. In certain embodiments, the chimeric Tim receptor of construct 5 or construct 5' contains the amino acid sequence of SEQ ID NO: 53.

[0140] In some embodiments, the chimeric Tim receptor of construct 6 or construct 6' contains amino acids 21-619 of SEQ ID NO: 54. In certain embodiments, the chimeric Tim receptor of construct 6 or construct 6' contains the amino acid sequence of SEQ ID NO: 54.

[0141] In some embodiments, the chimeric Tim receptor of construct 7 or construct 7' contains amino acids 21-625 of SEQ ID NO: 55. In certain embodiments, the chimeric Tim receptor of construct 7 or construct 7' contains the amino acid sequence of SEQ ID NO: 55.

[0142] In some embodiments, the chimeric Tim receptor of construct 8 or construct 8' contains amino acids 21-621 of SEQ ID NO: 56. In certain embodiments, the chimeric Tim receptor of construct 8 or construct 8' contains the amino acid sequence of SEQ ID NO: 56.

[0143] In some embodiments, the chimeric Tim receptor of construct 9 or construct 9' contains amino acids 21-415 of SEQ ID NO: 57. In certain embodiments, the chimeric Tim receptor of construct 9 or construct 9' contains the amino acid sequence of SEQ ID NO: 57.

[0144] In some embodiments, the chimeric Tim receptor of construct 10 or construct 10' contains amino acids 21-409 of SEQ ID NO: 58. In certain embodiments, the chimeric Tim receptor of construct 10 or construct 10' contains the amino acid sequence of SEQ ID NO: 58.

[0145] Further exemplary chimeric Tim receptors of this disclosure are described in Table 4.

[0146] Table 4. [Table 4]

[0147] Further exemplary chimeric Tim receptors are described in Table 5.

[0148] Table 5. [Table 5]

[0149] In some embodiments, the chimeric Tim receptor of this disclosure includes the constructs shown in Table 4. In some embodiments, the chimeric Tim receptor of this disclosure includes the constructs shown in Table 5.

[0150] In some embodiments, the chimeric Tim receptor of construct 11 or construct 11' contains amino acids 25-490 of SEQ ID NO: 59. In certain embodiments, the chimeric Tim receptor of construct 11 or construct 11' contains the amino acid sequence of SEQ ID NO: 59.

[0151] In some embodiments, the chimeric Tim receptor of construct 12 or construct 12' contains amino acids 25-495 of SEQ ID NO: 60. In certain embodiments, the chimeric Tim receptor of construct 12 or construct 12' contains the amino acid sequence of SEQ ID NO: 60.

[0152] In some embodiments, the chimeric Tim receptor of construct 13 or construct 13' contains amino acids 25-382 of SEQ ID NO: 61. In certain embodiments, the chimeric Tim receptor of construct 13 or construct 13' contains the amino acid sequence of SEQ ID NO: 61.

[0153] In some embodiments, the chimeric Tim receptor of construct 14 or construct 14' contains amino acids 25-609 of SEQ ID NO: 62. In certain embodiments, the chimeric Tim receptor of construct 14 or construct 14' contains the amino acid sequence of SEQ ID NO: 62.

[0154] In some embodiments, the chimeric Tim receptor of construct 15 or construct 15' contains amino acids 25-615 of SEQ ID NO: 63. In certain embodiments, the chimeric Tim receptor of construct 15 or construct 15' contains the amino acid sequence of SEQ ID NO: 63.

[0155] In some embodiments, the chimeric Tim receptor of construct 16 or construct 16' contains amino acids 25-638 of SEQ ID NO: 64. In certain embodiments, the chimeric Tim receptor of construct 16 or construct 16' contains the amino acid sequence of SEQ ID NO: 64.

[0156] In some embodiments, the chimeric Tim receptor of construct 17 or construct 17' contains amino acids 25-644 of SEQ ID NO: 65. In certain embodiments, the chimeric Tim receptor of construct 17 or construct 17' contains the amino acid sequence of SEQ ID NO: 65.

[0157] In some embodiments, the chimeric Tim receptor of construct 18 or construct 18' contains amino acids 25-640 of SEQ ID NO: 66. In certain embodiments, the chimeric Tim receptor of construct 18 or construct 18' contains the amino acid sequence of SEQ ID NO: 66.

[0158] In some embodiments, the chimeric Tim receptor of this disclosure is not one of the constructs in Table 4. In some embodiments, the chimeric Tim receptor of this disclosure is not one of the constructs in Table 5. In some embodiments, the chimeric Tim receptor of this disclosure does not have any one of the constructs 13, 14, 15, 16, and 17, or any combination thereof. In some embodiments, the chimeric Tim receptor of this disclosure does not have any one of the amino acid sequences of SEQ ID NOs. 61, 62, 63, 64, and 65, or any combination thereof.

[0159] Further exemplary chimeric Tim receptors of this disclosure are described in Table 6.

[0160] Table 6. [Table 6]

[0161] Further exemplary chimeric Tim receptors are described in Table 7.

[0162] Table 7. [Table 7]

[0163] In some embodiments, the chimeric Tim receptor of this disclosure includes the constructs shown in Table 6. In some embodiments, the chimeric Tim receptor of this disclosure includes the constructs shown in Table 7.

[0164] In some embodiments, the chimeric Tim receptor of construct 19 or construct 19' contains amino acids 25-628 of SEQ ID NO: 67. In certain embodiments, the chimeric Tim receptor of construct 19 or construct 19' contains the amino acid sequence of SEQ ID NO: 67.

[0165] In some embodiments, the chimeric Tim receptor of construct 20 or construct 20' contains amino acids 25-416 of SEQ ID NO: 68. In certain embodiments, the chimeric Tim receptor of construct 20 or construct 20' contains the amino acid sequence of SEQ ID NO: 68.

[0166] In some embodiments, the chimeric Tim receptor of construct 21 or construct 21' contains amino acids 25-422 of SEQ ID NO: 69. In certain embodiments, the chimeric Tim receptor of construct 21 or construct 21' contains the amino acid sequence of SEQ ID NO: 69.

[0167] Further exemplary chimeric Tim receptors are described in Table 8. In some embodiments, the chimeric Tim receptors of this disclosure include the constructs in Table 8. In some embodiments, the chimeric Tim receptors of this disclosure do not include chimeric Tim receptors having the amino acid sequence of SEQ ID NOs. 127, 138, 149, 157, or 158. In some embodiments, the chimeric Tim receptors of this disclosure do not include chimeric Tim receptors having the combination of components described for the constructs of SEQ ID NOs. 127, 138, 149, 157, or 158.

[0168] In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 127 or the amino acid sequence of SEQ ID NO: 127 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 128 or the amino acid sequence of SEQ ID NO: 128 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 129 or the amino acid sequence of SEQ ID NO: 129 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 130 or the amino acid sequence of SEQ ID NO: 130 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 131 or the amino acid sequence of SEQ ID NO: 131 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 132 or the amino acid sequence of SEQ ID NO: 132 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 133 or the amino acid sequence of SEQ ID NO: 133 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 134 or the amino acid sequence of SEQ ID NO: 134 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 135 or the amino acid sequence of SEQ ID NO: 135 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 136 or the amino acid sequence of SEQ ID NO: 136 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 137 or the amino acid sequence of SEQ ID NO: 137 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 138 or the amino acid sequence of SEQ ID NO: 138 lacking the signal sequence (amino acids 1-24).In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 139 or the amino acid sequence of SEQ ID NO: 139 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 140 or the amino acid sequence of SEQ ID NO: 140 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 141 or the amino acid sequence of SEQ ID NO: 141 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 142 or the amino acid sequence of SEQ ID NO: 142 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 143 or the amino acid sequence of SEQ ID NO: 143 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 144 or the amino acid sequence of SEQ ID NO: 144 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 145 or the amino acid sequence of SEQ ID NO: 145 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 146 or the amino acid sequence of SEQ ID NO: 146 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 147 or the amino acid sequence of SEQ ID NO: 147 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 148 or the amino acid sequence of SEQ ID NO: 148 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 149 or the amino acid sequence of SEQ ID NO: 149 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID NO: 150 or the amino acid sequence of SEQ ID NO: 150 lacking the signal sequence (amino acids 1-24).In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 151 or the amino acid sequence of SEQ ID NO: 151 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 152 or the amino acid sequence of SEQ ID NO: 152 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 153 or the amino acid sequence of SEQ ID NO: 153 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 154 or the amino acid sequence of SEQ ID NO: 154 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor includes the amino acid sequence of SEQ ID NO: 155 or the amino acid sequence of SEQ ID NO: 155 lacking the signal sequence (amino acids 1-24).

[0169] Table 8 [Table 8-1] [Table 8-2] [Table 8-3]

[0170] Polynucleotides, vectors, and host cells This disclosure provides nucleic acid molecules encoding any one or more of the chimeric Tim receptors described herein. Nucleic acids may refer to single-stranded or double-stranded DNA, cDNA, or RNA, and may include the positive and negative strands of complementary nucleic acids, including antisense DNA, cDNA, and RNA. Nucleic acids may be innate or synthetic forms of DNA or RNA. Nucleic acid sequences encoding a desired chimeric Tim receptor can be obtained or produced by, for example, screening a library from cells expressing the desired sequence or a portion thereof, using recombination methods known in the art and using standard techniques, as described in Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003), deriving a sequence from a vector known to contain the desired sequence or a portion thereof, or directly isolating the sequence or a portion thereof from cells or tissues containing the desired sequence or a portion thereof. Alternatively, the target sequence can be synthesized rather than cloned.

[0171] The polynucleotides encoding the chimeric Tim receptor compositions provided herein may originate from any animal, such as humans, primates, cattle, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, or combinations thereof. In certain embodiments, the polynucleotide encoding the chimeric Tim receptor is a polynucleotide from the same animal species as the host cell into which the polynucleotide is inserted.

[0172] The polynucleotide encoding the chimeric Tim receptor of this disclosure can be operably ligated to an expression regulatory sequence. The expression regulatory sequence may include appropriate transcription start, stop, promoter, and enhancer sequences; efficient RNA processing signals, e.g., splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and optionally sequences that improve protein secretion.

[0173] In certain embodiments, the polynucleotide encoding the chimeric Tim receptor includes a sequence encoding a signal peptide (also called a leader peptide or signal sequence) at its 5' end for targeting the precursor protein to the secretory pathway. The signal peptide may be cleaved from the N-terminus of the extracellular domain during cell processing and localization of the chimeric Tim receptor to the host cell membrane. The polypeptide from which the signal peptide sequence has been cleaved or removed may also be called a mature polypeptide. Examples of signal peptides that may be used in the chimeric Tim receptor of this disclosure include signal peptides derived from endogenous secretory proteins, such as GM-CSF (amino acid sequence of SEQ ID NO: 10), Tim1 (amino acid sequence of SEQ ID NO: 40), or Tim4 (amino acid sequences of SEQ ID NO: 11, 25, or 118). In certain embodiments, the polynucleotide sequence encodes a mature chimeric Tim receptor polypeptide, or the polypeptide sequence includes a mature chimeric Tim receptor polypeptide. With respect to sequences disclosed herein that include a signal peptide sequence, it will be understood by those skilled in the art that the signal peptide sequence may be replaced with another signal peptide capable of transporting the encoded protein to the extracellular membrane.

[0174] In certain embodiments, the chimeric Tim receptor encoding the polynucleotide of this disclosure is codon-optimized for efficient expression in target host cells containing the polynucleotide [see, for example, Scholten et al., Clin. Immunol. 119:135-145 (2006)]. As used herein, “codon-optimized” polynucleotides include heterologous polynucleotides having codons modified by silent mutations corresponding to the abundance of tRNA in the host cell of interest.

[0175] A single polynucleotide molecule may encode one, two, or more chimeric Tim receptors as described in any of the embodiments disclosed herein. A polynucleotide encoding two or more transgenes may include sequences (e.g., IRES, viral 2A peptide) that are positioned between each gene for multicistronic expression.

[0176] Polynucleotides encoding at least two transgenes (e.g., a chimeric Tim receptor and a CAR) provided in this disclosure may be used to constitute a tandem expression cassette. A tandem expression cassette refers to a component of a vector nucleic acid containing at least two transgenes under the control of the same set of regulatory sequences, or operably linked to the same set of regulatory sequences, for tandem or co-expression of at least two transgenes. Regulatory sequences that may be used in the tandem expression cassette of this disclosure include appropriate transcription start, stop, promoter, and enhancer sequences; efficient RNA processing signals, e.g., splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; sequences that improve protein secretion; or any combination thereof.

[0177] In one embodiment, the Disclosure provides a tandem expression cassette comprising a polynucleotide encoding the chimeric Tim receptor of the Disclosure and a polynucleotide encoding a cellular immunotherapy agent (e.g., CAR, TCR, etc.).

[0178] In certain embodiments, the tandem expression cassette may be constructed to optimize spatial and temporal control. For example, the tandem expression cassette may include promoter elements that optimize spatial and temporal control. In some embodiments, the tandem expression cassette includes a tissue-specific promoter or enhancer that enables specific induction of the tandem expression cassette to an organ, cell type (e.g., immune cells) or pathological microenvironment, such as tumor or infected tissue. An "enhancer" is an additional promoter element that can function cooperatively or independently to activate transcription. In certain embodiments, the tandem expression cassette includes a constitutive promoter. An exemplary constitutive promoter for use in the tandem expression cassette of this disclosure is the EF-1α promoter. In certain embodiments, the tandem expression cassette includes an inducible promoter. In certain embodiments, the tandem expression cassette includes a tissue-specific promoter.

[0179] The two transgenes contained within a tandem expression cassette can be in any order. For example, a tandem expression cassette containing a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR may be arranged as follows: 5' to 3': chimeric Tim receptor-CAR or CAR-chimeric Tim receptor.

[0180] In certain embodiments, a receptor comprising two or more polypeptide chains that associate to form a multimer or complex may be encoded by two or more polynucleotide molecules within a tandem expression construct. Exemplary multimeric receptors intended for expression in the tandem expression constructs of this disclosure include multi-chain CAR, TCR, TCR-CAR, and TRuC® constructs. Thus, exemplary tandem expression cassette embodiments encoding chimeric Tim receptors and TCRs may comprise a polynucleotide encoding the chimeric Tim receptor, a polynucleotide encoding the TCRα chain polypeptide, and a polynucleotide encoding the TCRβ chain polypeptide.

[0181] In certain embodiments, the tandem expression cassette of the present disclosure may include intrasequence ribosome entry sites (IRESs) or peptide cleavage sites, such as furin cleavage sites or viral 2A peptides, positioned between each polynucleotide contained within the tandem expression cassette to enable co-expression of multiple proteins from a single mRNA. For example, an IRES, furin cleavage site, or viral 2A peptide may be positioned between a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR within the tandem expression cassette. In another example, an IRES, furin cleavage site, or viral 2A peptide may be positioned between each of the polynucleotides encoding the chimeric Tim receptor, the polynucleotide encoding the TCRα chain polypeptide, and the polynucleotide encoding the TCRβ chain polypeptide. In certain embodiments, the viral 2A peptide may be porcine rhinitis virus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. An exemplary T2A peptide contains one of the amino acid sequences from SEQ ID NOs: 12, 28, 29, or 30. An exemplary P2A peptide contains the amino acid sequence from SEQ ID NOs: 13 or 31. An exemplary E2A peptide sequence contains the amino acid sequence from SEQ ID NOs: 14. An exemplary F2A peptide sequence contains the amino acid sequence from SEQ ID NOs: 15.

[0182] Certain embodiments of the tandem expression cassette of this disclosure include a polynucleotide encoding a CAR / or TCR specific to a target antigen (e.g., a tumor antigen) and a polynucleotide encoding a chimeric Tim receptor of this disclosure. Upon binding of target cells expressing the target antigen via the CAR / or TCR, cells modified to express such a tandem expression cassette induce apoptosis of the target cells. Apoptosis induces exposure of the target cells to a prophagocytic marker, e.g., phosphatidylserine, which can then target damaged or apoptotic cells for phagocytosis by the chimeric Tim receptor.

[0183] A polynucleotide encoding a desired chimeric Tim receptor can be inserted into a suitable vector, such as a viral vector, a non-viral plasmid vector, and other non-viral vectors, e.g., lipid-based DNA vectors, modified mRNA (modRNA), auto-amplified mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), for introduction into a host cell of interest (e.g., an immune cell). The polynucleotide encoding the chimeric Tim receptor of this disclosure can be cloned into any suitable vector, e.g., an expression vector, a replication vector, a probe-generating vector, or a sequencing vector. In certain embodiments, a polynucleotide encoding an extracellular domain, a polynucleotide encoding a transmembrane domain, and a polynucleotide encoding an intracellular signaling domain are joined together to form a single polynucleotide, which is then inserted into the vector. In other embodiments, the polynucleotide encoding the extracellular domain, the polynucleotide encoding the transmembrane domain, and the polynucleotide encoding the intracellular signaling domain may be inserted separately into the vector so that the expressed amino acid sequence produces a functional chimeric Tim receptor. A vector encoding a chimeric Tim receptor is referred to herein as a “chimeric Tim receptor vector.”

[0184] In certain embodiments, the vector contains a polynucleotide encoding one chimeric Tim receptor. In certain embodiments, the vector contains a polynucleotide encoding two or more chimeric Tim receptors. In certain embodiments, a single polynucleotide encoding two or more chimeric Tim receptors is cloned into a cloning site and expressed from a single promoter, and each chimeric Tim receptor sequence is separated from each other by an intrasequence ribosome entry site (IRES), a furin cleavage site, or a viral 2A peptide to allow co-expression of multiple genes from a single open reading frame (e.g., a multicistronic vector). In certain embodiments, the viral 2A peptide is porcine rhinitis virus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. An exemplary T2A peptide contains the amino acid sequence of SEQ ID NOs. 12, 28, 29, or 30. An exemplary P2A peptide contains the amino acid sequence of SEQ ID NO: 13 or 31. An exemplary E2A peptide sequence contains the amino acid sequence of SEQ ID NO: 14. An exemplary F2A peptide sequence contains the amino acid sequence of SEQ ID NO: 15.

[0185] In certain embodiments, the vector contains two or more polynucleotides, each encoding a chimeric Tim receptor. The two or more polynucleotides encoding the chimeric Tim receptor can be sequentially cloned into the vector at different cloning sites, and each chimeric Tim receptor is expressed under the regulation of a different promoter. In certain embodiments, vectors are utilized that enable long-term integration and transmission of the transgene to daughter cells. Examples include viral vectors, e.g., adenoviruses, adeno-associated viruses, vaccinia viruses, herpesviruses, cytomegaloviruses, poxviruses, or retroviruses, e.g., lentiviral vectors. Lentiviral-derived vectors are used to achieve long-term gene transfer and may have additional advantages over other vectors, including the ability to transduce non-proliferating cells, e.g., hepatocytes, and low immunogenicity.

[0186] In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapy agent (e.g., a chimeric antigen receptor, recombinant TCR, etc.). In certain embodiments, a single polynucleotide encoding the chimeric Tim receptor and the cellular immunotherapy agent (e.g., CAR) is cloned into a cloning site and expressed from a single promoter, and the chimeric Tim receptor sequence and the cellular immunotherapy agent (e.g., CAR) sequence are separated from each other by intra-sequence ribosome entry sites (IRES), furin cleavage sites, or viral 2A peptides to enable co-expression of multiple genes from a single open reading frame (e.g., a multicistronic vector). In certain embodiments, the viral 2A peptide is porcine rhinitis virus-1 (P2A), Tosea signalavirus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. Exemplary T2A peptides include the amino acid sequence of SEQ ID NOs. 12, 28, 29, or 30. An exemplary P2A peptide contains the amino acid sequence of SEQ ID NO: 13 or 31. An exemplary E2A peptide sequence contains the amino acid sequence of SEQ ID NO: 14. An exemplary F2A peptide sequence contains the amino acid sequence of SEQ ID NO: 15.

[0187] In certain embodiments, a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cell immunotherapy agent (e.g., CAR) binding protein are joined together to form a single polynucleotide, which is then inserted into a vector. In other embodiments, a polynucleotide encoding a CER and a polynucleotide encoding a CAR or TCR binding protein may be inserted separately into the vector at the same or different cloning sites so that the expressed amino acid sequences produce functional CER and CAR / or TCR. A vector encoding a tandem expression cassette is referred to herein as a “tandem expression vector.”

[0188] In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapy agent (e.g., CAR). The polynucleotides encoding the chimeric Tim receptor and the cellular immunotherapy agent (e.g., CAR) can be sequentially cloned into the vector at different cloning sites, and the chimeric Tim receptor and the cellular immunotherapy agent (e.g., CAR) are expressed under the regulation of different promoters.

[0189] Vectors encoding core viruses are referred to herein as “viral vectors.” There are numerous available viral vectors suitable for use with the compositions of this disclosure, including viral vectors identified for human gene therapy applications (see Pfeifer and Verme, Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectors include RNA virus-based vectors, such as retrovirus-derived vectors, e.g., Maloney murine leukemia virus (MLV)-derived vectors, and more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedivisna virus (sheep lentivirus). Methods using retroviral and lentiviral viral vectors, and methods for packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgenes, are known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Furthermore, constructs and expression systems for retroviral and lentiviral vectors are commercially available.

[0190] In certain embodiments, a viral vector is used to introduce a non-endogenous polynucleotide encoding a chimeric Tim receptor into a host cell. The viral vector may be a retroviral vector or a lentiviral vector. The viral vector may also contain a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selective 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 genetic marker for transduction, including a fluorescent protein (e.g., green, yellow), the extracellular domain of human CD2, or cleaved human EGFR (EGFRt or tEGFR; see Wang et al., Blood 118:1255, 2011). An exemplary tEGFR includes the amino acid sequence of SEQ ID NO: 16. If a viral vector genome contains multiple genes that are expressed in host cells as distinct proteins from a single transcript, the viral vector may also contain additional sequences between two (or more) genes that enable multicistronic expression. Examples of such sequences used in viral vectors include intra-sequence ribosome entry sites (IRESs), furin cleavage sites, viral 2A peptides (e.g., T2A, P2A, E2A, F2A), or any combination thereof.

[0191] Furthermore, for polynucleotide delivery, other viral vectors can be used, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; amplicon vectors; and DNA viral vectors, including herpes simplex virus (HSV)-derived vectors containing replication-deficient HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).

[0192] Furthermore, other viral vectors recently developed for gene therapy can be used in conjunction with the compositions and methods of this disclosure. Such vectors include baculovirus and alpha-virus-derived vectors (Jolly, D J. 1999. Emerging Viral Vectors. pp 209-40, 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).

[0193] In certain embodiments, a chimeric Tim receptor vector may be constructed to optimize spatial and temporal control. For example, a chimeric Tim receptor vector may include promoter elements that optimize spatial and temporal control. In some embodiments, a chimeric Tim receptor vector includes a tissue-specific promoter or enhancer that enables specific induction of the chimeric Tim receptor into an organ, cell type (e.g., immune cells) or pathological microenvironment, such as tumor or infected tissue. An "enhancer" is an additional promoter element that can function cooperatively or independently to activate transcription. In certain embodiments, a chimeric Tim receptor vector includes a constitutive promoter. In certain embodiments, a chimeric Tim receptor vector includes an inducible promoter. In certain embodiments, a chimeric Tim receptor vector includes a tissue-specific promoter.

[0194] In certain embodiments, the chimeric Tim receptor vector may include a gene encoding a homing receptor, e.g., CCR4 or CXCR4, to improve homing and antitumor activity in vivo.

[0195] If temporal control is desired, the chimeric Tim receptor vector may include an element that enables inducible depletion of the transduced cells. For example, such a vector may include an inducible suicide gene. The suicide gene may be an apoptotic gene or a gene that confers sensitivity to an agent (e.g., a drug). Exemplary suicide genes include chemoinducible caspase 9 (iCASP9) (U.S. Patent Publication No. 2013 / 0071414), chemoinducible Fas, or herpes simplex virus thymidine kinase (HSV-TK) that confers sensitivity to ganciclovir. In a further embodiment, the chimeric Tim receptor vector may be designed to express a known cell surface antigen that enables depletion of the transduced cells upon injection of the relevant antibody. Examples of cell surface antigens and their associated antibodies that can be used for the depletion of transduced cells include CD20 and rituximab, RQR8 (a mixed CD34 and CD20 epitope that enables CD34 selection and anti-CD20 depletion) and rituximab, as well as EGFR and cetuximab.

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

[0197] In certain embodiments, chimeric Tim receptor-modified host cells may also be modified to co-express one or more small GTPases. Rho GTPases, a family of small (approximately 21 kDa) signaling G proteins and also subfamilies of the Ras superfamily, regulate actin cytoskeletal organization in various cell types and promote pseudopod elongation and phagosome closure during phagocytosis (see, e.g., Castellano et al., 2000, J. Cell Sci. 113:2955-2961). Phagocytosis requires F-actin mobilization beneath the tethered cell or particle and F-actin rearrangement that allows for membrane elongation resulting in intracellular or intraparticle movement. Rho GTPases include RhoA, Rac1, Rac2, RhoG, and CDC42. Other small GTPases, e.g., Rap1, are involved in the regulation of complement-mediated phagocytosis. Co-expression of small GTPases and chimeric Tim receptors may promote target cell or particle internalization and / or phagosome formation by host cells. In some embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded in a separate vector from the chimeric Tim receptor-containing vector. In other embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded in the same vector as the chimeric Tim receptor. The GTPase and chimeric Tim receptor may be expressed under the control of different promoters in the same vector (e.g., at different multi-cloning sites). Alternatively, the chimeric Tim receptor and GTPase may be expressed under the control of a single promoter in a multi-cistronic vector. The polynucleotide sequences encoding the chimeric Tim receptor and the polynucleotide sequences encoding the small GTPase may be separated from each other by IRES or viral 2A peptides in a multi-cistronic vector. Exemplary 2A peptides include T2A (SEQ ID NO: 12), P2A (SEQ ID NO: 13), E2A (SEQ ID NO: 14), and F2A (SEQ ID NO: 15). Examples of GTPases that can be co-expressed with chimeric Tim receptors include Rac1, Rac2, Rab5 (also known as Rab5a), Rab7, Rap1, RhoA, RhoG, CDC42, or any combination thereof.In certain embodiments, the GTPase includes, or is, a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the Rac1 amino acid sequence of SEQ ID NO: 17, the Rab5 amino acid sequence of SEQ ID NO: 18, the Rab7 amino acid sequence of SEQ ID NO: 19, the Rap1 amino acid sequence of SEQ ID NO: 20, the RhoA amino acid sequence of SEQ ID NO: 21, the CDC42 amino acid sequence of SEQ ID NO: 22, or any combination thereof.

[0198] In certain embodiments, cells such as immune cells obtained from a subject may be modified into non-natural or recombinant cells (e.g., non-natural or recombinant immune cells) by introducing a polynucleotide encoding the chimeric Tim receptor described herein, thereby causing the cells to express a cell surface-localized chimeric Tim receptor. In certain embodiments, the host cells are immune cells, e.g., myeloid progenitor cells or lymphocyte progenitor cells. Exemplary immune cells that may be modified to include a polynucleotide encoding the chimeric Tim receptor or a vector containing a polynucleotide encoding the chimeric Tim receptor include T cells, natural killer cells, B cells, lymphocyte progenitor cells, antigen-presenting cells, dendritic cells, Langerhans cells, myeloid progenitor cells, mature myeloid cells, monocytes, or macrophages.

[0199] In certain embodiments, B cells are genetically modified to express one or more chimeric Tim receptors. The B cells possess certain properties that may make them beneficial as host cells, including the ability to transport to inflammatory sites, internalize and present antigens, co-stimulate T cells, and exhibit high proliferative and self-renewing (lifelong) capabilities. In certain embodiments, chimeric Tim receptor-modified B cells can digest phagocytosed target cells or phagocytosed target particles into smaller peptides, which they then present to T cells via MHC molecules. Antigen presentation by chimeric Tim receptor-modified B cells may contribute to the antigenic expansion of the immune response to non-targeting antigens. B cells include B cell lineage-committed progenitor or precursor cells (e.g., preproB cells, proB cells, and pre-B cells); immature and inactivated B cells; or mature and functional or activated B cells. In certain embodiments, B cells may be naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic cells, plasmablasts, memory B cells, or any combination thereof. Memory B cells may be distinguished from naive B cells by the expression of CD27, which is absent in naive B cells. In certain embodiments, B cells may 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 many sources, including blood, bone marrow, spleen, lymph nodes, or other tissues or fluids. B cell compositions may be enriched or purified.

[0200] In certain embodiments, T cells are genetically modified to express one or more chimeric Tim receptors. An exemplary T cell expresses CD4 + Helper, CD8 + Effector (cytotoxic), Naive (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-), Central Memory (CD45RO + CD62L + CD8 +These include T cells, effector memory cells (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), T memory stems, regulatory cells, mucosal-associated invariants (MAIT), γδ(gd), tissue-resident T cells, natural killer T cells, or any combination thereof. In certain embodiments, T cells may be primary cells or cell lines derived from humans, mice, rats, or other mammals. When obtained from mammals, T cells can be obtained from many sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cell compositions may be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In certain embodiments, T cells lack endogenous expression of the TCRα gene, the TCRβ gene, or both. Such T cells may naturally lack endogenous expression of TCRα and β chains, or may be modified to block their expression (e.g., T cells from transgenic mice that do not express TCRα and β chains, or cells engineered to inhibit the expression of TCRα and β chains), or to knock out the genes for TCRα chain, TCRβ chain, or both.

[0201] In certain embodiments, the host cell expressing the chimeric Tim protein of this disclosure on its cell surface is not a T cell or a T cell lineage cell, but a progenitor cell, stem cell, or cell modified to express anti-CD3 on its cell surface.

[0202] In certain embodiments, chimeric Tim receptor-modified host cells may also be modified to co-express cellular immunotherapy agents (e.g., CARs, TCRs, etc.). In some embodiments, the cellular immunotherapy agent includes a chimeric antigen receptor (CAR). A CAR is a recombinant receptor that generally includes: an extracellular domain containing a binding domain that binds to a target antigen; an intracellular signaling domain (e.g., an ITAM-containing intracellular signaling domain and an appropriate intracellular costimulatory domain); and a transmembrane domain located between and connecting the extracellular and intracellular signaling domains.

[0203] Suitable binding domains for use in CARs of this disclosure include any antigen-binding polypeptide. The binding domain may include, for example, an antibody or its antigen-binding fragments comprising a full-length heavy chain, Fab fragment, Fab', F(ab')2, sFv, VH domain, VL domain, dAb, VHH, CDR, and scFv. In certain embodiments, the CAR-binding domain is a mouse, chimeric, human, or humanized CAR-binding domain.

[0204] In certain embodiments, the binding domain of CAR targets cancer or tumor antigens. Exemplary antigens that CAR can target 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, and HPV. This includes 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.

[0205] In certain embodiments, the extracellular domain of the CAR provided in this disclosure may include an extracellular non-signaling spacer or linker domain. If included, such a spacer or linker domain may position the binding domain away from the host cell surface to further enable appropriate cell-to-cell contact, binding, and activation. The extracellular spacer domain is generally located between the extracellular binding domain and the transmembrane domain of the CAR. The length of the extracellular spacer may vary to optimize target molecule binding 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; PCT Publication WO2014 / 031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. The modified IgG4 hinge region is described in PCT Publication WO2014 / 031687, and the hinge region is incorporated herein by reference in its entirety. In certain embodiments, the extracellular spacer domain includes a modified IgG4 hinge region having the amino acid sequence of SEQ ID NO: 3.

[0206] Other examples of hinge regions that may be used in CARs described herein include hinge regions from the extracellular domains of type 1 membrane proteins, which may be wild-type or mutants, such as CD8a, CD4, CD28, and CD7. In certain embodiments, the extracellular spacer domain includes a CD8a hinge region having the amino acid sequence of SEQ ID NO: 70. In another particular embodiment, the extracellular spacer domain includes a CD28 hinge region having the amino acid sequence of SEQ ID NO: 32. In further embodiments, the extracellular spacer domain includes all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, for example, PCT Publication WO2014 / 031687, the spacer being incorporated herein by reference in its entirety). In yet another embodiment, the extracellular spacer domain may include a stalk region of type II C lectin (an extracellular domain located between the C lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.

[0207] The CARs of this disclosure include a transmembrane domain connecting an extracellular domain and an intracellular signaling domain, and located between them. The transmembrane domain is in the range of approximately 15 to 30 amino acids in length. The transmembrane domain is a hydrophobic alpha-helix that traverses the host cell membrane and fixes the CAR in the host cell membrane. The transmembrane domain may be fused directly to the binding domain or, if present, to the extracellular spacer domain. In certain embodiments, the transmembrane domain is derived from an endogenous membrane protein [e.g., receptors, cluster of differentiation (CD) molecules, enzymes, transporters, cell adhesion molecules, etc.]. The transmembrane domain may be selected from the same molecule as the extracellular domain or the intracellular signaling domain (e.g., the CAR includes a CD28 co-stimulatory signaling domain and a CD28 transmembrane domain). In certain embodiments, the transmembrane domain and the extracellular domain are each selected from different molecules. In other embodiments, the transmembrane domain and the intracellular signaling domain are each selected from different molecules. In yet another embodiment, the transmembrane domain, extracellular domain and intracellular signaling domain are each selected from different molecules.

[0208] Exemplary transmembrane domains for use in CARs of this disclosure include CD28, CD2, CD4, CD8a, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), and CD270(HVEM). The transmembrane domains include CD272(BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, and Zap70. An exemplary CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the transmembrane domain includes the CD8a transmembrane domain having the amino acid sequence of SEQ ID NO: 33.

[0209] The intracellular signaling domain of a CAR is an intracellular effector domain that, in response to the binding of the extracellular domain of the CAR to a target molecule (e.g., a cancer antigen), transmits a functional signal to the cell and can activate at least one of the normal effector functions or responses of immune cells engineered to express the CAR, such as T cells. In some embodiments, the CAR induces T cell function, e.g., cytolytic activity or T helper activity, e.g., secretion of cytokines or other factors. The intracellular signaling domain can be any portion of an intracellular signaling molecule that retains sufficient signaling activity. In some embodiments, the intracellular signaling domain is obtained from an antigen receptor component (e.g., TCR) or a costimulatory molecule. In some embodiments, the full-length intracellular signaling domain of an antigen receptor or costimulatory molecule is used. In some embodiments, a cleaved portion of the intracellular signaling domain of an antigen receptor or costimulatory molecule is used, provided that the cleaved portion retains sufficient signaling activity. In further embodiments, the intracellular signaling domain is a full-length or cleaved variant of the intracellular signaling domain of an antigen-receptor costimulatory molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

[0210] In some embodiments, the intracellular signaling domain of the CAR includes an immune receptor tyrosine-based activation motif (ITAM)-containing signaling domain. The ITAM-containing signaling domain typically contains at least one (1, 2, 3, 4 or more) ITAMs, which are YXXL / IX 6-8- Refers to the conserved motif YXXL / I. ITAM-containing signaling domains can initiate T cell activation signaling after antigen binding or ligand engagement. ITAM signaling domains include, for example, intracellular signaling domains for CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278(ICOS), DAP12, FcRγ, and CD66d. An exemplary CD3ζ signaling domain that may be used in the CARs of this disclosure includes the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 5.

[0211] The CAR intracellular signaling domain may include a costimulatory signaling domain that, when activated in conjunction with a major or classical (e.g., ITAM-driven) activating signal, promotes or enhances T cell responses, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. Co-stimulatory signaling domains for use in CAR include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, ZAP70, or any combination thereof. In some embodiments, the co-stimulatory signaling domain includes the OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137) signaling domain. An exemplary CD28 co-stimulatory signaling domain that may be used in the CAR of this disclosure includes the amino acid sequence of SEQ ID NO: 26 or 4. An exemplary 4-1BB co-stimulatory signaling domain includes the amino acid sequence of SEQ ID NO: 100. In certain embodiments, the CAR includes one, two, or more co-stimulatory signaling domains.

[0212] In some embodiments, the CAR is a recombinant receptor comprising an antibody-derived scFv-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is derived from the TCR.

[0213] In certain embodiments, the chimeric antigen receptor comprises an amino acid sequence derived from any mammalian species, including humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, their transgenic species, or any combination thereof. In certain embodiments, the chimeric antigen receptor is mouse, chimeric, human, or humanized.

[0214] In certain embodiments, the CAR is a first-generation CAR, a second-generation CAR, or a third-generation CAR. A first-generation CAR generally has an intracellular signaling domain including an intracellular signaling domain of CD3ζ, FcγRI, or another ITAM-containing activating domain to provide a T cell activation signal. A second-generation CAR further includes a co-stimulatory signaling domain (e.g., a co-stimulatory signaling domain from an endogenous T cell co-stimulatory receptor, e.g., CD28, 4-1BB, or ICOS). A third-generation CAR includes an ITAM-containing activating domain, a first co-stimulatory signaling domain, and a second co-stimulatory signaling domain.

[0215] In some embodiments, one or more of the extracellular domain, binding domain, linker, transmembrane domain, intracellular signaling domain, or costimulatory domain include junctional amino acids. “Junctional amino acids” or “junctional amino acid residues” refers to one or more (e.g., about 2 to 20) amino acid residues between two adjacent domains, motifs, regions, modules, or fragments of a protein, e.g., between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or at one or both ends of a linker connecting two domains, motifs, regions, modules, or fragments (e.g., between the linker and an adjacent binding domain or between the linker and an adjacent hinge). Junctional amino acids may arise from the construct design of the fusion protein (e.g., amino acid residues resulting from the use of restriction enzyme sites or self-cleaving peptide sequences during the construction of the polynucleotide encoding the fusion protein). For example, the transmembrane domain of a fusion protein may have one or more junctional amino acids at its amino terminus, carboxyl terminus, or both.

[0216] In certain embodiments, the manipulated host cells co-express a chimeric Tim receptor and an anti-CD72 CAR.

[0217] In some embodiments, the anti-CD72 CAR binding domain comprises (i) a heavy chain variable (VH) region comprising heavy chain complementarity determination region 1 (HCDR-1) containing the amino acid sequence shown in SEQ ID NO: 71; heavy chain complementarity determination region 2 (HCDR-2) containing the amino acid sequence shown in SEQ ID NO: 72; and heavy chain complementarity determination region 3 (HCDR-3) containing the amino acid sequence shown in SEQ ID NO: 73; and (ii) a light chain variable (VL) region comprising light chain complementarity determination region 1 (LCDR-1) containing the amino acid sequence shown in SEQ ID NO: 74; light chain complementarity determination region 2 (LCDR-2) containing the amino acid sequence shown in SEQ ID NO: 75; and light chain variable (VL) region comprising light chain complementarity determination region 3 (LCDR-3) containing the amino acid sequence shown in SEQ ID NO: 76; Or (iii) a heavy chain variable (VH) region including heavy chain complementarity determination region 1 (HCDR-1) containing the amino acid sequence shown in SEQ ID NO: 77; heavy chain complementarity determination region 2 (HCDR-2) containing the amino acid sequence shown in SEQ ID NO: 78; and heavy chain complementarity determination region 3 (HCDR-3) containing the amino acid sequence shown in SEQ ID NO: 79; and (iv)(ii) a light chain variable (VL) region including light chain complementarity determination region 1 (LCDR-1) containing the amino acid sequence shown in SEQ ID NO: 80; light chain complementarity determination region 2 (LCDR-2) containing the amino acid sequence shown in SEQ ID NO: 81; and light chain complementarity determination region 3 (LCDR-3) containing the amino acid sequence shown in SEQ ID NO: 82.

[0218] In some embodiments, the CAR binding domain includes (i) a VH region containing the amino acid sequence shown in SEQ ID NO: 83 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 83, and a segment containing the amino acid sequence shown in SEQ ID NO: 84 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 84. A VL region containing a column; or (ii) a VH region containing a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 85 or SEQ ID NO: 85, and a VL region containing a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 86 or SEQ ID NO: 86.

[0219] Exemplary binding domain, extracellular spacer domain, transmembrane domain, and intracellular signaling domain sequences, as well as exemplary anti-CD72 CAR sequences, for use in anti-CD72 CARs as disclosed herein are shown in Table 9 and described in the U.S. Provisional Patent Application filed August 14, 2020, entitled “Anti-CD72 Chimeric Receptors and Uses Thereof,” which is incorporated herein by reference in its entirety.

[0220] Table 9. [Table 9-1] [Table 9-2] [Table 9-3] Table 9-4 Table 9-5 Table 9-6 Table 9-7 Table 9-8 Table 9-9 Table 9-10 Table 9-11 Table 9-12 Table 9-13

[0221] In certain embodiments, chimeric Tim receptor-modified host cells co-express recombinant TCRs. Recombinant TCR proteins include “traditional” TCRs consisting of a heterodimer of α-chain polypeptide and β-chain polypeptide or a heterodimer of γ-chain polypeptide and δ-chain polypeptide, such as single-chain TCRs, single-domain TCRs, soluble TCR fusion TCR proteins, and TCR fusion constructs [TRuC®], as well as their binding fragments and fusion proteins. In certain embodiments, the tandem expression cassette includes a polynucleotide encoding a recombinant TCR beta chain containing a TCR beta variable region and a TCR beta constant region, and a polynucleotide encoding a recombinant TCR alpha chain containing a TCR alpha variable region and a TCR alpha constant region. In certain embodiments, the recombinant TCR is a high-affinity TCR. In one embodiment, the recombinant TCR is a high-affinity TCR.

[0222] In certain embodiments, the recombinant TCR-binding protein is a single-chain TCR (scTCR) containing Vα linked to Vβ by a flexible linker. In some embodiments, the scTCR contains a Vα-linker-Vβ polypeptide. In other embodiments, the scTCR contains a Vβ-linker-Vα polypeptide.

[0223] In certain embodiments, chimeric Tim receptor-modified host cells may also be modified to co-express a single-chain TCR (scTCR) fusion protein. The scTCR fusion protein comprises an scTCR-containing binding domain (a TCR Vα domain linked to a TCR Vβ domain), an appropriate extracellular spacer, a transmembrane domain, and an intracellular component comprising a single intracellular signaling domain providing a T cell activation signal (e.g., a CD3ζ ITAM-containing activation domain) and an appropriate co-stimulatory signaling domain (see Aggen et al., 2012, Gene Ther. 19:365-374; Stone et al., Cancer Immunol. Immunother. 2014, 63:1163-76).

[0224] In certain embodiments, chimeric Tim receptor-modified host cells may also be modified to co-express a T cell receptor-based chimeric antigen receptor (TCR-CAR). A TCR-CAR is a heterodimer fusion protein that typically contains a soluble TCR (a polypeptide chain containing Vα and Cα domains, and a polypeptide chain containing Vβ and Cβ domains), the VβCβ polypeptide chain being linked to a transmembrane domain and intracellular signaling components (e.g., an ITAM-containing activating domain and, as appropriate, a co-stimulatory signaling domain) (see, e.g., Walseng et al., 2017 Scientific Reports 7:10713).

[0225] In a particular embodiment, engineered host cells co-expressing a chimeric Tim receptor and a cellular immunotherapy agent (e.g., CAR, TCR, etc.) contain recombinant nucleic acids encoding the chimeric Tim receptor and recombinant nucleic acid molecules encoding the cellular immunotherapy agent on separate vectors within the engineered host cells.

[0226] In some embodiments, engineered host cells co-expressing a chimeric Tim receptor and a cellular immunotherapy agent (e.g., CAR, TCR, etc.) contain the recombinant nucleic acid encoding the chimeric Tim receptor and the recombinant nucleic acid molecule encoding the cellular immunotherapy agent on the same vector as the chimeric Tim receptor within the engineered host cells. The chimeric Tim receptor and the cellular immunotherapy agent may be expressed under the control of different promoters of the same vector (e.g., at different multi-cloning sites). Alternatively, the chimeric Tim receptor and the cellular immunotherapy agent may be expressed under the control of a single promoter in a multi-cistronic vector (e.g., a tandem expression vector). The polynucleotide sequences encoding the chimeric Tim receptor and the polynucleotide sequences encoding the cellular immunotherapy agent may be separated by IRES or viral 2A peptide in the multi-cistronic vector.

[0227] The tandem expression cassette, the tandem expression vector, and the engineered host cells containing them are described in International Patent Publication WO2019 / 191339, which is incorporated herein by reference in its entirety.

[0228] In certain embodiments, gene editing techniques are used to modify the host cell genome to include polynucleotides encoding the chimeric Tim receptor of this disclosure. Gene editing, or genome editing, is a genetic engineering technique in which DNA is inserted, replaced, or removed from the host cell genome using a genetically engineered endonuclease. The nuclease creates a specific double-strand break at a targeted locus in the genome. The host cell's endogenous DNA repair pathway then repairs the induced break(s), for example, by non-homologous ending joining (NHEJ) and homologous recombination. Exemplary endonucleases useful for gene editing include zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, CRISPR (clustered regularly interspaced short palindromic repeats) / Cas nuclease systems (e.g., CRISPR-Cas9), meganucleases, or combinations thereof. Methods for disrupting or knocking out genes or gene expression in immune cells, including B cells and T cells, using gene-editing endonucleases are publicly known in the art and are described, for example, in International Patent Publications WO2015 / 066262; WO2013 / 074916; WO2014 / 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.

[0229] In certain embodiments, the expression of endogenous genes in host 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 IGH, IGκ, IGλ, or any combination thereof. Examples of endogenous genes that can be inhibited, knocked down, or knocked out in T cells include TCR genes (TRA or TRB), HLA genes (HLA class I or HLA class II), 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, or PVRIG / CD112R), or any combination thereof. The expression of endogenous genes can be inhibited, knocked down, or knocked out at the gene level, transcriptional level, translational level, or a combination thereof. Methods for inhibiting, knocking down, or knocking out endogenous genes can be achieved, for example, by RNA interference agents (e.g., siRNA, shRNA, miRNA, etc.) or engineered endonucleases [e.g., CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), meganucleases] or any combination thereof. In certain embodiments, an endogenous B cell gene (e.g., IGH, IGκ, or IGλ) is knocked out by insertion of a polynucleotide encoding the chimeric Tim receptor of this disclosure into the locus of the endogenous B cell gene, such as via an engineered endonuclease. In certain embodiments, an endogenous T cell gene (e.g., a TCR gene, an HLA gene, or an immune checkpoint molecule gene) is knocked out by insertion of a polynucleotide encoding the chimeric Tim receptor of this disclosure into the locus of the endogenous T cell gene, such as via an engineered endonuclease.

[0230] In certain embodiments, host cells can be genetically modified to express one type of chimeric Tim receptor. In other embodiments, host cells may express at least two or more different chimeric Tim receptors.

[0231] Furthermore, this disclosure provides compositions comprising a population of chimeric Tim receptor-modified host cells. In certain embodiments, the population of chimeric Tim receptor-modified host cells may be a B cell population, a T cell population, a natural killer cell population, a lymphocyte precursor cell population, an antigen-presenting cell population, a dendritic cell population, a Langerhans cell population, a bone marrow precursor cell population, a mature bone marrow cell population, or any combination thereof. Moreover, a population of chimeric Tim receptor-modified host cells of a particular cell type may consist of one or more subtypes. For example, a B cell population may consist of chimeric Tim receptor-modified naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic cells, plasmablasts, memory B cells, or any combination thereof. In another example, a T cell population may consist of chimeric Tim receptor-modified CD4 + Helper T cells, CD8 + Effector (cytotoxic) T cells, naive (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-) T cells, central memory (CD45RO + CD62L + CD8 + It may consist of T cells, effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-) T cells, T memory stem cells, regulatory T cells, mucosa-associated invariant T cells (MAIT), γδ(gd) cells, tissue-resident T cells, natural killer T cells, or any combination thereof.

[0232] In one particular embodiment, the host cell population consists of cells, each expressing the same chimeric Tim receptor. In another embodiment, the host cell population consists of a mixture of two or more subpopulations of host cells, each subpopulation expressing a different chimeric Tim receptor or set of chimeric Tim receptors.

[0233] In certain embodiments, when preparing chimeric Tim receptor-modified host cells, such as B cells or T cells, one or more growth factor cytokines that promote the proliferation of host cells, such as B cells or T cells, may be added to the cell culture. The cytokines may be human or non-human cytokines. Exemplary growth factor cytokines that may be used to promote T cell proliferation include IL-2, IL-15, and others. Exemplary growth factor cytokines that may be used to promote B cell proliferation include CD40L, IL-2, IL-4, IL-15, IL-21, BAFF, and others.

[0234] Prior to genetic modification of host cells with a chimeric Tim receptor vector, a source of host cells (e.g., T cells, B cells, natural killer cells, etc.) is obtained from the subject (e.g., whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue), from which host cells are isolated using methods known in the art. A specific subset of host cells is collected according to known techniques such as affinity binding to an antibody, flow cytometry and / or immunomagnetic selection, and can be enriched or depleted by known techniques. After the enrichment and / or depletion steps and the introduction of the chimeric Tim receptor, in vitro expansion and proliferation of the desired modified host cells may be carried out according to known techniques or variations thereof that are apparent to those skilled in the art.

[0235] The chimeric Tim receptors of the present disclosure confer cytotoxic activity on host cells expressing the chimeric Tim receptor specific for phosphatidylserine. Thus, upon binding to phosphatidylserine exposed on the surface of target cells, host cells expressing the chimeric Tim receptor can induce apoptosis of the target cells. In certain embodiments, host cells expressing the chimeric Tim receptor induce apoptosis of target cells via release of granzyme, perforin, granulysin or any combination thereof; Fas ligand-Fas interaction; or both. In further embodiments, the chimeric Tim receptor further confers phosphatidylserine-specific phagocytic activity on host cells expressing the chimeric Tim receptor. In still further embodiments, host cells do not naturally exhibit a phagocytic phenotype prior to modification by the chimeric Tim receptor.

[0236] The chimeric Tim receptors of this disclosure may also be able to co-stimulate T cells via at least one signaling pathway. In certain embodiments, the chimeric Tim receptor provides a co-stimulatory signal to T cells via at least two different signaling pathways (e.g., via selected co-stimulatory signaling domains in the chimeric Tim receptor). For example, a chimeric Tim receptor comprising a CD28 co-stimulatory signaling domain may be able to provide a co-stimulatory signal via CD28 and Tim1. In certain embodiments, host immune cells expressing the chimeric Tim receptor exhibit reduced or inhibited immune cell exhaustion. In certain embodiments, the host immune cells are T cells or NK cells. In certain embodiments, exhausted T cells exhibit (a) increased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof; (b) decreased production of IFN-γ, IL-2, TNF-α, or any combination thereof; or both (a) and (b). In certain embodiments, exhausted NK cells exhibit (a) increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) decreased production of IFN-γ, TNF-α, or both; or both (a) and (b).

[0237] In certain embodiments, host cells expressing the chimeric Tim receptor exhibit an enhanced effector response (e.g., tumor-specific). In certain embodiments, the effector response may be enhanced T cell proliferation, cytokine production (e.g., IFN-γ, IL-2, TNF-α), cytotoxic activity, persistence, or any combination thereof. Host cells expressing the chimeric Tim receptor may be administered to a target alone or in combination with other therapeutic agents, such as CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, oncolytic viruses, electrotherapy, etc.

[0238] In certain embodiments, host cells expressing the chimeric Tim receptor exhibit a reduced immunosuppressive response to phosphatidylserine. Phosphatidylserine is one of the major apoptotic cell ligands that signals phagocytic cells to "eat." The removal of apoptotic cells by phagocytic cells generally reduces or inhibits the inflammatory response through decreased secretion of the anti-inflammatory cytokines IL-10 and TGF-β, as well as the inflammatory cytokines TNF-α, IL-1β, and IL-12. Therefore, phosphatidylserine can act as an immunosuppressive signal during the clearance of apoptotic cells. In certain embodiments, upon binding to phosphatidylserine, chimeric Tim receptor-modified host cells exhibit increased antigen-specific cytokine production (e.g., IFN-γ, IL-2, TNF-α), thereby reducing the immunosuppressive response to phosphatidylserine.

[0239] In some embodiments, T cells expressing the chimeric Tim receptor exhibit increased antigen capture, antigen processing, and / or antigen presentation activity, or enhance antigen capture, antigen processing, and / or antigen presentation activity. A method for measuring the ability of chimeric Tim receptor T cells to present target peptide antigens and induce target peptide-specific activation in target peptide-specific T cells is described in Example 2.

[0240] The expression of chimeric Tim receptors on host cells may be functionally characterized according to any of the many art-acceptable methods for assaying host cell (e.g., T cell) activity, including determining T cell binding, activation, or induction, and also determining antigen-specific T cell responses. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, and CTL activity (e.g., from pre-loaded target cells). 51This includes determining changes in T cell phenotypic marker expression and other measures of T cell function (by detecting Cr or europium release). Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and the references cited therein. Cytokine levels can be determined according to methods known in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and any combination thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from antigen-specific induction or stimulation of an immune response can be determined by isolating lymphocytes, such as circulating lymphocytes, from a sample of peripheral blood cells or lymph node cells, stimulating the cells with an antigen, and measuring cytokine production, cell proliferation, and / or cell viability by means of tritiated thymidine incorporation or non-radioactive assays such as MTT assays.

[0241] In certain embodiments, chimeric Tim receptor-modified host cells have a phagocytic index of approximately 20 to approximately 1,500 with respect to 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 or particles ingested per chimeric Tim receptor-modified host cell during the incubation period of a suspension of target cells or particles in culture medium and chimeric Tim receptor-modified host cells. The phagocytic index is calculated as [total number of phagocytosed target cells / total number of counted chimeric Tim receptor-modified cells (e.g., phagocytic frequency)] x [chimeric Tim receptor + [Average area of ​​target cells or particles stained per host cell x 100 (e.g., hybrid capture)] or [Total number of phagocytosed particles / Total number of chimeric Tim receptor-modified host cells counted] x [Number of chimeric Tim receptor-modified host cells containing phagocytosed particles / Number of chimeric Tim receptors counted] +The total number of cells can be calculated by multiplying by 100. In a particular embodiment, chimeric Tim receptor modified cells can be approximately 30-1,500; approximately 40-1,500; approximately 50-1,500; approximately 75-1,500; approximately 100-1,500; approximately 200-1,500; approximately 300-1,500; approximately 400-1,500; approximately 500-1,500; approximately 20-1,400; approximately 30-1,400; approximately 40-1,400; approximately 50-1,400; approximately 100-1,400; approximately 200-1,400; approximately 300- Approximately 1,400; Approximately 400-1,400; Approximately 500-1,400; Approximately 20-1,300; Approximately 30-1,300; Approximately 40-1,300; Approximately 50-1,300; Approximately 100-1,300; Approximately 200-1,300; Approximately 300-1,300; Approximately 400-1,300; Approximately 500-1,300; Approximately 20-1,200; Approximately 30-1,200; Approximately 40-1,200; Approximately 50-1,200; Approximately 100-1,200; Approximately 200-1,200; Approximately 300 ~approximately 1,200; approximately 400~approximately 1,200; approximately 500~approximately 1,200; approximately 20~approximately 1,100; approximately 30~approximately 1,100; approximately 40~approximately 1,100; approximately 50~approximately 1,100; approximately 100~approximately 1,100; approximately 200~approximately 1,100; approximately 300~approximately 1,100; approximately 400~approximately 1,100; or approximately 500~approximately 1,100; approximately 20~approximately 1,000; approximately 30~approximately 1,000; approximately 40~approximately 1,000; approximately 50~approximately 1,000; approximately 100~approximately 1,000; approximately 200~approximately 1,000; The phagocytic index is approximately 300 to 1,000; approximately 400 to 1,000; or approximately 500 to 1,000; approximately 20 to 750; approximately 30 to 750; approximately 40 to 750; approximately 50 to 750; approximately 100 to 750; approximately 200 to 750; approximately 300 to 750; approximately 400 to 750; or approximately 500 to 750; approximately 20 to 500; approximately 30 to 500; approximately 40 to 500; approximately 50 to 500; approximately 100 to 500; approximately 200 to 500; or approximately 300 to 500. In further embodiments, the incubation time is approximately 2 to 4 hours, approximately 2 hours, approximately 3 hours, or approximately 4 hours. In further embodiments, chimeric Tim receptor-modified cells exhibit a statistically significantly higher phagocytic index than cells transduced with cleaved EGFR controls.The phagocytic index can be calculated using methods known in the art, including quantification by flow cytometry or fluorescence microscopy, and further described in the examples and PCT application PCT / US2017 / 053553 (which is incorporated herein by reference in its entirety).

[0242] The host cells may be from animals, such as humans, primates, cattle, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, or combinations thereof. In a preferred embodiment, the animal is human. The host cells may be obtained from a healthy subject or from a subject having a disease associated with the expression or overexpression of an antigen.

[0243] How to use In one embodiment, the Disclosure provides a method for conferring or enhancing phosphatidylserine-specific cytotoxic activity of cells, comprising: introducing a nucleic acid molecule or chimeric Tim receptor vector encoding at least one chimeric Tim receptor as described in any of the embodiments described herein into host cells; and expressing at least one chimeric Tim receptor in the host cells, wherein at least one chimeric Tim receptor enhances the phosphatidylserine-specific cytotoxic activity of the host cells compared to host cells prior to modification for expressing the chimeric Tim receptor. In certain embodiments, the cytotoxic activity of host cells increases by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more compared to host cells before modification with the nucleic acid molecule encoding the chimeric Tim receptor or the chimeric Tim receptor vector. In some embodiments, the host cells are immune cells. In some embodiments, the host cells are T cells or NK cells. Methods for measuring the cytotoxic activity of host cells, particularly immune cells such as T cells and NK cells, include chromium (51Cr) release assays, β-gal or firefly luciferase release assays, and flow cytometry, which mediate targeted cell death and effector cell activity (see, e.g., Expert Rev. Vaccines, 2010, 9:601-616).

[0244] In certain embodiments, a method for conferring or enhancing the phosphatidylserine-specific cytotoxic activity of cells further comprises conferring or enhancing the phosphatidylserine-specific phagocytic activity of host cells expressing at least one chimeric Tim receptor. In certain such embodiments, the host cells do not naturally exhibit a phagocytic phenotype before modification with the chimeric Tim receptor. For example, in certain such embodiments, the phagocytic activity of the host cells is increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more compared to host cells before modification to express the chimeric Tim receptor vector. In certain embodiments, the host cells do not naturally possess phagocytic activity. In some embodiments, the host cells are immune cells. In some embodiments, the host cells are T cells or NK cells. Methods for measuring the phagocytic activity of host cells include those described in International Patent Publication WO2018 / 064076 (which is incorporated herein by reference in its entirety).

[0245] In another embodiment, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell expressing a chimeric Tim receptor as described in any embodiment provided herein may be used in a method for enhancing the effector function of a host cell. In certain embodiments, the enhanced effector function includes increased cytotoxic activity, increased antigen-specific cytokine production (e.g., IFN-γ, IL-2, TNF-α, or any combination thereof), increased anti-apoptotic signaling, increased persistence, increased growth, increased proliferation, or any combination thereof. In certain embodiments, the effector function of host cells is improved by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more compared to host cells not modified by a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cells are immune cells. In certain embodiments, the host cells are T cells or NK cells.

[0246] In another embodiment, host cells modified with the chimeric Tim receptor of the present disclosure may be used in methods for inhibiting or reducing immune cell exhaustion. In some embodiments, the immune cells are T cells or NK cells. In certain embodiments, reduced exhaustion in T cells includes (a) reduced expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof in T cells; (b) increased production of IFN-γ, IL-2, TNF-α, or any combination thereof in T cells; or both (a) and (b). In certain embodiments, reduced exhaustion in NK cells includes (a) reduced expression of PD-1, NKG2A, TIM3, or any combination thereof in NK cells; (b) increased production of IFN-γ, TNF-α, or both thereof in NK cells; or both (a) and (b). In certain embodiments, the expression of an immune checkpoint molecule is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in host immune cells expressing the chimeric Tim receptor compared to host immune cells not modified by the nucleic acid molecule encoding the chimeric Tim receptor or the chimeric Tim receptor vector. In certain embodiments, cytokine expression is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more in host immune cells expressing the chimeric Tim receptor compared to host immune cells not modified by the nucleic acid molecule encoding the chimeric Tim receptor or the chimeric Tim receptor vector.

[0247] In another embodiment, the chimeric Tim receptor, the polynucleotide encoding the chimeric Tim receptor, the chimeric Tim receptor vector, or the host cell expressing the chimeric Tim receptor described in any of the embodiments provided herein may be used in a method for reducing the immunosuppressive response to phosphatidylserine in a host cell. In certain embodiments, the immunosuppressive response includes the secretion of anti-inflammatory cytokines (e.g., IL-10, TGF-β, or both), a decrease in the secretion of inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-12), or both. In certain embodiments, the immunosuppressive response of host cells to phosphatidylserine is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% compared to host cells not modified by a nucleic acid molecule encoding a chimeric Tim receptor or a chimeric Tim receptor vector. In some embodiments, the host cells are immune cells. In certain embodiments, the host cells are T cells or NK cells.

[0248] In another embodiment, the chimeric Tim receptor, the polynucleotide encoding the chimeric Tim receptor, the chimeric Tim receptor vector, or the host cell expressing the chimeric Tim receptor described in any embodiment provided herein may be used in a method for removing target cells that hold surface-exposed phosphatidylserine, for example, for removing cancer cells that hold surface-presented phosphatidylserine. In a particular embodiment, the target cells are damaged cells, stressed cells, apoptotic cells, or necrotic cells (e.g., tumor cells) that hold surface-exposed phosphatidylserine. In a particular embodiment, the host cell expressing the chimeric Tim receptor clears the damaged target cells, stressed target cells, apoptotic target cells, or necrotic target cells that hold surface-exposed phosphatidylserine by inducing apoptosis, or by inducing both apoptosis and phagocytosis. The host cell expressing the chimeric Tim receptor may be administered to a target alone or in combination with other therapeutic agents, such as CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, oncolytic viruses, electropulse therapy, etc.

[0249] In another embodiment, the chimeric Tim receptor, the polynucleotide encoding the chimeric Tim receptor, the chimeric Tim receptor vector, or host cells expressing the chimeric Tim receptor as described in any embodiment provided herein may be used in methods to enhance the effects of therapeutic agents that induce cellular stress, injury, necrosis, or apoptosis. Certain therapies, such as chemotherapy, radiotherapy, UV phototherapy, electrotherapy, adoptive cell immunotherapy (e.g., CAR-T cells, TCRs), and oncolytic virus therapy, can induce cellular injury or cell death in tumor cells, diseased cells, and cells in their surrounding environment. Cells expressing the chimeric Tim receptor can be administered in combination with cytotoxic / cytotoxic therapies that bind to the phosphatidylserine moiety exposed on the outer leaflet of targeting cells, and can clear stressed, injured, diseased, apoptotic, and necrotic cells.

[0250] In another embodiment, the Disclosure provides a method for conferring or enhancing the antigen capture, antigen processing, and / or antigen presentation activity of cells, comprising: introducing a nucleic acid molecule or chimeric Tim receptor vector encoding at least one chimeric Tim receptor as described in any of the embodiments described herein into host cells; and expressing at least one chimeric Tim receptor in the host cells, the at least one chimeric Tim receptor improving the antigen capture, antigen processing, and / or antigen presentation activity of the host cells compared to host cells before modification for expressing the chimeric Tim receptor. In some embodiments, the chimeric Tim receptor comprises an ITAM-containing intracellular signaling domain and / or a co-stimulatory domain; and a TLR2 or TLR8 intracellular signaling domain. In some embodiments, the addition of a TLR signaling domain, e.g., a TLR2 intracellular signaling domain or a TLR8 intracellular signaling domain, to a chimeric Tim receptor design having traditional T cell signaling (e.g., CD28 and / or CD3ζ) improves T cell antigen capture, antigen processing, and / or antigen presentation.

[0251] In another embodiment, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell expressing a chimeric Tim receptor, as described in any of the embodiments provided herein, may be used in methods for treating subjects suffering from a disease, disorder, or undesirable condition. Embodiments of these methods involve administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising one or more chimeric Tim receptors, one or more polynucleotides encoding chimeric Tim receptors, a vector containing one or more polynucleotides encoding chimeric Tim receptors, or a population of genetically modified host cells expressing one or more chimeric Tim receptors, as described herein.

[0252] Diseases that can be treated with cells expressing the chimeric Tim receptor described herein include cancer and infectious diseases (viral infections, bacterial infections, fungal infections, and protozoan infections). 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 (Kitchen 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, 2011).

[0253] A wide variety of cancers, including solid tumors and leukemias, are suitable for the compositions and methods disclosed herein. Exemplary cancers that can be treated using the receptors, modified host cells and compositions described herein include adenocarcinomas of the breast, prostate, and colon; all forms of bronchogenic lung cancer; myeloid leukemia; melanoma; hepatocellular carcinoma; neuroblastoma; papilloma; apdoma; spondylolisthesis; branchiomas; malignant carcinoid syndromes; carcinoid heart disease; and carcinomas (e.g., Walker's carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pierce carcinoma, tubular carcinoma, Ehrlich carcinoma, Krebs II carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, sclerotic carcinoma, bronchiolar carcinoma, bronchogenic lung cancer, squamous cell carcinoma, and transitional cell carcinoma). Additional cancer types that can be treated using the receptors, modified host cells, and compositions described herein include histiocytic disorders; malignant histiocytosis; leukemia; Hodgkin's disease; immunoproliferative small; non-Hodgkin lymphoma; plasmacytoma; multiple myeloma; chronic myeloid leukemia (CML); acute myeloid leukemia (AML); plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; undifferentiated germ cell tumor; hamartoma; mesenchymaloma; mesonephroma; sarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; and trophoblastoma. Furthermore, the following types of cancer: adenoma; cholangiomas; cholesteatoma; cyclindroma; cystadenoma; cystadenoma; granulosa cell tumor; androblastoma; hepatocellular carcinoma; sweat adenoma; islet tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; sarcoma; rhabdomyomyoma; rhabdomyosarcoma; ependymoma; gangliomas; gliomas; medulloblastoma; meningioma; schwannomas; neuroblastomas; neuroepitheliomas; neurofibromas; neuromas; paragangliomas; and nonchrome-affinity paragangliomas are intended to be suitable for treatment using the receptors, modified host cells, and compositions described herein.Furthermore, the types of cancer that can be treated include angiokeratoma; angiolymphoid hyperplasia with eosinophilia; sclerosing hemangioma; hemangioma; glomus hemangioma; hemangioendothelioma; hemangioma; hemangioextortoidoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; phyllodes cystic sarcoma; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukemosarcoma; liposarcoma; lymphangiosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasm; neurofibromatosis; cervical dysplasia; and peritoneal cancer.

[0254] Examples of hyperproliferative disorders suitable for treatment using the receptors, modified host cells, and compositions described herein include B-cell cancers (B-cell tumors), including B-cell lymphomas [e.g., various forms of Hodgkin's disease, non-Hodgkin lymphoma (NHL), or central nervous system lymphoma], leukemias [e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, B-cell blastogenesis of chronic myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia], and myelomas [e.g., multiple myeloma]. Additional B-cell cancers that can be treated using the receptors, modified host cells, and compositions described herein include small lymphocytic lymphoma, B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytomyeloma, solitary plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodular 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 exudative lymphoma, Burkitt lymphoma / leukemia, B-cell proliferation with the potential for malignant transformation, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorders.

[0255] Infectious diseases include infectious diseases associated with infectious pathogens and encompass any of the following: various bacteria [e.g., pathogenic Escherichia coli (E. coli), Salmonella typhimurium, Pseudomonas aeruginosa, Bacillus anthracis, Clostridium botulinum, C. difficile, Clostridium perfringens, Helicobacter pylori, Vibrio cholerae, Listeria spp., Rickettsia spp., Chlamydia spp., etc.], mycobacteria, and parasites (including any known parasitic members of protozoa). Infectious viruses include eukaryotic viruses, such as adenoviruses, bunyaviruses, herpesviruses, papovaviruses, papillomaviruses (e.g., HPV), paramyxoviruses, picornaviruses, rhabdoviruses (e.g., rabies), orthomyxoviruses (e.g., influenza), poxviruses (e.g., vaccinia), reoviruses, retroviruses, lentiviruses (e.g., HIV), flaviviruses (e.g., HCV, HBV), and others. In certain embodiments, compositions comprising the chimeric Tim receptor described herein are used to treat infections caused by microorganisms that can establish a persistent infection in a subject.

[0256] The chimeric Tim receptors of this disclosure may be administered to a subject in a cell-bound form (e.g., gene therapy of a target cell population). Therefore, for example, the chimeric Tim receptors of this disclosure may be expressed on the surface of T cells, natural killer cells, natural killer T cells, B cells, lymphocyte precursor cells, antigen-presenting cells, dendritic cells, Langerhans cells, myeloid precursor cells, mature myeloid cells, or any combination thereof, including subsets thereof, and administered to a subject. In certain embodiments, a method of treating a subject includes administering an effective amount of chimeric Tim receptor-modified cells (i.e., recombinant cells expressing one or more chimeric Tim receptors). The chimeric Tim receptor-modified cells may be heterogeneous, syngeneic, allogeneic, or autologous to the subject.

[0257] Pharmaceutical compositions containing chimeric Tim receptor-modified cells may be administered in an appropriate manner to the disease or condition to be treated (or prevented), as determined by medical experts. The appropriate dose, preferred duration, and frequency of administration of the composition are determined by factors such as the patient's condition, size, weight, body surface area, age, sex, type and severity of the disease, the specific treatment to be administered, the specific form of the active ingredient, the time and method of administration, and other drugs administered concurrently. This disclosure provides pharmaceutical compositions comprising chimeric Tim receptor-modified cells and pharmaceutically acceptable carriers, diluents, or excipients. Preferred excipients include water, saline, dextrose, glycerol, and others, and combinations thereof. Other preferred infusion media may be any isotonic media formulations containing saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water, or Ringer's lactate solution.

[0258] A therapeutically effective amount of cells in a pharmaceutical composition is at least one cell (e.g., one chimeric Tim receptor-modified T cell), or more typically 10 2 More than a single cell, for example, up to 10 6 10 pieces, up to 10 7 10 pieces, up to 10 8 Individual cells, up to 10 9 Individual cells, up to 10 10 Individual cells or up to 10 11 10 cells or more. In a particular embodiment, there are about 10 cells. 6 pieces ~ about 10 10 individual cells / m 2 Within the range, preferably about 10 7 pieces ~ about 10 9 individual cells / m 2The cells are administered within a range of [specify range]. The number of cells depends on the cell types contained in the composition, as well as the intended end use of the composition. For example, a composition containing cells modified to contain chimeric Tim receptors contains a cell population containing about 5% to about 95% or more such cells. In certain embodiments, a composition containing chimeric Tim receptor 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 such cells. For the uses provided herein, cells generally exist in volumes of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the desired cell density is typically 10 4 It is higher than the number of cells / ml, and is generally 10 7 Higher than the number of cells / ml, generally 10 8 The number of cells / ml or higher. Cells may be administered as a single infusion or in multiple infusions over a time period. Repeated infusions of chimeric Tim receptor-modified cells may be isolated over days, weeks, months, or even years if a recurrence of disease or disease activity is present. The clinically relevant immune cell count is cumulatively 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 It can be distributed in multiple infusions equal to or exceeding the number of cells. A preferred dose for administering host cells containing the recombinant expression vector described herein is about 10 7 individual cells / m 2 , about 5x10 7 individual cells / m 2 , about 10 8 individual cells / m 2 , about 5x10 8 individual cells / m 2 , about 10 9 individual cells / m 2 , about 5x10 9 individual cells / m 2, about 10 10 individual cells / m 2 , about 5x10 10 individual cells / m 2 or about 10 11 individual cells / m 2 That is the case.

[0259] The chimeric Tim receptor compositions described herein may be administered intravenously, intraperitoneally, intranasally, intratumorally, intraboneally, intraly into the bone marrow, intraly into lymph nodes, and / or intracerebrospinal fluid.

[0260] Chimeric Tim receptor compositions may be administered to a subject in combination with one or more additional therapeutic agents. Examples of therapeutic agents that may be administered in combination with the chimeric Tim compositions described herein include radiotherapy, adoptive cell immunotherapy agents (e.g., recombinant TCR, high-affinity TCR, CAR, TCR-CAR, scTCR fusion protein, dendritic cell vaccine), antibody therapy, immune checkpoint molecule inhibitor therapy, UV phototherapy, electropulse therapy, high-intensity focused ultrasound therapy, oncolytic virus therapy or pharmaceutical therapy, including, for example, chemotherapeutic agents, therapeutic peptides, hormones, aptamers, antibiotics, antivirals, antifungals, anti-inflammatory agents, small molecule therapies, or any combination thereof. In certain embodiments, chimeric Tim receptor-modified host cells may clear stressed cells, damaged cells, apoptotic cells, necrotic cells, infected cells, and dead cells that present surface phosphatidylserine induced by one or more additional therapeutic agents.

[0261] In certain embodiments, the chimeric Tim receptor and adoptive cell immunotherapy agents (e.g., CAR, TCR-CAR, TCR, etc., as described above) are administered to the target in the same host cells or different host cells. In certain embodiments, the chimeric Tim receptor and adoptive cell immunotherapy agents are expressed in the same host cells from the same vector or from separate vectors. In certain embodiments, the chimeric Tim receptor and adoptive cell immunotherapy agents are expressed in the same host cells from a multicistronic vector. In certain embodiments, the chimeric Tim receptor is expressed in the same host cell type as the adoptive cell immunotherapy agent (e.g., the chimeric Tim receptor is expressed in CD4 T cells and the CAR / or TCR is expressed in CD4 T cells, or the chimeric Tim receptor is expressed in CD8 T cells and the CAR / or TCR is expressed in CD8 T cells). In other embodiments, the chimeric Tim receptor is expressed in a different host cell type than the adoptive immunotherapy agent (e.g., the chimeric Tim receptor is expressed in CD4 T cells and the CAR / or TCR is expressed in CD8 T cells). Cellular immunotherapy compositions, methods of preparation, and methods of use, including combinations of immune cells or cell subsets engineered with the chimeric Tim receptor and cell immunotherapy agents (e.g., CAR, TCR, etc.), are described in PCT International Publication WO2019 / 191340, which is incorporated herein by reference in its entirety.

[0262] Exemplary antigens that recombinant TCRs, high-affinity TCRs, CARs, TCR-CARs, or scTCR fusion proteins can target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, Survivin, α-fetoprotein, and tumor-specific neogenic antigens.

[0263] The CARs of this disclosure can target a variety of antigens, including viral antigens, bacterial antigens, fungal antigens, parasitic antigens, tumor antigens, and autoimmune disease antigens. Exemplary antigens that the CARs can target 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, and HPV. This includes 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.

[0264] Radiotherapy includes external beam radiation therapy (e.g., conventional external beam radiation, stereotactic radiation, three-dimensional conformal radiation therapy, intensity-modulated radiation therapy, intensity-modulated pendulum radiation therapy, particle beam therapy, proton beam therapy, and auger therapy), close-range radiation therapy, systematic radioisotope therapy, intraoperative radiation therapy, or any combination thereof.

[0265] Exemplary antibodies for use in combination with the chimeric Tim compositions described herein include rituxmab, pertuzumab, trastuzumab, alemtuzumab, ibritumomab tiuxetan, brentuximab vedotin, cetuximab, bevacizumab, absiximab, adalimumab, alefacept, basilizimab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, daclizumab, denosumab, efalizumab, golimumab, olaratumab, palivizumab, panitumumab, and tocilizumab.

[0266] Exemplary immune checkpoint molecule inhibitors that may be used in combination with the chimeric Tim compositions described herein include checkpoint inhibitors targeting 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 inhibitor may be an antibody, peptide, RNAi agent, or small molecule. A CTLA-4 specific antibody may be ipilimumab or tremelimumab. A PD-1 specific antibody may be pidilizumab, nivolumab, or pembrolizumab. Antibodies specific to PD-L1 may be durvalumab, atezolizumab, or avelumab.

[0267] Exemplary chemotherapeutic agents for use in combination with the chimeric Tim compositions described herein may include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folate antagonists, pyrimidine analogs, purine analogs, and glycosylation analogs), DNA synthesis inhibitors, DNA interaction agents (e.g., insertion agents), and DNA repair inhibitors.

[0268] Chemotherapy agents include nonspecific cytotoxic agents that inhibit mitosis or cell division, and molecular targeting therapies that block the growth and spread of cancer cells by targeting specific molecules (e.g., oncogenes) involved in tumor growth, progression, and metastasis. Exemplary nonspecific chemotherapeutic agents for use in combination with the expression cassette compositions described herein may include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folate antagonists, pyrimidine analogs, purine analogs, and glycosylation analogs), DNA synthesis inhibitors, DNA interaction agents (e.g., insertion agents), hypomethylating agents, and DNA repair inhibitors.

[0269] Examples of chemotherapeutic agents considered for use in combination therapies intended herein include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole [Arimidex®], bicalutamide [Casodex®], bleomycin sulfate [Blenoxane®], busulfan [Milleran®], busulfan injection [Busulfex®], capecitabine [Xeloda®], and N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine Carboplatin [Paraplatin®], Carmustine [BiCNU®], Chlorambucil [Leukeran®], Cisplatin [Platinol®], Cladribine [Leustatin®], Cyclophosphamide [Citoxane® or Neosar®], Cytarabine, Cytosine Arabinoside [Cytosar-U®], Cytarabine Liposome Injection [Depotite®], Dacarbazine [DTIC-Dome®], Dactino Mycin [Actinomycin D, Cosmegan], Daunorubicin hydrochloride [Cervidin®], Daunorubicin citrate liposome injection [DaunoXome®], Dexamethasone, Docetaxel [Taxotere®], Doxorubicin hydrochloride [Adriamycin®, Rubex®], Etoposide [Vepsid®], Fludarabine phosphate [Fludarabine®], 5-Fluorouracil [Adrucil®] ), Efudex (registered trademark), Flutamide (Eulexin (registered trademark)), Tezacitibine, Gemcitabine (Difluorodeoxycytidine), Hydroxyurea (Hydrea (registered trademark)), Idarubicin (Idamycin (registered trademark)), Ifosfamide (IFEX (registered trademark)), Irinotecan (Camptosar (registered trademark)), L-Asparaginase (ELSPAR (registered trademark)), Leucovorin calcium, Melphalan (Alkeran (registered trademark)),6-Mercaptopurine [Purinesol®], Methotrexate [Folex®], Mitoxantrone [Novantron®], Mylotarg, Paclitaxel [Taxol®], Phoenix (Yttrium 90 / MX-DTPA), Pentostatin, Polyfeprozan 20 Carmustine Implant [Gliadel®], FDARA Tamoxifen Citrate [Nolvadex®], Teni This includes Poside [Vumon (registered trademark)], 6-thioguanine, thiotepa, tirapazamine [Tirazone (registered trademark)], topotecan hydrochloride injection [Hycamptin (registered trademark)], vinblastine [Vervan (registered trademark)], vincristine [Oncovin (registered trademark)], ibrutinib, venetoclax, crizotinib, alectinib, brigatinib, ceritinib, and vinorelbine [Navelbine (registered trademark)].

[0270] Exemplary alkylating agents for use in combination therapies intended herein include nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosourea, and triazenes: uracil mustard [aminouracil mustard®, chlorethaminacil®, demethyldopan®, desmethyldopan®, haemanthamine®, Nordopan®, uracil nitrogen mustard®, uracillost®, uracilmostaza®, uramustin®, uramustine®], chlormethine [Mustargen®], cyclophosphamide [cytoxane®, neosal®, clafen] (Clafen) (registered trademark), Endoxan (registered trademark), Procytox (registered trademark), Revimmune (trademark)), Ifosfamide [Mitoxana (registered trademark)], Melphalan [Alkeran (registered trademark)], Chlorambucil [Leukeran (registered trademark)], Pipobroman [Amedel (registered trademark), Vercyte (registered trademark)], Triethylenemelamine [Hemel (registered trademark), Hexalen (registered trademark)] This includes Hexastat (registered trademark), triethylenethiophosphoramine, temozolomide (Temodal (registered trademark)), thiotepa (Thioplex (registered trademark)), busulfan (Busilvex (registered trademark), Milleran (registered trademark)), carmustine (BiCNU (registered trademark)), lomustine (CeeNU (registered trademark)), streptozosin (Zanosar (registered trademark)), and dacarbazine (DTIC-Dome (registered trademark)).Additional exemplary alkylating agents for use in combination therapies intended herein include oxaliplatin [Eloxatin®]; temozolomide [Temodar® and Temodal®]; dactinomycin [also known as actinomycin-D, Cosmegen®]; melphalan [also known as L-PAM, L-sarcolicin, and phenylalanine mustard, Alkeran®]; and altoretamine [Hexamethylmelamine (HMM:hex (Also known as amethylmelamine, Hexalen®); Carmustine [BiCNU®]; Bendamustine [Treanda®]; Busulfan [Busulfex® and Milleran®]; Carboplatin [Paraplatin®]; Lomustine [Also known as CCNU, CeeNU®]; Cisplatin [Also known as CDDP, Platinol® and Platinol®-AQ]; Chlorambucil [Leukeran®]; Cyclophospha Mido [Citoxane (registered trademark) and Neosal (registered trademark)]; Dacarbazine [also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome (registered trademark)]; Altretamine [also known as hexamethylmelamine (HMM), Hexalen (registered trademark)]; Ifosfamide [Ifex (registered trademark)]; Prednumustine; Procarbazine [Matulane (registered trademark)]; Mechloretamine [Nitrogen mustard, Mustine and Mechlore This includes, but is not limited to, thamine hydrochloride (also known as Mastergen®); streptozosin (Zanosar®); thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Citoxan®, Neosal®, Procitox®, Revimun®); and bendamustine HCl (Treanda®).

[0271] Exemplary platinum-based agents for use in combination therapies as envisioned herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin, and triplatin tetranitrate.

[0272] Exemplary hypomethylating agents for use in combination therapy include azacitidine and decitabine.

[0273] Exemplary molecular targeting inhibitors for use in combination with the chimeric Tim receptor compositions described herein include small molecules that target molecules involved in cancer cell proliferation and survival, such as receptor tyrosine kinase inhibitors, RAF inhibitors, BCL-2 inhibitors, ABL inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4 / 6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PDGFRA inhibitors, and RET inhibitors.

[0274] Exemplary molecular targeting therapies include hormone antagonists, signaling inhibitors, gene expression inhibitors (e.g., translation inhibitors), apoptosis inducers, angiogenesis inhibitors (e.g., VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g., EGF / EGFR pathway inhibitors), growth factor inhibitors, GTPase inhibitors, serine / threonine kinase inhibitors, transcription factor inhibitors, inhibitors of cancer-associated driver mutations, B-Raf inhibitors, RAF inhibitors, MEK inhibitors, mTOR inhibitors, and adenosine pathway inhibitors. These include EGFR inhibitors, PI3K inhibitors, BCL2 inhibitors, VEGFR inhibitors, MET inhibitors, MYC inhibitors, BCR-ABL inhibitors, ABL inhibitors, HER2 inhibitors, H-RAS inhibitors, K-RAS inhibitors, PDGFR inhibitors, ALK inhibitors, ROS1 inhibitors, BTK inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4 / 6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PARP inhibitors, PDGFRA inhibitors, and RET inhibitors. In certain embodiments, the use of molecular targeting therapy involves administering molecular targeting therapy specific to a molecular target to a subject identified as having a tumor having the molecular target (e.g., a driver oncogene). In certain embodiments, the molecular target has an activating mutation. In certain embodiments, the use of chimeric Tim receptor-modified cells in combination with molecular targeting inhibitors increases the magnitude of the antitumor response, the persistence of the antitumor response, or both. In certain embodiments, molecularly targeted therapies at lower doses than typical doses are used in combination with chimeric Tim receptor-modified cells.

[0275] Exemplary 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 (Prinomast) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), and ALN-VSP (ALN-VSP O2) (Alnylam). Pharmaceuticals), AMG386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Lidaforolimus / MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ197 (ArQule), ASA404 (Novartis / Antisoma), Atiprimod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (Bevacizumab) (Genentech), AZD2171 [Sediranib / Recentin] (AstraZeneca), BAY 57-9352 [Telatinib] (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim Pharmaceuticals), BIBW2992 (Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-MyersSquibb), BMS-582664 (brivanib) (Bristol-Myers Squibb), BMS-690514 (Bristol-Myers Squibb), calcitriol, CCI-779 (Torisel) (Wyeth), CDP-791 (ImClone Systems), ceflatonin (homohalintinin / HHT) (ChemGenex Therapeutics), celerex (celecoxib) (Pfizer), CEP-7055 (Cephalon / Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 [Metastat] (Collagenex Pharmaceuticals), Combretastatin (Oxigene), CP-751,871 (Figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Daiichi Sankyo), CT-322 [Angiocept] (Adnexus), Curcumin, Dalteparin (Fragmin) (Pfizer), Disulfiram (Antabuse), E7820 (Eisai Co., Ltd.), E7080 (Eisai Co., Ltd.), EMD 121974 (Cilenditide) (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 (Introgen Therapeutics), Iressa (ZD1839 / gefitinib), LBH589 [Faridak / Panobinostat] (Novartis), Lucentis (ranivizumab) (Genentech / Novartis), LY317615 (Enzastaurin) (Eli Lilly and Company), Macugen (pegaptanib) (Pfizer), MEDI522 [Abegrin] (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 Capsules (2ME2) ​​(EntreMed), Panzem NCD (2ME2) ​​(EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen Industries / Medigen Biotechnology), PKC412 (Novartis), Polypheno E (Green Tea Extract) (Polypheno E International, Inc.), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Vatalanib) (Novartis), PXD101 (Belinostat) (CuraGen (Corporation), RAD001 (everolimus) (Novartis), RAF265 (Novartis), regorafenib (BAY73-4506) (Bayer), levlimid (Celgene), retaane (Alcon)Research), SN38 (liposome formulation) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (pasireotide) (Novartis), squalamine (Genaera), suramin, sutent (Pfizer), tarceva (Genentech), TB-403 (Thrombogenics), tempostatin (Collard Biopharmaceuticals), tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), thalidomide (Celgene Corporation), tinzaparin sodium, TKI258 (Novartis), TRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (Afrivercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), beglin (VesGene) This includes, but is not limited to, bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), vorinostat (Merck), and ZSTK474.

[0276] Examples of B-Raf inhibitors include vemurafenib, dabrafenib, and encorafenib.

[0277] Examples of MEK inhibitors include binimetinib, cobimetinib, refametinib, selumetinib, and trametinib.

[0278] Exemplary BTK inhibitors include ibrutinib, pirtobrutinib (Loxo-305), tirabrutinib, trebrutinib, evobrutinib, fenebrutinib (GDC-0853), acalabrutinib, becabrutinib (SNS-062), ONO-4059, spebralutinib, zanubrutinib (BGB-3111), HM71224, and M7583.

[0279] Exemplary TRK inhibitors include entrectinib, larotrectinib, CH7057288, ONO-7579, LOXO-101, restaurtinib, and LOXO-195.

[0280] Exemplary c-KIT inhibitors include imatinb, sunitinb, and ponatinib.

[0281] Exemplary c-MET inhibitors include capmatinib, crizotinib, tivantinib, onartuzumab, INCB28060, AMG-458, savolitinib, and tepotinib.

[0282] Exemplary CDK4 / 6 inhibitors include palbociclib, ribociclib, abemaciclib, and trilaciclib.

[0283] Examples of FAK inhibitors include defactinib, GSK2256098, BI853520, and PF-00562271.

[0284] Examples of FGFR inhibitors include erdafitinib, pemigatinib, infiglatinib, logaratinib, AZD4547, BGJ398, FP-1039, and ARQ 087.

[0285] Examples of FLT-3 inhibitors include quizartinib, klenolanib, gilteritinib, midostaurin, and restaurtinib.

[0286] Examples of IDH1 inhibitors include ivosidenib, BAY-1436032, and AGI-5198.

[0287] An example of an IDH2 inhibitor is enasidenib.

[0288] Exemplary PARP inhibitors include talazoparib, niraparib, lucaparib, olaparib, veliparib, CEP9722, and E7016.

[0289] Examples of PDGFRA inhibitors include imatinib, regorafenib, klenolanib, and olaratumab.

[0290] Exemplary pan-RAF inhibitors include belvarafenib, LXH254, LY3009120, INU-152, and HM95573.

[0291] Exemplary RET inhibitors include lenvatinib, alectinib, vandetanib, cabozantinib, BLU-667, and LOXO-292.

[0292] Exemplary ROS1 inhibitors include ceritinib, lorlatinib, entrectinib, crizotinib, TPX-0005, and DS-6051b.

[0293] Exemplary vascular endothelial growth factor (VEGF) receptor inhibitors include bevacizumab [Avastin®], axitinib [Inlyta®]; brivanib alaninate [BMS-582664, (S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indole-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-yloxy)propan-2-yl)2-aminopropanoate]; sorafenib [Nexavar®]; pazopanib [Votrient®]; sunitinib malate [Sutent®]; sediranib (AZD2171, CAS 288383-20-1); balagatef (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Teratinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib [Gleevec®]; Ponatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Batalanib dihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib [Caprelsa (registered trademark) or AZD6474]; Motesanib diphosphate [AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indole-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide as described in PCT Publication WO02 / 066470]; Dovitinib dilactic acid (TKI258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Restaurtinib (CAS 111358-88-4);N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazine-5-yl)methyl)piperidine-3-ol (BMS690514); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrole-5-yl]methoxy]-4-quinazolinamine (XL647, CAS This includes, but is not limited to, 781613-23-8); 4-methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidine-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); and Afriversept [Eylea®].

[0294] Exemplary EGF pathway inhibitors include tilhostin 46, EKB-569, erlotinib [Tarceva®], gefitinib [Iressa®], erbitux, nimotuzumab, lapatinib [Tykerb®], cetuximab (anti-EGFR mAb), 188This includes, but is not limited to, Re-labeled nimotuzumab (anti-EGFR mAb) and compounds commonly and specifically disclosed in 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. Exemplary EGFR antibodies include, but are not limited to, cetuximab [Erbitux®]; panitumumab [Vectibix®]; matuzumab (EMD-72000); trastuzumab [Herceptin®]; nimotuzumab (hR3); zaltumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1).Exemplary epidermal growth factor receptor (EGFR) inhibitors include erlotinib hydrochloride [Tarceva®], ceritinib, brigutinib, osimeritinib, icotinib, gefitinib [Iressa®]; N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3''S'')-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®; vandetanib [Capre] Lusa (registered trademark); Lapatinib [Tykerb (registered trademark)]; (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazine-5-yl)methyl)piperidine-3-ol (BMS690514); Canertinib dihydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidine-4-amine (AEE788, CAS 497839-62-0); Mbritinib (TAK165); Peritinib (EKB569); Afatinib (BIBW2992); Neratinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazole-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-yl]carbamic acid, (3S)-3-morpholinyl methyl ester (BMS599626); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrole-5-yl]methoxy]-4-quinazolinamine (XL647, CAS This includes, but is not limited to, 781613-23-8); 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidine-6-yl]-phenol (PKI166, CAS 187724-61-4); and rocelitinib.

[0295] Exemplary mTOR inhibitors include rapamycin [Rapamune®] and its analogs and derivatives; SDZ-RAD; temsirolimus [Torisel®; also known as CCI-779]; ridafololimus [previously known as deferolimus]; (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 [Also known as hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, AP23573 and MK8669, described in PCT Publication WO03 / 064383]; everolimus [Afinitor® or RAD001]; rapamycin [AY22989, sirolimus®]; simapimod (CAS 164301-51-3); (5-{2,4-bis[(3S)-3-methylmorpholine-4-yl]pyrido[2,3-d]pyrimidine-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3-d]pyrimidine-7(8H)-one (PF04691502, CAS 1013101-36-4); and N 2 This includes, but is not limited to, -[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-, intramolecular salt (SF1126, CAS 936487-67-1).

[0296] Exemplary phosphoinositide 3-kinase (PI3K) inhibitors include duvelisib, idelalisib, 4-[2-(1H-indazole-4-yl)-6-[[4-(methylsulfonyl)piperazine-1-yl]methyl]thieno[3,2-d]pyrimidine-4-yl]morpholine (also known as GDC 0941 and described in PCT Publications WO09 / 036082 and WO09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinoline-3-yl)-2,3-dihydroimidazo[4,5-c]quinoline-1-yl]phenyl]propionitrile (BEZ 235 or NVP-BEZ) Also known as 235, described in PCT Publication WO06 / 122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidine-4-yl)pyridine-2-amine (also known as BKM120 or NVP-BKM120, described in PCT Publication WO2007 / 084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS This includes, but is not limited to, 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-(morpholine-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).Exemplary protein kinase B (PKB) or AKT inhibitors include 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridine-3(2H)-one (MK-2206, CAS 1032349-93-1); perifosine (KRX0401); 4-dodecyl-N-1,3,4-thiadiazole-2-ylbenzenesulfonamide (PHT-427, CAS 1191951-57-1); 4-[2-(4-amino-1,2,5-oxadiazole-3-yl)-1-ethyl-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridine-4-yl]-2-methyl-3-butin-2-ol (GSK690693, CAS 937174-76-0); 8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (paromid 529, P529 or SG-00529); Trisilbin (Tricirbine)(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-indazole-5-yl)-3-pyridinyl]oxy]methyl]benzeneethanamine (A674563, CAS This includes, but is not limited to, 552325-73-2); 4-[(4-chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidine-4-yl)-4-piperidineamine (CCT128930, CAS 885499-61-6); 4-(4-chlorophenyl)-4-[4-(1Hpyrazole-4-yl)phenyl]-piperidine (AT7867, CAS 857531-00-1); and alkexin (RX-0201, CAS 663232-27-7).

[0297] In certain embodiments, the tyrosine kinase inhibitor used in combination with chimeric Tim receptor-modified cells is an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, alectinib, brigatinib, dalantercept, entrectinib, and lorlatinib.

[0298] In certain embodiments in which chimeric Tim receptor-modified cells are administered in combination with one or more additional therapies, the one or more additional therapies may be administered in doses that would be considered less than therapeutic doses if administered otherwise as monotherapy. In such embodiments, the chimeric Tim receptor composition may provide additive or synergistic effects so that the one or more additional therapies can be administered in lower doses. The combination therapy includes the administration of the chimeric Tim receptor composition described herein before the additional therapy (e.g., 1 to 30 days before or earlier), concurrently with the additional therapy (on the same day), or after the additional therapy (e.g., 1 to 30 days after or later). In certain embodiments, the chimeric Tim receptor-modified cells are administered after the administration of one or more additional therapies. In further embodiments, chimeric Tim receptor-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 the administration of one or more additional therapies. In further embodiments, chimeric Tim receptor-modified cells are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after the administration of one or more additional therapies. If one or more additional therapies include multiple doses, chimeric Tim receptor-modified cells may be administered after the initial dose of one or more additional therapies, after the final dose of one or more additional therapies, or between multiple doses of one or more additional therapies.

[0299] In certain embodiments, the method of the present disclosure includes a depletion step. The depletion step for removing the chimeric Tim receptor from the subject may be performed after a sufficient time has elapsed to the therapeutic benefit in order to reduce toxicity to the subject. In such embodiments, the chimeric Tim receptor vector may include an inducible suicide gene, e.g., iCASP9, inducible Fas, or HSV-TK. Similarly, the chimeric Tim receptor vector may be designed for the expression of known cell surface antigens, e.g., CD20 or truncated EGFR (SEQ ID NO: 16), which promote the depletion of transduced cells through the infusion of an associated monoclonal antibody (mAb), e.g., rituximab for CD20 or cetuximab for EGFR. Alternatively, alemtuzumab targeting CD52 present on the surface of mature lymphocytes may be used to deplete transduced B cells, T cells, or natural killer cells.

[0300] Subjects that can be treated by the compositions and methods of this disclosure include animals, such as humans, primates, cattle, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. Subjects may be male or female and may be of any suitable age, including infants, young people, adolescents, adults, and elderly subjects. This disclosure relates, for example, to the following: [Section 1] (a)(i) Tim4 IgV domain and Tim1 mucin domain; or (ii) Tim1 IgV domain and Tim4 mucin domain An extracellular domain containing a binding domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain and an appropriate secondary intracellular signaling domain; (c) A transmembrane domain located between the extracellular domain and the intracellular signaling domain, and connecting them. Chimeric T cell immunoglobulins and mucin (Tim) receptors, including single-chain chimeric proteins. [Section 2] A chimeric Tim receptor as described in item 1, wherein the binding domain comprises a Tim4 IgV domain and a Tim1 mucin domain. [Section 3] The chimeric Tim receptor according to item 1 or 2, wherein the Tim4 IgV domain contains the amino acid sequence shown in SEQ ID NO: 34, or the Tim1 mucin domain contains the amino acid sequence shown in SEQ ID NO: 39, or both. [Section 4] A chimeric Tim receptor as described in item 1, wherein the binding domain comprises a Tim1 IgV domain and a Tim4 mucin domain. [Section 5] The chimeric Tim receptor described in item 4, wherein the Tim1 IgV domain contains the amino acid sequence shown in SEQ ID NO: 38, or the Tim4 mucin domain contains the amino acid sequence shown in SEQ ID NO: 35, or both. [Section 6] A chimeric Tim receptor according to item 1, 4, or 5, wherein the Tim1 IgV domain is a modified Tim1 IgV domain containing an R66G substitution in SEQ ID NO: 38. [Section 7] A chimeric Tim receptor as described in item 6, wherein the modified Tim1 IgV domain contains the amino acid sequence shown in SEQ ID NO: 41. [Section 8] A chimeric Tim receptor according to any one of items 1 to 7, further comprising an extracellular spacer domain. [Section 9] The chimeric Tim receptor described in item 8, wherein the extracellular spacer domain includes an IgG4 hinge region or a CD28 hinge region. [Section 10] The chimeric Tim receptor according to item 9, wherein the IgG4 hinge region comprises the amino acid sequence shown in SEQ ID NO: 3, or the CD28 hinge region comprises the amino acid sequence shown in SEQ ID NO: 32. [Section 11] A chimeric Tim receptor according to any one of items 1 to 10, wherein the transmembrane domain comprises a Tim1 transmembrane domain, a Tim4 transmembrane domain, or a CD28 transmembrane domain. [Section 12] The chimeric Tim receptor according to item 11, wherein the Tim1 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 8, the Tim4 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 6, 23, or 121, or the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7. [Section 13] A chimeric Tim receptor according to any one of items 1 to 12, wherein the primary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain. [Section 14] The chimeric Tim receptor according to item 13, wherein the Tim1 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 44, the Tim4 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 45, 124, or 125, the TRAF2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 48, the TRAF6 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 46, the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or 26, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 9, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 27, the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 122, or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 47. [Section 15] A chimeric Tim receptor according to any one of items 1 to 14, wherein the secondary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain. [Section 16] The chimeric Tim receptor according to item 15, wherein the Tim1 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 44, the Tim4 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 45, 124, or 125, the TRAF2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 48, the TRAF6 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 46, the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or 26, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 9, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 27, the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 122, or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 47. [Section 17] (i) The binding domain includes a Tim4 IgV domain and a Tim1 mucin domain, the primary intracellular signaling domain includes a TLR8 signaling domain, the secondary intracellular signaling domain includes a CD3ζ signaling domain, and the transmembrane domain includes a Tim1 transmembrane domain; (ii) The binding domain comprises a Tim4 IgV domain and a Tim1 mucin domain, the primary intracellular signaling domain comprises a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmembrane domain comprises a Tim1 transmembrane domain; or (iii) The binding domain comprises a Tim4 IgV domain and a Tim1 mucin domain, the primary intracellular signaling domain comprises a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmembrane domain comprises a CD28 transmembrane domain, The chimeric Tim receptor described in item 1. [Section 18] (i) The Tim4 IgV domain contains the amino acid sequence of SEQ ID NO: 34, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the TLR8 signaling domain contains the amino acid sequence of SEQ ID NO: 47, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27; and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (ii) The Tim4 IgV domain contains the amino acid sequence of SEQ ID NO: 34, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the CD28 signaling domain contains the amino acid sequence of SEQ ID NO: 4, the DAP12 signaling domain contains the amino acid sequence of SEQ ID NO: 9; and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; or (iii) The Tim4 IgV domain contains the amino acid sequence of SEQ ID NO: 34, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the CD28 signaling domain contains the amino acid sequence of SEQ ID NO: 4, the DAP12 signaling domain contains the amino acid sequence of SEQ ID NO: 9, and the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7, The chimeric Tim receptor described in item 17. [Section 19] (i) Does the single-chain chimeric protein contain amino acids 25-628 of SEQ ID NO: 67? (ii) The single-chain chimeric protein contains amino acids 25-416 of SEQ ID NO: 68; or (iii) The single-chain chimeric protein contains amino acids 25-422 of sequence number 69, Chimeric Tim receptor as described in item 17 or 18. [Section 20] (i) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 67? (ii) The single-chain chimeric protein contains the amino acid sequence of SEQ ID NO: 68; or (iii) A single-chain chimeric protein containing the amino acid sequence of SEQ ID NO: 69 A chimeric Tim receptor as described in any one of items 17-19. [Section 21] (a) an extracellular domain containing a binding domain including the Tim1 IgV domain and the Tim1 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain and an appropriate secondary intracellular signaling domain; (c) A transmembrane domain located between the extracellular domain and the intracellular signaling domain, and connecting them. Chimeric T cell immunoglobulin and mucin domain (Tim) receptors containing single-chain chimeric proteins. [Section 22] The chimeric Tim receptor as described in item 21, wherein the Tim1 IgV domain contains the amino acid sequence shown in SEQ ID NO: 38, or the Tim1 mucin domain contains the amino acid sequence shown in SEQ ID NO: 39, or both. [Section 23] The chimeric Tim receptor according to item 21 or 22, wherein the Tim1 IgV domain is a modified Tim1 IgV domain containing the R66G substitution in SEQ ID NO: 38. [Section 24] A chimeric Tim receptor as described in item 23, wherein the modified Tim1 IgV domain contains the amino acid sequence shown in SEQ ID NO: 41. [Section 25] A chimeric Tim receptor according to any one of items 21-24, further comprising an extracellular spacer domain. [Section 26] The chimeric Tim receptor as described in item 25, wherein the extracellular spacer domain includes an IgG4 hinge region or a CD28 hinge region. [Section 27] The chimeric Tim receptor according to item 26, wherein the IgG4 hinge region comprises the amino acid sequence shown in SEQ ID NO: 3, or the CD28 hinge region comprises the amino acid sequence shown in SEQ ID NO: 32. [Section 28] A chimeric Tim receptor as described in any one of items 21 to 27, wherein the transmembrane domain comprises a Tim1 transmembrane domain, a Tim4 transmembrane domain, or a CD28 transmembrane domain. [Section 29] The chimeric Tim receptor according to item 28, wherein the Tim1 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 8, the Tim4 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 6 or 23, or the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7. [Section 30] A chimeric Tim receptor as described in any one of items 21 to 29, wherein the primary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain. [Section 31] The chimeric Tim receptor according to item 30, wherein the Tim1 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 44, the Tim4 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 45, 124, or 125, the TRAF2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 48, the TRAF6 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 46, the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or 26, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 9, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 27, the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 122, or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 47. [Section 32] A chimeric Tim receptor according to any one of items 21 to 31, wherein the secondary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain. [Section 33] The chimeric Tim receptor according to item 32, wherein the Tim1 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 44, the Tim4 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 45, 124, or 125, the TRAF2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 48, the TRAF6 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 46, the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or 26, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 9, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 27, the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 122, or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 47. [Section 34] (i) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a Tim1 signaling domain, the secondary intracellular signaling domain contains a CD3ζ signaling domain, and the transmembrane domain contains a Tim1 transmembrane domain; (ii) The binding domain includes a Tim1 IgV domain and a Tim1 mucin domain, the primary intracellular signaling domain includes a Tim4 signaling domain, the secondary intracellular signaling domain includes a CD3ζ signaling domain, and the transmembrane domain includes a Tim1 transmembrane domain; (iii) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a CD28 signaling domain and the transmembrane domain contains a CD28 transmembrane domain; (iv) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a TRAF6 signaling domain and the transmembrane domain contains a Tim1 transmembrane domain; (v) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a TRAF6 signaling domain and the transmembrane domain contains a CD28 transmembrane domain; (vi) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a TRAF2 signaling domain and the transmembrane domain contains a Tim1 transmembrane domain; (vii) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a TRAF2 signaling domain and the transmembrane domain contains a CD28 transmembrane domain; (viii) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a TLR8 signaling domain, the secondary intracellular signaling domain contains a CD3ζ signaling domain, and the transmembrane domain contains a Tim1 transmembrane domain; (ix) The binding domain comprises a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain comprises a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmembrane domain comprises a CD28 transmembrane domain; or (x) The binding domain comprises a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain comprises a CD28 signaling domain; the secondary intracellular signaling domain comprises a DAP12 signaling domain; and the transmembrane domain comprises a Tim1 transmembrane domain. The chimeric Tim receptor described in item 21. [Section 35] (i) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the Tim1 signaling domain contains the amino acid sequence of SEQ ID NO: 44, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 4, and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (ii) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the Tim4 signaling domain contains the amino acid sequence of SEQ ID NO: 45, 124, or 125, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 5 or 27; and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (iii) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the CD28 signaling domain contains the amino acid sequence of SEQ ID NO: 4, and the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7; (iv) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the TRAF6 signaling domain contains the amino acid sequence of SEQ ID NO: 46, and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (v) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the TRAF6 signaling domain contains the amino acid sequence of SEQ ID NO: 46, and the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7; (vi) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the TRAF2 signaling domain contains the amino acid sequence of SEQ ID NO: 48, and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (vii) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the TRAF2 signaling domain contains the amino acid sequence of SEQ ID NO: 48, and the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7; (viii) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the TLR8 signaling domain contains the amino acid sequence of SEQ ID NO: 47, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27, and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (ix) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the CD28 signaling domain contains the amino acid sequence of SEQ ID NO: 4, the DAP12 signaling domain 9, and the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7; or (x) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the CD28 signaling domain contains the amino acid sequence of SEQ ID NO: 4, the DAP12 signaling domain contains the amino acid sequence of SEQ ID NO: 9, and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8. The chimeric Tim receptor described in item 34. [Section 36] (i) Does the single-chain chimeric protein contain amino acids 21-456 of SEQ ID NO: 49? (ii) Does the single-chain chimeric protein contain amino acids 21-471 of SEQ ID NO: 50? (iii) Does the single-chain chimeric protein contain amino acids 21-363 of SEQ ID NO: 51? (iv) Does the single-chain chimeric protein contain amino acids 21-590 of SEQ ID NO: 52? (v) Does the single-chain chimeric protein contain amino acids 21-596 of SEQ ID NO: 53? (vi) Does the single-chain chimeric protein contain amino acids 21-619 of sequence number 54? (vii) Does the single-chain chimeric protein contain amino acids 21-625 of sequence number 55? (viii) Does the single-chain chimeric protein contain amino acids 21-621 of sequence number 56? (ix) A single-chain chimeric protein containing amino acids 21-415 of SEQ ID NO: 57; or (x) The single-chain chimeric protein contains amino acids 21-409 of SEQ ID NO. 58, Chimeric Tim receptor as described in item 34 or 35. [Section 37] (i) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 49? (ii) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 50? (iii) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 51? (iv) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 52? (v) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 53? (vi) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 54? (vii) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 55? (viii) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 56? (ix) A single-chain chimeric protein containing the amino acid sequence of SEQ ID NO: 57; or (x) A single-chain chimeric protein contains the amino acid sequence of SEQ ID NO: 58, A chimeric Tim receptor as described in any one of items 34-36. [Section 38] (a)(i) Tim1 IgV domain and Tim1 mucin domain; (ii) Tim4 IgV domain and Tim4 mucin domain; (iii) Tim1 IgV domain and Tim4 mucin domain; or (iv) Tim4 IgV domain and Tim1 mucin domain An extracellular domain containing a binding domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain selected from the Tim1 signaling domain or the Tim4 signaling domain, and an appropriate secondary intracellular signaling domain; (c) A transmembrane domain located between the extracellular domain and the intracellular signaling domain, and connecting them. Chimeric T cell immunoglobulin and mucin domain (Tim) receptors containing single-chain chimeric proteins. [Section 39] (a) The Tim1 IgV domain contains the amino acid sequence shown in SEQ ID NO: 38, and the Tim1 mucin domain contains the amino acid sequence shown in SEQ ID NO: 39; (b) The Tim4 IgV domain contains the amino acid sequence shown in SEQ ID NO: 34, and the Tim4 mucin domain contains the amino acid sequence shown in SEQ ID NO: 35; (c) The Tim1 IgV domain contains the amino acid sequence shown in SEQ ID NO: 38, and the Tim4 mucin domain contains the amino acid sequence shown in SEQ ID NO: 35; or (d) The Tim4 IgV domain contains the amino acid sequence shown in SEQ ID NO: 34, and the Tim1 mucin domain contains the amino acid sequence shown in SEQ ID NO: 39. The chimeric Tim receptor described in item 38. [Section 40] The chimeric Tim receptor as described in section 38 or 39, wherein the Tim1 IgV domain is a modified Tim1 IgV domain containing the R66G substitution in SEQ ID NO: 38. [Section 41] A chimeric Tim receptor as described in item 40, wherein the modified Tim1 IgV domain comprises the amino acid sequence shown in SEQ ID NO: 41. [Section 42] A chimeric Tim receptor according to any one of items 38-41, further comprising an extracellular spacer domain. [Section 43] The chimeric Tim receptor described in item 42, wherein the extracellular spacer domain includes an IgG4 hinge region or a CD28 hinge region. [Section 44] The chimeric Tim receptor according to item 43, wherein the IgG4 hinge region comprises the amino acid sequence shown in SEQ ID NO: 3, or the CD28 hinge region comprises the amino acid sequence shown in SEQ ID NO: 32. [Section 45] A chimeric Tim receptor as described in any one of items 38 to 44, wherein the transmembrane domain comprises a Tim1 transmembrane domain, a Tim4 transmembrane domain, or a CD28 transmembrane domain. [Section 46] The chimeric Tim receptor according to item 45, wherein the Tim1 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 8, the Tim4 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 6 or 23, or the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7. [Section 47] A chimeric Tim receptor as described in any one of items 38 to 46, wherein the primary intracellular signaling domain includes a Tim1 signaling domain. [Section 48] A chimeric Tim receptor as described in item 47, comprising the Tim1 signaling domain with the amino acid sequence shown in SEQ ID NO: 44. [Section 49] A chimeric Tim receptor as described in any one of items 38 to 46, wherein the primary intracellular signaling domain includes a Tim4 signaling domain. [Section 50] A chimeric Tim receptor as described in item 49, comprising the amino acid sequence shown in SEQ ID NO: 45, 124, or 125 of the Tim4 signaling domain. [Section 51] A chimeric Tim receptor according to any one of items 38 to 50, wherein the secondary intracellular signaling domain includes a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain. [Section 52] The chimeric Tim receptor according to item 51, wherein the Tim1 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 44, the Tim4 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 45, 124, or 125, the TRAF2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 48, the TRAF6 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 46, the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or 26, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 9, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 27, the TLR2 signaling domain comprises the amino acid sequence of SEQ ID NO: 122, or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 47. [Section 53] (a)(i) an extracellular domain containing a binding domain including the Tim4 IgV domain and the Tim4 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain selected from the CD28 signaling domain, the CD3ζ signaling domain, and the 4-1BB signaling domain, and a secondary intracellular signaling domain selected from the TLR2 signaling domain or the TLR8 signaling domain; (c) A transmembrane domain located between the extracellular domain and the intracellular signaling domain, and connecting them. Chimeric T cell immunoglobulin and mucin domain (Tim) receptors containing single-chain chimeric proteins. [Section 54] The chimeric Tim receptor as described in item 53, wherein the Tim4 IgV domain contains the amino acid sequence shown in SEQ ID NO: 34, or the Tim4 mucin domain contains the amino acid sequence shown in SEQ ID NO: 35, or both. [Section 55] A chimeric Tim receptor as described in item 54, wherein the binding domain contains the amino acid sequence of SEQ ID NO: 2 or 42. [Section 56] A chimeric Tim receptor according to any one of items 53-55, further comprising an extracellular spacer domain. [Section 57] The chimeric Tim receptor as described in item 56, wherein the extracellular spacer domain includes an IgG4 hinge region or a CD28 hinge region. [Section 58] The chimeric Tim receptor according to item 57, wherein the IgG4 hinge region comprises the amino acid sequence shown in SEQ ID NO: 3, or the CD28 hinge region comprises the amino acid sequence shown in SEQ ID NO: 32. [Section 59] A chimeric Tim receptor as described in any one of items 53 to 58, wherein the transmembrane domain comprises a Tim1 transmembrane domain, a Tim4 transmembrane domain, or a CD28 transmembrane domain. [Section 60] The chimeric Tim receptor according to item 59, wherein the Tim1 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 8, the Tim4 transmembrane domain contains the amino acid sequence shown in SEQ ID NO: 6 or 23, or the CD28 transmembrane domain contains the amino acid sequence of SEQ ID NO: 7. [Section 61] A chimeric Tim receptor according to any one of claims 53 to 60, wherein the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 26, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 27, or the 4-1BB signaling domain comprises the amino acid sequence shown in SEQ ID NO: 100. [Section 62] A chimeric Tim receptor according to any one of claims 53 to 61, wherein the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 122, or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 47. [Section 63] A chimeric Tim receptor as described in any one of items 1 to 62, further comprising a tertiary intracellular signaling domain. [Section 64] The chimeric Tim receptor as described in item 63, wherein the tertiary intracellular signaling domain comprises a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3ζ signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain. [Section 65] The chimeric Tim receptor according to item 64, wherein the Tim1 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 44, the Tim4 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 45, 124, or 125, the TRAF2 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 48, the TRAF6 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 46, the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or 26, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 9, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 27, the TLR2 signaling domain comprises the amino acid sequence of SEQ ID NO: 122, or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 47. [Section 66] (a)(i) an extracellular domain containing a binding domain including the Tim4 IgV domain and the Tim4 mucin domain; (b) an intracellular signaling domain comprising a primary intracellular signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM); a secondary intracellular signaling domain comprising a co-stimulatory signaling domain, a Tim1 signaling domain, or a Tim4 signaling domain; and an intracellular signaling domain comprising a tertiary intracellular signaling domain comprising a TLR signaling domain; (c) A transmembrane domain located between the extracellular domain and the intracellular signaling domain, and connecting them. Chimeric T cell immunoglobulin and mucin domain (Tim) receptors containing single-chain chimeric proteins. [Section 67] The chimeric Tim receptor as described in item 66, wherein the ITAM-containing signaling domain is a CD3ζ signaling domain or a DAP12 signaling domain. [Section 68] A chimeric Tim receptor as described in section 66 or 67, wherein the co-stimulatory signaling domain is a 4-1BB signaling domain or a CD28 signaling domain. [Section 69] A chimeric Tim receptor according to any one of items 66-68, wherein the TLR signaling domain is either a TLR2 signaling domain or a TLR8 signaling domain. [Section 70] (i) The binding domain contains a Tim1 IgV domain and a Tim1 mucin domain; the primary intracellular signaling domain contains a Tim1 signaling domain, the secondary intracellular signaling domain contains a CD3ζ signaling domain, and the transmembrane domain contains a Tim1 transmembrane domain; (ii) The binding domain includes a Tim1 IgV domain and a Tim1 mucin domain, the primary intracellular signaling domain includes a Tim4 signaling domain, the secondary intracellular signaling domain includes a CD3ζ signaling domain, and the transmembrane domain includes a Tim1 transmembrane domain; (iii) The binding domain comprises a Tim4 IgV domain and a Tim4 mucin domain; the primary intracellular signaling domain comprises a Tim4 signaling domain, the secondary intracellular signaling domain comprises a CD3ζ signaling domain; and the transmembrane domain comprises a Tim4 transmembrane domain; or (iv) The binding domain comprises a Tim4 IgV domain and a Tim4 mucin domain; the primary intracellular signaling domain comprises a Tim1 signaling domain; the secondary intracellular signaling domain comprises a CD3ζ signaling domain; and the transmembrane domain comprises a Tim4 transmembrane domain. The chimeric Tim receptor described in item 38. [Section 71] (i) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the Tim1 signaling domain contains the amino acid sequence of SEQ ID NO: 44, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27; and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (ii) The Tim1 IgV domain contains the amino acid sequence of SEQ ID NO: 38, the Tim1 mucin domain contains the amino acid sequence of SEQ ID NO: 39, the Tim4 signaling domain contains the amino acid sequence of SEQ ID NO: 45, 124, or 125, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 5 or 27; and the Tim1 transmembrane domain contains the amino acid sequence of SEQ ID NO: 8; (iii) The Tim4 IgV domain contains the amino acid sequence of SEQ ID NO: 34, the Tim4 mucin domain contains the amino acid sequence of SEQ ID NO: 35, the Tim4 signaling domain contains the amino acid sequence of SEQ ID NO: 45, 124, or 125, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 5 or 27; and the Tim4 transmembrane domain contains the amino acid sequence of SEQ ID NO: 6; or (iv) The Tim4 IgV domain contains the amino acid sequence of SEQ ID NO: 34, the Tim4 mucin domain contains the amino acid sequence of SEQ ID NO: 35, the Tim1 signaling domain contains the amino acid sequence of SEQ ID NO: 44, the CD3ζ signaling domain contains the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27; and the Tim4 transmembrane domain contains the amino acid sequence of SEQ ID NO: 6 The chimeric Tim receptor described in item 70. [Section 72] (i) Does the single-chain chimeric protein contain amino acids 21-456 of SEQ ID NO: 49? (ii) Does the single-chain chimeric protein contain amino acids 21-471 of SEQ ID NO: 50? (iii) The single-chain chimeric protein contains amino acids 25-490 of SEQ ID NO: 59; or (iv) A single-chain chimeric protein containing amino acids 25-495 of SEQ ID NO. 60, Chimeric Tim receptor as described in item 70 or 71. [Section 73] (i) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 49? (ii) Does the single-chain chimeric protein contain the amino acid sequence of SEQ ID NO: 50? (iii) The single-chain chimeric protein contains the amino acid sequence of SEQ ID NO: 59; or (iv) A single-chain chimeric protein contains the amino acid sequence of SEQ ID NO. 60, A chimeric Tim receptor as described in any one of items 70 to 72. [Section 74] One of the chimeric T cell immunoglobulin and mucin domain (Tim) receptors listed in Tables 2-8. [Section 75] A chimeric Tim receptor as described in item 74, other than one or more constructs in Table 4. [Section 76] A chimeric Tim receptor as described in item 74 or 75, other than one or more constructs in Table 5. [Section 77] A polynucleotide encoding a chimeric Tim receptor as described in any one of items 1 to 76. [Section 78] A vector containing the polynucleotides described in item 77. [Section 79] Engineered cells comprising a chimeric Tim receptor as described in any one of items 1 to 76, a polynucleotide as described in item 77, or a vector as described in item 78. [Section 80] Immune cells, which are the modified cells described in item 79. [Section 81] T cells, which are the manipulated cells described in item 80. [Section 82] The manipulated cells described in section 81, which are CD4+ T cells, CD8+ T cells, or CD4+ / CD8+ T cells. [Section 83] Human cells, which are manipulated cells as described in any one of sections 79 to 82. [Section 84] A composition comprising a chimeric Tim receptor as described in any one of claims 1 to 76, a polynucleotide as described in claim 77, a vector as described in claim 78, or an engineered cell as described in any one of claims 79 to 83. [Section 85] The composition according to item 84, further comprising pharmaceutically acceptable excipients. [Section 86] A method for treating a disease in a subject, comprising administering a chimeric Tim receptor as described in any one of items 1 to 76, a polynucleotide as described in item 77, a vector as described in item 78, or engineered cells as described in any one of items 79 to 83, or a composition as described in item 84 or 85. [Section 87] The method described in paragraph 86, wherein the disease is cancer. [Section 88] Cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, or lung cancer; adenocarcinomas of the breast, prostate, and colon; bronchogenic lung cancer of all forms; myeloid leukemia; melanoma; hepatocellular carcinoma; neuroblastoma; papilloma; apdoma; spondylolisthesis; branchial cystoma; malignant carcinoid syndrome; carcinoid heart disease; and carcinomas (e.g., Walker's carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pierce carcinoma, tubular carcinoma, Ehrlich carcinoma, Krebs II carcinoma, Merkel cell carcinoma). Cancer, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, sclerotic carcinoma, bronchiolar carcinoma, bronchogenic lung cancer, squamous cell carcinoma and transitional cell carcinoma); histiocytic disorders; malignant histiocytosis; leukemia; Hodgkin's disease; immunoproliferative microcarcinoma; non-Hodgkin lymphoma; plasmacytoma; multiple myeloma; chronic myeloid leukemia (CML); plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; undifferentiated germ cell tumor; hamartoma; mesenchymal tumor; Mesonephroma; sarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; choriocarcinoma, adenoma; cholangiocarcinoma; cholesteatoma; cyclindroma; cystadenoma; cystadenoma; granulosa cell tumor; androblastoma; hepatocellular carcinoma; sweat adenoma; islet tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; sarcoma; rhabdomyomyoma; rhabdomyosarcoma; ependymoma; gangliomas; gliomas; medulloblastoma; meningioma; schwannoma; neuroblastoma; neuroepithelioma; neurofibroma Neuromas; paragangliomas; nonchrome affinity paragangliomas; angiokeratomas; angiolymphoid hyperplasia with eosinophilia; sclerosing hemangiomas; hemangiomatosis; glomus hemangiomas; hemangioendotheliomas; hemangiomas; hemangioexiocytes; hemangiosarcomas; lymphangiomas; lymphangiomyomas; lymphangiosarcomas; pinealomas; carcinosarcomas; chondrosarcomas; phyllodes cystic sarcoma; fibrosarcoma; hemangiosarcoma; leiomyosarcomas; leukemosarcoma; liposarcomas; lymphangiosarcoma; myxosarcomas; ovarian cancer; rhabdomyosarcoma; sarcomas; neoplasms; neurofibromatosis; cervical dysplasia and peritoneal cancer;B-cell lymphomas [e.g., various forms of Hodgkin's disease, non-Hodgkin's lymphoma (NHL), or central nervous system lymphoma], B-cell cancers, leukemias [e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, B-cell blastogenesis of chronic myeloid leukemia], and myelomas (e.g., multiple myeloma); small lymphocytic lymphomas, B-cell prelymphocytic leukemias, lymphoplasmacytic lymphomas, splenic marginal zone lymphomas, plasmacytic myelomas, and bone isolation. The method described in item 87, which includes plasmacytoma, extraskeletal plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodular 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 exudative lymphoma, Burkitt lymphoma / leukemia, B-cell proliferation with the potential for malignant transformation, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorders. [Section 89] The method according to any one of claims 86 to 88, further comprising the administration of an additional therapeutic agent. [Section 90] The method according to paragraph 89, wherein the additional therapeutic agent includes radiation therapy, cellular immunotherapy, antibodies, immune checkpoint molecule inhibitors, chemotherapy, hormone therapy, peptides, antibiotics, antivirals, antifungals, anti-inflammatory agents, UV light therapy, electropulsed light therapy, high-intensity focused ultrasound therapy, oncolytic virus therapy, small molecule therapy, or any combination thereof. [Section 91] The method according to paragraph 90, wherein the cell immunotherapy is a chimeric antigen receptor. [Section 92] The method according to item 89, wherein the additional therapeutic agent includes 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, 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, PDGFR inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4 / 6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PDGFRA inhibitors, or RET inhibitors. [Section 93] The method according to item 92, wherein the BTK inhibitor is ibrutinib, pirtobrutinib (Loxo-305), tirabrutinib, trebrutinib, evobrutinib, fenebrutinib (GDC-0853), acalabrutinib, ONO-4059, spebratinib, zanubrutinib (BGB-3111), HM71224, or M7583. [Section 94] A method for improving an effector response or antitumor efficacy in a subject, comprising administering a chimeric Tim receptor as described in any one of items 1 to 76, a polynucleotide as described in item 77, a vector as described in item 78, or engineered cells as described in any one of items 79 to 83, or a composition as described in item 84 or 85. [Examples]

[0301] [Example 1] CER-T cells induce cytotoxic effects against primed PTD-SER+ tumor cells. TLR-containing chimeric Tim4 receptor T cells are engineered to target cells presenting elevated levels of the cell membrane stress signal, phosphatidylserine (Ptd-Ser). Ptd-Ser is a phospholipid normally found on the inner leaflet of the plasma cell membrane. Upon activation of certain downstream signals (e.g., caspase 3 / 7 activation), Ptd-Ser is externalized to the outer cell membrane. Engagement by Ptd-Ser-specific receptors on professional phagocytic cells / antigen-presenting cells (APCs) triggers a multi-component signaling complex, which ultimately leads to actin cytoskeleton reorganization. Tim-4 (T cell immunoglobulin mucin-4) is one of several receptors that specifically bind to Ptd-Ser. Its expression in resident and peritoneal macrophages is involved in the clearance of apoptotic cells during normal tissue homeostasis. In dendritic cells (DCs), the Tim-4-Ptd-Ser interaction mediates antigen capture and phagocytosis for cross-priming of T cells.

[0302] Several strategies were developed to efficiently induce or prime Ptd-Ser on various tumor cell lines. This priming step uses standard care agents, such as targeted small molecule inhibitors, to induce cellular stress and / or apoptosis. Once induced, Ptd-Ser acts as a target for chimeric Tim4 receptor-T cell engagement, activation, and cytolytic functions. This two-step "priming-death" strategy is demonstrated using various small molecule inhibitor-chimeric Tim4 receptor-T cell product combinations, as well as combinations with engineered CAR T and TCR products.

[0303] In ovarian tumors, poly(ADP-ribose) polymerase (PARP) inhibitors, such as niraparib, are clinically approved drugs that target the DNA damage response pathway. Complete response is rare, and relapse after treatment is common. To induce Ptd-Ser exposure, BRCA-2 mutant Kuramochi cell lines were treated with therapeutic doses of the PARP inhibitor niraparib. Brief exposure to niraparib induced changes in membrane phospholipid symmetry in a dose-dependent manner and was effective in inhibiting the proliferation of Kuramochi cells (Figure 7A). The addition of the chimeric Tim4 receptor pCTX133 (Tim4-binding domain - TLR2 signaling domain - CD3z signaling domain) at a low effector-target ratio (1:1) improved the potency of niraparib in vitro compared to the transduced control, demonstrating the ability of pCTX133 to induce a direct cytotoxic effect on target cells (Figure 7B).

[0304] In mantle cell lymphoma, Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib, are clinically approved drugs that target pro-survival kinases. Complete response is rare, and relapse after treatment is common. Treatment of JeKo-1 MCL with ibrutinib involves exposure to Ptd-Ser, which is determined by immunohistochemistry using recombinant mouse Tim4 protein (Figure 8A). JeKo-1 mantle cell lymphoma cell lines were treated with 25 μM ibrutinib for 24 hours, followed by drug rinsing, and co-cultured with 0.5 μM ibrutinib with chimeric Tim4 receptor pCTX136 (Tim4-binding domain-CD28 signaling domain-CD3z signaling domain) or control cells at E:T ratios of 3:1, 2:1, or 1:1. Co-culturing with chimeric Tim4 receptor cells generally induced the removal of JeKo-1 target cells compared to treatment with ibrutinib or control T cells (Figure 8B).

[0305] Chimeric Tim-4 receptors possessing either a TLR2 or TLR8 intracellular signaling domain, and either a CD28 or CD3ζ intracellular signaling domain, were tested for their ability to promote tumor cell acquisition and induce cytotoxic and APC-like functions. To evaluate antigen acquisition, target cells were co-cultured with chimeric Tim-4 receptor T cells and evaluated by transmission electron microscopy (TEM) or flow cytometry. Target cells were treated with small molecule inhibitors to induce Ptd-Ser externalization, and lysosome uptake was evaluated using a pH indicator dye (pHrodo Red).

[0306] An alternative priming-death therapeutic strategy combines TLR-containing chimeric Tim4 receptor-T cells with chimeric antigen receptor-T (CAR)-T cells. This combination approach utilizes CARs to specifically target tumor cells, resulting in Ptd-Ser upregulation. CD19 CAR-T cell products (anti-CD19 scFv--CD28 costimulatory signaling domain-CD3ζ signaling domain; "1928z CAR") rapidly induce Ptd-Ser on CD19+ mantle cell lymphoma (MCL) cells in a dose-dependent manner (Figure 5A). In co-culture studies, the combination of 1928z CAR-T cells (pCTX184) and pCTX131 (Tim4-TLR8-CD3z) showed high efficacy as measured by incucyte and FACS. 1928z CAR T cells were combined with pCTX131(Tim4-TLR8-CD3z) cells at multiple CAR:CER ratios with a low effector:target ratio (Figure 5B, Figure 6A). pCTX156 is a cleavage-type EGFR (EGFRt) control. Increases in inflammatory cytokines, such as IFN-γ, were observed in the supernatant, in parallel with the observed increase in cytolytic function (Figure 5C). In addition, in co-culture studies, when measured by incucyte, the combination of 1928z CAR-T cells and pCTX131(Tim4-TLR8-CD3z) showed improved induction of cleavage-type caspases in target cells. 1928z CAR T cells were combined with pCTX131(Tim4-TLR8-CD3z) cells at multiple CAR:CER ratios with a low effector:target ratio (Figure 6B).

[0307] Therefore, this example demonstrates that chimeric Tim4 receptor-T cells containing pCTX133 and pCTX131 (TLR2 and TLR8) can induce cytolytic activity toward primed solid tumor and hematopoietic target cell lines expressing cell surface Ptd-Ser, and enhance small molecule and CAR-based therapeutic approaches. [Example 2]

[0308] Chimeric Tim4 receptor-T cells mediate antigen capture and presentation Activated T cells can express class II molecules, present antigen on the cell surface, and deliver co-stimulatory signals to other T cells, 10 indicating that they are capable of Ag processing and presentation. However, unlike professional APCs, T cells are limited by their inefficient capture of soluble antigens 9 . In contrast, APCs utilize constitutively expressed Ag uptake receptors to capture and phagocytose antigens for subsequent degradation and MHC loading 1213 . Capture of soluble antigens can be up to 10 3 -fold more efficient in the presence of surface receptors that bind Ag with high affinity 14 .

[0309] In dendritic cells (DCs), the Tim-4-Ptd-Ser interaction mediates antigen capture, phagocytosis, and concentration, enabling DCs to present antigen to T cells with high efficiency. Indeed, Tim-4 receptor blockade in a preclinical NSCLC model impairs the activation of tumor-specific CD8+ T cells and promotes tumor progression 12 . Furthermore, gene expression profiling shows downregulation of Tim-4 expression in advanced tumor cells, consistent with decreased antigen uptake, presentation, and T cell activation.

[0310] The experimental results presented herein demonstrate that T cells can be reoriented as antigen-presenting T cells for immunotherapy by enhancing their antigen uptake, capture, and co-stimulatory capabilities. Fusion of the human Tim4 phagocytic uptake receptor to intracellular signaling sequences that drive antigen uptake and antigen processing and presentation imparts enhanced APC capabilities to T cells. The modular design of the chimeric Tim receptor incorporates intracellular domains that drive multi-component signaling complexes, e.g., CD3ζ, CD28, 4-1BB, ITAM, and TLR signaling, to induce antigen degradation processes required to mediate cell activation, cytolysis, cytokine and chemokine secretion, adhesion and co-stimulatory molecule upregulation, and efficient T cell activation. 15 .

[0311] Chimeric Tim4 receptor-T cells were tested to determine whether they could capture and present soluble antigens and induce activation and proliferation of recombinant E7 restriction T cell clones in a co-culture system. First, Tim4 expression was confirmed on transduced CER T cells by flow cytometry. The Tim4-binding domain-CD28 transmembrane-CD28 signaling domain-CD3z signaling domain (CTX247) and the Tim4-binding domain-CD28 signaling domain-CD3z signaling domain-TLR2 signaling domain (CTX1107) were stained for Tim4 and EGFR, a transduction marker encoded on each vector. Tim4 expression was observed on CTX247 or CTX1107 transduced cells but not on mock transduced controls (Figure 4). The E7 restriction TCR targets the HPV16-derived E7 protein and has the TCRα and TCRβ chain sequences provided in SEQ ID NO: 156. E7 TCRs proliferate in response to APCs pulsed with E7 peptides via MHC class I. For autologous APCs, CD4+ and CD8+ chimeric Tim4 receptor T cell products transduced with different Tim-4 chimeric receptors were pulsed using a pool or medium of 15-mer peptides containing an 11-mer duplicate derived from HPV16-derived E7 protein. Chimeric Tim4 receptor T cells were pulsed with E7 peptides at 37°C for 4 hours to test their ability to induce E7-specific activation and proliferation. E7-TCR cell surface activation marker responses were evaluated by flow cytometry 24 hours after co-culture with chimeric Tim4 receptor T cell products. After a further 5 days of co-culture, proliferation responses were evaluated using Cell Trace (CT) Violet dilutions.

[0312] Figures 1B-1C show that pCTX1107 (Tim4 binding domain - CD28 intracellular signaling domain - CD3ζ intracellular signaling domain - TLR2 intracellular signaling domain) CER T cells were indeed stimulant to E7-specific T cells, while pCTX247 (Tim4 binding domain - CD28 intracellular signaling domain - CD3ζ intracellular signaling domain) or untransduced T cells were not stimulant, even when pulsed with high concentrations of E7 peptide. In these experiments, the only difference in construct design between pCTX247 and pCTX1107 was the addition of the TLR-2 intracellular sequence (Figure 1A), which implied amplification of T cell presentation of soluble antigens and TLR signaling in the induction of E7 TCRs.

[0313] Evidence that pCTX1107 is highly stimulating for E7-specific activation was also seen in CD25 and CD69 upregulation as measured by flow cytometry (Figure 2). Higher frequencies of E7-TCR-containing T cells expressed both activation markers after 24 hours of co-culture (Figure 2). E7 TCR-T cell surface activation markers CD25 and CD69 were upregulated by 41.2% and 23.1% compared to controls after 24 hours of co-culture with chimeric Tim4 receptor-T cells, and the percentage of mitotic E7-TCR-T cells up to 6 days was 44% for TIM4 / CD28 / CD3z / TLR2 chimeric Tim4 receptor T cells and 8% compared to controls.

[0314] Cell Trace Violet-labeled E7-specific TCR T cells were cultured for 4 days in a 1:2:2 ratio with untransduced T cells (UT), T cells transduced by the Tim4-CD28-CD3z construct (CTX247) or Tim4-CD28-CD3z-TLR2 construct (CTX1107), and JeKo-1 cells in the presence of a pool of 15-mer peptides (100 ng of each peptide) containing an 11aa duplicate from the HPV16 E7 protein. Double repeat cultures were incubated with HLA A, B, and C blocking antibodies (clone W6 / 32) or matched mouse IgG2a antibodies for the duration of the culture. Figure 10 shows the percentage of E7 TCR cells in culture among viable cells, as determined by flow cytometry based on staining of mouse TCRb+ cells. Activation of E7-specific TCR T cells mediated by chimeric Tim4 receptor antigen presentation is blocked by anti-HLA-I antibodies. [Example 3]

[0315] T cell transfection using chimeric Tim4 receptors Chimeric Tim4 receptors pCTX1183, pCTX1161, pCTX1189, pCTX1184, pCTX1163, pCTX1162, pCTX1190, pCTX1186, pCTX1187, pCTX1164, pCTX1185, and pCTX1165 (see Table 8) were transfected into Jurkat T lymphocyte cell lines (Figures 9A-9B).

[0316] References

[0317] Further embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications listed herein by reference and / or in the application datasheets, including, but not limited to, U.S. Provisional Patent Application No. 63 / 066,085 filed August 14, 2020, and U.S. Provisional Patent Application No. 63 / 226,643 filed July 28, 2021, are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified using concepts from various patents, applications, and publications, and where necessary to provide further embodiments.

[0318] These and other modifications may be made with respect to embodiments in light of the description detailed above. In general, the terms used in the following claims should not be construed as limiting the claims to specific embodiments disclosed in the specification and claims, but rather as including all possible embodiments along the entire scope of equivalents to which such claims are entitled. Thus, the claims are not limited by this disclosure.

Claims

1. (a) an extracellular domain containing a binding domain including a Tim4 IgV domain and a Tim4 mucin domain; (b) an intracellular signaling domain comprising a CD28 signaling domain, a CD3ζ signaling domain, and a TLR2 signaling domain; (c) CD28 transmembrane domain located between the extracellular domain and the intracellular signaling domain, and connecting them. A chimeric phagocytic receptor containing a single-chain chimeric protein.

2. The chimeric phagocytic receptor according to claim 1, wherein the intracellular signaling domain comprises a CD28 signaling domain, a CD3ζ signaling domain, and a TLR2 signaling domain, from the N-terminus to the C-terminus.

3. The chimeric phagocytic receptor according to claim 1 or 2, wherein the Tim4 IgV domain comprises the amino acid sequence shown in SEQ ID NO: 34, or the Tim4 mucin domain comprises the amino acid sequence shown in SEQ ID NO: 35, or both.

4. The chimeric phagocytic receptor according to claim 3, wherein the binding domain comprises the amino acid sequence of SEQ ID NO: 2 or 42.

5. A chimeric phagocytic receptor according to any one of claims 1 to 4, wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:

7.

6. A chimeric phagocytic receptor according to any one of claims 1 to 5, wherein the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 26, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 27, and / or the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO:

122.

7. A polynucleotide encoding a chimeric phagocytic receptor according to any one of claims 1 to 6.

8. A vector comprising the polynucleotide described in claim 7.

9. Engineered immune cells comprising a chimeric phagocytic receptor according to any one of claims 1 to 6, a polynucleotide according to claim 7, or a vector according to claim 8.

10. The manipulated immune cell according to claim 9, wherein the immune cell is a T cell.

11. The manipulated immune cells according to claim 10, wherein the T cells are CD4+ T cells, CD8+ T cells, or CD4+ / CD8+ T cells.

12. The manipulated immune cells according to any one of claims 9 to 11, wherein the immune cells are human immune cells.

13. A pharmaceutical composition comprising a polynucleotide according to claim 7, a vector according to claim 8, or an engineered cell according to any one of claims 9 to 12, and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, for use in a method of treating cancer in a subject.

15. Cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, adenocarcinoma of the breast, adenocarcinoma of the prostate, adenocarcinoma of the colon, bronchogenic lung cancer, myeloid leukemia, melanoma, hepatocellular carcinoma, neuroblastoma, papilloma, apdoma, spondylolysis, branchial tumor, malignant carcinoid syndrome, carcinoid heart disease, Walker's carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pierce carcinoma, tubular carcinoma, Ehrlich carcinoma, Krebs II carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung carcinoma, and oat cell carcinoma. Tumor, papillary carcinoma, sclerotic carcinoma, bronchiololoma, squamous cell carcinoma, transitional cell carcinoma, histiocytic disorder, malignant histiocytosis, Hodgkin's disease, non-Hodgkin lymphoma, plasmacytoma, multiple myeloma, chronic myeloid leukemia (CML), reticuloendotheliosis, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, chordoma, craniopharyngioma, undifferentiated germ cell tumor, hamartoma, mesenchymal tumor, mesonephroma, sarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastoma, adenoma, cholangiomas, cholesteatoma, cyclocolumbar cystoma Indroma, cystadenoma, cystadenoma, granulosa cell tumor, androblastoma, sweat adenoma, islet tumor, Leydig cell tumor, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, rhabdomyomas, rhabdomyosarcoma, ependymoma, gangliomas, gliomas, medulloblastoma, meningioma, schwannoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, nonchrome affinity paraganglioma, angiokeratomas, angiolymphoid hyperplasia with eosinophilia, sclerosing hemangioma, hemangioma, glomus hemangioma, hemangioendothelioma, hemangioma, hemangioextormioma, angiosarcoma, lymph Candidoma, lymphangiomyoma, lymphangiosarcoma, pineal glandoma, carcinosarcoma, chondrosarcoma, phyllodes cysticosarcoma, leukemosarcoma, sarcoma, neoplasm, neurofibromatosis, cervical dysplasia, peritoneal cancer, B-cell carcinoma, B-cell lymphoma, central nervous system lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), hairy cell leukemia, B-cell blastogenesis of chronic myeloid leukemia, myeloma, small lymphocytic lymphoma, B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytomyeloma, solitary plasmacytoma of bone, extraskeletal plasmacytoma,The pharmaceutical composition according to claim 14, which is an extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodular 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 exudative lymphoma, Burkitt lymphoma / leukemia, B-cell proliferation with the potential for malignant transformation, lymphomatoid granulomatosis, or post-transplant lymphoproliferative disorder.

16. The pharmaceutical composition according to claim 14 or 15, wherein the method further comprises the application of an additional therapeutic agent or additional therapy.

17. The pharmaceutical composition according to claim 16, wherein the additional therapeutic agent or additional therapy comprises radiation, cellular immunotherapy, antibodies, immune checkpoint molecule inhibitors, chemotherapy, hormone therapy, peptides, antibiotics, antivirals, antifungals, anti-inflammatory agents, UV light therapy, electropulse therapy, high-intensity focused ultrasound therapy, oncolytic virus therapy, small molecule therapy, or any combination thereof.

18. The pharmaceutical composition according to claim 17, wherein the cellular immunotherapy is the application of a chimeric antigen receptor.

19. The pharmaceutical composition according to claim 16, wherein the additional therapeutic agent comprises an angiogenesis inhibitor, a VEGF pathway inhibitor, a tyrosine kinase inhibitor, an EGF pathway inhibitor, a receptor tyrosine kinase inhibitor, a growth factor inhibitor, a GTPase inhibitor, a serine / threonine kinase inhibitor, a transcription factor inhibitor, a B-Raf inhibitor, a RAF inhibitor, a MEK inhibitor, an mTOR inhibitor, an EGFR inhibitor, an ALK inhibitor, a ROS1 inhibitor, a BCL-2 inhibitor, a PI3K inhibitor, a VEGF inhibitor, a BCR-ABL inhibitor, a MET inhibitor, a MYC inhibitor, an ABL inhibitor, a HER2 inhibitor, a BTK inhibitor, an H-RAS inhibitor, a K-RAS inhibitor, a PDGFR inhibitor, a TRK inhibitor, a c-KIT inhibitor, a c-MET inhibitor, a CDK4 / 6 inhibitor, a FAK inhibitor, an FGFR inhibitor, a FLT3 inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, a PDGFRA inhibitor, or a RET inhibitor.

20. The pharmaceutical composition according to claim 19, wherein the BTK inhibitor is ibrutinib, pirtobrutinib (Loxo-305), tirabrutinib (ONO-4059), trebrutinib, evobrutinib, fenebrutinib (GDC-0853), acalabrutinib, spebratinib, zanubrutinib (BGB-3111), HM71224, or M7583.

21. The pharmaceutical composition according to claim 13, for use in a method for improving effector response or antitumor efficacy in a subject.

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