JP2023537762A - Compositions and methods for treating cancer using chimeric TIM receptors in combination with inhibitors of poly(ADP-ribose) polymerase - Google Patents

Abstract

The present disclosure relates to combination therapy compositions and methods comprising chimeric Tim receptors, host cells engineered to contain chimeric Tim receptor molecules, and PAPR inhibitors.

Description

STATEMENT FOR SEQUENCE LISTING The Sequence Listing accompanying this application is provided in text form in lieu of a paper copy and is hereby incorporated herein by reference. The name of the text file containing the sequence listing is 200265_415WO_ST25. txt. The text file is 425 KB, was created on August 13, 2021 and has been submitted electronically via EFS-Web.

Upon exposure to antigen, naive antigen-specific CD8+ T cells promote their clonal expansion, differentiation, and development into functional effector T cells capable of killing cells expressing their cognate antigen (e.g., tumor cells). receive activation. After antigen clearance, the majority of effector T cells undergo apoptosis and a subset of surviving effector T cells differentiate into memory T cells that can confer long-term protection against antigen re-exposure. However, prolonged antigen exposure may cause T cell exhaustion and allow tumor cell persistence. T-cell exhaustion is a persistent TCR characterized by upregulated expression of immune checkpoint molecules (eg PD-1, CTLA-4, Tim-3), reduced effector function, poor proliferation, and metabolic disturbances. It refers to a dysfunctional state acquired by stimulated T cells. Engineered T cells expressing chimeric antigen receptors (CARs) can also develop exhaustion.

Figures 1A-1B show that the Kuramochi model of ovarian cancer is moderately sensitive to PARP inhibition. FIG. 1A shows the results of Kuramochi cells expressing the mCherry-NLS plasmid incubated with the PARP inhibitor niraparib. FIG. 1B shows the results of Kuramochi cells expressing the mCherry-NLS plasmid incubated with the PARP inhibitors niraparib, olaparib, talazoparib, veliparib, and rucaparib. Figures 2A-2C show that Kuramochi cells antigen-loaded with niraparib bind to Tim4, indicating increased levels of surface PS in response to PARP inhibition. FIG. 2A shows staining of niraparib-treated Kuramochi cells with Tim4-Fc chimera conjugated to Alexa488 and anti-mouse IgG2a antibody. FIG. 2B shows staining for the IgG control. FIG. 2C shows an increase in Kuramochi cell numbers in response to 1.56-12.5 μM niraparib. Figure 3 shows that niraparib has negligible effects on CD4/CD8 T cell health, cell number, or expansion. FIG. 4 shows the results of Kuramochi cells incubated with chimeric Tim receptor construct 13A, control T cells, and niraparib. Figure 5 shows that the combination of chimeric Tim4 receptor (CTX140) and niraparib treatment reduces Kuramochi cell numbers in vitro. Kuramochi cells were pre-incubated with 1.56 μM niraparib. CTX156 (tEGFR) is a transduction control. Figure 6 shows that the combination of chimeric Tim4 receptor (CTX137) and niraparib treatment reduces Kuramochi cell numbers in vitro. Kuramochi cells were pre-incubated with 25 μM niraparib and given a maintenance dose of 0.52 μM niraparib. CTX156 (tEGFR) is a transduction control. FIG. 7. Chimeric Tim4 receptor (CTX137) combinations with nirparib treatment at different doses (6.25 μM, 12.5 μM, or 25 μM pretreatment + 0.52 μM maintenance dose) and different effector:target cell ratios. reduces Kuramochi cell numbers in vitro. CTX156 (tEGFR) is a transduction control. Figures 8A-8B show that pCTX133, a TLR-2 containing chimeric Tim4 receptor, improves the efficacy of niraparib in an ovarian cancer model. Figure 8A: 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). Forty-eight hour samples were trypsinized and stained with Tim4-Fc followed by a fluorescently labeled secondary antibody against Tim4-Fc. Figure 8B: Kuramochi cells pretreated with 1.56 μM niraparib for approximately 20 hours were transduced with pCTX133 and donor 32-derived untransduced cells at a T cell:Kuramochi ratio of 2:1 and a final niraparib concentration of 1.56 μM. co-cultured with CD4 T cells. Samples treated with niraparib plus pCTX133 showed significantly fewer tumor cells after approximately 3 days in culture when compared to samples treated with niraparib alone or with niraparib plus untransduced T cells. All data were collected via IncuCyte. Figures 9A-B: CER pCTX247 (SEQ ID NO:257) in vitro antitumor activity in two ovarian cancer models. FIG. 9A: A2780-mCh-NLS cells pretreated with 25 μM niraparib for approximately 20 hours were treated with mock cells from CER247 and donor 21 at a 2:1 T cell:A2780 ratio and a final niraparib concentration of 6.25 μM. Co-cultured with 236 CD4/8 T cells. Samples treated with niraparib + CER247 showed significantly fewer tumor cells after about 5 days in culture when compared to samples treated with niraparib alone, CER247 alone, or niraparib + Mock236. FIG. 9B: Kuramochi-mCh-NLS cells pretreated with 25 μM niraparib for approximately 20 hours were treated with mock cells from CER247 and Donor 21 at a 2:1 T cell:A2780 ratio and a final niraparib concentration of 1.56 μM. Co-cultured with 236 CD4/8 T cells. Samples treated with niraparib + CER247 showed significantly fewer tumor cells after about 5 days in culture when compared to samples treated with niraparib alone, CER247 alone, or niraparib + Mock236. All data were collected via IncuCyte. Figure 10: CER pCTX797 (SEQ ID NO:258) in vitro antitumor activity in the A2780 ovarian cancer model. A2780-mCh-NLS cells pretreated with 0.52 μM niraparib for approximately 20 hours were treated with mock 236 from CER797 and donor 32 at a 2:1 T cell:A2780 ratio and a final niraparib concentration of 0.52 μM. Co-cultured with CD4/8 T cells. Samples treated with niraparib plus CER247 showed significantly fewer tumor cells after about 5 days in culture when compared to samples treated with niraparib alone, CER247 alone, or niraparib plus Mock236. All data were collected via IncuCyte. Figures 11A-11B: Phosphatidylserine is present on the cell surface in response to niraparib in two ovarian cancer models. FIG. 11A: A2780-mCh-NLS cells treated with 1.56 and 25 μM niraparib show significantly higher percentages of PS+ cells and Tim4− cells compared to samples treated with equivalent volumes of DMSO. Shows increased Fc MFI. Niraparib also has anti-tumor effects starting from concentrations of at least 1.56 μM niraparib. FIG. 11B: Kuramochi-mCh-NLS cells treated with 25 μM niraparib show significantly higher percentage of PS+ cells and increased Tim4-Fc MFI compared to samples treated with equivalent volume of DMSO. indicate. Niraparib also has anti-tumor effects starting from concentrations of at least 25 μM niraparib. All samples were analyzed via flow cytometry. CER shows synergy with additional PARP inhibitors (rucaparib and olaparib in addition to niraparib). A2780-mCh-NLS cells pretreated with 0.52 μM niraparib, rucaparib, or olaparib for approximately 20 hours were treated with CER247 and donor at a 2:1 T cell:A2780 ratio and a final niraparib concentration of 0.52 μM. were co-cultured with mock 236 CD4/8 T cells from 32. Samples treated with niraparib + CER247, rucaparib + CER247, or olaparib + CER247 were significantly more stable after approximately 5 days of culture when compared to samples treated with niraparib, rucaparib, or olaparib alone, CER247 alone, or niraparib + Mock236. showed significantly fewer tumor cells. All data were collected via IncuCyte.

In one aspect, the present disclosure provides a chimeric T cell immunoglobulin mucin protein (Tim) receptor, also referred to as a chimeric phagocytic receptor (CER), in combination with an inhibitor of poly(ADP-ribose) polymerase (PARP). A method for the treatment of cancer is provided. In another aspect, the disclosure provides pharmaceutical compositions or combinations comprising a chimeric Tim receptor and a PARP inhibitor. Chimeric Tim Receptors useful in the compositions and methods of the present disclosure confer phagocytic, cytotoxic, and/or antigen-presenting activity on chimeric Tim Receptor-modified host cells (e.g., T cells), is induced upon binding of the chimeric Tim receptor to its target antigen, phosphatidylserine. DNA-damaging agents such as PARP inhibitors promote externalization of phosphatidylserine that induces effector functions of chimeric Tim receptors such as cell damage and phagocytosis, cytotoxicity, co-stimulatory activity, antigen presentation, or a combination thereof. Thus, synergistic effects with chimeric Tim receptors can be demonstrated.

The chimeric tim receptors described herein are (a) an extracellular domain comprising a binding domain comprising (i) a Tim1 IgV domain or a Tim4 IgV domain; and (ii) a Tim1 mucin domain or a Tim4 mucin domain; b) an intracellular signaling domain comprising a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) located between and connecting the extracellular domain and the intracellular signaling domain. A single-chain chimeric protein comprising a transmembrane domain that

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

In certain embodiments, chimeric Tim receptors may also be able to co-stimulate T cells via signaling pathways distinct from the "classical" T cell co-stimulatory pathway (e.g., CD28). . For example, in addition to binding 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, a cytotoxic chimeric Tim receptor reduces or inhibits T-cell exhaustion or reduces or inhibits exhaustion by providing a co-stimulatory signal through at least one signaling pathway. damaged T cells can be restored. In certain embodiments, the cytotoxic chimeric Tim receptor is mediated through at least two different signaling pathways (e.g., through a selected co-stimulatory signaling domain in the cytotoxic chimeric Tim receptor and Tim1). ), providing co-stimulatory signals.

In certain embodiments, when expressed in a host cell, the chimeric Tim receptor of the present disclosure also confers phagocytic activity on the host cell. For example, in certain such embodiments, binding of a chimeric Tim receptor expressed in a host cell to a phosphatidylserine target can induce both cytolytic and phagocytic responses by the host cell. In certain embodiments of the modified host cells described herein, the host cell does not naturally display a phagocytic phenotype prior to modification with the chimeric Tim receptor.

In embodiments, administration of host cells modified with a chimeric Tim receptor of the present disclosure is a method for removing target cells bearing surface-exposed phosphatidylserine, e.g., for the treatment of cancer. can be used in 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 'scramblers' that rapidly expose phosphatidylserine on the cell surface, and scramblase binds to receptors such as Tim4 or Tim1. can be combined. Endogenous tumor-specific T effector cells can induce exposure of phosphatidylserine on the outer membrane of targeted tumor cells during cytolysis. Furthermore, certain cancer therapies (e.g., chemotherapy, radiotherapy, CAR-T cells, etc.) target tumor cells or cells in the tumor microenvironment by inducing apoptosis, cell stress, cell damage, etc. Exposure of phosphatidylserine on can be induced. engineered host cells expressing a chimeric Tim receptor of the present disclosure induce apoptosis in tumor cells that retain surface exposed phosphatidylserine, thereby damaging tumor cells that retain surface exposed phosphatidylserine; Stressed, apoptotic, or necrotic tumor cells can be cleared. In certain embodiments, host cells expressing the chimeric Tim receptors disclosed herein are induced by inducing apoptosis and by phagocytosis into damaged tumor cells that retain surface-exposed phosphatidylserine; Clear out stressed, apoptotic, or necrotic tumor cells. In another aspect, host cells modified with chimeric Tim receptors of the present disclosure can be used to enhance the efficacy of PARP inhibitors to induce cellular stress, injury, necrosis, or apoptosis. For example, administration of a PARP inhibitor can increase surface phosphatidylserine levels, resulting in a synergistic combination. Cells expressing the chimeric Tim receptors described herein bind to phosphatidylserine moieties exposed on the outer leaflet of damaged or dying cells resulting from administration of a PARP inhibitor and cells of the target cell. Lysis or both cell lysis and phagocytosis can be induced.

Engineered host cells comprising chimeric Tim receptors described herein in combination with PARP inhibitors can be used alone or for example, in CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, tumors. It can be administered to a subject in further combination with one or more additional therapeutic agents, including lytic viruses, electrical pulse therapy, and the like.

Before presenting the disclosure in more detail, it may be helpful to understand the disclosure to provide definitions of certain terms used herein.

In this description, any concentration range, percentage range, ratio range or integer range, unless otherwise indicated, refers to any integer value within the recited range and, where appropriate, fractions thereof (eg integer 10 should be understood to include fractions and hundredths). Also, any numerical range recited herein for any physical feature, such as polymer subunit, size or thickness, is understood to include any integer within the recited range, unless otherwise indicated. It should be. As used herein, the term "about" means ±20% of the indicated range, value or structure, unless otherwise indicated. It should be understood that the terms "a" and "an," as used herein, refer to "one or more" of the listed components. The use of alternatives (eg, "or") should be understood to mean either one, both, or any combination thereof. As used herein, the terms "include," "have," and "comprise" are used interchangeably, and the terms and variations thereof is intended to be construed as non-limiting.

Each term understood by those skilled in the art of antibody technology is given the meaning it has in the art, unless explicitly defined differently herein. The term "antibody" is used in its broadest sense and includes polyclonal and monoclonal antibodies. An "antibody" is an intact antibody comprising at least two heavy (H) and two light (L) chains interconnected by disulfide bonds and an intact antibody capable of or retaining the ability to bind a target molecule. can refer to the antigen-binding portion (or antigen-binding domain) of an antibody. Antibodies include naturally occurring, recombinantly produced, genetically engineered or modified forms of immunoglobulins, such as intrabodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multispecific antibodies (e.g. bispecific antibodies, diabodies , triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv, ADAPTIR). A monoclonal antibody or antigen-binding portion thereof may be a non-human, chimeric, humanized or human monoclonal antibody or antigen-binding portion thereof, preferably a humanized or human monoclonal antibody or antigen-binding portion thereof. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). An "antigen-binding portion" or "antigen-binding domain" of an intact antibody is meant to encompass an "antibody fragment", which refers to a portion of an intact antibody, which determines the antigenicity of the intact antibody. Refers to variable region or complementarity determining region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments, Fab'-SH, F(ab') ₂ , diabodies, linear antibodies, scFv antibodies, VH and antibody fragments formed from including, but not limited to, multispecific antibodies. A "Fab (fragment antigen binding)" is the portion of an antibody that binds to an antigen and comprises the variable region and CH1 of the heavy chain linked to the light chain via an interchain disulfide bond. The antibody can be of any class or subclass, including IgG and its subclasses ( _IgG1 , _IgG2 , _IgG3 , _IgG4 ), IgM, IgE, IgA and IgD.

The terms "variable region" or "variable domain" refer to the domains of an antibody heavy or light chain that are responsible for binding the antibody to antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain containing four conserved framework regions (FRs) and three including CDRs (see, eg, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind a particular antigen may be isolated using a VH or VL domain from the antibody that binds the antigen, and screening a library of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

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

As used herein, the terms "binding domain," "binding region," and "binding moiety" specifically and non-covalently bind, associate, coalesce, recognize target molecules (e.g., phosphatidylserine). Or refers to molecules, such as peptides, oligopeptides, polypeptides or proteins, that have the ability to combine. A binding domain includes any natural, synthetic, semi-synthetic or recombinantly produced binding partner for a biomolecule or other target of interest. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or functional binding domain or antigen-binding portion thereof. Exemplary binding domains include single chain antibody variable regions (e.g. domain antibodies, sFv, scFv, Fab), receptor ectodomains (e.g. Tim4), ligands (e.g. cytokines, chemokines) or specific ability to bind biomolecules. Synthetic polypeptides selected for are included.

"T cell receptor" (TCR) generally refers to T cells (T It refers to molecules found on the surface of lymphocytes (also called lymphocytes). TCRs generally consist of disulfide-linked heterodimers of highly variable α and β chains (also known as TCRα and TCRβ, respectively) in most T cells. In a small subset of T cells, the TCR consists of a heterodimer of gamma and delta chains (also known as TCRgamma and TCRdelta, 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 C-terminal cytoplasmic tail (Janeway et al., Immunobiology : The Immune System in Health and Disease, ^3rd Ed., Current Biology Publications, p. 4:33, 1997). The TCRs of this disclosure can be TCRs from a variety of animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. TCRs may be cell-associated (ie, having a transmembrane region or domain) or in soluble form. TCRs include recombinantly produced, genetically engineered, fusion or modified forms of TCRs including, for example, scTCRs, soluble TCRs, TCR fusion constructs [TRuC™; see US Patent Application Publication No. 2017/0166622]. be

The term "variable region" or "variable domain" of the TCR α-chain (Vα) and β-chain (Vβ) or Vγ and Vδ of the γδTCR are involved in the binding of the TCR to antigen. Native TCR _Vα and _Vβ generally have similar structures, with each variable domain containing four conserved FRs and three CDRs. The V _α domain is encoded by two separate DNA segments, the variable gene segment (V gene) and the junction gene segment (J gene); the V _β domain is encoded by three separate DNA segments, the variable gene segment. (V genes), diversity gene segments (D genes) and junctional gene segments (J genes). A single V _α or V _β domain may be sufficient to confer antigen binding specificity. A "major histocompatibility complex molecule (MHC molecule)" refers to a glycoprotein that delivers peptide antigens to the cell surface. MHC class I molecules are heterodimers consisting of a transmembrane α-chain (with three α-domains) and non-covalently associated β2-microglobulin. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides of cytosolic origin to the cell surface, where peptide:MHC complexes are recognized by CD8 ⁺ T cells. MHC class II molecules deliver peptides of vesicular origin to the cell surface where they are recognized by CD4 ⁺ T cells. The MHC molecules can be MHC molecules from various animal species including humans, mice, rats or other mammals.

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

A variety of assays, including Western blot, ELISA, and BIACORE® analysis, are known for identifying binding domains of the present disclosure that specifically bind to particular targets and for determining binding domain affinities. (see also, for example, Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; and U.S. Pat. Nos. 5,283,173, 5,468,614 or equivalents) ). As used ^herein , "specifically binds" means ^an affinity or K association or coalescence according to _a (i.e., the equilibrium association constant for a particular binding interaction in units of 1/M), while not significantly associating or coalescing with any other molecule or component in the sample point to

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

The term "epitope" or "antigenic epitope" includes cognate immune binding molecules such as antibodies or fragments thereof (e.g. scFv), T cell receptors (TCR), chimeric Tim receptors or other binding molecules, domains or Included are protein determinants within any molecule, structure, amino acid sequence or antigen to which a protein specifically binds. Epitopic determinants generally contain chemically active surface groupings of molecules such as amino acids or sugar side chains, and may have specific three dimensional structural characteristics, as well as specific charge characteristics. Epitopes can be linear epitopes or conformational epitopes.

As used herein, the term "PARP" (poly(ADP-ribose) polymerase) refers to a family of proteins, each of which has two ribose moieties and two phosphates per unit polymer. PARP1 and PARP2 are enzymes involved in DNA repair pathways.

As used herein, the term "Tim4" (T-cell immunoglobulin and mucin domain-containing protein 4), also known as "TimD4", is typically expressed on antigen-presenting cells such as macrophages and dendritic cells. Refers to expressed phosphatidylserine receptors. Tim4 mediates phagocytosis of apoptotic, necrotic, injured, injured, or stressed cells that present phosphatidylserine (PtdSer) on the outer surface (outer) leaflet of the plasma 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 comprises the amino acid sequence of SEQ ID NO:1.

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 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.

Tim4-binding domains include variable immunoglobulin (IgV)-like domains (referred to herein as "IgV domains") and mucin-like domains (referred to herein as "mucin domains"). 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.

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

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 comprises the amino acid sequence of SEQ ID NO:37.

A 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 contain a signal peptide. An exemplary human Tim1 signal peptide has the amino acid sequence of SEQ ID NO:40.

As used herein, an "effector domain" is a fusion protein or receptor capable of directly or indirectly promoting a biological or physiological response in cells expressing the effector domain upon receipt of an appropriate signal. is the intracellular portion of the In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when bound or directly binds to a target molecule that elicits a signal from the effector domain. An effector domain can 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.

As used herein, a "co-stimulatory signaling domain" refers to a primary or canonical (e.g., ITAM-driven) activation signal (e.g., provided by the CD3ζ intracellular signaling domain) An intracellular signaling domain of a costimulatory molecule that, when activated, promotes or enhances a T cell response, including T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof , or any functional part thereof. Costimulatory 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.

As used herein, an "immunoreceptor tyrosine-based activation motif (ITAM) activation domain" is naturally or endogenously present on an immune cell receptor or cell surface marker and has at least one Refers to an intracellular signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM), or a functional portion thereof. ITAM refers to the conserved motif of YXXL/IX _6-8 -YXXL/I. In certain embodiments, the ITAM signaling domain contains 1, 2, 3, 4 or more ITAMs. The ITAM signaling domain can initiate T cell activation signaling following antigen binding or ligand engagement. ITAM signaling domains include, for example, the intracellular signaling domains of CD3γ, CD3δ, CD3ε, CD3ζ, CD79a, and CD66d.

A "junction amino acid" or "junction amino acid residue" refers to one or more (eg, about 2-20) amino acid residues between two adjacent motifs, regions or domains of a polypeptide. Junctional amino acids can result from the construct design of the chimeric protein (eg, amino acid residues resulting from the use of restriction enzyme sites during construction of the nucleic acid molecule encoding the chimeric protein).

"Nucleic acid molecule" and "polynucleotide" can be in the form of RNA or DNA, including cDNA, genomic DNA and synthetic DNA. Nucleic acid molecules can consist of naturally occurring nucleotides (eg, deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (eg, α-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or substitutions of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidedates, phosphoramidates. Dates (phosphoramidates) and the like are included. A nucleic acid molecule may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense strand). A coding molecule may have the same coding sequence as a coding sequence known in the art, or different coding sequences that may encode the same polypeptide as a result of redundancy or degeneracy of the genetic code, or by splicing. may have

"Encoding" means either a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence in a biological process of a specific polynucleotide sequence, e.g., DNA, cDNA and mRNA sequences. It refers to the intrinsic properties that serve as templates for the synthesis of other polymers and macromolecules that possess, and the biological properties that result from them. Thus, a polynucleotide encodes a protein if transcription and translation of mRNA corresponding to that polynucleotide in a cell or other biological system produces the protein. Both the coding and non-coding strands can be referred to as encoding proteins or other products of the polynucleotide. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound 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 make up a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids joined together by peptide bonds. As used herein, the term refers to short chains, also commonly referred to in the art as e.g. peptides, oligopeptides and oligomers, and long chains, commonly referred to in the art as proteins, of which there are many types. refers to both "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. included. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

As used herein, the term "mature polypeptide" or "mature protein" refers to a protein that is secreted or bound to the cell membrane or within a particular organelle (e.g., endoplasmic reticulum, Golgi apparatus or endosome). It refers to a protein or polypeptide that localizes to and does not contain an N-terminal signal peptide.

A "signal peptide", also called a "signal sequence", "leader sequence", "leader peptide", "localization signal" or "localization sequence", is the N of newly synthesized proteins destined for the secretory pathway. It is a short peptide (usually 15-30 amino acids in length) present at the end. A signal peptide typically contains a short stretch 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, signal peptides induce the translocation of newly synthesized proteins into the endoplasmic reticulum, where they are cleaved by signal peptidases to create mature proteins, which then proceed to their proper destinations. do.

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

As used herein, the term "endogenous" or "native" refers to genes, proteins, compounds normally present in the host or host cell, including naturally occurring variants of the gene, protein, compound, molecule or activity. , refers to a molecule or activity.

As used herein, "homologous" or "homolog" refer to different species from a host cell, e.g., from the same host cell, from a different host cell, from a different organism, from a different strain. refers to a molecule or activity that is ancestrally related to a second gene or activity from . For example, the heterologous molecule, or the heterologous gene encoding the molecule, can be homologous to the native host cell molecule or the gene encoding the molecule, respectively, with altered structure, sequence, expression level or any combination thereof. may have

As used herein, a "heterologous" nucleic acid molecule, construct or sequence is a nucleic acid molecule or nucleic acid that is not native to the host cell, but may be homologous to the nucleic acid molecule or portion of the nucleic acid molecule from the host cell. Refers to a part of a molecule. The source of heterologous nucleic acid molecule, construct or sequence can be from a different genus or species. In some embodiments, the heterologous nucleic acid molecule is not naturally occurring. In certain embodiments, the heterologous nucleic acid molecule is added to the host cell or host genome (i.e., other than endogenous or native ), the added molecule may be integrated into the host cell genome, present as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), or present in multiple copies. . In addition, "heterologous" refers to a non-native enzyme, protein or other activity encoded by a non-endogenous nucleic acid molecule introduced into the host cell, even though the host cell encodes a homologous protein or activity. .

As used herein, the terms "engineered," "recombinant," "altered," or "non-naturally occurring" refer to organisms, microorganisms, cells, nucleic acid molecules or nucleic acid molecules that have been altered by the introduction of a heterologous nucleic acid molecule. refers to a vector, or to a cell or microorganism that has been genetically engineered by human intervention, i.e., modified by the introduction of a heterologous nucleic acid molecule, or whose expression of an endogenous nucleic acid molecule or gene is controlled or regulated Refers to a cell or microorganism that has been defused or altered to be constitutive, and such alterations or alterations can be introduced by genetic engineering. Human-generated genetic alterations include, for example, alterations that introduce nucleic acid molecules (which may include expression control elements such as promoters) that encode one or more proteins, chimeric receptors or enzymes; or the addition of other nucleic acid molecules. , deletions, substitutions; or other functional disruptions or additions to the genetic material of the cell. Exemplary modifications include modifications in the coding regions or functional fragments thereof, polypeptides heterologous or homologous to the reference or parent molecule. Additional exemplary modifications include, for example, modifications in non-coding regulatory regions where the modification alters expression of the gene or operon.

As used herein, the term "transgene" refers to a gene encoding a protein of interest (e.g., a chimeric Tim receptor) whose expression is desired in a host cell and transferred into the cell by genetic engineering techniques or It refers to polynucleotides. Transgenes can encode proteins of therapeutic interest and proteins that are reporters, tags, markers, suicide proteins, and the like. A transgene may be a transgene from a natural source, a modification of a natural gene, or it may be a recombinant or synthetic molecule. In certain embodiments, the transgene is a component of a vector.

The term "overexpressed" antigen or "overexpression" of an antigen refers to abnormally high levels of the antigen in cells. Overexpressed antigens or overexpression of antigens are often associated with disease states, such as those in hematological tumours, and in cells that form solid tumors within a particular tissue or organ of a subject. Solid tumors or hematologic tumors characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

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

A "conservative substitution" is recognized in the art as the substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (eg, WO 97/09433, published March 13, 1997, page 10; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975)). , pp. 71-77; see Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

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

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence required for expression of a gene product that is operably linked to the promoter/regulatory sequence. In some cases, this sequence may be the core promoter sequence, in other cases it may also include enhancer sequences and other regulatory elements required for expression of the gene product. A promoter/regulatory sequence can be, for example, a promoter/regulatory sequence that expresses a gene product in a tissue-specific manner.

A "constitutive" promoter encodes a gene product or, when operably linked to a specifying polynucleotide, directs the production of a gene product in a cell under most or all physiological conditions of the cell. is the resulting nucleotide sequence.

An "inducible" promoter encodes a gene product or, when operably linked to a specifying polynucleotide, is capable of producing a gene product in cells only when an inducer substantially corresponding to the promoter is present in the cell. is a nucleotide sequence resulting in production within.

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

The phrases "under transcriptional control" or "operably linked," as used herein, refer to a polynucleotide in which the promoter controls initiation of transcription by RNA polymerase and expression of the polynucleotide. It means existing in place and direction.

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 construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells. An "expression vector" is a vector capable of directing the expression of a protein encoded by one or more genes it carries when the vector is in the appropriate environment.

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

In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, closed-ended linear duplex (CELiD) DNA and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). If a non-viral delivery system is used, the delivery vehicle can be a liposome. Lipid formulations can be used to introduce nucleic acids into host cells in vitro, ex vivo or in vivo. The nucleic acid may be encapsulated inside the liposome, interspersed within the lipid bilayer of the liposome, or attached to the liposome via a linking molecule that associates with both the liposome and the nucleic acid; It may be contained in or complexed with micelles, or may be otherwise associated with lipids.

As used herein, the term "phagocytosis" refers to a receptor-mediated process in which endogenous or exogenous cells or particles greater than 100 nm in diameter are internalized by phagocytes or host cells of the present disclosure. Point. Phagocytosis is typically multi-step: (1) a target cell or particle via direct or indirect (via a bridging molecule) binding of a phagocytic receptor to a pro-phagocytic or antigenic marker on the target cell or particle; and (2) internalization or phagocytosis of the entire target cell or particle or portions thereof. In certain embodiments, internalization may occur via phagocytic or host cell cytoskeletal rearrangements to form phagosomes, which are membrane-bound compartments containing the internalized target. Phagocytosis may further involve maturation of the phagosome, which becomes more acidic and fuses with the lysosome (forming a phagolysosome), during which the phagocytosed target is degraded (e.g., "phagocytosis"). . Alternatively, phagosome-lysosome fusion may not be observed in phagocytosis. In yet another embodiment, phagosomes may regurgitate or excrete their contents into the extracellular environment prior to complete degradation. In some embodiments, phagocytosis refers to phagocytosis. In some embodiments, phagocytosis includes tethering, but not internalization, of target cells or particles by phagocytic cells of host cells of the present disclosure. In some embodiments, phagocytosis comprises tethering of target cells or particles and internalization of portions of target cells or particles by phagocytic cells of host cells of the present disclosure.

As used herein, the term "phagocytosis" refers to cells or large particles (0 .5 μm or larger) phagocytosis process. In certain embodiments, phagocytosis involves the formation of phagosomes that enclose the internalized target cell or particle, and phagosome fusion with lysosomes to form phagolysosomes whose contents are degraded. In certain embodiments, during phagocytosis, a phagocytic synapse is formed following binding of a chimeric Tim receptor expressed on a host cell of the present disclosure to phosphatidylserine expressed by a target cell or particle; An actin-rich phagocytic cup is generated at the working synapse; the phagocytic arm extends around the target cell or particle through cytoskeletal rearrangement; drawn into the host cell. As used herein, "phagocytosis" includes the process of "efferocytosis," which specifically refers to the phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.

The term "immune system cell" or "immune cell" means any cell of the immune system that is derived from hematopoietic stem cells of the bone marrow. Hematopoietic stem cells have two main lineages: myeloid progenitors (which give rise to myeloid cells, eg monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and lymphocytic progenitors [lymphocytic cells, eg T give rise to cells, B cells and natural killer (NK) cells]. Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8-binary negative T cells, γδ T cells, regulatory T cells, natural killer cells and dendritic cells. Macrophages and dendritic cells, sometimes called "antigen presenting cells" or "antigen presenting cells" (APCs), are the major histocompatibility complexes (MHC) on the surface of APCs complexed with peptides. A receptor is a specialized cell that can activate a T cell when it interacts with the TCR on the surface of the T cell.

The term "T cell" refers to cells of the T cell lineage. A "cell of T cell lineage" refers to a cell that exhibits at least one phenotypic characteristic of a T cell or its progenitor or progenitor that distinguishes the cell from other lymphoid and erythroid or myeloid lineage cells. Such phenotypic characteristics are expression of one or more T cell-specific proteins (e.g., CD3 ⁺ , CD4 ⁺ , CD8 ⁺ ), or T cell-specific physiological, morphological, functional or may include immunological features. For example, cells of the T-cell lineage include progenitor or progenitor cells committed to the T-cell lineage; CD25 ⁺ immature and inactivated T cells; cells that have undergone CD4 ^or CD8 lineage commitment ^; thymic progenitor cells that are positive; single positive CD4 ⁺ or CD8 ⁺ ; TCRαβ or TCRγδ; or mature and functional or activated T cells. The term "T cells" includes naive T cells (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-), central memory T cells (CD45RO ⁺ , CD62L ⁺ , CD8 ⁺ ), effector memory T cells (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), mucosa-associated invariant T (MAIT) cells, Tregs, natural killer T cells and tissue-resident T cells.

The term "B-cell" refers to cells of the B-cell lineage. A "B-lineage cell" refers to a cell that exhibits at least one phenotypic characteristic of a B-cell or its progenitor or progenitor that distinguishes the cell from other lymphoid and erythroid or myeloid lineage cells. Such phenotypic characteristics include expression of one or more proteins specific to B cells (e.g., CD19 ⁺ , CD72 + , CD24 + , CD20 ⁺ ), or physiological, morphological, or functional features specific to B cells. It may include therapeutic or immunological features. For example, cells of the B cell lineage can be progenitor or progenitor cells committed to the B cell lineage (e.g., pre-pro B cells, pro B cells and pre B cells); immature and inactivated B cells; can be active or activated B cells. Thus, "B cells" 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 ^- ).

The term "cytotoxic activity" for cells (e.g., T cells or NK cells) that express an immune receptor (e.g., TCR) or chimeric Tim receptor described in this disclosure on their surface is also referred to as "cytolytic activity." It means that the cell induces the target cell to undergo apoptosis upon antigen-specific signaling (eg, via the TCR, chimeric Tim receptor). In some embodiments, cytotoxic cells can induce apoptosis in target cells through the release of cytotoxins from granules, such as perforin, granzyme and granulysin. Perforin inserts into the target cell membrane, forming pores that allow water and salts to rapidly enter the target cell. Granzymes are serine proteases that induce apoptosis in target cells. Granulysin can also form pores in target cell membranes and is a pro-inflammatory molecule. In some embodiments, the cytotoxic cells are mediated through the interaction of Fas ligand, which is upregulated on T cells after antigen-specific signaling, with Fas molecules expressed on target cells. It can induce apoptosis in target cells. Fas is an apoptosis signaling receptor molecule on the surface of several different cells.

The term "exhaustion" for immune cells refers to poor effector function (e.g., reduced cytokine production, reduced cytotoxic activity), reduced proliferative capacity, increased expression of immune checkpoint molecules, and functional effector cells or memory cells. Refers to a state of immune cell dysfunction defined by a transcriptional state different from that of cells. In certain embodiments, exhausted immune cells become non-responsive to the presence of their target antigen. Immune cell exhaustion can result from chronic exposure to a target antigen (eg, as can result from chronic infection) or when it enters an immunosuppressive environment (eg, the 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) IFN-γ, IL-2, TNF - decreased production of 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) IFN-γ, TNF-α, or both or both (a) and (b).

A "disease" is a health condition in a subject in which the subject is unable to maintain homeostasis and, if the disease does not improve, the subject's health continues to deteriorate thereafter. In contrast, a "disorder" or "undesirable condition" in a subject means that the subject is able to maintain homeostasis, but the subject's health condition is lower than it would be if it were in the absence of the disorder or undesirable condition. It is also an undesirable health condition. A disorder or undesirable condition, if left untreated, does not necessarily result in a further decrease in the subject's health.

The term "cancer" as used herein is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Abnormal cells can form solid tumors or constitute hematologic malignancies. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various 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 and the like.

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

"Adoptive cell immunotherapy" or "adoptive immunotherapy" refers to the administration of natural or genetically engineered disease antigen-specific immune cells (eg, T cells). Adoptive cell immunotherapy can be performed using autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are genetically identical to the recipient). cells from a donor).

"Autologous" refers to any material (eg, organ, tissue, cell graft) derived from the same subject to which the material is later reintroduced.

"Allogeneic" refers to grafts derived from different subjects of the same species.

A “therapeutically effective amount” or “effective amount” of a chimeric protein of the disclosure or a cell expressing a chimeric protein (e.g., a chimeric Tim receptor or a cell expressing a chimeric Tim receptor) refers to the disease, disorder or desirability being treated. Refers to an amount of protein or cells sufficient to bring about an amelioration of one or more symptoms of a condition. When referring to an individual active ingredient, or cells expressing a single active ingredient, administered alone, a therapeutically effective dose refers to the sole effect of that ingredient or cells expressing that ingredient. When referring to a combination, a therapeutically effective dose is a dose of the active ingredient or ancillary active ingredients in combination with cells expressing the active ingredient that produces a therapeutic effect, whether administered sequentially or co-administered. Refers to combined quantities.

"Treat" or "treatment" or "ameliorate" refers to the medical management of a disease, disorder or undesirable condition in a subject. Generally, a suitable dose or therapeutic regimen involving host cells expressing a chimeric protein of the disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Treatment benefit or prevention/prevention benefit includes improved clinical outcome; reduction or alleviation of symptoms associated with the disease, disorder or undesirable condition; reduced incidence of symptoms; improved quality of life; longer disease-free state. a reduction in the extent of a disease, disorder or undesirable condition; stabilization of a disease state; slowing of disease progression; remission; survival;

The term "antitumor effect" can be evidenced by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy or an improvement in various physiological symptoms associated with cancerous conditions. refers to the effect of An "anti-tumor effect" may also be evidenced by prevention of hematological tumors or tumorigenesis.

"Autoimmune disease" refers to a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inappropriately exaggerated responses to self antigens. An autoimmune response can involve autoreactive B cells that generate autoantibodies, autoreactive T cells, or both. As used herein, an "autoantibody" is an antibody produced by a subject that binds to an autoantigen also produced by the subject.

Additional definitions are provided throughout this disclosure.

As noted above, the methods of the present disclosure are useful for treating cancer in a subject. Therapeutic agents of the disclosure (i.e., chimeric Tim receptor and PARP inhibitor), when administered together or sequentially, treat cancer in a subject (e.g., (i) arrest cancer development (ii) cause regression of the cancer; or (iii) alleviate symptoms resulting from the cancer).

In embodiments, the therapeutic methods disclosed herein comprise administering to a subject an effective amount of a chimeric Tim receptor and a PARP inhibitor, thereby treating cancer.

Chimeric Tim Receptor The present disclosure provides an extracellular domain comprising a Tim binding domain; an intracellular signaling domain comprising a first costimulatory signaling domain; located between the extracellular domain and the intracellular signaling domain. and a transmembrane domain connecting them. In certain embodiments, the extracellular domain of the chimeric Tim receptor described herein may comprise an extracellular spacer domain located between and connecting the binding domain and the transmembrane domain. .

When expressed in a host cell, a chimeric Tim receptor of the present disclosure can confer a phosphatidylserine-specific cytotoxic phenotype on an engineered host cell (e.g., when a host cell has phosphatidylserine on its surface). become cytotoxic to stressed, injured, injured, apoptotic, or necrotic cells that express In certain embodiments, chimeric Tim receptors induce apoptosis in targeted cells through the release of granzymes, perforins, granulysins, or any combination thereof. In further embodiments, cells expressing the chimeric Tim receptors described herein exhibit a phagocytic phenotype specific for phosphatidylserine presenting cells.

The intracellular signaling domain can include one or more effector domains that can transmit a functional signal to the cell in response to binding of the extracellular domain of the chimeric Tim receptor to phosphatidylserine. Signal transduction by the intracellular signaling domain is triggered by binding of the extracellular domain to phosphatidylserine. Signals transduced by the intracellular signaling domain promote effector functions of chimeric Tim receptor-containing cells. Examples of effector functions include cytotoxic activity, cytokine secretion, proliferation, anti-apoptotic signaling, persistence, expanded proliferation, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen presentation, or Any combination is included.

In certain embodiments, the intracellular signaling domain comprises a first intracellular signaling domain. In further embodiments, the intracellular signaling domain comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a first intracellular signaling domain, a second intracellular signaling domain, and a third intracellular signaling domain. The chimeric Tim receptors described in this disclosure provide co-stimulation to enhance cellular immune responses, reduce immune cell exhaustion, or both, while treating apoptotic, necrotic, injured, or stressed cells. It can be used in a variety of therapeutic methods where clearance of cells that have undergone cytotoxicity is beneficial.

The component parts of the fusion proteins of the present disclosure are further described in detail herein.

Extracellular Domain Tim4 binding domains suitable for use in the chimeric Tim4 receptors of the present disclosure can be any polypeptide or peptide derived from the Tim4 molecule that specifically binds phosphatidylserine. In certain embodiments, the Tim4 binding domain is derived from human Tim4. Exemplary human Tim4 molecules are found in Uniprot. Ref. Provided in Q96H15 (SEQ ID NO: 1). An exemplary human Tim4 binding domain comprises or consists of SEQ ID NO:2 or the amino acid sequence of amino acids 25-314 of SEQ ID NO:2. An exemplary mouse Tim4 binding domain comprises or consists of SEQ ID NO:24 or the amino acid sequence of amino acids 23-279 of SEQ ID NO:24. In certain embodiments, the Tim4 binding domain comprises at least about 75%, 80%, 85% of SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID NO:24. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% of an amino acid sequence comprising or from Become. In certain embodiments, the Tim4 binding domain has 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 alterations (e.g., deletions, additions, substitutions) containing amino acid sequences having

In certain embodiments, the extracellular domain may include an extracellular non-signaling spacer or linker domain. If included, such spacer or linker domains may position the binding domain away from the host cell surface to further permit proper 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 Tim4 receptor. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (e.g., Guest et al., J. Immunother. 28:203-11, 2005; see PCT Publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (eg, IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. A modified IgG ₄ hinge region is described in PCT Publication No. WO2014/031687, which is incorporated herein by reference in its entirety. In certain embodiments, the extracellular spacer domain comprises a modified _IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that can be used in the chimeric Tim receptors described herein include the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which can be wild type or mutants thereof. includes the hinge region from In some embodiments, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO:32. In a further embodiment, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from CH1 domain, CH2 domain, CH3 domain or combinations thereof (see e.g. PCT Publication No. WO2014/031687). Please, the spacer is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may comprise the stalk region of a type II C lectin (the extracellular domain located between the type C lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A and NKG2D.

In certain embodiments, the extracellular domain is human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, transgenic species thereof, or any thereof. including polynucleotide sequences from any mammalian species, including combinations of In certain embodiments, the extracellular domain is murine, human, or chimeric.

The intracellular signaling domain of the chimeric Tim4 receptor described herein is an intracellular effector domain that sends a functional signal to the cell in response to binding of the extracellular domain of the chimeric Tim4 receptor to phosphatidylserine. can be transmitted. Signals transduced by the intracellular signaling domain promote effector functions of chimeric Tim4 receptor-containing cells. Examples of effector functions include cytotoxic activity, cytokine secretion, proliferation, anti-apoptotic signaling, persistence, extended proliferation, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, or any combination thereof. is included.

In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. A costimulatory signaling domain can be any portion of a costimulatory signaling molecule that retains sufficient signaling activity. In some embodiments, full-length or full-length intracellular components of co-stimulatory signaling molecules are used. In some embodiments, a truncated portion of a costimulatory signaling molecule or intracellular component of a costimulatory signaling molecule is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the costimulatory signaling domain is a variant of a full or truncated portion of a costimulatory signaling molecule, provided that the variant retains sufficient signaling activity (i.e., functional is a mutated type).

In certain embodiments, the co-stimulatory signaling domain is 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), PD-1, CD273 (PD-L2), CD274 (PD-L1), B7 - contains H3 (CD276), ICOS (CD278), DAP10, LAT, LFA-1 (CD11a/CD18), LIGHT, NKG2C, SLP76, TRIM, or ZAP70 signaling domains. In certain embodiments, the co-stimulatory signaling domain is OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), or 4-1BB (CD137) signaling Contains domains. An exemplary CD28 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 118 or 119. An exemplary OX40 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:120. An exemplary CD2 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:121. An exemplary 4-1BB co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:122. An exemplary CD27 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:123. An exemplary ICAM-1 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:124. An exemplary LFA-1 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:125. An exemplary ICOS co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:126. An exemplary CD30 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:127. An exemplary CD40 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:128. An exemplary PD-1 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:129. An exemplary CD7 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:130. An exemplary LIGHT co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:131. An exemplary NKG2C co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:132. An exemplary B7-H3 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:133. In certain embodiments, the co-stimulatory signaling domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% of any one of SEQ ID NOs: 118-133 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the co-stimulatory signaling domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, Amino acid sequences with 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included. In certain embodiments, the intracellular signaling comprises a second co-stimulatory signaling domain. In preferred embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain are different.

In certain embodiments, the intracellular signaling domain further comprises an ITAM-containing activation domain. An ITAM-containing activation domain may orchestrate TCR signaling independently of the endogenous TCR complex. In certain embodiments, signaling through ITAM-containing activation domains results in mediation of T cell responses, including but not limited to proliferation, activation, differentiation, and the like. An ITAM-containing activation domain can be any portion of an ITAM-containing activation domain molecule that retains sufficient signaling activity. In some embodiments, full-length or full-length intracellular components of ITAM-containing activation domain molecules are used. In some embodiments, a cleaved portion of an ITAM-containing activation domain molecule or intracellular component of an ITAM-containing activation domain molecule is used, provided that the cleaved portion retains sufficient signaling activity. In further embodiments, the ITAM-containing activation domain is a variant of a full or truncated portion of an ITAM-containing activation domain molecule provided that the variant retains sufficient signaling activity (i.e., functional variant).

Examples of ITAM-containing activation domains that may be used in the chimeric Tim4 receptors of the present disclosure include those derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD278 (ICOS), and CD66d. In certain embodiments, the ITAM-containing activation domain is the CD3ζ signaling domain. An exemplary CD3ζ signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 134 or 135. An exemplary CD3γ signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:136. An exemplary CD3delta signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:137. An exemplary CD3ε signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:138. An exemplary CD5 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:139. An exemplary CD22 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:140. An exemplary CD79a signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:141. An exemplary CD66d signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:143. In certain embodiments, the ITAM-containing activation domain is at least about 75%, 80%, 85%, 90%, 91%, 92% of any one of SEQ ID NOS: 134-141, and 143 , 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the ITAM-containing activation domain comprises at least about 1, 2, 3, 4, 5, 6, 7, Amino acid sequences with 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included.

In another embodiment, the intracellular signaling domain comprises a CD28 co-stimulatory signaling domain and a CD3zeta signaling domain. In another embodiment, the intracellular signaling domain comprises a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain. In yet another embodiment, the intracellular signaling domain comprises a CD27 co-stimulatory signaling domain and a CD3zeta signaling domain. In another embodiment, the intracellular signaling domain comprises an ICOS co-stimulatory signaling domain and a CD3ζ signaling domain. In another embodiment, the intracellular signaling domain comprises the LFA-1 co-stimulatory signaling domain and the CD3ζ signaling domain. In another embodiment, the intracellular signaling domain comprises an OX40 co-stimulatory signaling domain and a CD3zeta signaling domain. In another embodiment, the intracellular signaling domain comprises a CD2 co-stimulatory signaling domain and a CD3zeta signaling domain. In yet another embodiment, the intracellular signaling domain comprises an ICAM-1 co-stimulatory signaling domain and a CD3ζ signaling domain.

The intracellular signaling domain can be derived from any mammalian species including human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, and transgenic species thereof.

The transmembrane domain of the chimeric Tim4 receptor connects and lies between the extracellular and intracellular signaling domains. A transmembrane domain is a hydrophobic alpha helix that spans the host cell membrane. A transmembrane domain may be fused directly to a binding domain or, if present, to an extracellular spacer domain. In certain embodiments, the transmembrane domain is derived from an integral membrane protein [eg, receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, 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, a chimeric Tim4 receptor can comprise a Tim4 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim4 receptor can include a CD28 transmembrane domain and a CD28 co-stimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, the extracellular domain, and All of the intracellular signaling domains are derived from different molecules. Examples of transmembrane domains that can be used in the chimeric Tim4 receptors of the present disclosure include Tim4, Tim1, CD3ζ, CD3γ, CD3δ, CD3ε, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37. , CD64, CD80, CD86, CD134, CD137, CD154, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3 A transmembrane domain from which is included. An exemplary Tim4 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 144 or 23. An exemplary Tim1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:8. An exemplary CD28 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:145. An exemplary 4-1BB transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:146. An exemplary OX40 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:147. An exemplary CD27 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:148. An exemplary ICOS transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:149. An exemplary CD2 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:150. An exemplary LFA-1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:151. An exemplary CD30 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:152. An exemplary CD40 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:153. An exemplary PD-1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:154. An exemplary CD7 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:155. An exemplary LIGHT transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:156. An exemplary NKG2C transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:157. An exemplary B7-H3 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:158. In certain embodiments, the transmembrane domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, It comprises or consists of an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the transmembrane domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, Includes amino acid sequences with 9 or 10 amino acid alterations (eg, deletions, additions, substitutions).

The transmembrane domain can be derived from any mammalian species including human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig and transgenic species thereof.

Exemplary components, constructs and chimeric Tim4 receptor sequences of this disclosure are provided in Table 1.

table 1.

A further embodiment of the chimeric Tim4 receptor described herein is an extracellular domain comprising a Tim4 binding domain; and an intracellular signaling domain comprising a first costimulatory signaling domain and an ITAM-containing activation domain. a transmembrane domain located between and connecting the extracellular and intracellular signaling domains; Contains chimeric proteins.

A suitable tim4 binding domain for use in the chimeric tim4 receptors of the present disclosure can be any polypeptide or peptide derived from the tim4 molecule that specifically binds phosphatidylserine. In certain embodiments, the Tim4 binding domain is derived from human Tim4. Exemplary human Tim4 molecules are found in Uniprot. Ref. Provided in Q96H15 (SEQ ID NO: 1). An exemplary human Tim4 binding domain comprises or consists of SEQ ID NO:2 or the amino acid sequence of amino acids 25-314 of SEQ ID NO:2. An exemplary mouse Tim4 binding domain comprises or consists of SEQ ID NO:24 or the amino acid sequence of amino acids 23-279 of SEQ ID NO:24. In certain embodiments, the Tim4 binding domain comprises at least about 75%, 80%, 85% of SEQ ID NO:2 or amino acids 25-314 of SEQ ID NO:2, or SEQ ID NO:24 or amino acids 23-279 of SEQ ID NO:24. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% of an amino acid sequence comprising or from Become. In certain embodiments, the Tim4 binding domain has 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 alterations (e.g., deletions, additions, substitutions) containing amino acid sequences having

In certain embodiments, the extracellular domain may include an extracellular non-signaling spacer or linker domain. If included, such spacer or linker domains may position the binding domain away from the host cell surface to further permit proper 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 Tim4 receptor. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (e.g., Guest et al., J. Immunother. 28:203-11, 2005; see PCT Publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (eg, IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. A modified IgG ₄ hinge region is described in PCT Publication No. WO2014/031687, which is incorporated herein by reference in its entirety. In certain embodiments, the extracellular spacer domain comprises a modified _IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that can be used in the chimeric Tim4 receptors described herein include extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which can be wild type or mutants thereof. includes the hinge region from In some embodiments, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO:32. In a further embodiment, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from CH1 domain, CH2 domain, CH3 domain or combinations thereof (see e.g. PCT Publication No. WO2014/031687). Please, the spacer is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may comprise the stalk region of a type II C lectin (the extracellular domain located between the type C lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A and NKG2D.

In certain embodiments, the extracellular domain is human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, transgenic species thereof, or any thereof. including polynucleotide sequences from any mammalian species, including combinations of In certain embodiments, the extracellular domain is murine, human, or chimeric.

In certain embodiments, the intracellular signaling domain comprises a costimulatory signaling domain and an ITAM-containing activation domain, wherein the ITAM-containing activation domain comprises a DAP12 signaling domain. A costimulatory signaling domain can be any portion of a costimulatory signaling molecule that retains sufficient signaling activity. In some embodiments, full-length or full-length intracellular components of co-stimulatory signaling molecules are used. In some embodiments, a truncated portion of a costimulatory signaling molecule or intracellular component of a costimulatory signaling molecule is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the costimulatory signaling domain is a variant of a full or truncated portion of a costimulatory signaling molecule, provided that the variant retains sufficient signaling activity (i.e., functional is a mutated type).

In certain embodiments, the co-stimulatory signaling domain is 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), PD-1, CD273 (PD-L2), CD274 (PD-L1), B7 - contains H3 (CD276), ICOS (CD278), DAP10, LAT, LFA-1 (CD11a/CD18), LIGHT, NKG2C, SLP76, or TRIM signaling domains. In certain embodiments, the co-stimulatory signaling domain is the OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) or 4-1BB (CD137) signaling domain including. An exemplary CD28 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:164 or 165. An exemplary OX40 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:166. An exemplary CD2 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:167. An exemplary 4-1BB co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:168. An exemplary CD27 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:169. An exemplary ICAM-1 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:170. An exemplary LFA-1 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:171. An exemplary ICOS co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:172. An exemplary CD30 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:173. An exemplary CD40 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:174. An exemplary PD-1 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:175. An exemplary CD7 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:176. An exemplary LIGHT co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:177. An exemplary NKG2C co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:178. An exemplary B7-H3 co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:179. In certain embodiments, the co-stimulatory signaling domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% of any one of SEQ ID NOs: 164-179. %, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the co-stimulatory signaling domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, Amino acid sequences with 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included. In certain embodiments, the intracellular signaling comprises a second co-stimulatory signaling domain. In preferred embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain are different.

The DAP12 signaling domain may oversee TCR signaling independently of the endogenous TCR complex. In certain embodiments, signaling through the DAP12 signaling domain results in mediation of T cell responses, including but not limited to proliferation, activation, differentiation, and the like. A DAP12 signaling domain can be any portion of the DAP12 molecule that retains sufficient signaling activity. In some embodiments, full-length or full-length intracellular components of the DAP12 molecule are used. In some embodiments, a truncated portion of DAP12 or an intracellular component of DAP12 is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the DAP12 signaling domain is a variant of a full or truncated portion of DAP12, provided that the variant retains sufficient signaling activity (ie, is a functional variant).

Exemplary human DAP12 molecules are found in Uniprot. Ref. O43914. An exemplary DAP12 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:180. In certain embodiments, the DAP12 signaling domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, It comprises or consists of amino acid sequences having 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the DAP12 signaling domain has 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 alterations (eg, deletions, additions, substitutions).

In another embodiment, the intracellular signaling domain comprises a CD28 co-stimulatory signaling domain and a DAP12 signaling domain. In another embodiment, the intracellular signaling domain comprises a 4-1BB co-stimulatory signaling domain and a DAP12 signaling domain. In yet another embodiment, the intracellular signaling domain comprises a CD27 co-stimulatory signaling domain and a DAP12 signaling domain. In another embodiment, the intracellular signaling domain comprises an ICOS co-stimulatory signaling domain and a DAP12 signaling domain. In another embodiment, the intracellular signaling domain comprises an LFA-1 co-stimulatory signaling domain and a DAP12 signaling domain. In another embodiment, the intracellular signaling domain comprises an OX40 co-stimulatory signaling domain and a DAP12 signaling domain. In yet another embodiment, the intracellular signaling domain comprises a CD2 co-stimulatory signaling domain and a DAP12 signaling domain. In yet another embodiment, the intracellular signaling domain comprises an ICAM-1 co-stimulatory signaling domain and a DAP12 signaling domain.

The intracellular signaling domain can be derived from mammalian species including human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, and transgenic species thereof.

In certain embodiments, the transmembrane domain is derived from an integral membrane protein [eg, receptors, surface antigen class (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, a chimeric Tim4 receptor can comprise a Tim4 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim4 receptor can include a CD28 transmembrane domain and a CD28 co-stimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, the extracellular domain, and All of the intracellular signaling domains are derived from different molecules. Examples of transmembrane domains that can be used in the chimeric Tim4 receptors of the present disclosure include Tim4, CD3ζ, CD3γ, CD3δ, CD3ε, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64. , CD80, CD86, CD134, CD137, CD154, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3 It contains a transmembrane domain. An exemplary Tim4 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 144 or 23. An exemplary CD28 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:181. An exemplary 4-1BB transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:182. An exemplary OX40 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:183. An exemplary CD27 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:184. An exemplary ICOS transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:185. An exemplary CD2 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:186. An exemplary LFA-1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:187. An exemplary CD30 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:188. An exemplary CD40 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:189. An exemplary PD-1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:190. An exemplary CD7 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:191. An exemplary LIGHT transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:192. An exemplary NKG2C transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:193. An exemplary B7-H3 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:194. In certain embodiments, the transmembrane domain is at least about 75%, 80%, 85%, 90%, 91%, 92% of any one of SEQ ID NOS:23, 144, and 181-194 , 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the transmembrane domain is at least about 1, 2, 3, 4, 5, 6, 7, 8 amino acid sequences of any one of SEQ ID NOS:23, 144, and 181-194 , 9, or 10 amino acid alterations (eg, deletions, additions, substitutions).

The transmembrane domain can be derived from any mammalian species, including humans, primates, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, and transgenic species thereof.

In certain embodiments, the chimeric Tim4 receptor is human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, transgenic species thereof, or any thereof. including polynucleotide sequences from any mammalian species, including combinations of In certain embodiments, the chimeric Tim4 receptor is a murine, chimeric, human or humanized chimeric Tim4 receptor.

It is understood that the direct fusion of one domain of the chimeric Tim4 receptor described herein to another does not preclude the presence of intervening junctional amino acids. Junctional amino acids can be natural or non-natural (eg, resulting from the construct design of the chimeric protein). For example, junctional amino acids can arise from restriction enzyme sites used for joining one domain to another or cloning a polynucleotide encoding a chimeric Tim4 receptor into a vector.

Exemplary components, constructs and chimeric Tim4 receptor sequences of this disclosure are provided in Table 2.

Table 2.

In embodiments, the chimeric tim receptor comprises (a) an extracellular domain comprising a binding domain comprising (i) a Tim1 IgV domain or a Tim4 IgV domain; and (ii) a Tim1 mucin domain or a Tim4 mucin domain; ) an intracellular signaling domain comprising a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) located between and connecting the extracellular domain and the intracellular signaling domain. A single-chain chimeric protein comprising a transmembrane domain and a transmembrane domain.

In certain embodiments, the binding domain is (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 the Tim1 mucin domain. In certain embodiments, the extracellular domain of the chimeric Tim receptor described herein may comprise an extracellular spacer domain located between and connecting the binding domain and the transmembrane domain. .

When expressed in a host cell, a chimeric Tim receptor of the present disclosure can confer a phosphatidylserine-specific cytotoxic phenotype on an engineered host cell (e.g., when a host cell has phosphatidylserine on its surface). become cytotoxic to stressed, injured, injured, apoptotic, or necrotic cells that express In certain embodiments, chimeric Tim receptors induce apoptosis in targeted cells through the release of granzymes, perforins, granulysins, or any combination thereof. In further embodiments, cells expressing the chimeric Tim receptors described herein exhibit a phagocytic phenotype specific for phosphatidylserine presenting cells.

An intracellular signaling domain may include one or more effector (also called "co-stimulatory signaling") domains that co-stimulate the modified host cell. Signal transduction by the intracellular signaling domain is triggered by binding of the extracellular domain to phosphatidylserine. In certain embodiments, the intracellular signaling domain comprises a first intracellular signaling domain. In further embodiments, the intracellular signaling domain comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a first intracellular signaling domain, a second intracellular signaling domain, and a third intracellular signaling domain. The chimeric Tim receptors described in this disclosure provide co-stimulation to enhance cellular immune responses, reduce immune cell exhaustion, or both, while treating apoptotic, necrotic, injured, or stressed cells. It can be used in a variety of therapeutic methods where clearance of cells that have undergone cytotoxicity is beneficial.

As described herein, chimeric Tim receptors contain an extracellular domain that includes a Tim binding domain. The Tim-binding domain confers specificity for phosphatidylserine (PtdSer), a phospholipid with a negatively charged head group and a component of cell membranes. In healthy cells, phosphatidylserine is found predominantly in the inner leaflet of the cell membrane. However, when cells are stressed, injured, or undergo apoptosis or necrosis, phosphatidylserine is exposed on the outer leaflet of the cell membrane. Phosphatidylserine can therefore be used as a marker to distinguish between stressed, damaged, apoptotic, necrotic, pyroptotic, or swollen cells. Binding of phosphatidylserine by the Tim-binding domain blocks interactions between phosphatidylserine and another molecule and can, for example, interfere with, reduce or eliminate certain functions of phosphatidylserine, such as signal transduction. In some embodiments, binding of phosphatidylserine may induce certain biological pathways or identify phosphatidylserine molecules or cells expressing phosphatidylserine for removal.

Tim-binding domains suitable for use in the chimeric Tim receptors of the present disclosure can be any polypeptide or peptide derived from Tim1 and/or Tim4 molecules that specifically binds phosphatidylserine. In embodiments, the tim-binding domain comprises an IgV domain derived from Tim1 or Tim4 and a mucin domain derived from Tim1 or Tim4. For example, a Tim-binding domain can include a Tim1 IgV domain and a Tim1 mucin domain. In another example, a Tim-binding domain can include a Tim1 IgV domain and a Tim4 mucin domain. In another example, a Tim-binding domain can include a Tim4 IgV domain and a Tim1 mucin domain. In another example, a Tim-binding domain can include a Tim4 IgV domain and a Tim4 mucin domain.

Phosphatidylserine binding is generally regulated by the IgV domain. The core phosphatidylserine binding domain is a four amino acid sequence in the IgV domain (eg, amino acids 95-98 of SEQ ID NO:34 or amino acids 92-95 of SEQ ID NO:38). The Tim4 binding domain minimally binds to cells with low phosphatidylserine density. The Tim1 binding domain binds more strongly to lower phosphatidylserine densities, resulting in a lower threshold for response. A summary of Tim1 and Tim4 binding to phosphatidylserine is provided in Table 3. 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 for phosphatidylserine can be modulated. Moreover, such a combination in the Tim-binding domain also provides a combination of Tim4 sensitivity to phosphatidylserine and Tim1 stability in protein expression.

Table 3.

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

In certain embodiments, the Tim-binding domain is derived from human Tim1 and/or Tim4. Exemplary human Tim1 molecules are found in Uniprot. Ref. Provided in 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 certain embodiments, the Tim1 binding domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, It comprises or consists of amino acid sequences having 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the Tim1 binding domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Amino acid sequences with 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included.

Exemplary human Tim4 molecules are found in Uniprot. Ref. Provided in Q96H15 (SEQ ID NO: 1). An exemplary human Tim4 binding domain comprises or consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:42. An exemplary mouse Tim4 binding domain comprises or consists of SEQ ID NO:24 or the amino acid sequence of amino acids 23-279 of SEQ ID NO:24. In certain embodiments, the Tim4 binding domain is at least about 75%, 80%, 85%, 90%, 91 for amino acids 23-279 of SEQ ID NO:2 or SEQ ID NO:42, or SEQ ID NO:24 or SEQ ID NO:24. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the Tim4-binding domain comprises at least about 1, 2, 3, 4, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5 or 5, respectively, than any other amino acid sequence of amino acids 23-279 of SEQ ID NO: 2 or SEQ ID NO: 42, or SEQ ID NO: 24 or SEQ ID NO: 24. Includes amino acid sequences with 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions).

In embodiments, the Tim-binding domain comprises an IgV domain from Tim1. 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 comprising an R66G substitution in SEQ ID NO:38. The R66G substitution (eg amino acids 68-70 of SEQ ID NO:34) confers an RGD domain in the Tim1 IgV domain that can regulate integrin binding as a co-receptor for phagocytosis. In certain embodiments, the modified Tim1 IgV domain comprises or consists of the amino acid sequence of SEQ ID NO:41. In some embodiments, this modified Tim1 domain may increase phagocytic activity while preserving Tim1 sensitivity. In certain embodiments, the Tim1 IgV domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, It comprises or consists of an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the Tim1 IgV has at least about 1, 2, 3, 4, 5, 6, 7, 8, Amino acid sequences with 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included.

In other embodiments, the Tim-binding domain comprises an IgV domain from Tim4. An exemplary human Tim4 IgV domain is provided in SEQ ID NO:34. In certain embodiments, the Tim4 IgV domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% comprising or consisting of amino acid sequences having %, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the Tim4 IgV domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, Amino acid sequences with 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included.

In embodiments, the Tim-binding domain comprises a mucin domain from Tim1. An exemplary human Tim1 mucin domain is provided in SEQ ID NO:39. In certain embodiments, the Tim1 mucin domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of SEQ ID NO:39 comprising or consisting of amino acid sequences having %, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the Tim1 mucin domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, Amino acid sequences with 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included.

In other embodiments, the Tim-binding domain comprises a mucin domain from Tim4. An exemplary human Tim4 mucin domain is provided in SEQ ID NO:35. In certain embodiments, the Tim4 mucin domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% comprising or consisting of amino acid sequences having %, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the Tim4 mucin domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, Amino acid sequences with 15, 16, 17, 18, 19, or 20 amino acid alterations (eg, deletions, additions, substitutions) are included.

In some embodiments, the Tim-binding domain comprises the Tim1 IgV domain and the Tim1 mucin domain. In certain embodiments, the Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:38 and the Tim1 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In another specific embodiment, the Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:38 with an R66G substitution and the Tim1 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In a further specific embodiment, the Tim1 IgV domain comprises the amino acid sequence shown in SEQ ID NO:41 and the Tim1 mucin domain comprises the amino acid sequence shown in SEQ ID NO:39. In some embodiments, the Tim1 IgV domain and the Tim1 mucin domain both comprise or consist of the amino acid sequence set forth in SEQ ID NO:37 or SEQ ID NO:43.

In some embodiments, the Tim-binding domain comprises the Tim4 IgV domain and the Tim4 mucin domain. In certain embodiments, the Tim4 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:34 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the Tim4 IgV domain and the Tim4 mucin domain both comprise or consist of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:42.

In some embodiments, the Tim-binding domain comprises the Tim1 IgV domain and the Tim4 mucin domain. In certain embodiments, the Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:38 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In another specific embodiment, the Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:38 with an R66G substitution and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In a further specific embodiment, the Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:41 and the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the Tim1 IgV domain further comprises the Tim1 signal sequence of SEQ ID NO:40.

In still further embodiments, the Tim binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain. In certain embodiments, the Tim4 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:34 and the Tim1 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:39. In some embodiments, the Tim4 IgV domain further comprises the Tim4 signal sequence of SEQ ID NO:11.

In certain embodiments, the extracellular domain may include an extracellular non-signaling spacer or linker domain. If included, such spacer or linker domains may position the binding domain away from the host cell surface to further permit proper cell/cell contact, binding and activation. When included in the chimeric receptors described herein, the extracellular spacer domain is generally positioned between the extracellular binding domain and the transmembrane domain of the chimeric Tim receptor. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (e.g., Guest et al., J. Immunother. 28:203-11, 2005; see PCT Publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (eg, IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. A modified IgG ₄ hinge region is described in PCT Publication No. WO2014/031687, which is incorporated herein by reference in its entirety. In certain embodiments, the extracellular spacer domain comprises a modified _IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO:3). Other examples of hinge regions that can be used in the chimeric Tim receptors described herein include the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which can be wild type or mutants thereof. includes the hinge region from In certain embodiments, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO:32. In a further embodiment, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from CH1 domain, CH2 domain, CH3 domain or combinations thereof (see e.g. PCT Publication No. WO2014/031687). Please, the spacer is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may comprise the stalk region of a type II C lectin (the extracellular domain located between the type C lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A and NKG2D.

In certain embodiments, the extracellular domain is human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, transgenic species thereof, or any of them. Includes amino acid sequences from any mammalian species, including combinations. In certain embodiments, the extracellular domain is murine, human or chimeric.

The intracellular signaling domain of the chimeric Tim Receptor described herein is an intracellular effector domain that sends a functional signal to the cell in response to binding of the extracellular domain of the chimeric Tim Receptor to phosphatidylserine. can be transmitted. Signals transduced by the intracellular signaling domain promote effector functions of chimeric Tim receptor-containing cells. Examples of effector functions include cytotoxic activity, cytokine secretion, proliferation, anti-apoptotic signaling, persistence, extended proliferation, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen capture, antigen processing, Antigen presentation, or any combination thereof is included.

Intracellular signaling domains include primary intracellular signaling domains. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain and a secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, a secondary intracellular signaling domain, and a tertiary intracellular signaling domain. Primary, secondary, and/or tertiary intracellular signaling domains may independently be any portion of a signaling molecule that retains sufficient signaling activity. In some embodiments, full-length signaling molecules or full-length intracellular components of signaling molecules are used. In some embodiments, truncated portions of signaling molecules or intracellular components of signaling molecules are used, provided that the truncated portions retain sufficient signaling activity. In some embodiments, the signaling domain is a variant of a whole or truncated portion of a signaling molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant). be).

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

In embodiments, the secondary intracellular signaling domain is 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, 4 -1BB signaling domain, TLR2 signaling domain, or TLR8 signaling domain.

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

In some embodiments, the primary intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling domain; the secondary intracellular signaling domain is a costimulatory signaling domain, Tim1 signal a transduction domain or a Tim4 signaling domain; and a tertiary intracellular signaling domain comprises a TLR signaling domain. ITAM-containing signaling domains generally include at least one (1, 2, 3, 4 or more) ITAMs that refer to the conserved motif of YXXL/IX _6-8 -YXXL/I contains ITAM-containing signaling domains can initiate T-cell activation signaling following antigen binding or ligand engagement. ITAM signaling domains include, for example, the intracellular signaling domains of CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP12, FcRγ, and CD66d. Costimulatory signaling domains, when activated in conjunction with primary or canonical (e.g., ITAM-driven) activation signals, induce T-cell responses such as T-cell activation, cytokine production, proliferation, differentiation, survival, Promotes or enhances effector functions or combinations thereof. Costimulatory 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 is OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), or 4-1BB (CD137) signaling Contains the transduction domain. The TLR signaling domain can be a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signaling domain. In some embodiments, the TLR signaling domain is a TLR2 signaling domain or a TLR8 signaling domain.

As used herein, the designations of primary, secondary, and tertiary intracellular signaling domains include the primary intracellular signaling domain at the N-terminus of the intracellular portion of the chimeric Tim receptor, the secondary cellular signaling domain in the middle, Including, but not limited to, intracellular signaling domains, and placement of tertiary intracellular signaling domains at the C-terminus. Therefore, 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 the secondary intracellular signaling domain is the intracellular domain of choice in the middle of the intracellular portion of the chimeric tim receptor (or at the C-terminus for those chimeric tim receptors with only two intracellular signaling domains). It does not limit the use of signaling domains. The designation of a tertiary intracellular signaling domain does not limit the use of the selected intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Accordingly, different arrangements of the primary, secondary, and/or tertiary intracellular signaling domains within the intracellular portion of the chimeric Tim receptor are contemplated.

An exemplary Tim1 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:44. Exemplary Tim4 signaling domains comprise or consist of the amino acid sequence of SEQ ID NO:45, SEQ ID NO:224, or SEQ ID NO:225. An exemplary TRAF2 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:48. An exemplary TRAF6 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:46. An exemplary CD28 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:4 or 26. An exemplary DAP12 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:9. An exemplary CD3ζ signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:27 or 5. An exemplary 4-1BB signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:122. An exemplary TLR2 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:222. An exemplary TLR8 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO:47.

In certain embodiments, the Tim1 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:44. In another specific embodiment, the Tim4 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:45, SEQ ID NO:224, or SEQ ID NO:225. In another specific embodiment, the TRAF2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:48. In another specific embodiment, the TRAF6 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:46. In another specific embodiment, the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:4. In another specific embodiment, the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:26. In another specific embodiment, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO:9. In another specific embodiment, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO:27. In another specific embodiment, the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO:5. In another specific embodiment, the 4-1BB signaling domain comprises the amino acid sequence of SEQ ID NO:122. In another specific embodiment, the TLR2 signaling domain comprises the amino acid sequence of SEQ ID NO:222. In another specific embodiment, the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO:47.

In embodiments, the primary, secondary, and/or tertiary signaling domains (if present) are at least about any one of SEQ ID NOS: 4, 5, 9, 26, 27, or 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity comprising or consisting of an amino acid sequence having In certain embodiments, the primary, secondary, and/or tertiary signaling domains are amino acids of any one of SEQ ID NOS: 4, 5, 9, 26, 27, 44-48, 122, and 222. 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). In embodiments, the primary signaling domain, the secondary, and/or the tertiary intracellular signaling domain are the same. In some embodiments, two or three of the primary, secondary, and tertiary intracellular signaling domains are different.

In certain embodiments, the intracellular signaling domain comprises a Tim1 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a Tim4 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TRAF6 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TRAF2 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TLR2 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TLR8 intracellular signaling domain.

In certain embodiments, the intracellular signaling domain comprises a Tim1 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In other embodiments, the intracellular signaling domain comprises a Tim4 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In other embodiments, the intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In other embodiments, the intracellular signaling domain comprises a TLR2 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In other embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a DAP12 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3zeta tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises 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 comprises a CD28 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain, and a CD3zeta tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises 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 comprises a TLR2 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ primary intracellular signaling domain and a TLR2 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TRAF6 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ primary intracellular signaling domain and a TRAF6 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3zeta tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises 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 comprises a CD28 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain, and a CD3zeta tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises 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 comprises a CD3ζ primary intracellular signaling domain and a TLR2 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TLR2 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ primary intracellular signaling domain and a TLR8 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ primary intracellular signaling domain and a TRAF6 secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a TRAF6 primary intracellular signaling domain and a CD3ζ secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a CD3zeta tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises 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 comprises a Tim4 primary intracellular signaling domain, a TLR8 secondary intracellular signaling domain, and a CD3zeta tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a CD3ζ secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain.

In certain embodiments, the intracellular signaling domains are a Tim1 primary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:44 and a CD3ζ secondary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:27 or 5. including. In another specific embodiment, the intracellular signaling domain comprises a Tim4 primary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:45, SEQ ID NO:224, or SEQ ID NO:225 and the amino acid sequence of SEQ ID NO:27 or 5 and a CD3ζ secondary intracellular signaling domain containing In further specific embodiments, the intracellular signaling domain is a TLR8 primary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:47 and a CD3ζ secondary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:27 or 5 including. In another specific embodiment, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:4 or 26 and a DAP12 secondary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:9. including domains. In some embodiments, the intracellular signaling domain comprises a combination of primary, secondary, and optionally tertiary intracellular signaling domain sequences shown in Table 10.

The intracellular signaling domain can be derived from mammalian species including human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig and transgenic species thereof.

The transmembrane domain of the chimeric Tim receptor connects and lies between the extracellular and intracellular signaling domains. A transmembrane domain is a hydrophobic alpha helix that spans the host cell membrane. A transmembrane domain may be fused directly to a binding domain or, if present, to an extracellular spacer domain. In certain embodiments, the transmembrane domain is derived from an integral membrane protein [eg, receptors, surface antigen class (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, a chimeric Tim receptor can comprise a Tim4 binding domain and a Tim4 transmembrane domain. In another example, a chimeric Tim receptor can include a CD28 transmembrane domain and a CD28 co-stimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are derived from different molecules; the transmembrane domain and the intracellular signaling domain are derived from different molecules; or the transmembrane domain, the extracellular domain, and All of the intracellular signaling domains are derived from different molecules. Examples of transmembrane domains that can be used in the chimeric Tim receptors of the present disclosure include transmembrane domains from Tim1, Tim4, and CD28. An exemplary Tim1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:8. An exemplary Tim4 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:6 or 23. An exemplary CD28 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:7. In certain embodiments, the transmembrane domain is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% of any one of SEQ ID NOs:6-8 or 23 , 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity. In certain embodiments, the transmembrane domain has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or amino acid sequences with 10 amino acid modifications (eg, deletions, additions, substitutions).

The transmembrane domain can be derived from any mammalian species, including humans, primates, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, and transgenic species thereof.

In certain embodiments, the chimeric Tim receptor is human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, transgenic species thereof, or any thereof. including polynucleotide sequences from any mammalian species, including combinations of In certain embodiments, the chimeric Tim Receptor is murine, chimeric, human or humanized.

It is understood that the direct fusion of one domain of the chimeric Tim receptor described herein to another does not preclude the presence of intervening junctional amino acids. Junctional amino acids can be natural or non-natural (eg, resulting from the construct design of the chimeric protein). For example, junctional amino acids can arise from restriction enzyme sites used for joining one domain to another or cloning a polynucleotide encoding a chimeric Tim receptor into a vector.

Exemplary Chimeric Tim Receptors The component parts of the chimeric Tim receptors disclosed herein can be selected and arranged in various combinations to provide desired specificity and effector phenotypes for host cells.

Exemplary chimeric Tim receptors of the present disclosure are set forth in Table 4.

Table 4.

Additional exemplary chimeric Tim receptors are set forth in Table 5.

Table 5.

In some embodiments, the chimeric Tim receptor of the present disclosure comprises the constructs of Table 4. In some embodiments, the chimeric Tim receptor of the present disclosure comprises the constructs of Table 5.

In some embodiments, the Construct 1 or Construct 1' chimeric Tim receptor comprises amino acids 21-456 of SEQ ID NO:49. In some embodiments, the Construct 1 or Construct 1' chimeric Tim Receptor comprises the amino acid sequence of SEQ ID NO:49.

In some embodiments, the Construct 2 or Construct 2' chimeric Tim receptor comprises amino acids 21-471 of SEQ ID NO:50. In some embodiments, the Construct 2 or Construct 2' chimeric Tim receptor comprises the amino acid sequence of SEQ ID NO:50.

In some embodiments, the Construct 3 or Construct 3' chimeric Tim receptor comprises amino acids 21-363 of SEQ ID NO:51. In some embodiments, the Construct 3 or Construct 3' chimeric Tim receptor comprises the amino acid sequence of SEQ ID NO:51.

In some embodiments, the Construct 4 or Construct 4' chimeric Tim receptor comprises amino acids 21-590 of SEQ ID NO:52. In some embodiments, the Construct 4 or Construct 4' chimeric Tim receptor comprises the amino acid sequence of SEQ ID NO:52.

In some embodiments, the Construct 5 or Construct 5' chimeric Tim Receptor comprises amino acids 21-596 of SEQ ID NO:53. In some embodiments, the Construct 5 or Construct 5' chimeric Tim Receptor comprises the amino acid sequence of SEQ ID NO:53.

In some embodiments, the Construct 6 or Construct 6' chimeric Tim receptor comprises amino acids 21-619 of SEQ ID NO:54. In some embodiments, the Construct 6 or Construct 6' chimeric Tim Receptor comprises the amino acid sequence of SEQ ID NO:54.

In some embodiments, the Construct 7 or Construct 7' chimeric Tim receptor comprises amino acids 21-625 of SEQ ID NO:55. In some embodiments, the Construct 7 or Construct 7' chimeric Tim Receptor comprises the amino acid sequence of SEQ ID NO:55.

In some embodiments, the Construct 8 or Construct 8' chimeric Tim receptor comprises amino acids 21-621 of SEQ ID NO:56. In some embodiments, the Construct 8 or Construct 8' chimeric Tim Receptor comprises the amino acid sequence of SEQ ID NO:56.

In some embodiments, the Construct 9 or Construct 9' chimeric Tim receptor comprises amino acids 21-415 of SEQ ID NO:57. In some embodiments, the Construct 9 or Construct 9' chimeric Tim receptor comprises the amino acid sequence of SEQ ID NO:57.

In some embodiments, the chimeric Tim receptor of Construct 10 or Construct 10' comprises amino acids 21-409 of SEQ ID NO:58. In some embodiments, the chimeric Tim receptor of Construct 10 or Construct 10' comprises the amino acid sequence of SEQ ID NO:58.

Additional exemplary chimeric Tim receptors of the present disclosure are set forth in Table 6.

Table 6.

Additional exemplary chimeric Tim receptors are set forth in Table 7.

Table 7.

In some embodiments, the chimeric Tim receptor of this disclosure comprises the constructs of Table 6. In some embodiments, the chimeric Tim receptor of this disclosure comprises the constructs of Table 7.

In some embodiments, the chimeric Tim receptor of Construct 11 or Construct 11' comprises amino acids 25-490 of SEQ ID NO:59. In some embodiments, the chimeric Tim receptor of Construct 11 or Construct 11' comprises the amino acid sequence of SEQ ID NO:59.

In some embodiments, the chimeric Tim receptor of Construct 12 or Construct 12' comprises amino acids 25-495 of SEQ ID NO:60. In some embodiments, the chimeric Tim receptor of Construct 12 or Construct 12' comprises the amino acid sequence of SEQ ID NO:60.

In some embodiments, the Construct 13 or Construct 13' chimeric Tim Receptor comprises amino acids 25-382 of SEQ ID NO:61. In some embodiments, the Construct 13 or Construct 13' chimeric Tim Receptor comprises the amino acid sequence of SEQ ID NO:61.

In some embodiments, the chimeric Tim receptor of construct 13A [TIM4 binding domain-CD28 TM-CD28costim-CD3ζ (amino acids 1-24 signal peptide)] comprises amino acids 25-383 of SEQ ID NO:71. In some embodiments, the chimeric Tim receptor of Construct 13A comprises the amino acid sequence of SEQ ID NO:71.

In some embodiments, the chimeric Tim receptor of Construct 14 or Construct 14' comprises amino acids 25-609 of SEQ ID NO:62. In some embodiments, the chimeric Tim receptor of Construct 14 or Construct 14' comprises the amino acid sequence of SEQ ID NO:62.

In some embodiments, the chimeric Tim receptor of Construct 15 or Construct 15' comprises amino acids 25-615 of SEQ ID NO:63. In some embodiments, the chimeric Tim receptor of Construct 15 or Construct 15' comprises the amino acid sequence of SEQ ID NO:63.

In some embodiments, the chimeric Tim receptor of Construct 16 or Construct 16' comprises amino acids 25-638 of SEQ ID NO:64. In some embodiments, the chimeric Tim receptor of Construct 16 or Construct 16' comprises the amino acid sequence of SEQ ID NO:64.

In some embodiments, the chimeric Tim receptor of Construct 17 or Construct 17' comprises amino acids 25-644 of SEQ ID NO:65. In some embodiments, the chimeric Tim receptor of Construct 17 or Construct 17' comprises the amino acid sequence of SEQ ID NO:65.

In some embodiments, the chimeric Tim receptor of Construct 18 or Construct 18' comprises amino acids 25-640 of SEQ ID NO:66. In some embodiments, the chimeric Tim receptor of Construct 18 or Construct 18' comprises the amino acid sequence of SEQ ID NO:66.

Additional exemplary chimeric Tim receptors of the present disclosure are set forth in Table 8.

Table 8.

Additional exemplary chimeric Tim receptors are set forth in Table 9.

Table 9.

In some embodiments, the chimeric Tim receptor of this disclosure comprises the constructs of Table 8. In some embodiments, the chimeric Tim receptor of this disclosure comprises the constructs of Table 9.

In some embodiments, the chimeric Tim receptor of Construct 19 or Construct 19' comprises amino acids 25-628 of SEQ ID NO:67. In some embodiments, the chimeric Tim receptor of Construct 19 or Construct 19' comprises the amino acid sequence of SEQ ID NO:67.

In some embodiments, the chimeric Tim receptor of Construct 20 or Construct 20' comprises amino acids 25-416 of SEQ ID NO:68. In some embodiments, the chimeric Tim receptor of Construct 20 or Construct 20' comprises the amino acid sequence of SEQ ID NO:68.

In some embodiments, the chimeric Tim receptor of Construct 21 or Construct 21' comprises amino acids 25-422 of SEQ ID NO:69. In some embodiments, the chimeric Tim receptor of Construct 21 or Construct 21' comprises the amino acid sequence of SEQ ID NO:69.

Additional exemplary chimeric Tim receptors are set forth in Table 10. In some embodiments, the chimeric Tim receptor of the present disclosure comprises the constructs of Table 10. In some embodiments, chimeric Tim receptors of the present disclosure do not include chimeric Tim receptors having the amino acid sequence of SEQ ID NOS:227, 238, 249, 72, or 73. In some embodiments, chimeric Tim receptors of the present disclosure do not include chimeric Tim receptors having a combination of components set forth for constructs of SEQ ID NOS:227, 238, 249, 72, or 73.

In some embodiments, the chimeric Tim4 receptor comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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).

Table 10.

Polynucleotides, Vectors and Host Cells In certain aspects, the disclosure provides nucleic acid molecules encoding any one or more of the chimeric Tim receptors described herein. Nucleic acid may refer to single- or double-stranded DNA, cDNA or RNA, and may include positive and negative strands of complementary nucleic acids, including antisense DNA, cDNA and RNA. Nucleic acids can be naturally occurring or synthetic forms of DNA or RNA. Nucleic acid sequences encoding desired chimeric Tim receptors can be found, for example, 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), using recombinant methods known in the art using standard techniques, for example by screening libraries from cells expressing the desired sequence or a portion thereof. , by deriving the sequence from a vector known to contain the desired sequence or portion thereof, or by isolating the sequence or portion thereof directly from cells or tissues containing the desired sequence or portion thereof. can be obtained or produced by Alternatively, the sequences of interest can be produced synthetically rather than cloned.

Polynucleotides encoding the chimeric Tim receptor compositions provided herein can be in any animal, including humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs or It can be derived from combinations thereof. In certain embodiments, a polynucleotide encoding a chimeric Tim receptor is a polynucleotide from the same animal species as the host cell into which it is inserted.

A polynucleotide encoding a chimeric Tim receptor of the disclosure can be operably linked to an expression control sequence. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; Kozak consensus sequences); sequences that improve protein stability; and optionally sequences that improve protein secretion.

In certain embodiments, the polynucleotide encoding the chimeric Tim receptor has a sequence encoding a signal peptide (also called leader peptide or signal sequence) at the 5' end for targeting the precursor protein to the secretory pathway. including. A signal peptide may be cleaved from the N-terminus of the extracellular domain during cellular processing and localization of the chimeric Tim receptor to the host cell membrane. A polypeptide with the signal peptide sequence truncated or removed may also be referred to as a mature polypeptide. Examples of signal peptides that can be used in the chimeric Tim receptors of the present disclosure include, for example, GM-CSF (amino acid sequence of SEQ ID NO: 10), Tim1 (amino acid sequence of SEQ ID NO: 40) or Tim4 (amino acid sequence of SEQ ID NO: 11 or 25). A signal peptide from an endogenous secretory protein is included, including the sequence ). In certain embodiments, the polynucleotide sequence encodes or comprises a mature chimeric Tim Receptor polypeptide. For 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. be.

In certain embodiments, chimeric Tim receptor encoding polynucleotides of the disclosure are codon-optimized for efficient expression in target host cells containing the polynucleotides (e.g., Scholten et al., Clin Immunol. 119:135-145 (2006)). As used herein, "codon-optimized" polynucleotides include heterologous polynucleotides that have altered codons by silent mutations that correspond to the abundance of the tRNA in the intended host cell.

A single polynucleotide molecule can encode one, two, or more chimeric Tim receptors according to any of the embodiments disclosed herein. Polynucleotides encoding two or more transgenes may contain sequences (eg, IRES, viral 2A peptide) interposed between each gene for multicistronic expression.

A polynucleotide encoding at least two transgenes provided in this disclosure (eg, a chimeric Tim receptor and a CAR) can be used to construct a tandem expression cassette. A tandem expression cassette comprises at least two transgenes under the control of or operably linked to the same set of regulatory sequences for tandem expression or co-expression of at least two transgenes. Refers to the component of the vector nucleic acid that contains it. Regulatory sequences that may be used in the tandem expression cassettes of the present disclosure include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; Sequences that improve translation efficiency (ie, Kozak consensus sequences); sequences that improve protein stability; sequences that improve protein secretion; or any combination thereof.

In one aspect, the disclosure provides a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim receptor of the disclosure and a polynucleotide encoding a cellular immunotherapeutic agent (eg, CAR, TCR, etc.).

In certain embodiments, tandem expression cassettes can be constructed to optimize spatial and temporal control. For example, a tandem expression cassette can contain promoter elements that optimize spatial and temporal control. In some embodiments, the tandem expression cassettes are tissue-specific, allowing specific induction of the tandem expression cassettes into organs, cell types (e.g., immune cells) or pathological microenvironments, e.g., tumors or infected tissues. contains a specific promoter or enhancer. An "enhancer" is an additional promoter element that can function either cooperatively or independently to activate transcription. In certain embodiments, a tandem expression cassette comprises a constitutive promoter. An exemplary constitutive promoter for use in the tandem expression cassettes of this disclosure is the EF-1α promoter. In certain embodiments, a tandem expression cassette comprises an inducible promoter. In certain embodiments, a tandem expression cassette includes a tissue-specific promoter.

The at least two transgenes contained within a tandem expression cassette can be in any order. For example, a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR is arranged 5′ to 3′: chimeric Tim receptor-CAR or CAR-chimeric Tim receptor. can be

In certain embodiments, a receptor comprising two or more polypeptide chains that associate to form a multimer or complex is combined with two or more polynucleotide molecules within a tandem expression construct. can be coded by Exemplary multimeric receptors contemplated for expression in the tandem expression constructs of this disclosure include multi-chain CAR, TCR, TCR-CAR and TRuC™ constructs. Thus, an exemplary tandem expression cassette embodiment encoding a chimeric Tim Receptor and a TCR is a polynucleotide encoding a chimeric Tim Receptor, a polynucleotide encoding a TCR α chain polypeptide and a polynucleotide encoding a TCR β chain polypeptide. can include

In certain embodiments, the tandem expression cassettes of the present disclosure are positioned between each polynucleotide contained within the tandem expression cassette to allow co-expression of multiple proteins from a single mRNA. may contain an internal ribosome entry site (IRES) or peptide cleavage site, such as a furin cleavage site or a viral 2A peptide. For example, an IRES, a furin cleavage site, or a viral 2A peptide can be placed between the polynucleotides encoding the chimeric Tim receptor and the CAR in the tandem expression cassette. In another example, the IRES, furin cleavage site or viral 2A peptide is between each of the polynucleotide encoding the chimeric Tim receptor, the polynucleotide encoding the TCR α chain polypeptide and the polynucleotide encoding the TCR β chain polypeptide. can be placed in In certain embodiments, the viral 2A peptide is porcine tescho virus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or is a variant of Exemplary T2A peptides comprise the amino acid sequence of any one of SEQ ID NOS: 12, 28, 29 or 30. An exemplary P2A peptide comprises the amino acid sequence of SEQ ID NO: 13 or 31. An exemplary E2A peptide sequence comprises the amino acid sequence of SEQ ID NO:14. An exemplary F2A peptide sequence comprises the amino acid sequence of SEQ ID NO:15.

Certain embodiments of the tandem expression cassettes of the disclosure comprise a polynucleotide encoding a CAR/or TCR specific for a target antigen (e.g., a tumor antigen) and a polynucleotide encoding a chimeric Tim receptor of the disclosure including. Upon binding of target cells expressing target antigens by CAR/or TCR, cells modified to express such tandem expression cassettes induce apoptosis of target cells. Apoptosis induces the exposure of pro-phagocytic markers, such as phosphatidylserine, on target cells, which can then target damaged or apoptotic cells for phagocytosis by chimeric Tim receptors.

Polynucleotides encoding the desired chimeric Tim Receptor are placed in suitable vectors, including viral vectors, non-viral plasmid vectors and non-viral vectors, including lipid-based vectors, for introduction into the desired host cells (e.g., immune cells). DNA vectors, modified mRNA (modRNA), self-amplified mRNA, CELiD and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). A polynucleotide encoding a chimeric Tim receptor of the present disclosure can be cloned into any suitable vector, including expression vectors, replication vectors, probe generation vectors or sequencing vectors. 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 into a single polynucleotide. , which is then inserted into the vector. In other embodiments, the extracellular domain-encoding polynucleotide, the transmembrane domain-encoding polynucleotide, and the intracellular signaling domain-encoding polynucleotide are such that the expressed amino acid sequence is a functional chimeric Tim receptor. can be separately inserted into vectors so as to produce A vector encoding a chimeric Tim receptor is referred to herein as a "chimeric Tim receptor vector."

In certain embodiments, the vector comprises a polynucleotide encoding one chimeric Tim receptor. In certain embodiments, the vector comprises one 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, each chimeric tim receptor sequence , separated from each other by internal ribosome entry sites (IRES), furin cleavage sites or viral 2A peptides to allow co-expression of multiple genes from a single open reading frame (e.g., multicistronic vectors) . In certain embodiments, the viral 2A peptide is porcine tescho virus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or is a variant of Exemplary T2A peptides comprise the amino acid sequence of SEQ ID NOs: 12, 28, 29 or 30. An exemplary P2A peptide comprises the amino acid sequence of SEQ ID NO: 13 or 31. An exemplary E2A peptide sequence comprises the amino acid sequence of SEQ ID NO:14. An exemplary F2A peptide sequence comprises the amino acid sequence of SEQ ID NO:15.

In certain embodiments, the vector comprises two or more polynucleotides, each polynucleotide encoding a chimeric Tim receptor. Two or more polynucleotides encoding chimeric Tim Receptors can be sequentially cloned into a vector at different cloning sites, each chimeric Tim Receptor being expressed under the control of a different promoter. . In certain embodiments, vectors are utilized that allow long-term integration of the transgene and its spread to daughter cells. Examples include viral vectors such as adenovirus, adeno-associated virus, vaccinia virus, herpes virus, cytomegalovirus, poxvirus or retrovirus, such as lentiviral vectors. Lentiviral-derived vectors are used to achieve long-term gene transfer and may have additional advantages over vectors, including the ability to transduce non-proliferating cells, such as hepatocytes, and low immunogenicity. .

A vector encoding a core virus is referred to herein as a "viral vector." There are a number of available viral vectors suitable for use with the compositions of the present disclosure, including those identified for human gene therapy applications (Pfeifer and Verme, Ann. Rev. Genomics Hum. Genet 2:177, 2001). Suitable viral vectors include RNA virus-based vectors such as retrovirus-derived vectors such as Maloney murine leukemia virus (MLV)-derived vectors, and more complex retroviral-derived vectors such as lentiviruses. A derived vector is included. HIV-1 derived vectors belong to this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV and maedi visnavirus (ovine lentivirus). Methods for using retroviral and lentiviral viral vectors and packaging cells for transducing viral particles containing chimeric receptor transgenes into mammalian host cells are known in the art. Yes, e.g. 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. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

In certain embodiments, viral vectors are used to introduce a non-endogenous polynucleotide encoding a chimeric Tim receptor into host cells. Viral vectors can be retroviral or lentiviral vectors. Viral vectors may also contain nucleic acid sequences that encode markers for transduction. Transduction markers for viral vectors are known in the art, selectable markers that can confer drug resistance, or detectable markers, such as fluorescent markers, or cells that can be detected by methods such as flow cytometry. Contains surface proteins. In certain embodiments, the viral vector comprises a genetic marker for transduction comprising a fluorescent protein (e.g., green, yellow), the extracellular domain of human CD2 or truncated human EGFR (EGFRt or tEGFR; Wang et al., Blood 118:1255, 2011). An exemplary tEGFR comprises the amino acid sequence of SEQ ID NO:16. When the viral vector genome contains multiple genes that are expressed in the host cell as separate proteins from a single transcript, viral vectors also have two (or more) genes that allow multicistronic expression. Additional sequences between genes may also be included. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides (eg T2A, P2A, E2A, F2A), or any combination thereof. .

Also, for polynucleotide delivery, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; amplicon vectors, herpes simplex virus (HSV: Other viral vectors can be used, including DNA viral vectors, including vectors derived from Herpes simplex virus (Krisky et al., Gene Ther. 5: 1517, 1998).

Other viral vectors recently developed for gene therapy can also be used 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 (eg sleeping beauty or other transposon vectors).

In certain embodiments, chimeric Tim receptor vectors can be constructed to optimize spatial and temporal control. For example, chimeric Tim acceptor vectors can contain promoter elements that optimize spatial and temporal control. In some embodiments, the chimeric Tim Receptor vector allows specific targeting of the chimeric Tim Receptor to an organ, cell type (e.g., immune cell) or pathological microenvironment, such as a tumor or infected tissue. contains tissue-specific promoters or enhancers that An "enhancer" is an additional promoter element that can function either cooperatively or independently to activate transcription. In certain embodiments, the chimeric Tim acceptor vector contains a constitutive promoter. In certain embodiments, the chimeric Tim acceptor vector contains an inducible promoter. In certain embodiments, the chimeric Tim receptor vector contains a tissue-specific promoter.

In certain embodiments, chimeric Tim receptor vectors may include genes encoding homing receptors, such as CCR4 or CXCR4, to improve homing and anti-tumor activity in vivo.

If temporal control is desired, the chimeric Tim receptor vector can contain elements that allow for inducible depletion of transduced cells. For example, such vectors may contain an inducible suicide gene. A suicide gene can be an apoptotic gene or a gene that confers sensitivity to an agent (eg, drug). Exemplary suicide genes include chemo-inducible caspase 9 (iCASP9) (U.S. Patent Publication No. 2013/0071414), chemo-inducible Fas, or herpes simplex virus thymidine kinase (HSV-TK) that confers sensitivity to ganciclovir. is included. In further embodiments, chimeric Tim receptor vectors can be designed to express known cell surface antigens that allow depletion of transduced cells upon injection of the relevant antibody. Examples of cell surface antigens and their associated antibodies that can be used for depletion of transduced cells include CD20 and rituximab, RQR8 (mixed CD34 and CD20 epitopes allowing CD34 selection and anti-CD20 depletion) and rituximab. , as well as EGFR and cetuximab.

Inducible vector systems, such as the tetracycline (Tet)-On vector system, which activates transgene expression with doxycycline (Heinz et al., Hum. Gene Ther. 2011, 22:166-76), are also known to be inducible. can be used for chimeric Tim receptor expression. Inducible chimeric tim receptor expression is also mediated via retention using a streptavidin-based selective hook (RUSH) system anchored to the endoplasmic reticulum membrane through hooks and streptavidin-binding proteins introduced into the chimeric tim receptor structure. Here, addition of biotin to the system results in the release of chimeric Tim receptors from the endoplasmic reticulum (Agaugue et al., 2015, Mol. Ther. 23(Suppl. 1):S88). .

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

In certain embodiments, B cells are genetically modified to express one or more chimeric Tim receptors. B cells are beneficial as host cells including trafficking to sites of inflammation, ability to internalize and present antigens, ability to co-stimulate T cells, hyperproliferation and self-renewal (sustaining for life). Possibly has certain properties. In certain embodiments, chimeric Tim receptor-modified B cells digest phagocytosed target cells or phagocytosed target particles into smaller peptides and present them to T cells via MHC molecules. can be done. Antigen presentation by chimeric Tim receptor-modified B cells may contribute to antigenic extension of the immune response to non-targeted antigens. B cells are progenitor or progenitor cells committed to the B cell lineage (e.g., pre-pro B cells, pro B cells and pre B cells); immature and inactivated B cells; or mature and functional or activated B cells. Contains cells. In certain embodiments, the B cells are naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic cells, plasmablasts, memory B cells, or can be any combination of Memory B cells can be distinguished from naive B cells by the expression of CD27, which is absent on naive B cells. In certain embodiments, B cells can be primary cells or cell lines from humans, mice, rats, or other mammals. B cell lineages are well known in the art. When obtained from a mammal, B cells can be obtained from many sources including blood, bone marrow, spleen, lymph nodes or other tissues or fluids. B-cell compositions can be enriched or purified.

In certain embodiments, T cells are genetically modified to express one or more chimeric Tim receptors. Exemplary T cells include CD4 ⁺ helper, CD8 ⁺ effector (cytotoxic), naive (CD45 RA +, CCR7 +, CD62L +, CD27 +, CD45RO-), central memory (CD45RO ⁺ , CD62L ⁺ , CD8 ⁺ ), effector Memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-), T memory stem, regulatory, mucosa-associated invariant (MAIT), γδ(gd), tissue resident T cells, natural killer T cells or any thereof includes a combination of In certain embodiments, T cells can be primary cells or cell lines from humans, mice, rats, or other mammals. When obtained from a mammal, T cells can be obtained from many sources including blood, bone marrow, lymph nodes, thymus or other tissues or fluids. T cell compositions can be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In certain embodiments, the T cells lack endogenous expression of the TCRα gene, the TCRβ gene, or both. Such T cells may naturally lack endogenous expression of the TCRα and β chains, and may be induced to block expression (e.g., T cells from transgenic mice that do not express TCRα and β chains, or TCRα and β chains), or modified to knock out genes for the TCR α chain, the TCR β chain, or both.

In certain embodiments, a host cell that expresses a chimeric Tim protein of the present disclosure on its cell surface is not a T cell or cell of the T cell lineage, but a progenitor that has been engineered to express cell surface anti-CD3. A cell that is a cell, a stem cell or a cell.

In certain embodiments, gene editing methods are used to modify the host cell genome to contain a polynucleotide encoding a chimeric Tim receptor of the present disclosure. Gene editing, or genome editing, is a genetic engineering method in which DNA is inserted, replaced, or removed from the genome of a host cell using genetic engineering endonucleases. Nucleases create specific double-strand breaks at targeted loci in the genome. The host cell's endogenous DNA repair pathways then repair the induced break(s) by, for example, non-homologous end joining (NHEJ) and homologous recombination. Exemplary endonucleases useful for gene editing include zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, clustered regularly interspaced short p alindromic repeats: cluster Regularly arranged short palindromic repeats)/Cas nuclease systems (eg CRISPR-Cas9), meganucleases or combinations thereof. Methods of using gene-editing endonucleases to disrupt or knock out genes or gene expression in immune cells, including B cells and T cells, are known in the art, e.g. WO2014/059173; Cheong et al., Nat. Comm. 2016 7:10934; Chu et al., Proc. Natl. Acad. The method from each is incorporated herein by reference in its entirety.

In certain embodiments, expression of the host cell's endogenous gene 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 genes or HLA class II genes), 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. Endogenous gene expression can be inhibited, knocked down or knocked out at the gene level, transcription level, translation level or a combination thereof. Methods of inhibiting, knocking down or knocking out endogenous genes include, for example, RNA interfering agents (eg siRNA, shRNA, miRNA, etc.) or engineered endonucleases [eg CRISPR/Cas nuclease systems, zinc finger nucleases (ZFNs), transcriptional activators. beta-like effector nucleases (TALENs), meganucleases] or any combination thereof. In certain embodiments, an endogenous B cell gene (e.g., IGH, IGκ or IGλ) is isolated from a polynucleotide encoding a chimeric Tim receptor of the present disclosure, such as through an engineered endonuclease. The gene is knocked out by insertion into the locus. In certain embodiments, an endogenous T cell gene (e.g., a TCR gene, HLA gene or immune checkpoint molecule gene) is transfected, such as via an engineered endonuclease, to a poly(polyethylene) encoding chimeric Tim receptor of the present disclosure. It is knocked out by insertion of a nucleotide into the endogenous T cell gene locus.

In certain embodiments, host cells can be genetically modified to express one chimeric Tim receptor. In other embodiments, the host cell may express at least two or more different chimeric Tim receptors.

The disclosure also provides compositions comprising populations of chimeric Tim receptor modified host cells. In certain embodiments, the population of chimeric Tim receptor-modified host cells is a B cell population, a T cell population, a natural killer cell population, a lymphocyte progenitor cell population, an antigen presenting cell population, a dendritic cell population, a Langerhans It can be a cell population, a myeloid progenitor cell population, a mature myeloid cell population or any combination thereof. Furthermore, a population of chimeric Tim receptor-modified host cells of a particular cell type may consist of one or more subtypes. For example, the B cell population may be 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 can consist of any combination of In another example, the T cell population is 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 ⁺ ) 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.

In certain embodiments, the host cell population consists of cells that each express the same chimeric Tim receptor. In other embodiments, 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.

In certain embodiments, when preparing chimeric Tim receptor-modified host cells, e.g., B cells or T cells, one or more growth factor cytokines that promote proliferation of the host cells, e.g., B cells or T cells, are , can be added to the cell culture. Cytokines can be human or non-human cytokines. Exemplary growth factor cytokines that can be used to promote T cell proliferation include IL-2, IL-15 and others. Exemplary growth factor cytokines that can be used to promote B cell proliferation include CD40L, IL-2, IL-4, IL-15, IL-21, BAFF and others.

Prior to genetic modification of host cells with chimeric Tim receptor vectors, a source of host cells (e.g., T cells, B cells, natural killer cells, etc.) is obtained from a subject (e.g., whole blood, peripheral blood mononuclear cells, Bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural fluid, spleen tissue) from which host cells are isolated using methods known in the art. Particular host cell subsets can be collected according to known techniques such as affinity binding to antibodies, flow cytometry and/or immunomagnetic selection, and enriched or depleted by known techniques. After the enrichment and/or depletion steps and introduction of the chimeric Tim receptor, in vitro expansion of the desired modified host cells may be performed according to known techniques or variations thereof that will be apparent to those skilled in the art.

The chimeric Tim receptors of the present disclosure confer cytotoxic activity against host cells expressing phosphatidylserine-specific chimeric Tim receptors. Thus, upon binding to phosphatidylserine exposed on the surface of target cells, host cells expressing the chimeric Tim receptor can induce apoptosis of target cells. In certain embodiments, a host cell expressing a chimeric Tim Receptor is able to target cells via release of granzyme, perforin, granulysin, or any combination thereof; Fas ligand-Fas interaction; or both. induces apoptosis. 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, the host cell does not naturally display a phagocytic phenotype prior to modification with the chimeric Tim receptor.

A chimeric Tim receptor of the present disclosure may also be capable of co-stimulating T cells via at least one signaling pathway. In certain embodiments, the chimeric Tim Receptor co-activates T cells via at least two different signaling pathways (e.g., via selected co-stimulatory signaling domains in the chimeric Tim Receptor). Provides a stimulatory signal. For example, a chimeric Tim receptor containing a CD28 co-stimulatory signaling domain may be able to provide co-stimulatory signals through CD28 and Tim1. In certain embodiments, host immune cells expressing chimeric Tim receptors exhibit reduced or inhibited immune cell exhaustion. In certain embodiments, 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) IFN-γ, IL-2, TNF - decreased production of 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) IFN-γ, TNF-α, or both or both (a) and (b).

In certain embodiments, host cells expressing chimeric Tim receptors exhibit enhanced effector responses (eg, tumor-specific). In certain embodiments, the effector response is enhanced T cell proliferation, cytokine production (eg, IFN-γ, IL-2, TNF-α), cytotoxic activity, persistence, or any combination thereof. be.

In certain embodiments, host cells expressing a 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". Elimination of apoptotic cells by phagocytic cells generally promotes inflammation through decreased secretion of the anti-inflammatory cytokines IL-10 and TGF-β, and the pro-inflammatory cytokines TNF-α, IL-1β, and IL-12. Reduce or prevent response. Phosphatidylserine may therefore 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 (eg, IFN-γ, IL-2, TNF-α), thereby , reduce the immunosuppressive response to phosphatidylserine.

Expression of chimeric Tim receptors on host cells host cell (e.g., T cell) activity, including determination of T cell binding, activation or induction, and also determination of T cell responses that are antigen specific. may be functionally characterized according to any of a number of art-accepted methods for assaying. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, CTL activity (e.g., by detecting ⁵¹ Cr or Europium release from preloaded target cells), T cell phenotypic marker expression and determination of other measures of T cell function. Techniques 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); See Reed, Science 281:1309 (1998) and 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 (eg, intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from antigen-specific induction or stimulation of an immune response involves isolating lymphocytes, such as circulating lymphocytes in a sample of cells from peripheral blood cells or lymph nodes, stimulating the cells with an antigen, It can be determined by measuring cytokine production, cell proliferation and/or cell viability, such as by incorporation of tritiated thymidine or non-radioactive assays such as the MTT assay.

In certain embodiments, the chimeric Tim receptor-modified host cell has a phagocytic index of about 20 to about 1,500 for target cells. A "phagocytic index" is the number of ingested target cells or A measure of phagocytic activity of transduced host cells determined by counting the number of particles. The phagocytosis index is [total number of target cells phagocytosed/total number of chimeric Tim receptor-modified cells counted (e.g., frequency of phagocytosis)] x [mean target cell or particle staining per chimeric Tim receptor ⁺ host cell. area x 100 (e.g. hybrid capture)] or [total number of particles phagocytosed/total number of chimeric Tim receptor modified host cells counted] x [number of chimeric Tim receptor modified host cells containing phagocytosed particles/counted total number of chimeric Tim receptor ⁺ cells]×100. In certain embodiments, the chimeric Tim receptor-modified cells are about 30 to about 1,500; about 40 to about 1,500; about 50 to about 1,500; about 75 to about 1,500; about 1,500; about 200 to about 1,500; about 300 to about 1,500; about 400 to about 1,500; about 500 to about 1,500; about 40 to about 1,400; about 50 to about 1,400; about 100 to about 1,400; about 200 to about 1,400; about 300 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300; about 500 to about 1,300; about 20 to about 1,200; about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200; about 300 to about 1,200; about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 300 to about 1,100; about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about 30 to about 1,000; from about 50 to about 1,000; from about 100 to about 1,000; from about 200 to about 1,000; from about 300 to about 1,000; from about 400 to about 1,000; about 20 to about 750; about 30 to about 750; about 40 to about 750; about 50 to about 750; about 100 to about 750; about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; has an exponent. In further embodiments, the incubation time is from about 2 hours to about 4 hours, about 2 hours, about 3 hours, or about 4 hours. In still further embodiments, the chimeric Tim receptor-modified cells exhibit a statistically significantly higher phagocytic index than cells transduced with a truncated EGFR control. Phagocytosis index is known in the art and further described in the Examples and PCT Application No. PCT/US2017/053553, which is incorporated herein by reference in its entirety, including quantification by flow cytometry or fluorescence microscopy. can be calculated using the method

The host cell can be from an animal, such as a human, primate, bovine, equine, ovine, canine, feline, murine, rat, rabbit, guinea pig, porcine, or combinations thereof. In preferred embodiments, the animal is human. Host cells may be obtained from a healthy subject or a subject with a disease associated with antigen expression or overexpression.

PARP Inhibitors Any suitable PARP inhibitor can be used in the compositions and methods of this disclosure. Exemplary PARP inhibitors include talazoparib, niraparib, rucaparib, olaparib (AZ2281, KU59436), veliparib (ABT888), CEP9722, E7016, AG014699, MK4827, BMN-673, and pamiparib (BGB-290). included.

In certain embodiments, the PARP inhibitor comprises niraparib. In certain embodiments, the PARP inhibitor comprises talazoparib. In certain embodiments, the PARP inhibitor comprises rucaparib. In certain embodiments, the PARP inhibitor comprises Olaparib. In certain embodiments, the PARP inhibitor comprises veliparib. In certain embodiments, the PARP inhibitor comprises CEP9722. In certain embodiments, the PARP inhibitor comprises E7016. In certain embodiments, the PARP inhibitor comprises AG014699. In certain embodiments, the PARP inhibitor comprises MK4827. In certain embodiments, the PARP inhibitor comprises BMN-673. In certain embodiments, the PARP inhibitor comprises pamiparib.

Methods of Treatment A chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a chimeric Tim receptor in combination with a PARP inhibitor according to any of the embodiments provided herein can be used in methods of treating a subject suffering from a disease, disorder or undesirable condition. These method embodiments comprise (i) a therapeutically effective amount of one or more chimeric tim receptors described in this description, polynucleotides encoding one or more chimeric tim receptors, one or more or a host cell population genetically modified to express one or more chimeric Tim Receptors; and (ii) a therapeutically effective amount of administering to the subject a pharmaceutical composition comprising a PARP inhibitor.

The chimeric Tim receptor compositions described herein can be administered prior to PARP inhibitor therapy (eg, from 1 day to 30 days or more prior to PARP inhibitor therapy) and concurrently (on the same day) with PARP inhibitor therapy. , or after PARP inhibitor therapy (eg, 1 to 30 days or more after PARP inhibitor therapy). In certain embodiments, chimeric Tim receptor-modified cells are administered after administration of one or more additional therapies. In a further embodiment, the chimeric Tim receptor-modified cell is administered a PARP inhibitor at 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 later. In still further embodiments, the chimeric Tim receptor-modified cell is administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of PARP inhibitor therapy. When the PARP inhibitor therapy comprises multiple doses, the chimeric Tim receptor-modified cells are administered after the first dose of one or more additional therapies, after the final dose of one or more additional therapies, or after one or more Multiple doses of additional therapy may be administered in between.

Diseases that can be treated with cells expressing the chimeric Tim receptor described in this disclosure include cancer. Adoptive immunotherapy and gene therapy have been used to treat 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).

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 adenocarcinoma of the breast, prostate and colon; all forms of bronchogenic lung cancer; melanoma; hepatoma; neuroblastoma; papilloma; apdoma; Brown-Pierce carcinoma, ductal carcinoma, Ehrlich carcinoma, Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung carcinoma, oat cell carcinoma, papillary carcinoma, sclerotic carcinoma, bronchiolar carcinoma, bronchogenic lung carcinoma, squamous cell carcinoma 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; non-Hodgkin's lymphoma; plasmacytoma; multiple myeloma; chronic myelogenous leukemia (CML); acute myelogenous leukemia (AML); Chondrosarcoma; Fibroma; Fibrosarcoma; Giant cell tumor; Histiocytoma; Lipoma; Liposarcoma; Mesothelioma; mesonephroma; sarcoma; amelocytoma; cementoma; odontoma; teratoma; thymoma; In addition, also the following types of cancer: adenoma; cholangiomas; cholesteatoma; cyclindroma; cystadenoma; cystadenoma; Leydig cell tumor; papilloma; Sertoli tumor; thecacytoma; leimyoma; glioma; medulloblastoma; meningioma; schwannoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; It is contemplated that tumors are suitable for treatment using the receptors, modified host cells and compositions described herein. Also, types of cancers that may be treated include angiokeratoma; angiolymphoid hyperplasia with eosinophilia; sclerosing hemangiomas; angiomatosis; glomus hemangioma; Perithelium; angiosarcoma; lymphangioma; lymphangiosarcoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasm; neurofibromatosis; cervical dysplasia;

In some embodiments, the compositions and methods of this disclosure are useful for treating solid tumors. In some embodiments, the solid tumor cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, or prostate cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the ovarian cancer is advanced ovarian cancer. In some embodiments, prostate cancer. You have advanced prostate cancer. In some embodiments, the solid tumor cancer is melanoma. In some embodiments, the solid tumor cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer.

In additional embodiments, the cancer is a breast cancer (BRCA) mutated cancer. In certain embodiments, the cancer is a BRCA1 mutated cancer, a BRCA2 mutated cancer, or both.

A chimeric Tim receptor of the disclosure can be administered to a subject in a cell-bound form (eg, gene therapy of a target cell population). Thus, for example, the chimeric Tim receptors of the present disclosure can be used for T cells, including subsets thereof, natural killer cells, natural killer T cells, B cells, lymphocyte progenitor cells, antigen presenting cells, dendritic cells, Langerhans cells, The myeloid progenitor cells, mature myeloid cells, or any combination thereof may be expressed on the surface and administered to the subject. In certain embodiments, the method of treating a subject comprises administering an effective amount of chimeric Tim receptor-modified cells (ie, recombinant cells expressing one or more chimeric Tim receptors). Chimeric Tim receptor modified cells can be xenogeneic, syngeneic, allogeneic or autologous to the subject.

Chimeric Tim receptor-modified cells and PARP inhibitors can be administered in a manner appropriate to the disease or condition to be treated (or prevented), as determined by those skilled in the medical arts. A suitable dose, suitable duration and frequency of administration of the composition will depend on the patient's condition, size, weight, body surface area, age, sex, type and severity of the disease, the particular therapy to be administered, the particular nature of the active ingredient. It is determined by factors such as form, time and method of administration and other drugs administered at the same time. The disclosure provides pharmaceutical compositions comprising chimeric Tim receptor modified cells and a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients include water, saline, dextrose, glycerol and the like and combinations thereof. Other suitable infusion vehicles can be any isotonic vehicle formulation including saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water or Ringer's lactate.

A therapeutically effective amount of cells in the pharmaceutical composition is at least one cell (eg, one chimeric Tim receptor-modified T cell), or more typically more than 10 ² cells, eg, up to 10 ⁶ , up to 10 ⁷ , up to 10 ⁸ cells, up to 10 ⁹ cells, up to 10 ¹⁰ cells or up to 10 ¹¹ cells or more. In certain embodiments, cells are administered within the range of about 10 ⁶ to about 10 ¹⁰ cells/m ² , preferably within the range of about 10 ⁷ to about 10 ⁹ cells/m ² . be done. The number of cells will depend on the end use for which the composition is intended as well as the cell types included in the composition. For example, compositions comprising cells that have been modified to contain chimeric Tim receptors include cell populations containing from about 5% to about 95% or more of such cells. In certain embodiments, compositions comprising chimeric Tim receptor-modified cells are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the uses provided herein, cells are generally present in volumes of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Desired cell densities are therefore typically higher than 10 ⁴ cells/ml, generally higher than 10 ⁷ cells/ml, generally higher than 10 ⁸ cells/ml or higher than that. Cells can be administered as a single infusion or in multiple infusions over a range of times. Repeated infusions of chimeric Tim receptor-modified cells can be separated by days, weeks, months or even years if there is recurrence of disease or disease activity. Clinically relevant immune cell numbers can be allocated to multiple infusions equal to or greater than 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ or 10 ¹¹ cells cumulatively. Preferred doses for administration of host cells containing the recombinant expression vectors described herein are about 10 ⁷ cells/m ² , about 5×10 ⁷ cells/m ² , about 10 ⁸ cells/m 2 , ^m2 , about ^5x108 cells/ ^m2 , about ¹⁰⁹ cells/ ^m2 , about ^5x109 cells/ ^m2 , about ¹⁰¹⁰ cells/ ^m2 , about ^5x1010 cells/m2 m ² or about 10 ¹¹ cells/m ² .

The chimeric Tim receptor and PARP inhibitors described herein are administered intravenously, intraperitoneally, intranasally, intratumorally, into bone marrow, into lymph nodes, and/or into cerebrospinal fluid can be

In one aspect, a method of the present disclosure comprises introducing into a host cell a nucleic acid molecule encoding at least one chimeric Tim Receptor or a chimeric Tim Receptor vector according to any of the embodiments described herein. and conferring or enhancing the phosphatidylserine-specific cytotoxic activity of the cell comprising expressing at least one chimeric Tim receptor in the host cell, wherein the at least one chimeric Tim receptor is a chimeric Tim receptor enhances the phosphatidylserine-specific cytotoxic activity of the host cell compared to the host cell prior to modification to express the antibody. In certain embodiments, the host cell cytotoxic activity is 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. In some embodiments, host cells are immune cells. In some embodiments, 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, which mediate target cell death and effector cell activity. , including flow cytometric methods (see, eg, Expert Rev. Vaccines, 2010, 9:601-616).

In certain embodiments, the methods of the disclosure comprising conferring or enhancing phosphatidylserine-specific cytotoxic activity of a cell are performed by phosphatidylserine-specific phagocytosis of a host cell expressing at least one chimeric Tim receptor. Further comprising providing or enhancing activity. In certain such embodiments, the host cell does not naturally display a phagocytic phenotype prior to modification with the chimeric Tim receptor. For example, in certain such embodiments, the phagocytic activity of the host cell is at least about 10%, 15%, 20% compared to the host cell prior to modification to express the chimeric Tim receptor vector. , 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 certain embodiments, the host cell does not naturally have phagocytic activity. In some embodiments, host cells are immune cells. In some embodiments, host cells are T cells or NK cells. Methods for measuring host cell phagocytic activity include those described in PCT/US2017/053553, which is incorporated herein by reference in its entirety.

In another aspect, expressing a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a chimeric Tim receptor according to any of the embodiments provided herein A host cell can be used in a method to enhance the effector function of a host cell. In certain embodiments, the improved effector function is increased cytotoxic activity, increased antigen-specific cytokine production (eg, IFN-γ, IL-2, TNF-α, or any combination thereof) , increased anti-apoptotic signaling, increased persistence, increased proliferation, increased proliferation, or any combination thereof. In certain embodiments, the effector function of the host cell is 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. In some embodiments, host cells are immune cells. In certain embodiments, host cells are T cells or NK cells.

In another aspect, host cells modified with chimeric Tim receptors of the present disclosure can 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, the reduced exhaustion in T cells is (a) reduced expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof in T cells; (b) IFN in T cells. - increased production of gamma, IL-2, TNF-α, or any combination thereof; or both (a) and (b). In certain embodiments, the reduced exhaustion in NK cells is (a) reduced expression of PD-1, NKG2A, TIM3, or any combination thereof in NK cells; (b) IFN-γ in NK cells. , TNF-α, or both; or both (a) and (b). In certain embodiments, the expression of the immune checkpoint molecule increases the expression of the chimeric Tim Receptor relative to host immune cells that have not been modified by the nucleic acid molecule encoding the chimeric Tim Receptor or the chimeric Tim Receptor vector. at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80% %, 85%, 90%, 95%, or 100%. In certain embodiments, expression of the cytokine increases host immunity expressing the chimeric Tim Receptor relative to host immune cells that have not been modified by the chimeric Tim Receptor-encoding nucleic acid molecule or chimeric Tim Receptor vector. at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% in cells %, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more.

In another aspect, expressing a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a chimeric Tim receptor according to any of the embodiments provided herein A host cell can be used in a method of reducing an immunosuppressive response to phosphatidylserine in a host cell. In certain embodiments, the immunosuppressive response is the secretion of anti-inflammatory cytokines (eg, IL-10, TGF-β, or both), inflammatory cytokines (eg, TNF-α, IL-1β, and IL-1β). -12), or both. In certain embodiments, the host cell's immunosuppressive response to phosphatidylserine is at least about 5% greater than a host cell that has not been modified by a chimeric Tim Receptor-encoding nucleic acid molecule or chimeric Tim Receptor vector. %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% reduction. In some embodiments, host cells are immune cells. In certain embodiments, host cells are T cells or NK cells.

In yet another aspect, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or expressing a chimeric Tim receptor according to any of the embodiments provided herein Host cells that carry surface-exposed phosphatidylserine can be used in methods to eliminate target cells that retain surface-exposed phosphatidylserine, such as cancer cells that retain surface-displayed phosphatidylserine. In certain embodiments, target cells are damaged cells, stressed cells, apoptotic cells, necrotic cells (eg, tumor cells) that retain surface-exposed phosphatidylserine. In certain embodiments, a host cell expressing a chimeric Tim receptor is a target of injury that retains surface-exposed phosphatidylserine through inducing apoptosis, or both apoptosis and phagocytosis. Clear cells, stressed target cells, apoptotic target cells, or necrotic target cells. Host cells expressing chimeric Tim receptors can be used alone or with other therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, oncolytic viruses, electrical pulse therapy, etc. Can be administered to a subject in combination.

In another aspect, expressing a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a chimeric Tim receptor according to any of the embodiments provided herein Host cells can be used in methods to enhance the efficacy of therapeutic agents that induce cellular stress, injury, necrosis, or apoptosis. Certain therapies, such as chemotherapy, radiotherapy, UV light therapy, electrical pulse therapy, adoptive cell immunotherapy (e.g. CAR-T cells, TCR) and oncolytic virus therapy, treat tumor cells, disease cells and their can induce cell damage or cell death to cells in the surrounding environment. Cells expressing chimeric Tim receptors are administered in combination with a cytotoxic/cytotoxic therapy that binds to phosphatidylserine moieties exposed on the outer leaflet of targeted cells, and stressed cells, injured cells, Diseased, apoptotic and necrotic cells can be cleared.

A chimeric Tim receptor and PARP inhibitor can be administered to a subject in combination with one or more additional therapeutic agents. Examples of therapeutic agents that can be administered in combination with the chimeric Tim compositions described herein include radiotherapy, adoptive cell immunotherapy agents (eg, recombinant TCR, high affinity TCR, CAR, TCR-CAR, scTCR fusion proteins). , dendritic cell vaccine), antibody therapy, immune checkpoint molecule inhibitor therapy, UV light therapy, electrical pulse therapy, high-intensity focused ultrasound therapy, oncolytic virus therapy or pharmaceutical therapy, such as chemotherapeutic agents, treatment therapeutic peptides, hormones, aptamers, antibiotics, antivirals, antifungals, anti-inflammatory agents, small molecule therapies, or any combination thereof. In certain embodiments, the chimeric Tim receptor-modified host cell presents surface phosphatidylserine induced by one or more additional therapeutic agents. It can clear cells, infected cells, dead cells.

In certain embodiments, a vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapeutic agent (eg, chimeric antigen receptor, recombinant TCR, etc.). In certain embodiments, a single polynucleotide encoding a chimeric tim receptor and a cellular immunotherapeutic agent (e.g., CAR) is cloned into a cloning site and expressed from a single promoter to produce a chimeric tim receptor Sequences and cellular immunotherapeutic agents (e.g., CAR) sequences include an internal ribosome entry site (IRES), to allow co-expression of multiple genes from a single open reading frame (e.g., multicistronic vectors) They are separated from each other by furin cleavage sites or viral 2A peptides. In certain embodiments, the viral 2A peptide is porcine tessho virus-1 (P2A), Tosea acignavirus (T2A), equine rhinitis A virus (E2A), foot and mouth disease virus (F2A), or variants thereof. is. Exemplary T2A peptides comprise the amino acid sequence of SEQ ID NOs: 12, 28, 29 or 30. An exemplary P2A peptide comprises the amino acid sequence of SEQ ID NO: 13 or 31. An exemplary E2A peptide sequence comprises the amino acid sequence of SEQ ID NO:14. An exemplary F2A peptide sequence comprises the amino acid sequence of SEQ ID NO:15.

In certain embodiments, a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapeutic agent (e.g., CAR) binding protein are conjugated together into a single polynucleotide, which is then conjugated to a vector. is inserted into In other embodiments, the polynucleotide encoding the CER and the polynucleotide encoding the CAR or TCR binding protein are the same or different such that the expressed amino acid sequence produces a functional CER and CAR/or TCR. It can be inserted separately into the vector at the cloning site. A vector encoding a tandem expression cassette is referred to herein as a "tandem expression vector."

In certain embodiments, a vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapeutic agent (eg, CAR). A polynucleotide encoding a chimeric Tim receptor and a cellular immunotherapeutic agent (e.g., CAR) can be sequentially cloned into a vector at different cloning sites, wherein the chimeric Tim receptor and cellular immunotherapeutic agent (e.g., CAR) are Expressed under the control of different promoters.

In certain embodiments, chimeric Tim receptor engineered host cells can also be engineered to co-express one or more small GTPases. Rho GTPases, a family of small (~21 kDa) signaling G proteins and also a subfamily of the Ras superfamily, regulate actin cytoskeletal organization in various cell types, pseudopodia elongation and phagosome closure during phagocytosis. (see, eg, Castellano et al., 2000, J. Cell Sci. 113:2955-2961). Phagocytosis requires F-actin recruitment beneath tethered cells or particles and F-actin rearrangement to allow membrane elongation leading to cell or particle internalization. Rho GTPases include RhoA, Rac1, Rac2, RhoG and CDC42. Other small GTPases, such as Rap1, are involved in regulating complement-mediated phagocytosis. Co-expression of a small GTPase and a chimeric Tim receptor can facilitate target cell or particle internalization and/or phagosome formation by a host cell. In some embodiments, the GTPase-encoding recombinant nucleic acid molecule is encoded on a separate vector from the chimeric Tim receptor-containing vector. In other embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on the same vector as the chimeric Tim receptor. A GTPase and a chimeric Tim receptor can be expressed under the control of different promoters in the same vector (eg at different multiple cloning sites). Alternatively, a chimeric Tim receptor and GTPase can be expressed under the control of a single promoter in a multicistronic vector. The polynucleotide sequence encoding the chimeric Tim receptor and the polynucleotide sequence encoding the small GTPase can be separated from each other by an IRES or viral 2A peptide in a multicistronic vector. Exemplary 2A peptides include T2A (SEQ ID NO:12), P2A (SEQ ID NO:13), E2A (SEQ ID NO:14), F2A (SEQ ID NO:15). Examples of GTPases that can be co-expressed with the chimeric Tim receptor include Racl, Rac2, Rab5 (also called Rab5a), Rab7, Rapl, RhoA, RhoG, CDC42 or any combination thereof. In certain embodiments, the GTPases are 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 , 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% with the CDC42 amino acid sequence of SEQ ID NO: 22 or any combination thereof , includes or is a sequence that is at least 99%, at least 99.5% or 100% identical.

In certain embodiments, chimeric Tim receptor engineered host cells can also be engineered to co-express a cellular immunotherapeutic agent (eg, CAR, TCR, etc.). In some embodiments, the cellular immunotherapeutic agent comprises a chimeric antigen receptor (CAR). CARs generally include: an extracellular domain that includes a binding domain that binds to a target antigen; an intracellular signaling domain (e.g., including an ITAM-containing intracellular signaling domain and an appropriate intracellular co-stimulatory domain); It is a recombinant receptor containing a transmembrane domain located between and connecting the ectodomain and the intracellular signaling domain.

Suitable binding domains for use in the CARs of the present disclosure include any antigen-binding polypeptide. A binding domain can comprise an antibody or antigen-binding fragment thereof, including, for example, full-length heavy chains, Fab fragments, Fab', F(ab') ₂ , sFv, VH domains, VL domains, dAbs, VHHs, CDRs and scFvs. In certain embodiments, the CAR binding domain is a murine, chimeric, human or humanized CAR binding domain.

In certain embodiments, the CAR binding domain targets a cancer or tumor antigen. Exemplary antigens that a 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, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, WT1, VEGF-alpha, VEGFR1 , IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, EphrinA2, EphrinB2, Lewis formula A antigen, Lewis formula Y antigen, MAGE, Included are MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2 and GD2.

In certain embodiments, the extracellular domains of CARs provided in this disclosure may include extracellular non-signaling spacer or linker domains. If included, such spacer or linker domains may position the binding domain away from the host cell surface to further permit proper cell-cell contact, binding and activation. An extracellular spacer domain is generally located between the extracellular binding domain and the transmembrane domain of the CAR. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (e.g., Guest et al., J. Immunother. 28:203-11, 2005; see PCT Publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (eg, IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. A modified IgG ₄ hinge region is described in PCT Publication No. WO2014/031687, which is incorporated herein by reference in its entirety. In certain embodiments, the extracellular spacer domain comprises a modified _IgG4 hinge region having the amino acid sequence of SEQ ID NO:3.

Other examples of hinge regions that can be used in the CARs described herein include hinges from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which can be wild-type or mutants thereof. area is included. In certain embodiments, the extracellular spacer domain comprises a CD8a hinge region having the amino acid sequence of SEQ ID NO:70. In another specific embodiment, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO:32. In a further embodiment, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from CH1 domain, CH2 domain, CH3 domain or combinations thereof (see e.g. PCT Publication No. WO2014/031687). Please, the spacer is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may comprise the stalk region of a type II C lectin (the extracellular domain located between the type C lectin domain and the transmembrane domain). Type II C lectins include CD23, CD69, CD72, CD94, NKG2A and NKG2D.

The CARs of this disclosure comprise a transmembrane domain that connects and is located between the extracellular domain and the intracellular signaling domain. Transmembrane domains range in length from about 15 amino acids to about 30 amino acids. The transmembrane domain is a hydrophobic alpha helix that traverses the host cell membrane and anchors the CAR in the host cell membrane. A transmembrane domain may be fused directly to a binding domain or, if present, to an extracellular spacer domain. In certain embodiments, the transmembrane domain is derived from an integral membrane protein [eg, receptor, cluster of differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, etc.]. The transmembrane domain may be selected from the same molecule as the extracellular domain or the intracellular signaling domain (eg, CAR includes CD28 co-stimulatory signaling domain and CD28 transmembrane domain). In certain embodiments, the transmembrane domain and extracellular domain are each selected from different molecules. In other embodiments, the transmembrane domain and intracellular signaling domain are each selected from different molecules. In still other embodiments, the transmembrane domain, extracellular domain and intracellular signaling domain are each selected from different molecules.

Exemplary transmembrane domains for use in the CARs of the present 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) , CD270 (HVEM), 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 membranes Contains a penetrating domain. An exemplary CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:7. In certain embodiments, the transmembrane domain comprises a CD8a transmembrane domain having the amino acid sequence of SEQ ID NO:33.

The intracellular signaling domain of the CAR is an intracellular effector domain that transduces a functional signal to the cell and activates the CAR in response to binding of the extracellular domain of the CAR to a target molecule (e.g., cancer antigen). The immune cells that have been engineered to express can activate at least one of the normal effector functions or responses of eg T cells. In some embodiments, the CAR induces T cell function, eg, cytolytic or T helper activity, eg, secretion of cytokines or other factors. An 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 derived from an antigen receptor component (eg, TCR) or co-stimulatory molecule. In some embodiments, full-length intracellular signaling domains of antigen receptors or co-stimulatory molecules are used. In some embodiments, a truncated portion of the intracellular signaling domain of the antigen receptor or co-stimulatory molecule is used, provided that the truncated portion retains sufficient signaling activity. In a further embodiment, the intracellular signaling domain is a full-length or truncated variant of the intracellular signaling domain of the antigen receptor co-stimulatory molecule, provided that the variant retains sufficient signaling activity. (i.e., is a functional variant).

In certain embodiments, the intracellular signaling domain of the CAR comprises an immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling domain. ITAM-containing signaling domains generally contain at least one (1, 2, 3, 4 or more) ITAMs, which are conserved YXXL/IX _6-8 -YXXL/I It refers to the motif that was created. ITAM-containing signaling domains can initiate T-cell activation signaling following antigen binding or ligand engagement. ITAM signaling domains include, for example, the intracellular signaling domains of CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP12, FcRγ and CD66d. Exemplary CD3ζ signaling domains that may be used in the CARs of this disclosure include the amino acid sequences of SEQ ID NOs:27 or 5.

CAR intracellular signaling domains, when activated in conjunction with primary or canonical (e.g., ITAM-driven) activation signals, induce T-cell responses such as T-cell activation, cytokine production, proliferation, differentiation, survival, Costimulatory signaling domains that promote or enhance effector functions or combinations thereof may be included. Costimulatory 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 certain embodiments, the co-stimulatory signaling domain is the OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) or 4-1BB (CD137) signaling domain including. Exemplary CD28 co-stimulatory signaling domains that may be used in the CARs of the present disclosure comprise the amino acid sequence of SEQ ID NO:26 or 4. An exemplary 4-1BB co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:122. In certain embodiments, the CAR comprises one, two or more co-stimulatory signaling domains.

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

In certain embodiments, the chimeric antigen receptor is human, primate, bovine, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, transgenic species thereof, or any thereof. Amino acid sequences from any mammalian species, including combinations of In certain embodiments, the chimeric antigen receptor is murine, chimeric, human or humanized.

In certain embodiments, the CAR is a first generation CAR, second generation CAR or third generation CAR. First generation CARs generally have intracellular signaling domains, including those of CD3ζ, FcγRI or other ITAM-containing activation domains, to provide T cell activation signals. Second generation CARs further comprise costimulatory signaling domains (eg, costimulatory signaling domains from endogenous T cell costimulatory receptors such as CD28, 4-1BB or ICOS). Third generation CARs contain an ITAM-containing activation domain, a first costimulatory signaling domain and a second costimulatory signaling domain.

In some embodiments, one or more of the extracellular domain, binding domain, linker, transmembrane domain, intracellular signaling domain, or co-stimulatory domain comprises a junctional amino acid. A "junction amino acid" or "junction amino acid residue" is defined between two adjacent domains, motifs, regions, modules or fragments of a protein, e.g. between a binding domain and an adjacent linker, a transmembrane domain and an adjacent extracellular or intracellular domains, or at one or both ends of a linker that joins two domains, motifs, regions, modules or fragments (e.g., between a linker and an adjacent binding domain or between a linker and an adjacent hinge). It refers to one or more (eg, about 2-20) amino acid residues. Junction amino acids may result from the construct design of the fusion protein (eg, amino acid residues resulting from the use of restriction enzyme sites or self-cleaving peptide sequences during construction of the polynucleotide encoding the fusion protein). For example, the transmembrane domain of the fusion protein can have one or more junctional amino acids at the amino terminus, carboxy terminus, or both.

Exemplary binding domain, extracellular spacer domain, transmembrane domain, and intracellular signaling domain sequences for use in the CARs of the present disclosure are shown in Table 11.

Table 11.

In certain embodiments, chimeric Tim receptor-modified host cells co-express a recombinant TCR. Recombinant TCR proteins include "traditional" TCRs consisting of heterodimers of α and β chain polypeptides or heterodimers of γ and δ chain polypeptides, e.g. Included are binding fragments and fusion proteins thereof, including TCRs, single domain TCRs, soluble TCR fusion TCR proteins and TCR fusion constructs [TRuC™]. In certain embodiments, the tandem expression cassette is a polynucleotide encoding a recombinant TCR beta chain comprising the TCR beta variable region and the TCR beta constant region and a recombinant TCR comprising the TCR alpha variable region and the TCR alpha constant region and a polynucleotide encoding the alpha chain. In certain embodiments, the recombinant TCR is a high affinity TCR. In one embodiment, the recombinant TCR is a high affinity TCR.

In certain embodiments, the recombinant TCR binding protein is a single chain TCR (scTCR) comprising Vα joined to Vβ by a flexible linker. In some embodiments, the scTCR comprises a Vα-linker-Vβ polypeptide. In other embodiments, the scTCR comprises a Vβ-linker-Vα polypeptide.

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

In certain embodiments, chimeric Tim receptor-modified host cells can also be modified to co-express a T-cell receptor-based chimeric antigen receptor (TCR-CAR). TCR-CARs are heterodimeric fusion proteins that generally comprise a soluble TCR (a polypeptide chain comprising Vα and Cα domains, and a polypeptide chain comprising Vβ and Cβ domains), where the VβCβ polypeptide chain is , linked to transmembrane domains and intracellular signaling components (e.g. ITAM-containing activation domains and optionally co-stimulatory signaling domains) (see e.g. Walseng et al., 2017 Scientific Reports 7:10713) .

In certain embodiments, an engineered host cell that co-expresses a chimeric Tim Receptor and a cellular immunotherapeutic agent (e.g., CAR, TCR, etc.) is a recombinant nucleic acid encoding a chimeric Tim Receptor and a cellular immunotherapeutic agent. A recombinant nucleic acid molecule encoding an agent is contained on a separate vector within an engineered host cell.

In some embodiments, an engineered host cell that co-expresses a chimeric Tim Receptor and a cellular immunotherapeutic agent (e.g., CAR, TCR, etc.) is a recombinant nucleic acid encoding a chimeric Tim Receptor and a cellular immunotherapeutic agent. A recombinant nucleic acid molecule encoding an agent is contained on the same vector as the chimeric Tim receptor in an engineered host cell. A chimeric Tim receptor and a cellular immunotherapeutic agent can be expressed under the control of different promoters in the same vector (eg, in different multiple cloning sites). Alternatively, a chimeric Tim receptor and a cellular immunotherapeutic agent can be expressed under the control of a single promoter in a multicistronic vector (eg, a tandem expression vector). A polynucleotide sequence encoding a chimeric Tim receptor and a polynucleotide sequence encoding a cellular immunotherapeutic agent can be separated by an IRES or viral 2A peptide in a multicistronic vector.

Tandem expression cassettes, tandem expression vectors, and engineered host cells containing same are described in International Application Publication No. WO2019/191339, which is hereby incorporated by reference in its entirety.

Host cells expressing chimeric Tim receptors can be used alone or with other therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiotherapy, chemotherapy, small molecules, oncolytic viruses, electrical pulse therapy, etc. They can be administered to a subject in combination.

In certain embodiments, a chimeric Tim receptor and an adoptive cell immunotherapy agent (eg, CAR, TCR-CAR, TCR, etc. described above) are administered to a subject in the same host cell or different host cells. In certain embodiments, the chimeric Tim receptor and the adoptive cell immunotherapy agent are expressed in the same host cell from the same vector or from separate vectors. In certain embodiments, a chimeric Tim receptor and an adoptive cell immunotherapy agent are expressed in the same host cell from a multicistronic vector. In certain embodiments, the chimeric Tim Receptor is expressed in the same host cell type as the adoptive immunotherapeutic agent (e.g., the chimeric Tim Receptor is expressed in CD4 T cells and the CAR/or TCR is expressed in CD4 T cells). expressed on T cells, or chimeric Tim receptor expressed on CD8 T cells and CAR/or TCR expressed on CD8 T cells). In other embodiments, the chimeric Tim Receptor is expressed in a different host cell type than the adoptive immunotherapeutic agent (e.g., the chimeric Tim Receptor is expressed in CD4 T cells and the CAR/or TCR is expressed in CD8 T cells). cells). Cellular immunotherapeutic compositions, methods of making, and methods of use comprising combinations of chimeric Tim receptor engineered immune cells or cell subsets and cellular immunotherapeutic agents (e.g., CAR, TCR, etc.) are herein incorporated by reference in their entirety. It is described in PCT International Publication No. WO2019/191340, which is incorporated herein.

Exemplary antigens that a recombinant TCR, high affinity TCR, CAR, TCR-CAR, or scTCR fusion protein may target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, Included are HPV E7, survivin, alpha-fetoprotein and tumor-specific neoantigens.

CARs of the present disclosure can target a variety of antigens, including viral antigens, bacterial antigens, fungal antigens, parasite antigens, tumor antigens, autoimmune disease antigens. Exemplary antigens that a 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, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, WT1, VEGF-alpha, VEGFR1 , IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, EphrinA2, EphrinB2, Lewis formula A antigen, Lewis formula Y antigen, MAGE, Included are MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD2 and GD2.

Radiotherapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy, intensity modulated radiation therapy, intensity modulated pendulum radiation therapy, particle beam therapy, proton therapy and auger therapy), proximity Radiation therapy, systematic radioisotope therapy, perioperative radiation therapy or any combination thereof are included.

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

Exemplary inhibitors of immune checkpoint molecules that may be for use in combination with the chimeric Tim compositions described herein include PD-L1, PD-L2, CD80, CD86, B7-H3, B7- H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, Included are checkpoint inhibitors that target LAIR-1, PVRIG/CD112R, or any combination thereof. In certain embodiments, immune checkpoint inhibitors can be antibodies, peptides, RNAi agents, or small molecules. The CTLA-4 specific antibody can be ipilimumab or tremelimumab. The PD-1 specific antibody can be pidilizumab, nivolumab or pembrolizumab. The PD-L1 specific antibody can be durvalumab, atezolizumab or avelumab.

Exemplary chemotherapeutic agents for use in combination with the chimeric Tim compositions described herein include alkylating agents, platinum-based agents, cytotoxic agents, inhibitors of chromatin function, topoisomerase inhibitors, microtubule May include inhibitors, DNA damaging agents, antimetabolites (eg folic acid antagonists, pyrimidine analogs, purine analogs and sugar modified analogs), DNA synthesis inhibitors, DNA interactors (eg intercalating agents) and DNA repair inhibitors. .

Chemotherapeutic agents include nonspecific cytotoxic agents that inhibit mitosis or cell division, and cancer cell growth by targeting specific molecules (e.g., oncogenes) involved in tumor growth, progression and metastasis. and molecularly targeted therapies to block spread. Exemplary non-specific chemotherapeutic agents for use in combination with the expression cassette compositions described herein include alkylating agents, platinum-based agents, cytotoxic agents, inhibitors of chromatin function, topoisomerase inhibitors. , microtubule inhibitors, DNA damaging agents, antimetabolites (e.g. folic acid antagonists, pyrimidine analogs, purine analogs and sugar modified analogs), DNA synthesis inhibitors, DNA interacting agents (e.g. intercalating agents), hypomethylating agents and DNA repair inhibitors.

Examples of chemotherapeutic agents contemplated for use in the combination therapy contemplated herein include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole [Arimidex®], bicalutamide [Casodex®] ], bleomycin sulfate [Blenoxane®], busulfan [Millelan®], busulfan injection [Busulfex®], capecitabine [Xeloda®], N4-pentoxycarbonyl-5 - deoxy-5-fluorocytidine, carboplatin [Paraplatin®], carmustine [BiCNU®], chlorambucil [Leukeran®], cisplatin [Platinol®], cladribine [Leustatin®] )], cyclophosphamide [Cytoxan® or Neosar®], cytarabine, cytosine arabinoside [Cytosar-U®], cytarabine liposome injection [Depot Cytos ( ®], dacarbazine [DTIC-Dome®], dactinomycin [actinomycin D, Cosmegan], daunorubicin hydrochloride [Servidin®], daunorubicin citrate liposome injection [DaunoXome ) (registered trademark)], dexamethasone, docetaxel [Taxotere®], doxorubicin hydrochloride [Adriamycin®, Rubex®], etoposide [Vepside®], fludarabine phosphate [Fludara®], 5-fluorouracil [Adrucil®, Efudex®], flutamide [Eulexin®], tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea [Hydrea®], idarubicin [Idamycin®], ifosfamide [IFEX®], irinotecan [Camptosar®], L-asparaginase [ ELSPAR®], leucovorin calcium, melphalan [Alkeran®], 6-mercaptopurine [Purinesol®], methotrexate [Folex®], mitoxantrone [Novantron ( ®], Mylotarg, Paclitaxel [Taxol®], Phoenix (Yttrium 90/MX-DTPA), Pentostatin, Polyfeprozane 20 Carmustine Implant [Gliadel®], fdabra tamoxifen citrate [Nolvadex ®], teniposide [Vumon®], 6-thioguanine, thiotepa, tirapazamine [Tirazone®], topotecan hydrochloride injection [Hycamptin®] , vinblastine [Velvan®], vincristine [Oncovin®], ibrutinib, venetoclax, crizotinib, alectinib, brigatinib, ceritinib and vinorelbine [Navelbine®].

Exemplary alkylating agents for use in combination therapy contemplated herein include nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: uracil mustard [aminouracil mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil Nitrogen Mustard®, Uracilost®, Uracilmostaza®, Uramustin®, Uramustine®], Chlormethine [Mustargen ® ], cyclophosphamide [Cytoxan ® , Neosal ® , Clafen ® , Endoxan ® , Procytox ® , Levimune (Revimmune)], ifosfamide [Mitoxana®], melphalan [Alkeran®], chlorambucil [Leukelan®], pipobroman [Amedale®, Vercite ( Vercyte®], triethylenemelamine [Hemel®, Hexalen®, Hexastat®], triethylenethiophosphoramine, temozolomide [Temodar® )], thiotepa [Thioplex®], busulfan [Busilvex®, Milleran®], carmustine [BiCNU®], lomustine [CeeNU® ], streptozocin [Zanosar®] and dacarbazine [DTIC-Dome®]. Additional exemplary alkylating agents for use in combination therapy contemplated herein include oxaliplatin [Eloxatine®]; temozolomide [Temodar® and Temodal ®]; dactinomycin [also known as actinomycin-D, Cosmogen®]; melphalan [L-PAM, also known as L-sarcolysin and phenylalanine mustard, Alkeran®]; altretamine [also known as hexamethylmelamine (HMM), Hexalene®]; 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]; Leukelan®]; cyclophosphamide [Cytoxan® and Neosal®]; dacarbazine [DTIC, also known as DIC and imidazolecarboxamide, DTIC-Dome®]; altretamine [hexamethyl ifosfamide [Ifex®]; prednumustine; procarbazine [Matulane®]; mechlorethamine [nitrogen mustard, also known as muscin and mechlorethamine hydrochloride, Mastergen®]; streptozocin [Zanosar®]; thiotepa [thiophosphoamide, also known as TESPA and TSPA, Thioplex® ]; cyclophosphamide [Endoxan®, Cytoxan®, Neosal®, Procytox®, Levimune®]; and bendamustine HCl [Treanda®]. include, but are not limited to:

Exemplary platinum-based agents for use in combination therapy contemplated herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin and triplatin tetranitrate. ) is included.

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

Exemplary molecularly targeted inhibitors for use in combination with the chimeric Tim receptor compositions described herein include, for example, 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 Included are small molecules that target molecules involved in cancer cell proliferation and survival, including

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 driver mutations associated with cancer, B-Raf inhibitors, RAF inhibitors , MEK inhibitors, mTOR inhibitors, adenosine pathway inhibitors, EGFR inhibitors, PI3K inhibitors, BCL2 inhibitors, VEGFR inhibitors, MET inhibitors, MYC inhibitors, BCR-ABL inhibitors, ABL inhibitors, HER2 inhibitors agent, H-RAS inhibitor, K-RAS inhibitor, PDGFR inhibitor, ALK inhibitor, ROS1 inhibitor, BTK inhibitor, TRK inhibitor, c-KIT inhibitor, c-MET inhibitor, CDK4/6 inhibitor agents, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PARP inhibitors, PARP inhibitors, PDGFRA inhibitors and RET inhibitors. In certain embodiments, the use of molecular targeting therapy comprises administering a molecular targeting therapy specific for a molecular target to a subject identified as having a tumor with a molecular target (e.g., a driver oncogene). . In certain embodiments, the molecular target has an activating mutation. In certain embodiments, use of chimeric Tim receptor-modified cells in combination with a molecularly targeted inhibitor increases the magnitude of the anti-tumor response, the persistence of the anti-tumor response, or both. In certain embodiments, lower than typical doses of molecular targeting therapy are used in combination with chimeric Tim receptor-modified cells.

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 (Prinomastat) (Agouron Pharmaceuticals), AGX 1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP O2) (Alnylam Pharmaceuticals), AMG386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine e), AP23573 (ridaforolimus/MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ197 (ArQule), ASA404 (Novartis/Antisoma), Atiprimod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Ph. pharmaceuticals), AV-951 (Aveo Pharmaceuticals) , Avastin (bevacizumab) (Genentech), AZD2171 [cediranib/Recentin] (AstraZeneca), BAY 57-9352 [Telatinib] (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ing Elheim Pharmaceuticals), BIBW2992 ( Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-Myers Squibb), BMS-582664 (brivanib) (Bristol-Myers Squibb), BMS-690514 (Bristol-Myers Squibb), Cal Citriol, CCI-779 (Toricel) (Wyeth) , CDP-791 (ImClone Systems), Ceflatonin (Homoharringtonine/HHT) (ChemGenex Therapeutics), Celebrex (celecoxib) (Pfizer), CEP-7055 (Cephalon/Sanofi), CHIR-265 (Chiron Corp.) oration), NGR - TNF, COL-3 [Metastat] (Collagenex Pharmaceuticals), combretastatin (Oxigene), CP-751,871 (figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Pfizer) Ichisankyo), CT-322 [Angiocept] (Adnexus), Curcumin, Dalteparin (Fragmin) (Pfizer), Disulfiram (Antabuse), E7820 (Eisai Co., Ltd.), E7080 (Eisai Co., Ltd.), EMD 121974 (Cilengitide) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (ImClone/Bristol-Myers Squibb), EZN-2208 (Enzon P pharmaceuticals), EZN -2968 (Enzon Pharmaceuticals), GC1008 (Genzyme), Genistein, GSK1363089 [Foretinib] (GlaxoSmithKline), GW786034 (Pazopanib) (GlaxoSmithKline), GT-11 1 (Vascular Biogenics Ltd.). ), IMC-1121B (ramucirumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (Introgen Therapeutics), Ressa (ZD1839/gefitinib), LBH589 [Faridak/Panobinostt] (Novartis), Lucentis (ranibizumab) (Genentech/Novartis), LY317615 (enzastaurin) (Eli Lilly and Company), Macugen (pegaptanib) (Pfizer), MEDI522 [ avegulin ( Abegrin)] (MedImmune), MLN518 (tandutinib) (Millennium), Neovastat (AE941/benephine) (Aeterna Zentaris), Nexavar (Bayer/Onyx), NM-3 (Genzyme Corporation), Noscapine ( Cougar Biotechnology) , NPI-2358 (Nereus Pharmaceuticals), OSI-930 (OSI), Palomid 529 (Paloma Pharmaceuticals, Inc.), Panzem Capsules (2ME2) (EntreMed), Panzem NCD (2ME2) (EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen Industries/Medigen Biotechnology), PKC412 (Novartis), Polyphenon E (green tea extract) (Polypheno E International, Inc), P PI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Vatalanib) (Novartis), PXD101 (Velinostat) (CuraGen Corporation), RAD001 (Everolimus) (Novartis), RAF265 (Novartis), Regorafenib (BA Y73-4506) (Bayer), Revlimid (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 (Aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis ), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat (Merck) and ZSTK474.

Exemplary B-Raf inhibitors include vemurafenib, dabrafenib and encorafenib.

Exemplary MEK inhibitors include binimetinib, cobimetinib, lefametinib, selumetinib and trametinib.

Exemplary BTK inhibitors include ibrutinib, Loxo-305, tirabrutinib, GDC-0853, acalabrutinib, ONO-4059, pebrutinib, BGB-3111, HM71224 and M7583.

Exemplary TRK inhibitors include entrectinib, larotrectinib, CH7057288, ONO-7579, LOXO-101, lestaurtinib and LOXO-195.

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

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

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

Exemplary FAK inhibitors include defactinib, GSK2256098, BI853520 and PF-00562271.

Exemplary FGFR inhibitors include Erdafitinib, Pemigatinib, Infigratinib, Rogaratinib, AZD4547, BGJ398, FP-1039 and ARQ 087.

Exemplary FLT-3 inhibitors include quizartinib, crenolanib, gilteritinib, midostaurin and lestaurtinib.

Exemplary IDH1 inhibitors include ivosidenib, BAY-1436032 and AGI-5198.

Exemplary IDH2 inhibitors include enasidenib.

Exemplary PDGFRA inhibitors include imatinib, regorafenib, crenolanib and olalatumab.

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

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

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

Exemplary vascular endothelial growth factor (VEGF) receptor inhibitors are bevacizumab [Avastin®], axitinib [Inlyta®]; brivanib alaninate [BMS-582664, (S )-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-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®]; Vargatef (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); ); vatalanib dihydrochloride (PTK787, CAS 212141-51-0); brivanib (BMS-540215, CAS 649735-46-6); vandetanib [Caprelsa® or AZD6474]; motesanib diphosphate [AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl) described in PCT publication WO 02/066470 ) amino]-3-pyridinecarboxamide]; Dovitinib dilactic acid (TKI258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 84 9217 -68-1); lestaurtinib (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]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα )-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-methyl-3-[[1-methyl-6- (3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); and Aflibercept [Eylea®].

Exemplary EGF pathway inhibitors include Tyrphostin 46, EKB-569, Erlotinib [Tarceva®], Gefitinib [Iressa®], Erbitux, Nimotuzumab, Lapatinib [Tikerb®], Cetuximab (anti EGFR mAb), ¹⁸⁸ Re-labeled nimotuzumab (anti-EGFR mAb) and 10767, WO97/30034 , WO 97/49688, WO 97/38983 and WO 96/33980 generally and specifically disclosed. Exemplary EGFR antibodies include cetuximab [Erbitux®]; panitumumab [Vectibix®]; matuzumab (EMD-72000); trastuzumab [Herceptin®]; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1), but not limited to h-R3; Exemplary epidermal growth factor receptor (EGFR) inhibitors include erlotinib hydrochloride [Tarceva®], ceritinib, brigtinib, osimeritinib, icotinib, gefitinib [Iressa®]; 4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2 -butenamide, Tovok®; vandetanib [Caprelsa®]; lapatinib [Tykerb®]; (3R,4R)-4-amino-1-((4-((3- methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); canertinib dihydrochloride (CI-1033); 6-[ 4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839 Pelitinib (EKB569); Afatinib (BIBW2992); Neratinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]- 1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester ( BMS599626); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl] Methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidine-6 -yl]-phenol (PKI166, CAS 187724-61-4); rocelitinib;

Exemplary mTOR inhibitors include rapamycin [Rapamune®] and analogs and derivatives thereof; SDZ-RAD; temsirolimus [Toricel®; also known as CCI-779]; ridaforolimus [deferolimus previously known as (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-pentoxo-11,36-dioxa-4- azatricyclo[30.3.1.0 ^4,9 ]hexatriacont-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, AP23573 and also known as MK8669 , described in PCT Publication No. WO 03/064383]; everolimus [Afinitor® or RAD001]; rapamycin [AY22989, Sirolimus®]; simapimod (CAS 164301-51-3); (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2- Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N ² -[1,4-dioxo-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl ]Methoxy]butyl]-L-arginylglycyl-L-α-aspartyl L-serine-, inner salt (SF1126, CAS 936487-67-1).

Exemplary phosphoinositide 3-kinase (PI3K) inhibitors are duvelisib, idelalisib, 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl] thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941 and described in PCT publications WO09/036082 and WO09/055730); 2-methyl-2-[ 4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (BEZ 235 or NVP -BEZ 235 and described in PCT Publication No. WO 06/122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridine-2- Amine (also known as BKM120 or NVP-BKM120 and described in PCT Publication No. WO 2007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)- 5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 958852-01-2); (1E,4S,4aR,5R,6aS,9aR)- 5-(acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)- 4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8); and 8-phenyl-2- (morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6), including but not limited to; 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]naphthyridin-3(2H)-one (MK-2206, CAS 1032349-93-1); perifosine (KRX0401); 4-dodecyl-N-1,3,4-thiadiazole-2- yl-benzenesulfonamide (PHT-427, CAS 1191951-57-1); 4-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-[ (3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-yl]-2-methyl-3-butyn-2-ol (GSK690693, CAS 937174-76-0) 8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (Palomide 529, P529 or SG-00529); 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-indazol-5-yl)-3-pyridinyl]oxy]methyl]-benzeneethanamine (A674563, CAS 552325-73-2); 4-[( 4-chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinamine (CCT128930, CAS 885499-61-6); 4-(4-chlorophenyl)- 4-[4-(1Hpyrazol-4-yl)phenyl]-piperidine (AT7867, CAS 857531-00-1); and Archexin (RX-0201, CAS 663232-27-7), but It is not limited to these.

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

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 are otherwise administered as monotherapy. can be administered at doses that can be considered sub-therapeutic. In such embodiments, chimeric Tim receptor compositions may provide additive or synergistic effects such that one or more additional therapies may be administered at lower doses. Combination therapy may be administered prior to (e.g., 1 to 30 days prior to or more than) the additional therapy, concurrently (same day) with the additional therapy, or after the additional therapy (e.g., the administration of a chimeric Tim receptor composition described herein after 1 day to 30 days or later). In certain embodiments, chimeric Tim receptor-modified cells are administered after administration of one or more additional therapies. In a further embodiment, the chimeric Tim receptor-modified cell is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 administrations of one or more additional therapies. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days later. In still further embodiments, the chimeric Tim receptor-modified cells are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of the one or more additional therapies. When the one or more additional therapies comprise multiple doses, the chimeric Tim receptor-modified cells are administered after the first dose of the one or more additional therapies, after the final dose of the one or more additional therapies, Or, one or more additional therapies may be administered among multiple doses.

In certain embodiments, methods of the present disclosure include a depletion step. The depletion step to remove the chimeric Tim receptor from the subject can be performed after a period of time sufficient for therapeutic benefit to reduce toxicity to the subject. In such embodiments, the chimeric Tim receptor vector may contain an inducible suicide gene such as iCASP9, inducible Fas or HSV-TK. Similarly, chimeric Tim receptor vectors are known cell surface antigens, such as May be designed for expression of CD20 or truncated EGFR (SEQ ID NO: 16). Alemtuzumab, which targets CD52 present on the surface of mature lymphocytes, can also be used to deplete transduced B cells, T cells or natural killer cells.

Subjects that can be treated by the compositions and methods of the present disclosure include animals such as humans, primates, cows, 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 infant, juvenile, adolescent, adult and geriatric subjects.

These and other changes can be made to the embodiments in light of the above detailed description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims; Rather, such claims are to be construed to include all possible embodiments along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

[Example 1]
Evaluation of Tim-CER-T Cells for Synthetic Lethal PARP Inhibitor Synergy in Ovarian Cancer Kuramochi ovarian cancer cells expressing the mCherry-NLS plasmid were incubated with the PARP inhibitor niraparib at 0.78-100 μM. Kuramochi cell numbers per well were tracked via imaging. At t=50, the number of Kuramochi cells in culture decreases monotonically with increasing niraparib concentration. The results are shown in FIG. 1A.

Similar experiments were performed with various doses of the PARP inhibitors niraparib, olaparib, talazoparib, veliparib and rucaparib, and Kuramochi cell numbers were plotted at t=50 h. Talazoparib and niraparib showed the most sensitive _EC50 values, followed by rucaparib and olaparib. Veliparib has the smallest effect on the number of Kuramochi cells detected among the five PARP inhibitors tested. Niraparib and rucaparib almost eliminate the Kuramochi culture by t=50 h. The results are shown in FIG. 1B.
[Example 2]

PARP inhibitors upregulate surface phosphatidylserine and have negligible effects on CD4/CD8 T cells Incubated with an anti-mouse IgG2a antibody conjugated to Alexa488. The number of Tim4+ Kuramochi cells (ie, green objects) is significantly increased over IgG-only controls in response to even incubation with 1.56 μM niraparib (FIGS. 2A-2B). Kuramochi cell numbers increased in response to 1.56-12.5 μM niraparib (FIG. 2C), indicating that PARP inhibition induces surface phosphatidylserine without killing Kuramochi cells.

Co-cultures of primary human CD4+ and CD8+ T cells were incubated with various concentrations of niraparib and T cell viability, diameter and density were measured on days 1, 2 and 3 after incubation with niraparib. Corresponding measurements were made using a ViCell cell counter on days 7, 8 and 9 after activation with TransAct. T cell viability, mean diameter and expanded proliferation on days 7-9 show negligible changes as a function of niraparib concentration (Fig. 3).
[Example 3]

Chimeric Tim Receptor/Niraparib Synergistically Kills Kuramochi Cells In Vitro at Different Doses Kuramochi cells expressing the mCherry-NLS plasmid were incubated with 25, 12.5, or 6.25 μM niraparib for 20 hours. The next day, the niraparib concentration was reduced to 0.52 μM and the primary cells expressing chimeric Tim receptor construct 13A (TIM4 binding domain-CD28 transmembrane domain-CD28 co-stimulatory domain-CD3zeta signaling domain) (SEQ ID NO: 162). Co-cultures of human CD4+ and CD8+ T cells were added to Kuramochi cultures. T cell cultures expressing the chimeric Tim receptor construct 13A significantly reduce the number of Kuramochi cells in culture, lower than the mock-transduced control (Control-T), depending on the experimental conditions. The number of Kuramochi cells expressing mCherry-NLS was monitored via IncuCyte. The results are shown in FIG. Combination therapy with chimeric Tim receptor construct 13A and niraparib synergistically kills Kuramochi cells at different doses.

Kuramochi cells expressing the mCherry-NLS plasmid were incubated with 1.56 μM niraparib for approximately 20 hours. The next day, the medium was removed from the cells and replaced with fresh medium containing 0 μM niraparib and the chimeric Tim receptor CTX140 (Tim4 binding domain-CD28 hinge-CD28 transmembrane-CD28 co-stimulatory domain-DAP12) (SEQ ID NO: 195) or primary human CD4+ and CD8+ T cells expressing CTX156 (tEGFR control) were added to Kuramochi cultures. The number of Kuramochi cells expressing mCherry-NLS was monitored via IncuCyte. The results are shown in FIG. T cell cultures expressing the chimeric Tim receptor construct CTX140 significantly reduce the number of Kuramochi cells in culture, lower than mock-transduced controls.

2500 Kuramochi ovarian cancer cells expressing the mCherry-NLS plasmid were plated on day 0. 25, 12.5, or 6.25 μM niraparib was added to the cell cultures after approximately 4 hours. Approximately 20 hours after the first niraparib dose, primary human CD4+ and CD8+ T cells were added as well as a maintenance dose of 0.52 μM niraparib. Results for 25 μM niraparib pretreatment are shown in FIG. Results for 25, 12.5, or 6.25 μM niraparib pretreatment at various effector:target (E:T) ratios are shown in FIG. T cell cultures expressing the chimeric Tim receptor construct CTX137 significantly reduce the number of Kuramochi cells in culture, lower than mock-transduced controls.

To induce Ptd-Ser exposure, the BRCA-2 mutant Kuramochi cell line was treated with a therapeutic dose of the PARP inhibitor niraparib. Brief exposure to niraparib induces changes in membrane phospholipid symmetry in a dose-dependent manner and is effective in inhibiting proliferation of Kuramochi cells (Fig. 8A). Addition of the chimeric Tim4 receptor pCTX133 (Tim4-TLR2-CD3z) at a low effector:target ratio (1:1) enhanced the potency of niraparib in vitro compared to the transduced control, indicating that the target cells demonstrated the ability of pCTX133 to elicit a direct cytotoxic effect against (Fig. 8B).
[Example 4]

Chimeric Tim Receptor/Niraparib Synergistically Kills A2780 Cells T cell transduction and culture: CD4 and CD8 T cells were isolated from frozen healthy donor PBMC using Miltenyi CD4+ and CD8+ isolation kits. Subsequently, CD4 and CD8 T cells were mixed at a 1:1 ratio and treated with cell serum replacement, L-glutamine, GlutaMAX, and IL-2, IL-7 and IL-15 cytokines, and TransAct diluted 1:50. were incubated overnight in flat-bottomed 24-well plates in OpTmizer medium supplemented with 16-24 hours after activation with TransAct, T cells were transduced with the appropriate virus at MOI=10 with 8 μg/mL polybrene and 1:50 TransAct in flat-bottom 96-well plates. Approximately 24 hours after transduction, T cells were transferred to 24-well GRex plates in OpTmizer medium supplemented with cell serum replacement, L-glutamine, GlutaMAX, and IL-2, IL-7 and IL-15 cytokines. moved. T cells were counted and replenished with fresh medium approximately every two days thereafter.

Cell line engineering: A2780 (human ovarian cancer cell line) and Kuramochi (BRCA-deficient human ovarian cancer cell line) cells were transduced with a lentiviral vector encoding the mCherry-nuclear localization sequence (NLS) gene. and subsequently sorted according to mCherry expression. The A2780-mCherry-NLS cell line was clonally sorted, expanded and banked, while the Kuramochi-mCherry-NLS cells were expanded and banked in bulk. Cell lines were thawed and maintained in culture in RPMI 1640 supplemented with 10% FBS until approximately 25 passages.

Co-culture assay: On day 0, 2,500 A2780-mCh-NLS or Kuramochi-mCh-NLS cells were plated in NUNC Edge 96-well plates and niraparib (free base), all in RPMI 1640 + 10% FBS. , at the appropriate concentration, was added after about 4 hours. After about 20 hours, the medium was removed and niraparib (free bases) and the appropriate number of T cells. When A2780-mCh-NLS cells were used, the supernatant was centrifuged and resuspended in T cell medium to preserve any suspended components of A2780 cells. When appropriate, plates were immediately transferred to the IncuCyte.

Flow cytometry measurement of surface PS: A2780-mCh-NLS cells were treated with 1.56 or 25 μM niraparib or an equivalent volume of DMSO (control). After 48 hours, samples were trypsinized and stained with Tim4-Fc followed by a fluorescently labeled secondary antibody against Tim4-Fc. Samples were analyzed immediately via flow cytometry.

As shown in Figures 9A-B, T cells modified with the chimeric Tim4 receptor pCTX247 (Tim4 binding domain-CD28 transmembrane-CD28 signaling domain-CD3zeta signaling domain; Demonstrate in vitro anti-tumor activity in cancer cell lines. As shown in FIG. 10, T cells modified with the chimeric Tim4 receptor pCTX797 (Tim4 binding domain-CD28 transmembrane-CD28 signaling domain-CD3zeta signaling domain; SEQ ID NO: 258) were induced to: Demonstrate in vitro anti-tumor activity.

As shown in FIGS. 11A-11B, phosphatidylserine is present on the cell surface in response to niraparib in two ovarian cancer models.

As shown in FIG. 12, the chimeric Tim4 receptor pCTX797 (Tim4 binding domain-CD28 transmembrane-CD28 signaling domain-CD3zeta signaling domain; SEQ ID NO:258) compared to the truncated EGFR (EGFRt) control (pCTX236). show synergy with additional PARP inhibitors (rucarparib, olaparib, and niraparib).

The various embodiments described above can be combined to provide further embodiments. Without limitation, U.S. Provisional Patent Application No. 63/066,150 filed Aug. 14, 2020, U.S. Provisional Patent Application No. 63/087,049 filed Oct. 2, 2020 and Jul. 28, 2021 All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign Patent applications and non-patent publications are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, using concepts of the various patents, applications and publications to provide still further embodiments.

These and other changes can be made to the embodiments in light of the above detailed description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims. , is to be construed to include all possible embodiments along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims (88)

A method for treating cancer in a subject in need thereof, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer, or melanoma, the method comprising: an effective amount of
(a)(i) a Tim1 IgV domain or a Tim4 IgV domain; and
(ii) an extracellular domain comprising a binding domain, comprising a Tim1 mucin domain or a Tim4 mucin domain;
(b) an intracellular signaling domain comprising a primary intracellular signaling domain and optionally a secondary intracellular signaling domain;
(c) a chimeric T-cell immunoglobulin and mucin (Tim) receptor comprising a single-chain chimeric protein comprising a transmembrane domain located between and connecting an extracellular domain and an intracellular signaling domain; and a method comprising administering to a subject a poly (ADP-ribose) polymerase (PARP) inhibitor. 2. The method of claim 1, wherein the breast cancer is triple-negative breast cancer. 2. The method of claim 1, wherein the ovarian cancer is advanced ovarian cancer. 2. The method of claim 1, wherein the prostate cancer is advanced prostate cancer. 2. The method of claim 1, wherein the lung cancer is non-small cell lung cancer. 6. The method of any one of claims 1-5, wherein the cancer is a breast cancer gene (BRCA) mutated cancer. 7. The method of claim 6, wherein the cancer is a BRCA1 mutated cancer, a BRCA2 mutated cancer, or both. 8. The method of any one of claims 1-7, wherein the PARP inhibitor is talazoparib, niraparib, rucaparib, olaparib, veliparib, CEP9722, E7016, AG014699, MK4827, BMN-673, pamiparib, or a combination thereof. . The method of any one of claims 1-8, wherein the PARP inhibitor comprises niraparib. The method of any one of claims 1-8, wherein the PARP inhibitor comprises talazoparib. The method of any one of claims 1-8, wherein the PARP inhibitor comprises rucaparib. The method of any one of claims 1-8, wherein the PARP inhibitor comprises olaparib. The method of any one of claims 1-8, wherein the PARP inhibitor comprises veliparib. The method of any one of claims 1-8, wherein the PARP inhibitor comprises CEP9722. The method of any one of claims 1-8, wherein the PARP inhibitor comprises E7016. The method of any one of claims 1-8, wherein the PARP inhibitor comprises AG014699. The method of any one of claims 1-8, wherein the PARP inhibitor comprises MK4827. The method of any one of claims 1-8, wherein the PARP inhibitor comprises BMN-673. The method of any one of claims 1-8, wherein the PARP inhibitor comprises pamiparib. 20. The method of any one of claims 1-19, further comprising administering an additional therapeutic agent. Additional therapeutic agents include radiation, cellular immunotherapy, antibodies, immune checkpoint molecule inhibitors, chemotherapy, hormone therapy, peptides, antibiotics, antivirals, antifungals, anti-inflammatory agents, UV light therapy, electricity. 21. The method of claim 20, comprising pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, small molecule therapy, or combinations thereof. 22. The method of claim 21, wherein the cellular immunotherapy is chimeric antigen receptor or T cell receptor. Additional therapeutic agents are 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 inhibitor, BCR-ABL inhibitor, MET inhibitor, MYC inhibitor, ABL inhibitor, HER2 inhibitor, BTK inhibitor, H-RAS inhibitor, K-RAS inhibitor, PDGFR inhibitor, TRK inhibitor, c -KIT inhibitor, c-MET inhibitor, CDK4/6 inhibitor, FAK inhibitor, FGFR inhibitor, FLT3 inhibitor, IDH1 inhibitor, IDH2 inhibitor, PDGFRA inhibitor, or RET inhibitor. 22. The method according to 20 or 21. The method of any one of claims 1-23, wherein the binding domain comprises the Tim1 IgV domain and the Tim1 mucin domain. 24. The method of any one of claims 1-23, wherein the binding domain comprises the Tim4 IgV domain and the Tim4 mucin domain. 24. The method of any one of claims 1-23, wherein the binding domain comprises the Tim1 IgV domain and the Tim4 mucin domain. 24. The method of any one of claims 1-23, wherein the binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain. 27. The method of any one of claims 1-24 or 26, wherein the Tim1 IgV domain comprises the amino acid sequence shown in SEQ ID NO:38. 29. The method of any one of claims 1-24, 26 or 28, wherein the Timl IgV domain is a modified Timl IgV domain comprising the R66G substitution in SEQ ID NO:38. 30. The method of claim 29, wherein the modified Timl IgV domain comprises the amino acid sequence shown in SEQ ID NO:41. 31. The method of any one of claims 1-25 or 27-30, wherein the Tim1 mucin domain comprises the amino acid sequence shown in SEQ ID NO:39. 28. The method of any one of claims 1-23, 25 or 27, wherein the Tim4 IgV domain comprises the amino acid sequence shown in SEQ ID NO:34. 27. The method of any one of claims 1-23, 25 or 26, wherein the Tim4 mucin domain comprises the amino acid sequence shown in SEQ ID NO:35. The transmembrane domain is Tim4 transmembrane domain, Tim1 transmembrane domain, CD28 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, CD27 transmembrane domain, ICOS transmembrane domain, CD2 transmembrane domain, LFA-1 1-, comprising a transmembrane domain, a CD30 transmembrane domain, a CD40 transmembrane domain, a PD-1 transmembrane domain, a CD7 transmembrane domain, a LIGHT transmembrane domain, an NKG2C transmembrane domain, or a B7-H3 transmembrane domain 34. The method of any one of 33. The Tim4 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 144 or 23; the Tim1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 8; the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 145 4-1BB transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 146; OX40 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 147; CD27 the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 148; the ICOS transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 149; the CD2 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 150 the LFA-1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 151; the CD30 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 152; the CD40 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 153; the PD-1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 154; the CD7 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 155 the LIGHT transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 156; the NKG2C transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 157; or the B7-H3 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:158. 35. The method of any one of claims 1-34, wherein the transmembrane domain comprises a Tim1 transmembrane domain, a Tim4 transmembrane domain, or a CD28 transmembrane domain. Tim1 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:8; Tim4 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:6 or 23; or CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:7 43. The method of claim 42. 38. The method of any one of claims 1-37, wherein the chimeric Tim receptor further comprises an extracellular spacer domain. 39. The method of claim 38, wherein the extracellular spacer domain comprises an IgG4 hinge region or a CD28 hinge region. 40. The method of claim 39, 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. 4-1BB costimulatory signaling domain; CD27 costimulatory signaling domain; ICOS costimulatory signaling domain; LFA-1 costimulatory signaling domain; 41. The method of any one of claims 1-40, comprising an OX40 costimulatory signaling domain; a CD2 costimulatory signaling domain; or an ICAM-1 costimulatory signaling domain. whether the CD28 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 118 or 119; whether the 4-1BB costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 122; ICOS co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 172; LFA-1 co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 171; OX40 co-stimulatory the signaling domain comprises the amino acid sequence of SEQ ID NO: 166; the CD2 co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 167; or the ICAM-1 co-stimulatory signaling comprises the amino acid sequence of SEQ ID NO: 170 42. The method of claim 41 . 43. The method of any one of claims 1-42, wherein the secondary signaling domain comprises a CD3zeta signaling domain. 44. The method of claim 43, wherein the CD3zeta signaling domain comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27. 4-1BB costimulatory signaling domain; CD27 costimulatory signaling domain; ICOS costimulatory signaling domain; LFA-1 costimulatory signaling domain; 41. The method of any one of claims 1-40, comprising an OX40 costimulatory signaling domain; a CD2 costimulatory signaling domain; or an ICAM-1 costimulatory signaling domain. whether the CD28 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 118 or 119; whether the 4-1BB costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 122; ICOS co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 172; LFA-1 co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 171; OX40 co-stimulatory the signaling domain comprises the amino acid sequence of SEQ ID NO:166; the CD2 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:167; or the ICAM-1 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:170 46. The method of claim 45, comprising: 47. The method of any one of claims 1-42, 45, or 46, wherein the secondary signaling domain comprises the DAP12 signaling domain. 48. The method of claim 47, wherein the DAP12 signaling domain comprises the amino acid sequence of SEQ ID NO:180. the primary intracellular signaling domain is 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 CD3zeta signaling domain, a TLR2 signaling domain, or TLR8 41. The method of any one of claims 1-40, comprising a signaling domain. The Tim1 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:44, the Tim4 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:45, 224 or 225, or the TRAF2 signaling domain comprises the amino acid sequence set forth 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 or the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO:5, the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO:222; or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO:222; 50. The method of claim 49, comprising the amino acid sequence shown in 47. the secondary intracellular signaling domain is 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 51. The method of any one of claims 1-42, 49 or 50, comprising a TLR8 signaling domain. 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, 224 or 225, or the TRAF2 signaling domain comprises the amino acid sequence shown in SEQ ID NO:48 the TRAF6 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:46; the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:4 or 26; the DAP12 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:9 or the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO:5, the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO:222; or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO:222; 52. The method of claim 51, comprising the amino acid sequence shown in 47. (i) the binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain, the primary intracellular signaling domain comprises the TLR8 signaling domain, the secondary intracellular signaling domain comprises the CD3ζ signaling domain, and the transmembrane domain comprises contains a Tim1 transmembrane domain;
(ii) the binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain, the primary intracellular signaling domain comprises the CD28 signaling domain, the secondary intracellular signaling domain comprises the DAP12 signaling domain, and the transmembrane domain comprises 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, and the secondary intracellular signaling domain comprises DAP12 signaling domain, and the transmembrane domain comprises the CD28 transmembrane domain;
The method of any one of claims 1-52. (i) the Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO:34, the Tim1 mucin domain comprises the amino acid sequence of SEQ ID NO:39, the TLR8 signaling domain comprises the amino acid sequence of SEQ ID NO:47, and the CD3ζ signaling domain comprises SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO:27; and the Tim1 transmembrane domain comprises the amino acid sequence of SEQ ID NO:8;
(ii) the Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO:34, the Tim1 mucin domain comprises the amino acid sequence of SEQ ID NO:39, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO:4, and the DAP12 signaling domain comprises the amino acid sequence of SEQ ID NO: and the Tim1 transmembrane domain comprises the amino acid sequence of SEQ ID NO:8; or (iii) the Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO:34 and the Tim1 mucin domain comprises the amino acid sequence of SEQ ID NO:39. wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO:4, the DAP12 signaling domain comprises the amino acid sequence of SEQ ID NO:9; and the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:7;
54. The method of claim 53. (i) whether the single-chain chimeric protein comprises amino acids 25-628 of SEQ ID NO:67;
(ii) the single-chain chimeric protein comprises amino acids 25-416 of SEQ ID NO:68; or (iii) the single-chain chimeric protein comprises amino acids 25-422 of SEQ ID NO:69.
55. A method according to claim 53 or 54. (i) whether the single-chain chimeric protein comprises the amino acid sequence of SEQ ID NO:67;
(ii) the single-chain chimeric protein comprises the amino acid sequence of SEQ ID NO:68; or (iii) the single-chain chimeric protein comprises the amino acid sequence of SEQ ID NO:69.
The method of any one of claims 53-55. the chimeric receptor
(i) a binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; a primary intracellular signaling domain comprising the Tim1 signaling domain, a secondary intracellular signaling domain comprising the CD3ζ signaling domain, and a membrane comprising the Tim1 transmembrane domain; a penetrating domain;
(ii) a binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain, a primary intracellular signaling domain comprising the Tim4 signaling domain, a secondary intracellular signaling domain comprising the CD3ζ signaling domain; and a membrane comprising the Tim1 transmembrane domain. penetrating domain;
(iii) a binding domain comprising the Tim1 IgV domain and a Tim1 mucin domain; a primary intracellular signaling domain comprising the CD28 signaling domain, and a transmembrane domain comprising the CD28 transmembrane domain;
(iv) a binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; a primary intracellular signaling domain comprising the TRAF6 signaling domain, and a transmembrane domain comprising the Tim1 transmembrane domain;
(v) a binding domain comprising the Tim1 IgV domain and a Tim1 mucin domain; a primary intracellular signaling domain comprising the TRAF6 signaling domain, and a transmembrane domain comprising the CD28 transmembrane domain;
(vi) a binding domain comprising the Tim1 IgV domain and a Tim1 mucin domain; a primary intracellular signaling domain comprising the TRAF2 signaling domain, and a transmembrane domain comprising the Tim1 transmembrane domain;
(vii) a binding domain comprising the Tim1 IgV domain and a Tim1 mucin domain; a primary intracellular signaling domain comprising the TRAF2 signaling domain, and a transmembrane domain comprising the CD28 transmembrane domain;
(viii) a binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; a primary intracellular signaling domain comprising the TLR8 signaling domain, a secondary intracellular signaling domain comprising the CD3ζ signaling domain, and a membrane comprising the Tim1 transmembrane domain; penetrating domain;
(ix) a binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; a primary intracellular signaling domain comprising the CD28 signaling domain, a secondary intracellular signaling domain comprising the DAP12 signaling domain, and a membrane comprising the CD28 transmembrane domain; or (x) a binding domain comprising a Tim1 IgV domain and a Tim1 mucin domain; a primary intracellular signaling domain comprising a CD28 signaling domain, a secondary intracellular signaling domain comprising a DAP12 signaling domain, and a Tim1 transmembrane 53. The method of any one of claims 1-52, comprising a transmembrane domain comprising domain. A chimeric Tim4 receptor is
(i) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a Tim1 signaling domain comprising the amino acid sequence of SEQ ID NO:44, a CD3ζ signal comprising the amino acid sequence of SEQ ID NO:4. a transduction domain and a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ii) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a Tim4 signaling domain comprising the amino acid sequence of SEQ ID NO:45, 224 or 225, SEQ ID NO:5 or SEQ ID NO: a CD3ζ signaling domain comprising the 27 amino acid sequence; and a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(iii) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a CD28 signaling domain comprising the amino acid sequence of SEQ ID NO:4, and a CD28 comprising the amino acid sequence of SEQ ID NO:7 a transmembrane domain;
(iv) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, TRAF6 signaling comprising the amino acid sequence of SEQ ID NO:46 domain, and a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(v) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a TRAF6 signaling domain comprising the amino acid sequence of SEQ ID NO:46, and a CD28 comprising the amino acid sequence of SEQ ID NO:7 a transmembrane domain;
(vi) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a TRAF2 signaling domain comprising the amino acid sequence of SEQ ID NO:48, and a Tim1 comprising the amino acid sequence of SEQ ID NO:8 a transmembrane domain;
(vii) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a TRAF2 signaling domain comprising the amino acid sequence of SEQ ID NO:48, and a CD28 comprising the amino acid sequence of SEQ ID NO:7 a transmembrane domain;
(viii) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a TLR8 signaling domain comprising the amino acid sequence of SEQ ID NO:47, an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27 and a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ix) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a CD28 signaling domain comprising the amino acid sequence of SEQ ID NO:4, a DAP12 signaling domain 9, and SEQ ID NO:7 or (x) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a CD28 signaling domain comprising the amino acid sequence of SEQ ID NO:4. , the DAP12 signaling domain comprising the amino acid sequence of SEQ ID NO:9, and the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:8. A chimeric Tim receptor is
(i) a single-chain chimeric protein comprising amino acids 21-456 of SEQ ID NO:49;
(ii) a single-chain chimeric protein comprising amino acids 21-471 of SEQ ID NO:50;
(iii) a single chain chimeric protein comprising amino acids 21-363 of SEQ ID NO:51;
(iv) a single-chain chimeric protein comprising amino acids 21-590 of SEQ ID NO:52;
(v) a single-chain chimeric protein comprising amino acids 21-596 of SEQ ID NO:53;
(vi) a single-chain chimeric protein comprising amino acids 21-619 of SEQ ID NO:54;
(vii) a single-chain chimeric protein comprising amino acids 21-625 of SEQ ID NO:55;
(viii) a single chain chimeric protein comprising amino acids 21-621 of SEQ ID NO:56;
(ix) a single chain chimeric protein comprising amino acids 21-415 of SEQ ID NO:57; or (x) a single chain chimeric protein comprising amino acids 21-409 of SEQ ID NO:58. described method. A chimeric Tim receptor is
(i) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:49;
(ii) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:50;
(iii) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:51;
(iv) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:52;
(v) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:53;
(vi) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:54;
(vii) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:55;
(viii) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:56;
60. The method of claim 59, comprising (ix) a single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:57; or (x) a single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:58. A chimeric Tim receptor is
(i) a binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; a primary intracellular signaling domain comprising the Tim1 signaling domain, a secondary intracellular signaling domain comprising the CD3ζ signaling domain, and a membrane comprising the Tim1 transmembrane domain; penetrating domain;
(ii) a binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; a primary intracellular signaling domain comprising the Tim4 signaling domain, a secondary intracellular signaling domain comprising the CD3ζ signaling domain; and a membrane comprising the Tim1 transmembrane domain. penetrating domain;
(iii) a binding domain comprising the Tim4 IgV domain and the Tim4 mucin domain; a primary intracellular signaling domain comprising the Tim4 signaling domain, a secondary intracellular signaling domain comprising the CD3ζ signaling domain; and a membrane comprising the Tim4 transmembrane domain. or (iv) a binding domain comprising the Tim4 IgV domain and the Tim4 mucin domain; a primary intracellular signaling domain comprising the Tim1 signaling domain, a secondary intracellular signaling domain comprising the CD3ζ signaling domain; and a Tim4 transmembrane domain. 53. The method of any one of claims 1-52, comprising a transmembrane domain comprising domain. A chimeric Tim receptor is
(i) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a Tim1 signaling domain comprising the amino acid sequence of SEQ ID NO:44, an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27 and a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(ii) a Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO:38, a Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO:39, a Tim4 signaling domain comprising the amino acid sequence of SEQ ID NO:45, 224 or 225, SEQ ID NO:5 or SEQ ID NO: a CD3ζ signaling domain comprising the 27 amino acid sequence; and a Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:8;
(iii) a Tim4 IgV domain comprising the amino acid sequence of SEQ ID NO:34, a Tim4 mucin domain comprising the amino acid sequence of SEQ ID NO:35, a Tim4 signaling domain comprising the amino acid sequence of SEQ ID NO:45, 224 or 225, SEQ ID NO:5 or SEQ ID NO: and a Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO:5; or (iv) a Tim4 IgV domain comprising the amino acid sequence of SEQ ID NO:34, Tim4 comprising the amino acid sequence of SEQ ID NO:35. a mucin domain, a Tim1 signaling domain comprising the amino acid sequence of SEQ ID NO:44, a CD3ζ signaling domain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and a Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO:6. 62. The method of Paragraph 61. A chimeric Tim receptor is
(i) a single-chain chimeric protein comprising amino acids 21-456 of SEQ ID NO:49;
(ii) a single-chain chimeric protein comprising amino acids 21-471 of SEQ ID NO:50;
(iii) a single chain chimeric protein comprising amino acids 25-490 of SEQ ID NO:59; or (iv) a single chain chimeric protein comprising amino acids 25-495 of SEQ ID NO:60. A chimeric Tim receptor is
(i) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:49;
(ii) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO:50;
(iii) a single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:59; or (iv) a single chain chimeric protein comprising the amino acid sequence of SEQ ID NO:60. A chimeric Tim receptor is
(i) a single-chain chimeric protein comprising the amino acid sequence of SEQ ID NO: 195 or amino acids 25-473 of SEQ ID NO: 195;
(ii) a single chain chimeric protein comprising the amino acid sequence of SEQ ID NO: 196 or amino acids 25-446 of SEQ ID NO: 196;
(iii) a single chain chimeric protein comprising the amino acid sequence of SEQ ID NO: 197 or amino acids 25-434 of SEQ ID NO: 197; or (iv) a single chain chimeric protein comprising the amino acid sequence of SEQ ID NO: 198 or amino acids 25-428 of SEQ ID NO: 198 53. The method of any one of claims 1-52, comprising A chimeric Tim receptor is
(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 selected from a CD28 signaling domain, a CD3ζ signaling domain, and a 4-1BB signaling domain, and a secondary intracellular signal selected from a TLR2 signaling domain or a TLR8 signaling domain; an intracellular signaling domain comprising a transduction domain;
(c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. 67. The method of claim 66, wherein the Tim4 IgV domain comprises the amino acid sequence set forth in SEQ ID NO:34, the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID NO:35, or both. 68. The method of claim 67, wherein the binding domain comprises the amino acid sequence of SEQ ID NO:2 or 42. 69. The method of any one of claims 66-68, wherein the chimeric Tim receptor further comprises an extracellular spacer domain. 70. The method of claim 69, wherein the extracellular spacer domain comprises an IgG4 hinge region or a CD28 hinge region. 71. The method of claim 70, wherein the IgG4 hinge region comprises the amino acid sequence set forth in SEQ ID NO:3 or the CD28 hinge region comprises the amino acid sequence set forth in SEQ ID NO:32. 72. The method of any one of claims 66-71, wherein the transmembrane domain comprises a Tim1 transmembrane domain, a Tim4 transmembrane domain, or a CD28 transmembrane domain. The Tim1 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:8, the Tim4 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:6 or 23, or the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:7 73. The method of claim 72. 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 74. The method of any one of claims 66-73, comprising the amino acid sequence shown in SEQ ID NO:100. 75. The method of any one of claims 66-74, wherein the TLR2 signaling domain comprises the amino acid sequence shown in SEQ ID NO:222 or the TLR8 signaling domain comprises the amino acid sequence shown in SEQ ID NO:47. A chimeric Tim receptor is
(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; a signaling domain; and an intracellular signaling domain comprising a tertiary intracellular signaling domain comprising a TLR signaling domain;
(c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. 77. The chimeric Tim receptor of claim 76, wherein the ITAM-containing signaling domain is the CD3zeta signaling domain or the DAP12 signaling domain. 78. The chimeric Tim receptor of claim 76 or 77, wherein the co-stimulatory signaling domain is the 4-1BB signaling domain or the CD28 signaling domain. 79. The chimeric Tim Receptor of any one of claims 76-78, wherein the TLR signaling domain is a TLR2 signaling domain or a TLR8 signaling domain. 80. The method of any one of claims 1-79, wherein the single chain protein comprises an amino acid sequence shown in any of Tables 1, 2 or 4-10. 81. The method of any one of claims 1-80, wherein the chimeric Tim receptor is administered as a polynucleotide encoding a single chain protein. 82. The method of claim 81, wherein the chimeric Tim Receptor is administered as a vector comprising a polynucleotide. 83. The method of claim 82, wherein the chimeric Tim Receptor is administered as an engineered cell containing the chimeric Tim Receptor, polynucleotide, or vector. 84. The method of claim 83, wherein the cells are immune cells. 85. The method of claim 83 or 84, wherein the cells are T cells. 86. The method of any one of claims 83-85, wherein the cells are CD4+ T cells, CD8+ T cells, or CD4+/CD8+ T cells. 86. The method of any one of claims 83-85, wherein the cells are human cells. 88. Any one of claims 1-87, wherein the chimeric Tim Receptor is administered as a composition comprising the chimeric Tim Receptor, a polynucleotide, a vector, or an engineered cell, and a pharmaceutically acceptable excipient. The method described in section.