CN117015396A

CN117015396A - Compositions and methods for treating cancer with chimeric TIM receptors in combination with poly (ADP-ribose) polymerase inhibitors

Info

Publication number: CN117015396A
Application number: CN202180070365.XA
Authority: CN
Inventors: D·M·科瑞; N·基普尼斯; G·奥多诺霍
Original assignee: Cero Therapeutics Inc
Current assignee: Cero Therapeutics Holdings Inc
Priority date: 2020-08-14
Filing date: 2021-08-13
Publication date: 2023-11-07

Abstract

The present disclosure relates to combination therapeutic compositions and methods comprising a chimeric Tim receptor, a host cell modified to comprise a chimeric Tim receptor molecule, and a PARP inhibitor.

Description

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

Statement regarding sequence listing

The sequence listing associated with the present application is provided in text form to replace paper copies and is hereby incorporated by reference into this specification. The text file containing the sequence listing is named 200265_415wo_st25.Txt. The text file is 425KB, created at 2021, 8, 13 and submitted electronically via the EFS-Web.

Background

Upon exposure to antigen, the primary antigen-specific cd8+ T cells undergo activation, which promotes their clonal expansion, differentiation, and development into functional effector T cells that can kill cells expressing the cognate antigen (e.g., tumor cells). After antigen clearance, most effector T cells undergo apoptosis, and a subset of surviving effector T cells differentiate into memory T cells, which can provide long-term protection against antigen re-exposure. However, prolonged antigen exposure may lead to T cell depletion, enabling tumor cell survival. T cell depletion refers to a dysfunctional state obtained by T cells undergoing sustained TCR stimulation, characterized by upregulation of immune checkpoint molecules (e.g., PD-1, CTLA-4, tim-3) expression, impaired effector function, dysproliferation, and metabolic defects. Engineered T cells expressing Chimeric Antigen Receptors (CARs) may also develop depletion.

Drawings

Figures 1A-1B show that Kuramochi model of ovarian cancer is moderately sensitive to PARP inhibition. FIG. 1A shows the results of Kuramochi cells expressing mCherry-NLS plasmid incubated with the PARP inhibitor nilaparib. FIG. 1B shows the results of incubation of Kuramochi cells expressing mCherry-NLS plasmid with the PARP inhibitors nilaparib, olaparib, tazopanib, velipatib and Lu Kapa.

Figures 2A-2C show binding of Kuramochi cells stimulated with nilaparib to Tim4, indicating an increase in the level of surface PS in response to PARP inhibition. FIG. 2A shows staining of Nilapatinib treated Kuramochi cells by Tim4-Fc chimera and anti-mouse IgG2A antibody conjugated to Alexa 488. Fig. 2B shows staining of IgG controls. FIG. 2C shows an increase in the number of Kuramochi cells in response to 1.56-12.5. Mu.M Nilapatinib.

FIG. 3 shows that Nilapatinib has a negligible effect on the health, cell size, or expansion of CD4/CD 8T cells.

FIG. 4 shows the results of Kuramochi cells incubated with chimeric Tim receptor construct 13A, control T cells and nilaparib.

Figure 5 shows that the combination of chimeric Tim4 receptor (CTX 140) and nilaparib treatment reduced Kuramochi cell numbers in vitro. Kuramochi cells were previously incubated with 1.56 μm nilaparib. CTX156 (tgfr) is a transduction control.

Figure 6 shows that the combination of chimeric Tim4 receptor (CTX 137) and nilaparib treatment reduced Kuramochi cell numbers in vitro. Kuramochi cells were previously incubated with 25 μm nilaparib and a maintenance dose of 0.52 μm nilaparib was administered. CTX156 (tgfr) is a transduction control.

FIG. 7 shows that the combination of chimeric Tim4 receptor (CTX 137) and Nilapatinib treatment with different doses (6.25. Mu.M, 12.5. Mu.M or 25. Mu.M pretreatment+0.52. Mu.M maintenance dose) and different effector to target cell ratios reduced the number of Kuramochi cells in vitro. CTX156 (tgfr) is a transduction control.

Fig. 8A-8B: pCTX133, a chimeric Tim4 receptor containing TLR-2, enhances the efficacy of nilaparib in ovarian cancer models. Fig. 8A: flow cytometry measurement of surface PtdSer. Kuramochi cells were treated with 1.56 or 25 μm nilaparib or with an equivalent volume of DMSO (control). After 48 hours, the samples were trypsinized and stained with Tim4-Fc followed by fluorescent-labeled secondary antibodies to Tim 4-Fc. Fig. 8B: kuramochi cells pretreated with 1.56 μm nilaparib for about 20 hours were co-cultured with pCTX133 from donor 32 and uninduced CD 4T cells at a 2:1 ratio of T cells to Kuramochi and a final nilaparib concentration of 1.56 μm. Samples treated with nilaparib+pctx 133 showed significantly fewer tumor cells in culture after about 3 days when compared to samples treated with nilaparib alone or with nilaparib+uninduced T cells. All data were collected via IncuCyte.

FIGS. 9A-9B, in vitro antitumor Activity of CER pCTX247 (SEQ ID NO: 257) in two ovarian cancer models. Fig. 9A: A2780-mCh-NLS cells pretreated with 25. Mu.M nilaparib for about 20 hours were co-cultured with CER 247 and MOCK 236CD4/8T cells from donor 21 at a 2:1 ratio of T cells to A2780 and final nilaparib concentration of 6.25. Mu.M. Samples treated with nilaparib+cer 247 showed significantly fewer tumor cells after about 5 days of culture when compared to samples treated with nilaparib alone, CER 247 alone, or nilaparib+mock 236. Fig. 9B: kumamachi-mCh-NLS cells pretreated with 25. Mu.M nilaparib for about 20 hours were co-cultured with CER 247 and MOCK 236CD4/8T cells from donor 21 at a 2:1 ratio of T cells to A2780 and final nilaparib concentration of 1.56. Mu.M. Samples treated with nilaparib+cer 247 showed significantly fewer tumor cells after about 5 days of culture when compared to samples treated with nilaparib alone, CER 247 alone, or nilaparib+mock 236. All data were collected via IncuCyte.

Fig. 10: in vitro anti-tumor Activity of CER pCTX797 (SEQ ID NO: 258) in A2780 ovarian cancer model. A2780-mCh-NLS cells pretreated with 0.52. Mu.M nilaparib for about 20 hours were co-cultured with CER 797 and MOCK 236CD4/8T cells from donor 32 at a 2:1 ratio of T cells to A2780 and final nilaparib concentration of 0.52. Mu.M. Samples treated with nilaparib+cer 247 showed significantly fewer tumor cells after about 5 days of culture when compared to samples treated with nilaparib alone, CER 247 alone, or nilaparib+mock 236. All data were collected via IncuCyte.

Fig. 11A-11B: in both models of ovarian cancer, phosphatidylserine is presented on the cell surface in response to nilaparib. Fig. 11A: A2780-mCh-NLS cells treated with 1.56 and 25. Mu.M nilaparib showed significantly higher proportion of PS+ cells and Tim4-Fc MFI increase compared to samples treated with equal volumes of DMSO. Nilapatinib also begins to have an antitumor effect at a concentration of at least 1.56 μm of Nilapatinib. Fig. 11B: kuramochi-mCh-NLS cells treated with 25. Mu.M Nilapatinib showed significantly higher proportion of PS+ cells and Tim4-Fc MFI increase compared to samples treated with an equal volume of DMSO. Nilapatinib also begins to have an antitumor effect at a concentration of at least 25 μm Nilapatinib. All samples were analyzed via flow cytometry.

Fig. 12: CER shows a synergistic effect with additional PARP inhibitors (in addition to nilaparib, lu Kapa ni and olaparib). A2780-mCh-NLS cells pretreated with 0.52. Mu.M nilaparib, lu Kapa Ni or Olaparib for about 20 hours were co-cultured with CER247 and MOCK 236CD4/8T cells from donor 32 at a 2:1 ratio of T cells to A2780 and final nilaparib concentration of 0.52. Mu.M. Samples treated with nilaparib+cer 247, lu Kapa rib+cer 247 or olaparib+cer 247 showed significantly fewer tumor cells after about 5 days of culture when compared to samples treated with nilaparib, lu Kapa rib or olaparib alone, CER247 or nilaparib+mock 236. All data were collected via IncuCyte.

Detailed Description

In one aspect, the present disclosure provides methods of treating cancer using chimeric T cell immunoglobulin mucin (Tim) receptors (also known as chimeric phagocytic receptors (CER)) in combination with poly (ADP-ribose) polymerase (PARP) inhibitors. In another aspect, the present disclosure provides a pharmaceutical composition or combination comprising a chimeric Tim receptor and a PARP inhibitor. The chimeric Tim receptors useful in the compositions and methods of the present disclosure confer phagocytic, cytotoxic, and/or antigen presenting activity on host cells (e.g., T cells) modified with the chimeric Tim receptor, wherein the cytotoxic activity is induced upon binding of the chimeric Tim receptor to its target antigen phosphatidylserine. DNA damaging agents such as PARP inhibitors may act synergistically with chimeric Tim receptors by promoting cellular damage and the externalization of phosphatidylserine, which induces effector functions of the chimeric Tim receptor such as phagocytosis, cytotoxicity, co-stimulatory activity, antigen presentation, or combinations thereof.

Chimeric Tim receptors described herein include single chain chimeric proteins comprising: (a) An extracellular domain comprising a binding domain, the 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, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

In certain embodiments, the extracellular domain of a chimeric Tim receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain.

In certain embodiments, the chimeric Tim receptor is also capable of costimulating T cells via a signaling pathway that is different from the "classical" T cell costimulatory pathway (e.g., CD 28). For example, in addition to binding phosphatidylserine, tim4 is also a ligand for Tim1, tim1 being expressed on the surface of activated T cells. Tim1 is also capable of binding to phosphatidylserine. Tim 4-induced Tim1 signaling has been found to co-stimulate T cell proliferation and survival (Hartt Meyers et al 2005, nature immunology (Nat. Immunol.)), 6:455. Thus, in certain embodiments, the cytotoxic chimeric Tim receptor can reduce or inhibit T cell depletion, or restore depleted T cells, by providing a costimulatory signal via at least one signaling pathway. In certain embodiments, the cytotoxic chimeric Tim receptor provides a costimulatory signal via at least two different signaling pathways (e.g., via a costimulatory signaling domain selected from the cytotoxic chimeric Tim receptor and Tim 1).

In certain embodiments, the chimeric Tim receptors of the present disclosure also confer phagocytic activity on a host cell when expressed in 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 a cytolytic and phagocytic response by the host cell. In particular embodiments of the modified host cells described herein, the host cells do not naturally exhibit a phagocytic phenotype prior to modification with the chimeric Tim receptor.

In embodiments, administration of host cells modified with the chimeric Tim receptors of the present disclosure can be used in methods of eliminating target cells with surface exposed phosphatidylserine, e.g., for treating cancer. In normal healthy cells, phosphatidylserine is located in the inner leaflet of the plasma membrane. However, certain cellular events (such as injury, apoptosis, necrosis, and stress) activate a "promiscuous enzyme" which rapidly exposes phosphatidylserine to the cell surface where it binds to receptors such as Tim4 or Tim 1. Endogenous tumor-specific effector T cells can induce exposure of phosphatidylserine on the outer membrane of targeted tumor cells during cell lysis. In addition, certain cancer therapies (e.g., chemotherapy, radiation therapy, CAR-T cells, etc.) can induce phosphatidylserine exposure to targeted tumor cells or cells in the tumor microenvironment by inducing apoptosis, cellular stress, cellular injury, etc. Engineered host cells expressing the disclosed chimeric Tim receptors can clear damaged, stressed, apoptotic or necrotic tumor cells bearing surface-exposed phosphatidylserine by inducing apoptosis in tumor cells bearing surface-exposed phosphatidylserine. In certain embodiments, host cells expressing the chimeric Tim receptors disclosed herein clear damaged, stressed, apoptotic, or necrotic tumor cells with surface-exposed phosphatidylserine by inducing apoptosis and by phagocytosis. In another aspect, host cells modified with the chimeric Tim receptors of the present disclosure can be used to enhance the effect of PARP inhibitors that induce cellular stress, injury, necrosis, or apoptosis. For example, the administration of PARP inhibitors may increase the level of surface phosphatidylserine, resulting in synergistic combinations. Cells expressing a chimeric Tim receptor as described herein can bind to a phosphatidylserine moiety on the outer leaflet of a damaged or dead cell that is exposed by the administration of a PARP inhibitor and induce cytolysis or both cytolysis and phagocytosis of the targeted cell.

The combination of engineered host cells comprising a chimeric Tim receptor and a PARP inhibitor according to the present description may be administered to a subject alone or in further combination with one or more additional therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapy, small molecules, oncolytic viruses, electric pulse therapy, and the like.

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

In this description, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range and to include fractions thereof (e.g., tenths and hundredths of integers) as appropriate. Furthermore, any numerical range recited herein in connection with any physical feature, such as a polymer subunit, size, or thickness, should be understood to include any integer within the range, unless otherwise indicated. As used herein, unless otherwise indicated, the term "about" means ± 20% of the indicated range, value, or structure. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the recited components. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof. As used herein, the terms "comprising," "having," and "including" are used synonymously, and these terms and variants thereof are intended to be construed as non-limiting.

Unless the context clearly indicates otherwiseThe term is understood by those skilled in the art of antibodies in the sense given in the art. The term "antibody" is used in the broadest sense and encompasses polyclonal antibodies and monoclonal antibodies. An "antibody" may refer to an intact antibody that includes at least two heavy (H) and two light (L) chains linked to each other by disulfide bonds, as well as an antigen-binding portion (or antigen-binding domain) of the intact antibody that has or retains the ability to bind to a target molecule. Antibodies can be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, e.g., intracellular antibodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem bis-scFV, tandem tri-scFV, ADAPTIR). The monoclonal antibody or antigen binding portion thereof may be non-human, chimeric, humanized or human, preferably humanized or human. Antibodies are described, for example, in Harlow et al: the structure and function of immunoglobulins is reviewed in the laboratory Manual (Antibodies: A Laboratory Manual), chapter 14, (Cold spring harbor laboratory (Cold Spring Harbor Laboratory), cold spring harbor (Cold Spring Harbor), 1988). The "antigen binding portion" or "antigen binding domain" of an intact antibody is intended to encompass such "antibody fragments": which represents a portion of an intact antibody and refers to the epitope variable region or complementarity determining region of the intact antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') ₂ And Fv fragment, fab '-SH, F (ab') ₂ Bifunctional antibodies, linear antibodies, scFv antibodies, VH and multispecific antibodies formed from antibody fragments. "Fab" (antigen binding fragment) is a portion of an antibody that binds to an antigen and comprises the variable region of the heavy chain linked to the light chain via an interchain disulfide bond and CH1. Antibodies can be of any class or subclass, including IgG and subclasses thereof (IgG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ ) IgM, igE, igA and IgD.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three CDRs. (see, e.g., kit et al, kuby Immunology, 6 th edition, w.h. frieman company (w.h. freeman and co.) (page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated from antibodies that bind that antigen using VH or VL domains to screen libraries of complementary VL or VH domains, respectively. See, e.g., portolano et al, J.Immunol.), 150:880-887 (1993); clarkson et al, nature, 352:624-628 (1991).

The terms "complementarity determining region" and "CDR," synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous amino acid sequences within the variable region of an antibody that confer antigen specificity and/or binding affinity. In general, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region.

As used herein, the terms "binding domain," "binding region," and "binding moiety" refer to a molecule (such as a peptide, oligopeptide, polypeptide, or protein) that has the ability to specifically and non-covalently bind, associate (unite), recognize, or combine a target molecule (e.g., phosphatidylserine). The binding domain comprises any naturally occurring, synthetic, semisynthetic 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 a functional binding domain or antigen binding portion thereof. Exemplary binding domains include single chain antibody variable regions (e.g., domain antibodies, sFv, scFv, fab), receptor ectodomains (e.g., tim 4), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their ability to specifically bind to a biological molecule.

"T cell receptor" (TCR) refers to a cell that is present on the surface of a T cell (also known as a T lymphocyte) and is generally responsible for recognitionMolecules of antigens bound to Major Histocompatibility Complex (MHC) molecules. In most T cells, TCRs are typically composed of heterodimers of disulfide-linked highly variable alpha and beta chains (also referred to as tcra and tcrp, respectively). In a small fraction of T cells, TCRs consist of heterodimers of gamma and delta chains (also referred to as tcrgamma and tcrdelta, respectively). Each chain of TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region and a short cytoplasmic tail at the C-terminus (see Janeway et al, immunobiology: immune system in health and disease (Immunobiology: the Immune System in Health and Disease), 3 rd edition, contemporary biological publications (Current Biology Publications), page 4: 33, 1997). The TCRs of the present disclosure may be from a variety of animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. TCRs may be cell-bound (i.e., have a transmembrane region or domain) or soluble in form. TCRs comprise recombinantly produced, genetically engineered, fused or modified forms of TCRs, including, for example, sctcrs, soluble TCRs, TCR fusion constructs (trucs ^TM The method comprises the steps of carrying out a first treatment on the surface of the See, U.S. patent publication No. 2017/0166622).

The terms vy and vδ of the "variable region" or "variable domain" or γδ TCR of the TCR α chain (vα) and β chain (vβ) are involved in the binding of the TCR to the antigen. V of native TCR _α And V _β Typically have a similar structure, where each variable domain includes four conserved FR and three CDRs. V (V) _α The domains are encoded by two separate DNA segments: a variable gene segment (V gene) and a junction gene segment (J gene); v (V) _β The domains are encoded by three separate DNA segments: variable gene segments (V genes), diverse gene segments (D genes), and junction gene segments (J genes). Single V _α Or V _β The domain may be sufficient to confer antigen binding specificity. "major histocompatibility complex molecule" (MHC molecule) refers to a glycoprotein that delivers peptide antigens to the cell surface. MHC class I molecules are heterodimers composed of a membrane spanning the alpha chain (containing three alpha domains) and non-covalently associated beta 2 microglobulin. MHC class II molecules are defined by twoTransmembrane glycoproteins α and β, both of which are transmembrane. Each chain has two domains. MHC class I molecules deliver cytosolic derived peptides to the cell surface where the peptides: MHC complex is surrounded by CD8 ⁺ T cell recognition. MHC class II molecules deliver peptides derived from the vesicle system to the cell surface where they are recognized by cd4+ T cells. MHC molecules may be from a variety of animal species, including humans, mice, rats or other mammals.

"chimeric antigen receptor" (CAR) refers to a chimeric protein comprising two or more distinct domains and can act as a receptor when expressed on the surface of a cell. CARs generally consist of an extracellular domain (comprising a binding domain that binds a target antigen), an optional extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain (e.g., a T cell activation motif containing an immune receptor tyrosine activation motif (ITAM), and optionally an intracellular co-stimulatory domain). In certain embodiments, the intracellular signaling domain of the CAR has a T cell activation domain (e.g., cd3ζ) and an intracellular co-stimulatory domain (e.g., CD 28) that contains ITAM. In certain embodiments, the CAR is synthesized as a single polypeptide chain, or encoded by a nucleic acid molecule as a single-chain polypeptide.

Various assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, and determining affinity of the binding domains, such as western blotting, ELISA, and Analysis (see, e.g., scatchard et al, new York academy of sciences annual book (Ann. N.Y. Acad. Sci.) 51:660, 1949, and U.S. Pat. Nos. 5,283,173, 5,468,614 or equivalent). As used herein, "specifically bind" refers to binding domains or fusion proteins thereof to 10 or greater ⁵ M ^-1 Affinity or K of (2) _a (i.e., the equilibrium association constant of a particular binding interaction, where the units are 1/M) associates or associates with a target molecule, but does not significantly associate or associate with any other molecule or component in the sample.

The terms "antigen" and "Ag" refer to molecules capable of inducing an immune response. The immune response induced may be involved in antibody production, activation of specific immunocompetent cells, or both. Macromolecules (including proteins, glycoproteins and glycolipids) can be used as antigens. The antigen may be derived from recombinant DNA or genomic DNA. As contemplated herein, an antigen need not be encoded (i) by only the full length nucleotide sequence of the gene or (ii) by the "gene" at all. The antigen may be produced or synthesized, or the antigen may be 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" encompasses any molecule, structure, amino acid sequence, or protein determinant within an antigen that is specifically bound by a cognate immune binding molecule, such as an antibody or fragment thereof (e.g., scFv), T Cell Receptor (TCR), chimeric Tim receptor, or other binding molecule, domain, or protein. Epitope 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. The epitope may be a linear epitope or a conformational epitope.

As used herein, the term "PARP" (poly (ADP-ribose) polymerase) refers to a family of proteins, wherein each protein has two ribose moieties and two phosphate esters 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 referred to as "TimD4", refers to phosphatidylserine receptors that are typically expressed on antigen presenting cells such as macrophages and dendritic cells. Tim4 mediates phagocytosis by apoptotic, necrotic, injured or stressed cells that present phosphatidylserine (PtdSer) on the outer (external) leaflet of the cell membrane. Tim4 is also capable of binding to Tim1 expressed on the surface of T cells and inducing proliferation and survival. In certain embodiments, tim4 refers to human Tim4. An exemplary human Tim4 protein includes the amino acid sequence of SEQ ID NO. 1.

As used herein, the term "Tim4 binding domain" refers to the N-terminal immunoglobulin folding domain of Tim4, which has 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.

The Tim4 binding domain comprises a variable immunoglobulin (IgV) -like domain (referred to herein as an "IgV domain") and a mucin-like domain (referred to herein as an "mucin domain"). An exemplary human Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO. 34, and an exemplary human Tim4 mucin domain comprises the amino acid sequence of SEQ ID NO. 35. In certain embodiments, the Tim4 binding domain does not comprise 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" (protein 1 containing T cell immunoglobulin and mucin domains) refers to a phosphatidylserine receptor expressed on the surface of T cells. As described above, tim1 is also capable of binding to Tim4 expressed on the surface of antigen presenting cells. In certain embodiments, tim1 refers to human Tim1. An exemplary human Tim1 protein includes the amino acid sequence of SEQ ID NO. 36.

As used herein, the term "Tim1 binding domain" refers to the N-terminal immunoglobulin folding domain of Tim1 that selectively binds PtdSer. An exemplary human Tim1 binding domain includes the amino acid sequence of SEQ ID NO. 37.

The Tim1 binding domain comprises 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 comprise 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 an intracellular portion of a fusion protein or receptor that, upon receipt of an appropriate signal, can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule that triggers a signal from the effector domain. When the effector domain contains one or more signaling domains or motifs, such as an immune receptor tyrosine activation motif (ITAM), it may directly promote a cellular response. In other embodiments, the effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote the cellular response.

As used herein, a "costimulatory signaling domain" refers to an intracellular signaling domain of a costimulatory molecule, or a functional portion thereof, that, when activated together with a primary or classical (e.g., ITAM-driven) activation signal (e.g., provided by a cd3ζ intracellular signaling domain), promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. Co-stimulatory signaling domains include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), 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-activating motif (ITAM) activation domain" refers to an intracellular signaling domain or functional portion thereof that is naturally or endogenously present on an immune cell receptor or cell surface marker and contains at least one immunoreceptor tyrosine-activating motif (ITAM). ITAM means YXXL/I-X _6-8 Conserved motifs of YXXL/I. In certain embodiments, the ITAM signaling domain contains one, two, three, four, or more ITAMs. The ITAM signaling domain may initiate T cell activation signaling following antigen binding or ligand binding. ITAM signaling domains comprise, for example, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD79a and CD66 d.

"connecting amino acid" or "connecting amino acid residues" refers to the polypeptide of two adjacent motifs, region or domain between one or more (e.g., about 2-20) amino acid residues. The linking amino acids may be from the construct design of the chimeric protein (e.g., amino acid residues generated using restriction enzyme sites during construction of the nucleic acid molecule encoding the chimeric protein).

"nucleic acid molecules" and "polynucleotides" may be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. The nucleic acid molecule may consist of naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., the alpha-enantiomeric form of a naturally-occurring nucleotide), or a combination of both. The modified nucleotide may have a modification or substitution of a sugar moiety or a pyrimidine or purine base moiety. Nucleic acid monomers may be linked by phosphodiester bonds or analogues of such bonds. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenites, phosphorodiselenates, phosphoroanilide, phosphorophosphoramidates, and the like. The nucleic acid molecule may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding strand (antisense strand). The coding molecules may have the same coding sequence as known in the art, or may have a different coding sequence which may encode the same polypeptide due to redundancy or degeneracy of the genetic code, or by splicing.

"coding" refers to the inherent properties of specific polynucleotide sequences (e.g., DNA, cDNA, and mRNA sequences) to serve as templates for the synthesis of other polymers and macromolecules having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological properties that result therefrom during bioprocessing. Thus, if transcription and translation of mRNA corresponding to a polynucleotide produces a protein in a cell or other biological system, the polynucleotide encodes the protein. Both coding and non-coding strands may be referred to as encoding proteins or other products of polynucleotides. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" encompasses 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. The 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 the protein sequence or peptide sequence. A polypeptide comprises any peptide or protein consisting of two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as proteins, of many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide comprises a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, the term "mature polypeptide" or "mature protein" refers to a protein or polypeptide secreted or localized in the cell membrane or within certain cellular organelles (e.g., endoplasmic reticulum, golgi, or endosomes), and does not comprise an N-terminal signal peptide.

"Signal peptide", also known as "signal sequence", "leader peptide", "localization signal" or "localization sequence", is a short peptide (typically 15-30 amino acids in length) that is present at the N-terminus of a newly synthesized protein and is transported to the secretory pathway. The signal peptide typically comprises a short, hydrophilic, positively charged amino acid at the N-terminus, a central hydrophobic domain of 5-15 residues and a C-terminal region with a cleavage site for the signal peptidase. In eukaryotes, the signal peptide facilitates the transport of the newly synthesized protein to the endoplasmic reticulum where it is cleaved by the signal peptidase to produce the mature protein, which then enters its appropriate destination.

The term "chimeric" refers to any nucleic acid molecule or protein that is non-endogenous and includes sequences that bind or join together (typically do not bind or join together in nature). For example, a chimeric nucleic acid molecule may include regulatory sequences and coding sequences from different sources, or regulatory sequences and coding sequences from the same source but arranged in a manner different from that found in nature.

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

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

As used herein, a "heterologous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to the host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of the heterologous nucleic acid molecule, construct or sequence may 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 (i.e., non-endogenous or native) to the host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, etc., wherein the added molecule may be integrated into the host cell genome or present as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies. Furthermore, "heterologous" refers to a non-native enzyme, protein, or other activity encoded by a non-endogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity.

As used herein, the term "engineered", "recombinant", "modified" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule or vector that has been modified by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism that has been genetically engineered by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, wherein such alterations or modifications may be introduced by genetic engineering. A human-produced genetic alteration may comprise, for example, the introduction of a modification of a nucleic acid molecule encoding one or more proteins, chimeric receptors or enzymes (which may comprise an expression control element, such as a promoter), or the addition, deletion, substitution of other nucleic acid molecules, or other functional disruption or addition of genetic material of a cell. Exemplary modifications include those in the coding region of a heterologous or homologous polypeptide from a reference or parent molecule, or a functional fragment thereof. Other exemplary modifications include, for example, modifications in non-coding regulatory regions, wherein the modifications alter expression of the gene or operon.

As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., chimeric Tim receptor), whose expression in a host cell is desired, and which has been transferred into the cell by genetic engineering techniques. The transgene may encode a protein of therapeutic interest, as a reporter, tag, marker, suicide protein, or the like. The transgene may be from a natural source, a modified or recombinant molecule of a natural gene, or a synthetic molecule. In certain embodiments, the transgene is a component of a vector.

The term "overexpression" or "overexpression" of an antigen refers to an abnormally high level of antigen expression in a cell. The overexpression of an antigen or antigen is often associated with a disease state, such as in hematological malignancies and cells that form solid tumors within a particular tissue or organ of a subject. Solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

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

"conservative substitutions" are considered in the art to be amino acid substitutions of one amino acid for another having similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published 3/13 1997; lehninger, biochemistry, second edition; walsh Publishers, inc.) New York (1975), pages 71-77; lewis, genes IV, oxford university Press (Oxford University Press), new York and Cell Press (Cell Press), cambridge, massachusetts (1990), page 8).

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

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

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

An "inducible" promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded by or specified by a gene, results in the production of a gene product in a cell, essentially only if the cell is a cell of the tissue type corresponding to the promoter.

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

A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. The vector may be, for example, a plasmid, cosmid, virus or phage. 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" refers to a vector that, when present in an appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector.

In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma retrovirus vectors, and lentiviral vectors. A "retrovirus" is a virus having an RNA genome. "Gamma retrovirus" refers to a genus of the family retrovirus. Examples of gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "lentivirus" refers to a genus of retrovirus capable of infecting dividing cells and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus (FIV), bovine Immunodeficiency Virus (BIV), 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-amplified mRNA, closed-end linear duplex (CELiD) DNA, and transposon mediated gene transfer (PiggyBac). In the case of non-viral delivery systems, the delivery vehicle may be a liposome. The lipid formulation may be used to introduce nucleic acids into host cells in vitro, ex vivo, or in vivo. The nucleic acid may be encapsulated within the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with the liposome and the nucleic acid, contained within or complexed with the micelle, or otherwise associated with the lipid.

As used herein, the term "phagocytosis" refers to receptor-mediated processes in which endogenous or exogenous cells or particles having a diameter greater than 100nm are internalized by the phagocytes or host cells of the present disclosure. Phagocytosis typically consists of multiple steps: (1) Binding the target cell or particle via direct or indirect (via bridging molecules) binding of the 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 a portion thereof. In certain embodiments, internalization may occur via cytoskeletal rearrangement of the phagocyte or host cell to form phagosome, i.e., a membrane-bound compartment containing the internalized target. Phagocytosis may further comprise maturation of phagosome, wherein phagosome becomes more and more acidic and fuses with lysosomes (to form phagolysosomes), whereby the target being phagocytosed is degraded (e.g., "phagocytosis"). Alternatively, phagosome-lysosomal fusion may not be observed during phagocytosis. In yet other embodiments, the phagosome may reflux or drain its contents into the extracellular environment before complete degradation. In some embodiments, phagocytosis refers to phagocytosis. In some embodiments, phagocytes include the binding of target cells or particles by phagocytes of host cells of the present disclosure, but do not include internalization. In some embodiments, phagocytosis comprises the binding of a target cell or particle by a phagocyte of a host cell of the present disclosure and internalization of a portion of the target cell or particle.

As used herein, the term "phagocytosis" refers to the process of phagocytosis of cells or large particles (> 0.5 μm) in which binding of target cells or particles, phagocytosis of target cells or particles, and degradation of internalized target cells or particles occurs. In certain embodiments, phagocytosis comprises the formation of phagosome that encompasses internalized target cells or particles, and the phagosome fuses with lysosomes to form phagosome, wherein the contents of the phagosome are degraded. In certain embodiments, during phagocytosis, a chimeric Tim receptor expressed on a host cell of the present disclosure forms a phagocytic synapse upon binding to phosphatidylserine expressed by a target cell or particle; producing an actin-rich phagocytic cup at the phagocytic synapse; phagocytic arms extend around target cells or particles through cytoskeletal rearrangement; and eventually, the target cell or particle is pulled into the phagocytic cell or host cell by the force generated by the motor protein. As used herein, "phagocytosis" encompasses the process of "cytoburial", which specifically refers to phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.

The term "immune system cell" or "immune cell" refers to any cell of the immune system derived from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells produce two major lineages, myeloid progenitor cells (producing myeloid cells, such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (producing lymphoid cells, such as T cells, B cells, and Natural Killer (NK) cells). Exemplary immune system cells include cd4+ T cells, cd8+ T cells, CD4-CD 8-double negative T cells, γδ T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells may also be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when the Major Histocompatibility Complex (MHC) receptor on the surface of APCs complexed with a peptide interacts with a TCR on the surface of the T cells.

The term "T cell" refers to a cell of the T cell lineage. By "cells of the T cell lineage" is meant cells that exhibit at least one phenotypic characteristic of a T cell or precursor or progenitor cell thereof, which distinguishes the cell from other lymphoid cells and cells of the erythroid or myeloid lineage. Such phenotypic characteristics may include one or more proteins specific for T cells (e.g., CD3 ⁺ 、CD4 ⁺ 、CD8 ⁺ ) Or a physiological, morphological, functional or immunological characteristic specific for T cells. For example, cells of the T cell lineage can be progenitor or precursor cells committed to the T cell lineage; CD25 ⁺ Immature and unactivated T cells; cells that have undergone CD4 or CD8 lineage targeting; CD4 ⁺ CD8 ⁺ Double positive thymus progenitor cells; single positive CD4 ⁺ Or CD8 ⁺ The method comprises the steps of carrying out a first treatment on the surface of the Tcrαβ or tcrγδ; or mature and functional or activated T cells. The term "T cell" encompasses naive T cells (cd45ra+, ccr7+, cd62l+, cd27+, CD45 RO-), central memory T cells (CD 45 RO) ⁺ 、CD62L ⁺ 、CD8 ⁺ ) Effector memory T cells (cd45ra+, CD45RO-, CCR7-, CD62L-, CD 27-), mucosa-associated invariant T (MAIT) cells, tregs, natural killer T cells and tissue resident T cells.

The term "B cell" refers to a cell of the B cell lineage. By "cells of the B cell lineage" is meant cells that exhibit at least one phenotypic characteristic of B cells or precursors or progenitors thereof, which distinguish the cells from other lymphoid cells and cells of the erythroid or myeloid lineage. Such phenotypic characteristics may include one or more proteins specific for B cells (e.g., CD19 ⁺ 、CD72+、CD24+、CD20 ⁺ ) Or a physiological, morphological, functional or immunological characteristic specific for B cells. For example, the cells of the B cell lineage can be progenitor cells or precursor cells committed to the B cell lineage (e.g., pre-progenitor-B cells, and pre-B cells); immature and unactivated B cells or mature and functional or activated B cells. Thus, "B cells" encompass naivePrimary B cells, plasma cells, regulatory B cells, border zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablasts, and memory B cells (e.g., CD 27) ⁺ 、IgD ^- )。

The term "cytotoxic activity", also referred to as "cytolytic activity", with respect to cells (e.g., T cells or NK cells) expressing on their surface an immune receptor (e.g., TCR) or chimeric Tim receptor according to the present disclosure, means that upon antigen-specific signaling (e.g., via TCR, chimeric Tim receptor), the cells induce the target cells to undergo apoptosis. In some embodiments, the cytotoxic cells may induce apoptosis in the target cells via release of cytotoxins, such as perforins, granzymes, and granulysins, from the particles. Perforin is inserted into the target cell membrane and forms pores that allow water and salts to quickly enter the target cell. Granzymes are serine proteases that induce apoptosis in target cells. Granulysin is also capable of forming pores in the target cell membrane and is a pro-inflammatory molecule. In some embodiments, cytotoxic cells can induce apoptosis in target cells via the interaction of Fas ligand with Fas molecules expressed on the target cells, which are up-regulated on T cells following antigen specific signaling. Fas is an apoptotic signaling receptor molecule on the surface of many different cells.

The term "depletion" in reference to an immune cell refers to a state of immune cell dysfunction, which is defined as a state of poor effector function (e.g., reduced cytokine production, reduced cytotoxic activity), reduced proliferative capacity, increased expression of an immunocheckpoint molecule, and transcription that is different from a functional effector or memory cell. In certain embodiments, the depleted immune cells become unresponsive to the presence of their target antigens. Immune cell depletion may be due to chronic exposure to the target antigen (e.g., possibly due to chronic infection) or when it enters an immunosuppressive environment (e.g., tumor microenvironment). In certain embodiments, immune cell depletion refers to T cell depletion, NK cell depletion, or both. In certain embodiments, the depleted T cells exhibit: (a) Increased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof; (b) Reduced production of IFN-gamma, IL-2, TNF-alpha, or any combination thereof; or both (a) and (b). In certain embodiments, the depleted NK cells exhibit: (a) Increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) reduced production of IFN- γ, TNF- α, or both; or both (a) and (b).

A "disease" is a state of health of a subject, wherein the subject is unable to maintain homeostasis, and wherein the subject's health continues to deteriorate if the disease is not improved. Conversely, a "disorder" or "undesired condition" of a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is not as good as in the absence of the disorder or undesired condition. The disorder or condition does not necessarily result in a further decrease in the health of the subject if not treated.

As used herein, the term "cancer" is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Abnormal cells may form solid tumors or constitute hematological malignancies. Cancer cells can spread locally or through the blood stream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal 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 encompass a living organism (e.g., a mammal) in which an immune response may be elicited. Examples of subjects include humans, primates, cattle, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, and transgenic species thereof.

"adoptive cell immunotherapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy may be autologous (immune cells from the recipient), allogeneic (immune cells from a donor of the same species) or syngeneic (immune cells from a donor genetically identical to the recipient).

By "autologous" is meant any material (e.g., organ, tissue, graft of cells) from the same subject that is subsequently reintroduced into the subject.

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

A "therapeutically effective amount" or "effective amount" of a chimeric protein or a cell expressing a chimeric protein of the present disclosure (e.g., a chimeric Tim receptor or a cell expressing a chimeric Tim receptor) refers to an amount of a protein or cell sufficient to result in an improvement in one or more symptoms of the disease, disorder, or undesired condition being treated. When referring to a single active ingredient administered alone or a cell expressing a single active ingredient, a therapeutically effective dose refers to the effect of the ingredient administered alone or the cell expressing the ingredient. When referring to a combination, a therapeutically effective dose refers to the combined amount of the active ingredients or combined co-active ingredients and the cells expressing the active ingredients that produce a therapeutic effect, whether administered sequentially or simultaneously.

"treatment" or "amelioration" refers to the medical management of a disease, disorder, or undesired condition in a subject. Typically, the appropriate dose or treatment regimen comprising host cells expressing the chimeric proteins of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventative benefits include improved clinical outcome; alleviating or alleviating symptoms associated with a disease, disorder, or undesired condition; reducing the occurrence of symptoms; improving the quality of life; prolonging disease-free status; reducing the extent of a disease, disorder, or undesired condition; stabilizing the disease state; delay disease progression; relief; survival; prolonging the life cycle; or any combination thereof.

The term "anti-tumor effect" refers to a biological effect that may be manifested as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancer condition. "anti-tumor effects" may also be manifested by prevention of hematological malignancies or neoplasia.

An "autoimmune disease" refers to a condition caused by an autoimmune response. Autoimmune diseases are the result of an excessive response to self-antigens. The autoimmune response may involve autoreactive B cells, autoreactive T cells, or both that produce the autoantibody. 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 described above, the methods of the present disclosure can be used to treat cancer in a subject. The therapeutic agents of the present disclosure (i.e., chimeric Tim receptor and PARP inhibitor), when administered together or sequentially, treat cancer in a subject (e.g., either (i) prevent the progression of cancer, (ii) cause regression of cancer, or (iii) alleviate symptoms caused by cancer).

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

Chimeric Tim receptors

The present disclosure provides a chimeric Tim receptor comprising a single-chain chimeric protein comprising: an extracellular domain comprising a Tim binding domain; an intracellular signaling domain comprising a first costimulatory signaling domain; and a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. In certain embodiments, the extracellular domain of a chimeric Tim receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain.

When expressed in a host cell, the chimeric Tim receptors of the present disclosure can confer a modified host cell phosphatidylserine specific cytotoxic phenotype (e.g., a host cell becomes cytotoxic to stressed, damaged, injured, apoptotic, or necrotic cells expressing phosphatidylserine on its surface). In certain embodiments, the chimeric Tim receptor induces apoptosis in the targeted cells via release of granzyme, perforin, granulysin, or any combination thereof. In further embodiments, cells expressing a chimeric Tim receptor according to the present description exhibit a phagocytic phenotype specific for phosphatidylserine presenting cells.

The intracellular signaling domain may comprise one or more effector domains capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the chimeric Tim receptor to phosphatidylserine. Signaling through the intracellular signaling domain is triggered by the binding of the extracellular domain to phosphatidylserine. The signal transduced by the intracellular signaling domain promotes effector function of the cells containing the chimeric Tim receptor. Examples of effector functions include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, amplification, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen presentation, or any combination thereof.

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. Chimeric Tim receptors according to the present disclosure can be used in a variety of therapeutic methods, where scavenging apoptotic, necrotic, injured or stressed cells is beneficial, while providing co-stimulation that enhances cellular immune responses, reduces immune cell depletion, or both.

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

Extracellular domain

The Tim4 binding domain of the chimeric Tim4 receptor suitable for use in the present disclosure can be any polypeptide or peptide derived from a Tim4 molecule that specifically binds phosphatidylserine. In certain embodiments, the Tim4 binding domain is derived from human Tim4. An exemplary human Tim4 molecule is provided in Uniprot reference number Q96H15 (SEQ ID NO: 1). Exemplary human Tim4 binding domains include or consist of the amino acid sequence of SEQ ID NO. 2 or amino acids 25-314 of SEQ ID NO. 2. Exemplary mouse Tim4 binding domains include or consist of the amino acid sequence of SEQ ID NO:24 or amino acids 23-279 of SEQ ID NO: 24. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to amino acids 25-314 of SEQ ID NO. 2 or amino acids 23-279 of SEQ ID NO. 24 or SEQ ID NO. 24. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO. 2 or amino acids 25-314 of SEQ ID NO. 2 or amino acids 23-279 of SEQ ID NO. 24 or SEQ ID NO. 24.

In certain embodiments, the extracellular domain optionally comprises an extracellular non-signaling spacer or a linker domain. Such spacer or linker domains, if included, can position the binding domain at a location remote from the surface of the host cell to further achieve proper cell/cell contact, binding and activation. When included in a chimeric receptor as described herein, the extracellular spacer domain is typically 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, the selected binding epitope, the binding domain size, and affinity (see, e.g., guest et al, journal of immunotherapy (j. Immunother.), 28:203-11, 2005; pct publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., igG1, igG2, igG3, igG4, igA, igD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Altered IgG is described in PCT publication No. WO 2014/031687 ₄ A hinge region, which is incorporated herein by reference in its entirety. In particular embodiments, the extracellular spacer domain comprises a modified IgG ₄ A hinge region having the amino acid sequence of ESKYGPPCPPCP (SEQ ID NO: 3). Other examples of hinge regions that may be used for the chimeric Tim4 receptors described herein include hinge regions from extracellular regions of type 1 membrane proteins (e.g., CD8a, CD4, CD28, and CD 7), which may be wild-type or variants thereof. In some embodiments, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO. 32. In further embodiments, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT publication WO2014/031687, which is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may include a stem region of a type II C-lectin (an extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins comprise CD23, CD69, CD72, CD94, NKG2A and NKG2D.

In certain embodiments, the extracellular domain comprises a polynucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, transgenic species thereof, or any combination thereof. In certain embodiments, the extracellular domain is murine, human, or chimeric.

The intracellular signaling domain of the chimeric Tim4 receptor as described herein is an intracellular effector domain and is capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the chimeric Tim4 receptor to phosphatidylserine. The signal transduced by the intracellular signaling domain promotes effector function of cells containing the chimeric Tim4 receptor. Examples of effector functions include cytotoxic activity, cytokine secretion, proliferation, anti-apoptotic signaling, persistence, amplification, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, or any combination thereof.

In certain embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. The costimulatory signaling domain may be any portion of a costimulatory signaling molecule that retains sufficient signaling activity. In some embodiments, full length or full length intracellular components of the costimulatory signaling molecule are used. In some embodiments, a truncated portion of a costimulatory signaling molecule, or an 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 the entire or truncated portion of the costimulatory signaling molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

In certain embodiments, the costimulatory signaling domain comprises the CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), CD226, CD270 (HVEM), PD-1, CD273 (PD-L2), CD274 (PD-L1), B7-H3 (CD 276), ICOS (CD 278), DAP10, LAT, LFA-1 (CD 11a/CD 18), LIGHT, NKG2C, SLP76, TRIM, or ZAP70 signaling domain. In particular embodiments, the costimulatory signaling domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), or 4-1BB (CD 137) signaling domain. Exemplary CD28 costimulatory signaling domains include or consist of the amino acid sequence of SEQ ID NO:118 or 119. An exemplary OX40 co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO. 120. An exemplary CD2 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 121. An exemplary 4-1BB costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 122. An exemplary CD27 co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO. 123. An exemplary ICAM-1 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 124. An exemplary LFA-1 co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO. 125. An exemplary ICOS co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO. 126. An exemplary CD30 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 127. An exemplary CD40 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 128. An exemplary PD-1 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 129. An exemplary CD7 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 130. An exemplary LIGHT costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID No. 131. An exemplary NKG 2C-costimulatory-signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 132. An exemplary B7-H3 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 133. In certain embodiments, the costimulatory signaling domain comprises or consists of an amino acid sequence that has at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any of SEQ ID NOs 118-133. In certain embodiments, the costimulatory signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of any of SEQ ID NOs 118-133. In certain embodiments, intracellular signaling comprises a second costimulatory signaling domain. In a preferred embodiment, the first and second co-stimulatory signaling domains are different.

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

Examples of ITAM-containing activation domains that can be used for the chimeric Tim4 receptors of the present disclosure include those derived from cd3ζ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD278 (ICOS), and CD66 d. In a particular embodiment, the ITAM-containing activation domain is a CD3 zeta signaling domain. Exemplary CD3 zeta signaling domains include or consist of the amino acid sequence of SEQ ID NO 134 or 135. An exemplary CD3 gamma signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 136. An exemplary CD3δ signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 137. An exemplary CD3 epsilon signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 138. Exemplary CD5 signaling domains include or consist of the amino acid sequence of SEQ ID NO 139. Exemplary CD22 signaling domains include or consist 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, an activation domain comprising ITAM comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs 134-141 and 143. In certain embodiments, an activation domain comprising ITAM comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of any of SEQ ID NOs 134-141 and 143.

In another embodiment, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a CD3 zeta signaling domain. In another embodiment, the intracellular signaling domain comprises a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain. In yet other embodiments, the intracellular signaling domain comprises a CD27 costimulatory signaling domain and a CD3 zeta signaling domain. In another embodiment, the intracellular signaling domain comprises an ICOS costimulatory signaling domain and a CD3 zeta signaling domain. In another embodiment, the intracellular signaling domain comprises an LFA-1 costimulatory signaling domain and a CD3 zeta signaling domain. In another embodiment, the intracellular signaling domain comprises an OX40 costimulatory signaling domain and a CD3 zeta signaling domain. In yet other embodiments, the intracellular signaling domain comprises a CD2 costimulatory signaling domain and a CD3 zeta signaling domain. In yet another embodiment, the intracellular signaling domain comprises an ICAM-1 costimulatory signaling domain and a CD3 zeta signaling domain.

The intracellular signaling domain may be from mammalian species, including humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, and transgenic species thereof.

The transmembrane domain of the chimeric Tim4 receptor connects and is located between the extracellular domain and the intracellular signaling domain. The transmembrane domain is a hydrophobic alpha helix that passes through the host cell membrane. The transmembrane domain may be fused directly to the binding domain or extracellular spacer domain, if present. In certain embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptor, cluster of Differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, etc.). In one embodiment, the transmembrane domain is selected from the same molecules as the molecules from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimeric Tim4 receptor can include a Tim4 binding domain and a Tim4 transmembrane domain. In another example, the chimeric Tim4 receptor can include a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are from different molecules; the transmembrane domain and the intracellular signaling domain are from different molecules; or the transmembrane domain, extracellular domain and intracellular signaling domain are all from different molecules. Examples of transmembrane domains that may be used for the chimeric Tim4 receptor of the present disclosure include transmembrane domains from 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. Exemplary Tim4 transmembrane domains include or consist 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. Exemplary CD28 transmembrane domains include or consist 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. Exemplary OX40 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO: 147. Exemplary CD27 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 148. Exemplary ICOS transmembrane domains include or consist of the amino acid sequence of SEQ ID NO: 149. Exemplary CD2 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 150. Exemplary LFA-1 transmembrane domains include or consist 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. Exemplary CD40 transmembrane domains include or consist 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. Exemplary CD7 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO: 155. Exemplary LIGHT transmembrane domains include or consist of the amino acid sequence of SEQ ID No. 156. Exemplary NKG 2C-transmembrane domains comprise or consist of the amino acid sequence of SEQ ID NO. 157. Exemplary B7-H3 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 158. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs 8, 23 and 144-158. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of any of SEQ ID NOs 8, 23, and 144-158.

The transmembrane domain may be from any mammalian species, including human, primate, bovine, equine, caprine, ovine, canine, feline, murine, rat, rabbit, guinea pig, porcine, and transgenic species thereof.

Exemplary components, configurations, and chimeric Tim4 receptor sequences of the present disclosure are provided in table 1.

Table 1.

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Additional embodiments of the chimeric Tim4 receptors described herein include single-chain chimeric proteins comprising: an extracellular domain comprising a Tim4 binding domain; an intracellular signaling domain comprising a first costimulatory signaling domain and an ITAM-containing activation domain, wherein the ITAM-containing activation domain comprises a DAP12 signaling domain; and a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

In certain embodiments, the extracellular domain optionally comprises an extracellular non-signaling spacer or a linker domain. Such spacer or linker domains, if included, can position the binding domain at a location remote from the surface of the host cell to further achieve proper cell/cell contact, binding and activation. When included in a chimeric receptor as described herein, the extracellular spacer domain is typically 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 based on the selected target molecule, the selected binding epitope, the binding domain size, and affinityTarget molecule binding (see, e.g., guest et al, journal of immunotherapy, 28:203-11, 2005; PCT publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., igG1, igG2, igG3, igG4, igA, igD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Altered IgG is described in PCT publication No. WO 2014/031687 ₄ A hinge region, which is incorporated herein by reference in its entirety. In particular embodiments, the extracellular spacer domain comprises a modified IgG ₄ A hinge region having the amino acid sequence of ESKYGPPCPPCP (SEQ ID NO: 3). Other examples of hinge regions that may be used for the chimeric Tim4 receptors described herein include hinge regions from extracellular regions of type 1 membrane proteins (e.g., CD8a, CD4, CD28, and CD 7), which may be wild-type or variants thereof. In some embodiments, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO. 32. In further embodiments, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT publication WO2014/031687, which is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may include a stem region of a type II C-lectin (an extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins comprise CD23, CD69, CD72, CD94, NKG2A and NKG2D.

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. The costimulatory signaling domain may be any portion of a costimulatory signaling molecule that retains sufficient signaling activity. In some embodiments, full length or full length intracellular components of the costimulatory signaling molecule are used. In some embodiments, a truncated portion of a costimulatory signaling molecule, or an 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 the entire or truncated portion of the costimulatory signaling molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

In certain embodiments, the costimulatory signaling domain comprises the CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), CD226, CD270 (HVEM), PD-1, CD273 (PD-L2), CD274 (PD-L1), B7-H3 (CD 276), ICOS (CD 278), DAP10, LAT, LFA-1 (CD 11a/CD 18), LIGHT, NKG2C, SLP76, or TRIM signaling domain. In particular embodiments, the costimulatory signaling domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), or 4-1BB (CD 137) signaling domain. Exemplary CD28 costimulatory signaling domains include or consist of the amino acid sequence of SEQ ID NO. 164 or 165. An exemplary OX40 co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO. 166. An exemplary CD2 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 167. An exemplary 4-1BB costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 168. An exemplary CD27 co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO. 169. An exemplary ICAM-1 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 170. An exemplary LFA-1 co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO: 171. An exemplary ICOS co-stimulatory signaling domain includes or consists of the amino acid sequence of SEQ ID NO. 172. An exemplary CD30 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 173. An exemplary CD40 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 174. An exemplary PD-1 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 175. An exemplary CD7 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 176. Exemplary LIGHT co-stimulatory signaling domains include or consist of the amino acid sequence of SEQ ID NO. 177. An exemplary NKG 2C-costimulatory-signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 178. An exemplary B7-H3 costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 179. In certain embodiments, the costimulatory signaling domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any of SEQ ID NOs 164-179. In certain embodiments, the costimulatory signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of any of SEQ ID NOs 164-179. In certain embodiments, intracellular signaling comprises a second costimulatory signaling domain. In a preferred embodiment, the first and second co-stimulatory signaling domains are different.

The DAP12 signaling domain may reproduce TCR signaling independently of the endogenous TCR complex. In certain embodiments, signaling via the DAP12 signaling domain results in the mediation of T cell responses, including but not limited to proliferation, activation, differentiation, and the like. The DAP12 signaling domain may be any moiety in the DAP12 molecule that retains sufficient signaling activity. In some embodiments, the full length or full length intracellular component of the DAP12 molecule is used. In some embodiments, DAP12 or a truncated portion of 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 the intact or truncated portion of DAP12, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

Exemplary human DAP12 molecules are provided in Uniprot reference 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 comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO. 180. In certain embodiments, the DAP12 signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO. 180.

In another embodiment, the intracellular signaling domain comprises a CD28 costimulatory signaling domain and a DAP12 signaling domain. In another embodiment, the intracellular signaling domain comprises a 4-1BB costimulatory signaling domain and a DAP12 signaling domain. In yet other embodiments, 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 costimulatory 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 other embodiments, 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 costimulatory signaling domain and a DAP12 signaling domain.

In certain embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptor, cluster of Differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, etc.). In one embodiment, the transmembrane domain is selected from the same molecules as the molecules from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimeric Tim4 receptor can include a Tim4 binding domain and a Tim4 transmembrane domain. In another example, the chimeric Tim4 receptor can include a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are from different molecules; the transmembrane domain and the intracellular signaling domain are from different molecules; or the transmembrane domain, extracellular domain and intracellular signaling domain are all from different molecules. Examples of transmembrane domains that may be used for the chimeric Tim4 receptor of the present disclosure include transmembrane domains from Tim4, CD3 ζ, CD3 γ, CD3 δ, CD3 epsilon, 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. An exemplary Tim4 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO 144 or 23. Exemplary CD28 transmembrane domains include or consist 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 includes 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. Exemplary ICOS transmembrane domains include or consist of the amino acid sequence of SEQ ID NO: 185. Exemplary CD2 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 186. Exemplary LFA-1 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 187. Exemplary CD30 transmembrane domains include or consist 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. Exemplary CD7 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 191. Exemplary LIGHT transmembrane domains include or consist of the amino acid sequence of SEQ ID No. 192. An exemplary NKG 2C-transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO. 193. Exemplary B7-H3 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO: 194. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs 23, 144 and 181-194. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of any of SEQ ID NOs 23, 144, and 181-194.

In certain embodiments, the chimeric Tim4 receptor comprises a polynucleotide sequence from any mammalian species including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, transgenic species thereof, or any combination thereof. In certain embodiments, the chimeric Tim4 receptor is murine, chimeric, human, or humanized.

It is to be understood that direct fusion of one domain of the chimeric Tim4 receptor described herein to another domain does not preclude the presence of an intermediate linking amino acid. The linking amino acids may be natural or unnatural (e.g., from the construct design of the chimeric protein). For example, the linking amino acids may be from restriction enzyme sites used to link one domain to another domain or to clone a polynucleotide encoding a chimeric Tim4 receptor into a vector.

Exemplary components, configurations, and chimeric Tim4 receptor sequences of the present disclosure are provided in table 2.

Table 2.

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In embodiments, the chimeric Tim receptor comprises a single chain chimeric protein comprising: (a) An extracellular domain comprising a binding domain, the 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, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

In particular embodiments, the binding domain comprises: (i) a Tim1 IgV domain and a Tim1 mucin domain; (ii) a Tim4 IgV domain and a Tim4 mucin domain; (iii) a Tim1 IgV domain and a Tim4 mucin domain; or (iv) a Tim4 IgV domain and a Tim1 mucin domain. In certain embodiments, the extracellular domain of a chimeric Tim receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain.

The intracellular signaling domain may comprise one or more effector (also referred to as "costimulatory signaling") domains that costimulatory the modified host cell. Signaling through the intracellular signaling domain is triggered by the 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. Chimeric Tim receptors according to the present disclosure can be used in a variety of therapeutic methods, where scavenging apoptotic, necrotic, injured or stressed cells is beneficial, while providing co-stimulation that enhances cellular immune responses, reduces immune cell depletion, or both.

As described herein, the chimeric Tim receptor includes an extracellular domain that includes a Tim binding domain. The Tim binding domain confers phosphatidylserine (PtdSer) specificity, a negatively charged head group of phospholipids and is a component of the cell membrane. In healthy cells, phosphatidylserine is preferentially found in the inner leaflet of the cell membrane. However, when cells are stressed, damaged or undergo apoptosis or necrosis, phosphatidylserine is exposed on the outer leaflet of the cell membrane. Thus, phosphatidylserine can be used as a marker to distinguish stressed cells, damaged cells, apoptotic cells, necrotic cells, pyroapoptotic cells, or distending cells. Binding of phosphatidylserine via the Tim binding domain can block interactions between phosphatidylserine and another molecule, and for example, interfere with, reduce, or eliminate certain functions of phosphatidylserine (e.g., signal transduction). In some embodiments, the binding of phosphatidylserine may induce certain biological pathways or recognize phosphatidylserine molecules or cells expressing phosphatidylserine for elimination.

The Tim binding domain of the chimeric Tim receptor suitable for use in the present disclosure can be any polypeptide or peptide derived from a Tim1 and/or Tim4 molecule that specifically binds phosphatidylserine. In embodiments, the Tim binding domain comprises an IgV domain from Tim1 or Tim4 and a mucin domain from Tim1 or Tim 4. For example, a Tim binding domain can include a Tim1IgV domain and a Tim1 mucin domain. In another example, the Tim binding domain can include a Tim1IgV domain and a Tim4 mucin domain. In another example, the 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 typically regulated by IgV domains. The core phosphatidylserine binding domain is the four amino acid sequence in the IgV domain (e.g., amino acids 95-98 of SEQ ID NO:34 or amino acids 92-95 of SEQ ID NO: 38). The Tim4 binding domain binds minimally to cells of low phosphatidylserine density. The Tim1 binding domain binds more strongly to lower phosphatidylserine densities, resulting in lower response thresholds. A summary of the binding of Tim1 and Tim4 to phosphatidylserine is provided in table 3. The binding affinity of the binding domain to phosphatidylserine can be modulated by combining a Tim1IgV domain and a Tim4 mucin domain, or a Tim4 IgV domain and a Tim1 mucin domain. Furthermore, this combination of Tim binding domains also provides a combination of sensitivity of Tim4 to phosphatidylserine and stability in protein expression of Tim 1.

Table 3.

	Low PtdSer	Moderate PtdSer	High PtdSer	Cooperativity of
					Tim-1	Moderate bonding	Strong binding	Strong binding
Tim-4	Minimum binding	Moderate bonding	Strong binding	Is that

Furthermore, the RGD domain in the IgV domain (e.g., amino acids 68-70 of SEQ ID NO: 34) may regulate integrin binding as a phagocytic co-receptor.

In certain embodiments, the Tim binding domain is derived from human Tim1 and/or Tim4. An exemplary human Tim1 molecule (SEQ ID NO: 36) is provided in Uniprot reference Q96D 42. Exemplary human Tim1 binding domains include or consist of the amino acid sequence of SEQ ID NO. 37 or SEQ ID NO. 43. In certain embodiments, a Tim1 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO. 37 or SEQ ID NO. 43. In certain embodiments, a Tim1 binding domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO. 37 or SEQ ID NO. 43.

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

In embodiments, the Tim binding domain comprises an IgV domain from Tim 1. 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 that includes the R66G substitution in SEQ ID NO: 38. R66G substitution (e.g., amino acids 68-70 of SEQ ID NO: 34) confers an RGD domain in the Tim1 IgV domain that may regulate integrin binding as a phagocytic co-receptor. In particular embodiments, the modified Tim1 IgV domain comprises or consists of the amino acid sequence of SEQ ID NO. 41. In some embodiments, such modified Tim1 domains can increase phagocytic activity while maintaining Tim1 sensitivity. In certain embodiments, a Tim1 IgV domain comprises or consists of an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:38, SEQ ID NO:38 with R66G substitution, or SEQ ID NO: 41. In certain embodiments, tim1 IgV comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO:38, SEQ ID NO:38 with R66G substitution, or SEQ ID NO: 41.

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

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

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

In some embodiments, the Tim binding domain comprises a Tim1 IgV domain and a Tim1 mucin domain. In a particular embodiment, 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 shown in SEQ ID NO:38 with the R66G substitution and the Tim1 mucin domain comprises the amino acid sequence shown in SEQ ID NO: 39. In further specific embodiments, the Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID NO. 41 and the Tim1 mucin domain comprises the amino acid sequence set forth in SEQ ID NO. 39. In some embodiments, the Tim1 IgV domain and the Tim1 mucin domain together 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 a Tim4 IgV domain and a Tim4 mucin domain. In a particular embodiment, 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 together 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 a Tim1 IgV domain and a Tim4 mucin domain. In a particular embodiment, 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 a particular embodiment, the Tim1 IgV domain comprises the amino acid sequence shown in SEQ ID NO:38 with the R66G substitution and the Tim4 mucin domain comprises the amino acid sequence shown in SEQ ID NO: 35. In further specific embodiments, 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 includes the Tim1 signal sequence of SEQ ID NO. 40.

In still further embodiments, the Tim binding domain comprises a Tim4 IgV domain and a Tim1 mucin domain. In a particular embodiment, 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 includes the Tim4 signal sequence of SEQ ID NO. 11.

In certain embodiments, the extracellular domain optionally comprises an extracellular non-signaling spacer or a linker domain. Such spacer or linker domains, if included, can position the binding domain at a location remote from the surface of the host cell to further achieve proper cell/cell contact, binding and activation. When included in a chimeric receptor as described herein, the extracellular spacer domain is typically located 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, the selected binding epitope, the binding domain size, and affinity (see, e.g., guest et al, journal of immunotherapy, 28:203-11, 2005; pct publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., igG1, igG2, igG3, igG4, igA, igD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Described in PCT publication number WO 2014/031687 The altered IgG ₄ A hinge region, which is incorporated herein by reference in its entirety. In particular embodiments, the extracellular spacer domain comprises a modified IgG ₄ A hinge region having the amino acid sequence of ESKYGPPCPPCP (SEQ ID NO: 3). Other examples of hinge regions that may be used for the chimeric Tim receptors described herein include hinge regions from extracellular regions of type 1 membrane proteins (e.g., CD8a, CD4, CD28, and CD 7), which may be wild-type or variants thereof. In a particular embodiment, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO. 32. In further embodiments, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT publication WO2014/031687, which is incorporated herein by reference in its entirety). In further embodiments, the extracellular spacer domain may include a stem region of a type II C-lectin (an extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins comprise CD23, CD69, CD72, CD94, NKG2A and NKG2D.

In certain embodiments, the extracellular domain comprises an amino acid sequence from any mammalian species, including human, primate, cow, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, transgenic species thereof, or any combination thereof. In certain embodiments, the extracellular domain is murine, human, or chimeric.

The intracellular signaling domain of the chimeric Tim receptor as described herein is an intracellular effector domain and is capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the chimeric Tim receptor to phosphatidylserine. The signal transduced by the intracellular signaling domain promotes effector function of the cells containing the chimeric Tim receptor. Examples of effector functions include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, amplification, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen capture, antigen processing, antigen presentation, or any combination thereof.

The intracellular signaling domain comprises a primary intracellular signaling domain. 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. The primary intracellular signaling domain, the secondary intracellular signaling domain, and/or the tertiary intracellular signaling domain may independently be any portion of a signaling molecule that retains sufficient signaling activity. In some embodiments, a full-length signaling molecule or a full-length intracellular component of a signaling molecule is used. In some embodiments, a truncated portion of the signaling molecule or an intracellular component of the signaling molecule is used, provided that the truncated portion retains sufficient signaling activity. In some embodiments, the signaling domain is a variant of a complete or truncated portion of a signaling molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

In some embodiments, the primary intracellular signaling domain comprises a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3 zeta signaling domain, a 4-1BB signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

In embodiments, the secondary intracellular signaling domain comprises a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3 zeta signaling domain, a 4-1BB signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

In some embodiments, the tertiary intracellular signaling domain comprises a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3 zeta signaling domain, a 4-1BB signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

In some embodiments, the primary intracellular signaling domain comprises a signaling domain comprising an immune receptor tyrosine activation motif (ITAM); the secondary intracellular signaling domain comprises a costimulatory signaling domain, a Tim1 signaling domain, or a Tim4 signaling domain; and the tertiary intracellular signaling domain comprises a TLR signaling domain. The signaling domain containing ITAM typically contains at least one (one, two, three, four or more) ITAM, which refers to YXXL/I-X _6-8 Conserved motifs of YXXL/I. The signaling domain containing ITAM can initiate T cell activation signaling following antigen binding or ligand binding. The ITAM signaling domain comprises intracellular signaling domains such as cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP12, fcrγ, and CD66 d. The costimulatory signaling domain, when activated with a primary or classical (e.g., ITAM-driven) activation signal, promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. Co-stimulatory signaling domains for chimeric Tim receptors include, for example, CD27, CD28, CD40L, GITR, NKG C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), 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 costimulatory signaling domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), or 4-1BB (CD 137) signaling domain. The TLR signaling domain may 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 intracellular signaling domain, secondary intracellular signaling domain, and tertiary intracellular signaling domain include, but are not limited to, an arrangement of primary intracellular signaling domain at the N-terminus, intermediate secondary intracellular signaling domain, and tertiary intracellular signaling domain at the C-terminus of the intracellular portion of the chimeric Tim receptor. Thus, the naming of the primary intracellular signaling domain does not limit the use of the selected intracellular signaling domain at the N-terminus of the intracellular portion of the chimeric Tim receptor. The naming of the secondary intracellular signaling domains does not limit the use of the selected intracellular signaling domain in the middle of the intracellular portion of the chimeric Tim receptor (or at the C-terminus for those chimeric Tim receptors having only two intracellular signaling domains). The naming of tertiary intracellular signaling domains does not limit the use of selected intracellular signaling domains at the C-terminus of the intracellular portion of the chimeric Tim receptor. Thus, different arrangements of primary, secondary and/or tertiary intracellular signaling domains in 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 include or consist of the amino acid sequences of SEQ ID NO. 45, SEQ ID NO. 224, or SEQ ID NO. 225. Exemplary TRAF2 signaling domains include or consist of the amino acid sequence of SEQ ID NO. 48. Exemplary TRAF6 signaling domains include or consist of the amino acid sequence of SEQ ID NO. 46. Exemplary CD28 signaling domains include or consist 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. Exemplary CD3 zeta signaling domains include or consist 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. Exemplary TLR2 signaling domains include or consist 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 a particular embodiment, 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 shown in SEQ ID NO. 48. In another specific embodiment, the TRAF6 signaling domain comprises the amino acid sequence shown 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 particular embodiment, the DAP12 signaling domain comprises the amino acid sequence shown in SEQ ID NO. 9. In another specific embodiment, the CD3 zeta signaling domain comprises the amino acid sequence shown in SEQ ID NO. 27. In another specific embodiment, the CD3 zeta 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, either the TLR8 signaling domain comprises the amino acid sequence set forth in SEQ ID NO. 47.

In embodiments, the primary signaling domain, secondary signaling domain, and/or tertiary signaling domain (if present) comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any of SEQ ID NOs 4, 5, 9, 26, 27, or 44-48. In certain embodiments, the primary signaling domain, the secondary signaling domain, and/or the tertiary signaling domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of any of SEQ ID NOs 4, 5, 9, 26, 27, 44-48, 122, and 222. In embodiments, the primary signaling domain, the secondary signaling domain, and/or the tertiary signaling domain are the same. In some embodiments, two or three of the primary intracellular signaling domain, the secondary intracellular signaling domain, and the tertiary intracellular signaling domain 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 CD3 zeta secondary intracellular signaling domain. In other embodiments, the intracellular signaling domain comprises a Tim4 primary intracellular signaling domain and a CD3 zeta secondary intracellular signaling domain. In other embodiments, the intracellular signaling domain comprises a TLR8 primary intracellular signaling domain and a CD3 zeta secondary intracellular signaling domain. In other embodiments, the intracellular signaling domain comprises a TLR2 primary intracellular signaling domain and a CD3 zeta 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 CD3 zeta 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 cd3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3 zeta 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 cd3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3 zeta 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 CD3 zeta 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 CD3 zeta 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 CD3 zeta 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 CD3 zeta 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 cd3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3 zeta 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 cd3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 primary intracellular signaling domain, a CD3 zeta 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 CD3 zeta 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 CD3 zeta 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 CD3 zeta 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 cd3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a CD3 zeta 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 cd3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a Tim4 primary intracellular signaling domain, a CD3 zeta secondary intracellular signaling domain, and a TLR8 tertiary intracellular signaling domain.

In a particular embodiment, the intracellular signaling domain comprises a Tim1 primary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO. 44 and a CD3 zeta secondary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO. 27 or 5. 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 a CD3 ζ secondary intracellular signaling domain comprising the amino acid sequence of SEQ ID NO. 27 or 5. In further specific embodiments, the intracellular signaling domain comprises 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. In another particular 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. In some embodiments, the intracellular signaling domain comprises a combination of a primary intracellular signaling domain, a secondary intracellular signaling domain, and optionally a tertiary intracellular signaling domain sequence as shown in table 10.

The transmembrane domain of the chimeric Tim receptor connects and is located between the extracellular domain and the intracellular signaling domain. The transmembrane domain is a hydrophobic alpha helix that passes through the host cell membrane. The transmembrane domain may be fused directly to the binding domain or extracellular spacer domain, if present. In certain embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptor, cluster of Differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, etc.). In one embodiment, the transmembrane domain is selected from the same molecules as the molecules from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, a chimeric Tim receptor can include a Tim4 binding domain and a Tim4 transmembrane domain. In another example, the chimeric Tim receptor can include a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are from different molecules; the transmembrane domain and the intracellular signaling domain are from different molecules; or the transmembrane domain, extracellular domain and intracellular signaling domain are all from different molecules. Examples of transmembrane domains that can be used for the chimeric Tim receptors of the present disclosure include transmembrane domains from Tim1, tim4 and CD 28. An exemplary Tim1 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO. 8. Exemplary Tim4 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 6 or 23. Exemplary CD28 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 7. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs 6-8 or 23. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of any of SEQ ID NOs 6-8 or 23.

In certain embodiments, the chimeric Tim receptor comprises a polynucleotide sequence from any mammalian species including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, transgenic species thereof, or any combination thereof. In certain embodiments, the chimeric Tim receptor is murine, chimeric, human, or humanized.

It is to be understood that direct fusion of one domain of the chimeric Tim receptor described herein to another domain does not preclude the presence of an intermediate linking amino acid. The linking amino acids may be natural or unnatural (e.g., from the construct design of the chimeric protein). For example, the linking amino acids may be from restriction enzyme sites used to link one domain to another domain or to clone a polynucleotide encoding a chimeric Tim receptor into a vector.

Exemplary chimeric Tim receptors

The components of the chimeric Tim receptors as disclosed herein can be selected and arranged in various combinations to provide the desired specificity and effector phenotype to the host cell.

Exemplary chimeric Tim receptors of the present disclosure are described in table 4.

Table 4.

Additional exemplary chimeric Tim receptors are described in table 5.

Table 5.

In some embodiments, the chimeric Tim receptors of the disclosure include the constructs of table 4. In some embodiments, the chimeric Tim receptors of the disclosure include the constructs of table 5.

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

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

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

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

In some embodiments, construct 5 or the chimeric Tim receptor of construct 5' includes amino acids 21-596 of SEQ ID NO. 53. In some embodiments, construct 5 or the chimeric Tim receptor of construct 5' comprises the amino acid sequence of SEQ ID No. 53.

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

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

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

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

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

Additional exemplary chimeric Tim receptors of the present disclosure are described in table 6.

Table 6.

Additional exemplary chimeric Tim receptors are described in table 7.

Table 7.

In some embodiments, the chimeric Tim receptors of the disclosure include the constructs of table 6. In some embodiments, the chimeric Tim receptors of the disclosure include the constructs of table 7.

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

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

In some embodiments, construct 13 or the chimeric Tim receptor of construct 13' includes amino acids 25-382 of SEQ ID NO. 61. In some embodiments, construct 13 or the chimeric Tim receptor of construct 13' includes the amino acid sequence of SEQ ID NO. 61.

In some embodiments, the chimeric Tim receptor of construct 13A (TIM 4 binding domain-CD 28 TM-CD28costim-CD3 zeta (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 includes the amino acid sequence of SEQ ID NO: 71.

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

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

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

In some embodiments, construct 17 or the chimeric Tim receptor of construct 17' includes amino acids 25-644 of SEQ ID NO. 65. In some embodiments, construct 17 or the chimeric Tim receptor of 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' includes amino acids 25-640 of SEQ ID NO. 66. In some embodiments, construct 18 or the chimeric Tim receptor of construct 18' includes the amino acid sequence of SEQ ID NO. 66.

Additional exemplary chimeric Tim receptors of the present disclosure are described in table 8.

Table 8.

Additional exemplary chimeric Tim receptors are described in table 9.

Table 9.

In some embodiments, the chimeric Tim receptors of the disclosure include the constructs of table 8. In some embodiments, the chimeric Tim receptors of the disclosure include the constructs of table 9.

In some embodiments, construct 19 or the chimeric Tim receptor of construct 19' includes amino acids 25-628 of SEQ ID NO. 67. In some embodiments, construct 19 or the chimeric Tim receptor of 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, construct 20 or the chimeric Tim receptor of 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' includes 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 described in table 10. In some embodiments, the chimeric Tim receptors of the disclosure include the constructs of table 10. In some embodiments, the chimeric Tim receptor of the present disclosure does not comprise a chimeric Tim receptor having the amino acid sequence of SEQ ID NOs 227, 238, 249, 72, or 73. In some embodiments, the chimeric Tim receptor of the present disclosure does not comprise a chimeric Tim receptor having a combination of components as described for constructs 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

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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 comprise the positive and negative strands of nucleic acid complementary to each other, including antisense DNA, cDNA and RNA. The nucleic acid may be DNA or RNA in naturally occurring or synthetic form. Nucleic acid sequences encoding the desired chimeric Tim receptor can be obtained or generated by recombinant methods known in the art using standard techniques, such as by screening libraries from cells expressing the desired sequence or portions thereof, by deriving the sequence from vectors known to contain the sequence, or by isolating the sequence or portions thereof directly from cells or tissues containing the sequence, as described, for example, in Sambrook et al (1989 and 2001; molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press), new York) and Ausubel et al (molecular biology recent protocol (Current Protocols in Molecular Biology), 2003). Alternatively, the sequence of interest may be synthetically produced, rather than cloned.

Polynucleotides encoding the chimeric Tim receptor compositions provided herein can be from any animal, such as human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, or a combination thereof. In certain embodiments, the polynucleotide encoding the chimeric Tim receptor is from the same animal species as the host cell into which the polynucleotide is inserted.

Polynucleotides encoding the chimeric Tim receptors of the disclosure may be operably linked to expression control sequences. Expression control sequences may comprise appropriate transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; a sequence that stabilizes cytoplasmic mRNA; sequences that increase translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and sequences that may enhance protein secretion.

In certain embodiments, the polynucleotide encoding the chimeric Tim receptor includes a sequence encoding a signal peptide (also referred to as a leader peptide or signal sequence) at the 5' end for targeting the precursor protein to the secretory pathway. During cellular processing and localization of the chimeric Tim receptor on the host cell membrane, the signal peptide is optionally cleaved from the N-terminus of the extracellular domain. The polypeptide from which the signal peptide sequence is cleaved or removed may also be referred to as a mature polypeptide. Examples of signal peptides that can be used for the chimeric Tim receptor of the present disclosure include signal peptides derived from endogenous secreted proteins, including, 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). In certain embodiments, the polynucleotide sequence encodes a mature chimeric Tim receptor polypeptide, or the polypeptide sequence comprises a mature chimeric Tim receptor polypeptide. It will be appreciated by those skilled in the art that for the sequences disclosed herein comprising a signal peptide sequence, the signal peptide sequence may be replaced with another signal peptide capable of transporting the encoded protein to the extracellular membrane.

In certain embodiments, the chimeric Tim receptor encoding the polynucleotides of the present disclosure is codon optimized for efficient expression in a target host cell comprising the polynucleotide (see, e.g., scholten et al, clinical immunology (clin. Immunol.)), 119:135-145 (2006)). As used herein. "codon-optimized" polynucleotides include heterologous polynucleotides whose codons are modified with silent mutations corresponding to the abundance of tRNA in the host cell of interest.

According to any of the embodiments disclosed herein, a single polynucleotide molecule may encode one, two or more chimeric Tim receptors. Polynucleotides encoding more than one transgene may include sequences (e.g., IRES, viral 2A peptide) disposed between each gene for polycistronic expression.

Polynucleotides encoding at least two transgenes (e.g., chimeric Tim receptor and CAR) provided in the present disclosure can be used to make up a tandem expression cassette. Tandem expression cassette refers to a component of a vector nucleic acid comprising at least two transgenes under the control of, or operably linked to, the same set of regulatory sequences used to tandem or co-express the at least two transgenes. Regulatory sequences that can be used in the tandem expression cassettes of the present disclosure include suitable transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; a sequence that stabilizes cytoplasmic mRNA; sequences that increase translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; a sequence that enhances protein secretion, or any combination thereof.

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

In certain embodiments, tandem expression cassettes can be constructed to optimize spatial and temporal control. For example, a tandem expression cassette may contain promoter elements to optimize spatial and temporal control. In some embodiments, the tandem expression cassette comprises a tissue-specific promoter or enhancer that is capable of specifically inducing the tandem expression cassette to an organ, cell type (e.g., immune cell), or pathological microenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can act synergistically or independently to activate transcription. In certain embodiments, the tandem expression cassette comprises a constitutive promoter. An exemplary constitutive promoter for the tandem expression cassette of the present disclosure is the EF-1. Alpha. Promoter. In certain embodiments, the tandem expression cassette comprises an inducible promoter. In certain embodiments, the tandem expression cassette comprises a tissue specific promoter.

The at least two transgenes contained in the tandem expression cassette may be in any order. For example, a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR may be arranged from 5 'to 3' as: chimeric Tim receptor-CAR or CAR-chimeric Tim receptor.

In certain embodiments, a receptor comprising two or more polypeptide chains that associate to form a multimer or complex may be encoded by two or more polynucleotide molecules in a tandem expression construct. Exemplary multimeric receptors contemplated for expression in the tandem expression constructs of the present disclosure comprise a multiplex CAR, TCR, TCR-CAR and a TRuC ^TM A construct. Thus, exemplary tandem expression cassette embodiments encoding a chimeric Tim receptor and a TCR may include polynucleotides encoding the chimeric Tim receptor, polynucleotides encoding a TCR alpha chain polypeptide, and polynucleotides encoding a TCR beta chain polypeptide.

In certain embodiments, the tandem expression cassettes of the present disclosure may include an Internal Ribosome Entry Site (IRES) or a peptide cleavage site, such as a furin cleavage site or a viral 2A peptide, disposed between each polynucleotide contained within the tandem expression cassette, to allow co-expression of multiple proteins from a single mRNA. For example, an IRES, furin cleavage site, or viral 2A peptide can be disposed within the tandem expression cassette between a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a CAR. In another example, an IRES, furin cleavage site, or viral 2A peptide may be disposed between a polynucleotide encoding a chimeric Tim receptor, a polynucleotide encoding a TCR alpha chain polypeptide, and a polynucleotide encoding a TCR beta chain polypeptide. In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), thosea asigna virus (T2A), equine rhinitis a virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. Exemplary T2A peptides include the amino acid sequence of any one of SEQ ID NOs 12, 28, 29 or 30. Exemplary P2A peptides include the amino acid sequence of SEQ ID NO. 13 or 31. Exemplary E2A peptide sequences include the amino acid sequence of SEQ ID NO. 14. Exemplary F2A peptide sequences include the amino acid sequence of SEQ ID NO. 15.

Certain embodiments of the tandem expression cassettes of the present disclosure include polynucleotides encoding CARs/or TCRs that are specific for a target antigen (e.g., a tumor antigen) and polynucleotides encoding chimeric Tim receptors of the present disclosure. When target cells expressing a target antigen are bound by the CAR and/or TCR, cells modified to express such tandem expression cassettes induce apoptosis of the target cells. Apoptosis induces exposure of a pro-phagocytic marker (such as phosphatidylserine) on target cells, which can then target damaged or apoptotic cells for phagocytosis by the chimeric Tim receptor.

Polynucleotides encoding the desired chimeric Tim receptor can be inserted into suitable vectors, such as viral vectors, non-viral plasmid vectors, and non-viral vectors, e.g., lipid-based DNA vectors, modified mRNA (modRNA), self-amplified mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac), for introduction into host cells of interest (e.g., immune cells). Polynucleotides encoding the chimeric Tim receptors of the present disclosure can be cloned into any suitable vector, such as an expression vector, a replication vector, a probe-generating vector, or a sequencing vector. In certain embodiments, the polynucleotide encoding the extracellular domain, the polynucleotide encoding the transmembrane domain, and the polynucleotide encoding the intracellular signaling domain are ligated together into a single polynucleotide, and then inserted into a vector. In other embodiments, the polynucleotide encoding the extracellular domain, the polynucleotide encoding the transmembrane domain, and the polynucleotide encoding the intracellular signaling domain, respectively, may be inserted into a vector such that the expressed amino acid sequence results in a functional chimeric Tim receptor. Vectors encoding chimeric Tim receptors are referred to herein as "chimeric Tim receptor vectors".

In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim receptor. In certain embodiments, the vector comprises a polynucleotide encoding two or more chimeric Tim receptors. In certain embodiments, a single polynucleotide encoding two or more chimeric Tim receptors is cloned into a cloning site and expressed from a single promoter, wherein each chimeric Tim receptor sequence is separated from each other by an Internal Ribosome Entry Site (IRES), a furin cleavage site, or a viral 2A peptide, to allow co-expression of multiple genes from a single open reading frame (e.g., a polycistronic vector). In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), thosea asigna virus (T2A), equine rhinitis a virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. Exemplary T2A peptides include the amino acid sequences of SEQ ID NO. 12, 28, 29 or 30. Exemplary P2A peptides include the amino acid sequence of SEQ ID NO. 13 or 31. Exemplary E2A peptide sequences include the amino acid sequence of SEQ ID NO. 14. Exemplary F2A peptide sequences include 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 may be cloned sequentially into a vector at different cloning sites, wherein each chimeric Tim receptor is expressed under the control of a different promoter. In certain embodiments, vectors are used that allow for long-term integration and propagation of transgenes to daughter cells. Examples include viral vectors, such as adenovirus, adeno-associated virus, vaccinia virus, herpes virus, cytomegalovirus, poxvirus, or retroviral vectors, such as lentiviral vectors. Lentiviral derived vectors can be used to achieve long term gene transfer and have additional advantages over vectors, including the ability to transduce non-proliferating cells (e.g., hepatocytes) and low immunogenicity.

The vector encoding the core virus is referred to herein as a "viral vector". There are a number of viral vectors available for use in the compositions of the present disclosure, including those identified for use in human gene therapy applications (see Pfeifer and Verme, annual reviews of genomics and human genetics (an. Rev. Genomics hum. Genet.)) 2:177, 2001. Suitable viral vectors include RNA virus-based vectors, such as retroviral derived vectors, e.g., a maroney Murine Leukemia Virus (MLV) derived vector, and more complex retroviral derived vectors, e.g., lentiviral derived vectors. HIV-1 derived vectors belong to this class. Other examples include lentiviral vectors from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (sheep lentivirus). Methods for transducing mammalian host cells with viral particles containing chimeric receptor transgenes using retroviral and lentiviral vectors and packaging cells are known in the art and have been previously described, for example, in U.S. patent 8,119,772; walchli et al, public science library, complex (PLoS One) 6:327930, 2011; zhao et al, J.Immunol.) (174:4415, 2005; engels et al, human gene therapy (hum. Gene Ther.) 14:1155, 2003; frecha et al, molecular therapy (mol. Ther.) 18:1748, 2010; verhoeyen et al, methods of molecular biology (Methods mol. Biol.) 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

In certain embodiments, the viral vector is used to introduce a non-endogenous polynucleotide encoding a chimeric Tim receptor into a host cell. The viral vector may be a retroviral vector or a lentiviral vector. Viral vectors may also comprise nucleic acid sequences encoding markers for transduction. Transduction markers for viral vectors are known in the art and comprise selectable markers that may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that may be detected by methods such as flow cytometry. In particular embodiments, the viral vector further comprises a gene marker for transduction comprising a fluorescent protein (e.g., green, yellow), an extracellular domain of human CD2, or truncated human EGFR (EGFR t or tEGFR; see Wang et al, blood (Blood) 118:1255, 2011). Exemplary tEGFR includes the amino acid sequence of SEQ ID NO. 16. When the viral vector genome comprises multiple genes to be expressed in a host cell as independent proteins from a single transcript, the viral vector may also comprise additional sequences between the two (or more) genes that allow for polycistronic expression. Examples of such sequences for viral vectors include an Internal Ribosome Entry Site (IRES), a furin cleavage site, a viral 2A peptide (e.g., T2A, P2A, E2A, F a), or any combination thereof.

Other viral vectors may also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV) -based vectors; vectors derived from Herpes Simplex Virus (HSV) include amplicon vectors, replication defective HSV and attenuated HSV (Krisky et al, gene therapy 5:1517, 1998).

Other viral vectors recently developed for gene therapy use may also be used with the compositions and methods of the present disclosure. Such vectors include vectors derived from baculovirus and alpha-virus. (Jolly, D J.1999, emerging viral vectors (Emerging Viral Vectors), pages 209-40, friedmann T. Edit, development of human Gene therapy (The Development of Human Gene Therapy), new York: cold spring harbor laboratory (New York: cold Spring Harbor Lab)), or plasmid vectors (e.g., sleep bearing or other transposon vectors).

In certain embodiments, chimeric Tim receptor vectors can be constructed to optimize spatial and temporal control. For example, chimeric Tim receptor vectors can contain promoter elements to optimize spatial and temporal control. In some embodiments, the chimeric Tim receptor vector comprises a tissue-specific promoter or enhancer capable of specifically inducing the chimeric Tim receptor to an organ, cell type (e.g., immune cell), or pathological microenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can act synergistically or independently to activate transcription. In certain embodiments, the chimeric Tim receptor vector comprises a constitutive promoter. In certain embodiments, the chimeric Tim receptor vector comprises an inducible promoter. In certain embodiments, the chimeric Tim receptor vector comprises a tissue-specific promoter.

In certain embodiments, the chimeric Tim receptor vector can comprise a gene encoding a homing receptor, such as CCR4 or CXCR4, to enhance homing and anti-tumor activity in vivo.

Where time control is required, the chimeric Tim receptor vector may contain elements that allow for inducible elimination of the transduced cells. For example, such a vector may comprise an inducible suicide gene. Suicide genes may be apoptotic genes or genes that confer sensitivity to agents (e.g., drugs). Exemplary suicide genes include chemically inducible cysteine protease 9 (iCASP 9) (U.S. patent publication No. 2013/007434), chemically inducible Fas, or herpes simplex virus thymidine kinase (HSV-TK), which confers sensitivity to ganciclovir. In further embodiments, the chimeric Tim receptor vector can be designed to express a known cell surface antigen that is capable of clearing transduced cells upon infusion of the relevant antibody. Examples of cell surface antigens and their related antibodies that can be used to remove transduced cells include CD20 and rituximab, RQR8 (combined CD34 and CD20 epitopes, allowing for CD34 selection and anti-CD 20 deletion) and rituximab, as well as EGFR and cetuximab.

Inducible vector systems, such as the tetracycline (Tet) -On vector system (Heinz et al, human Gene therapy, 2011, 22:166-76) that activates transgene expression with doxycycline, may also be used for inducible chimeric Tim receptor expression. Inducible chimeric Tim receptor expression can also be achieved via retention using RUSH (retention using a selective hook) systems based on streptavidin anchored to the endoplasmic reticulum membrane by hooks and streptavidin binding proteins incorporated into the chimeric Tim receptor structure, wherein the addition of biotin to the system results in release of the chimeric Tim receptor from the endoplasmic reticulum (Agaugue et al 2015, molecular therapy 23 (supplement 1): S88).

In certain embodiments, a cell (e.g., an immune cell) obtained from a subject can be engineered into a non-natural or recombinant cell (e.g., a non-natural or recombinant immune cell) by introducing a polynucleotide encoding a chimeric Tim receptor as described herein, whereby the cell expresses a cell surface-localized chimeric Tim receptor. In certain embodiments, the host cell is an immune cell, such as a myeloid progenitor cell or lymphoid progenitor cell. Exemplary immune cells that can be modified to include a polynucleotide encoding a chimeric Tim receptor or a vector including a polynucleotide encoding a chimeric Tim receptor include T cells, natural killer cells, B cells, lymphoid precursor cells, antigen presenting cells, dendritic cells, langerhans cells, bone marrow precursor cells, mature bone marrow cells, monocytes or macrophages.

In certain embodiments, the B cells are genetically modified to express one or more chimeric Tim receptors. B cells have certain properties that may be advantageous as host cells, including: trafficking to the site of inflammation, internalization and presentation of antigen, co-stimulation of T cells, high proliferation and self-renewal (life-long duration). In certain embodiments, the B cells modified with the chimeric Tim receptor are capable of digesting phagocytic target cells or phagocytic target particles into smaller peptides and presenting them to T cells via MHC molecules. Antigen presentation by B cells modified with chimeric Tim receptors may aid in antigen diffusion of immune responses against non-targeted antigens. B cells include progenitor or precursor cells committed to the B cell lineage (e.g., pre-progenitor-B cells, and pre-B cells); immature and unactivated B cells; or mature and functional or activated B cells. In certain embodiments, the B cell may be a primary B cell, a plasma cell, a regulatory B cell, a border region B cell, a follicular B cell, a lymphoplasmacytoid cell, a plasmablast cell, a memory B cell, or any combination thereof. Memory B cells can be distinguished from primary B cells based on the lack of expression of CD27 on the primary B cells. In certain embodiments, the B cells may be primary cells or cell lines derived from humans, mice, rats, or other mammals. B cell lines are well known in the art. If obtained from a mammal, the B cells may be obtained from a variety of sources, including blood, bone marrow, spleen, lymph nodes or other tissues or fluids. The B cell composition may be enriched or purified.

In certain embodiments, the T cells are genetically modified to express one or more chimeric Tim receptors. Exemplary T cells comprise CD4 ⁺ Helper cells, CD8 ⁺ Effector cells (cytotoxicity), primordial cells (CD45RA+, CCR7+, CD62L+, CD27+, CD45 RO-), central memory cells (CD 45 RO-) ⁺ 、CD62L ⁺ 、CD8 ⁺ ) Effector memory cells (CD45RA+, CD45RO-, CCR7-, CD62L-, CD 27-), T memory stem cells, regulatory cells, mucosa-associated invariant cells (MAIT), γδ (gd), tissue resident T cells, natural killer T cells, or any combination thereof. In certain embodiments, the T cell may be a primary cell or cell line derived from a human, mouse, rat, or other mammal. If obtained from a mammal, T cells may be obtained from a variety of 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 (Leukemia), 21:230, 2000. In certain embodiments, the T cell lacks endogenous expression of the TCR a gene, the TCR a 0 gene, or both. Such T cells may naturally lack endogenous expression of TCR a and β chains, or may have been modified to block expression (e.g., T cells from transgenic mice that do not express TCR a and β chains or cells that have been manipulated to inhibit expression of TCR a and β chains) or to knock out TCR a chains, TCR β chains, or both genes.

In certain embodiments, the host cell expressing the chimeric Tim proteins of the disclosure on the cell surface is not a T cell or a cell of the T cell lineage, but a progenitor cell, stem cell, or a cell modified to express cell surface anti-CD 3.

In certain embodiments, the gene editing methods are used to modify the host cell genome to include a polynucleotide encoding a chimeric Tim receptor of the disclosure. Gene editing or genome editing is a genetically engineered method in which DNA is inserted, replaced, or removed from the genome of a host cell using a genetically engineered endonuclease. Nucleases produce a specific double strand break at a targeted site in the genome. The endogenous DNA repair pathway of the host cell then repairs the induced break, for example, by non-homologous end joining (NHEJ) and homologous recombination. Exemplary endonucleases that can be used for gene editing include Zinc Finger Nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas nuclease systems (e.g., CRISPR-Cas 9), homing endonucleases, or combinations thereof. Methods of disrupting or knocking out genes or gene expression in immune cells including B cells and T cells using gene editing endonucleases are known in the art and are described, for example, in PCT publication No. WO 2015/066262; WO2013/074916; WO 2014/059173; cheong et al, nat. Communication (Nat. Comm.) 2016 7:10934; chu et al, proc. Natl. Acad. Sci. USA, 2016 113:12514-12519; the methods from each of these documents are incorporated herein by reference in their entirety.

In certain embodiments, expression of an endogenous gene of the host cell is inhibited, knocked down, or knocked out. Examples of endogenous genes that can be suppressed, knocked down or knocked out in B cells include IGH, igκ, igλ, or any combination thereof. Examples of endogenous genes that can be suppressed, knocked down, or knocked out in T cells include TCR genes (TRA or TRB), HLA genes (HLA class I genes or HLAII class I 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/CD 112R), or any combination thereof. Expression of the endogenous gene may 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 an endogenous gene can be accomplished, for example, by an RNA interfering agent (e.g., siRNA, shRNA, miRNA, etc.) or an engineered endonuclease (e.g., CRISPR/Cas nuclease system, zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), homing endonuclease), or any combination thereof. In certain embodiments, an endogenous B cell gene (e.g., IGH, igκ, or igλ) is knocked out, such as via an engineered endonuclease, by inserting a polynucleotide encoding a chimeric Tim receptor of the disclosure into a locus of the endogenous B cell gene. In certain embodiments, an endogenous T cell gene (e.g., a TCR gene, an HLA gene, or an immune checkpoint molecule gene) is knocked out, such as via an engineered endonuclease, by inserting a polynucleotide encoding a chimeric Tim receptor of the present disclosure into a locus of the endogenous T cell gene.

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

The present disclosure also provides a composition comprising a population of host cells modified with a chimeric Tim receptor. In certain embodiments, the population of host cells modified by the chimeric Tim receptor can be a population of B cells, a population of T cells, a population of natural killer cells, a population of lymphoid precursor cells, a population of antigen presenting cells, a population of dendritic cells, a population of langerhans cells, a population of bone marrow precursor cells, a population of mature bone marrow cells, or any combination thereof. Furthermore, a population of host cells of a particular cell type modified by a chimeric Tim receptor may be composed of one or more subtypes. For example, the population of B cells may consist of primary B cells modified with a chimeric Tim receptor, plasma cells, regulatory B cells, border zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablasts, memory B cells, or any combination thereof. In another example, the population of T cells can be composed of CD4 modified by a chimeric Tim receptor ⁺ Helper T cells, cd8+ effector (cytotoxic) T cells, naive (cd45ra+, ccr7+, cd62l+, cd27+, CD45 RO-) T cells, central memory (CD 45 RO) ⁺ 、CD62L ⁺ 、CD8 ⁺ ) T cells, effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD 27-) 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 population of host cells consists of cells each expressing the same chimeric Tim receptor. In other embodiments, the population of host cells consists of a mixture of two or more subpopulations of host cells, wherein each subpopulation expresses a different chimeric Tim receptor or a set of chimeric Tim receptors.

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

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

The chimeric Tim receptors of the present disclosure confer host cell cytotoxic activity that expresses a chimeric Tim receptor specific for phosphatidylserine. Thus, host cells expressing chimeric Tim receptors are able to induce apoptosis of target cells upon binding to phosphatidylserine exposed on the surface of the target cells. In certain embodiments, host cells expressing the chimeric Tim receptor induce apoptosis of the target cell via: releasing granzyme, perforin, granulysin, or any combination thereof; fas ligand-Fas interaction; or both. In further embodiments, the chimeric Tim receptor further confers phosphatidylserine-specific phagocytic activity to a host cell expressing the chimeric Tim receptor. In still further embodiments, the host cell does not naturally exhibit a phagocytic phenotype prior to modification with the chimeric Tim receptor.

The chimeric Tim receptors of the present disclosure are also capable of costimulating T cells via at least one signaling pathway. In certain embodiments, the chimeric Tim receptor provides a costimulatory signal to the T cell via at least two different signaling pathways (e.g., via a costimulatory signaling domain selected in the chimeric Tim receptor). For example, a chimeric Tim receptor comprising a CD28 costimulatory signaling domain may be capable of providing a costimulatory signal via CD28 and Tim 1. In certain embodiments, host immune cells expressing the chimeric Tim receptor exhibit a reduction or inhibition of immune cell depletion. In certain embodiments, the host immune cell is a T cell or NK cell. In certain embodiments, the depleted T cells exhibit: (a) Increased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof; (b) Reduced production of IFN-gamma, IL-2, TNF-alpha, or any combination thereof; or both (a) and (b). In certain embodiments, the depleted NK cells exhibit: (a) Increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) reduced production of IFN- γ, TNF- α, or both; or both (a) and (b).

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

In certain embodiments, host cells expressing the chimeric Tim receptor exhibit a reduced immunosuppressive response to phosphatidylserine. Phosphatidylserine is one of the major apoptotic cell ligands that signals phagocytes of "eat me". Clearance of apoptotic cells by phagocytes generally reduces or prevents inflammatory responses via secretion of anti-inflammatory cytokines IL-10 and TGF-beta and reduction of secretion of inflammatory cytokines TNF-alpha, IL-1β and IL-12. Thus, phosphatidylserine may act as an immunosuppressive signal during the clearance of apoptotic cells. In certain embodiments, host cells modified with a chimeric Tim receptor exhibit increased antigen-specific cytokine production (e.g., IFN- γ, IL-2, TNF- α) upon binding to phosphatidylserine, thereby reducing an immunosuppressive response to phosphatidylserine.

Expression of chimeric Tim receptors on host cells can be functionally characterized according to any of a number of art accepted methods for determining host cell (e.g., T cell) activity, including determining T cell binding, activation or induction and also including determining antigen-specific T cell responses. Examples include determining T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, CTL activity (e.g., by detecting a target cell from a preload ⁵¹ Cr or europium release), changes in T cell phenotype marker expression, and other measurements of T cell function. Methods for performing these and similar assays can be found, for example, in Lefkovits (handbook of immunological methods: technical comprehensive (Immunology Methods Manual: the Comprehensive Sourcebook of Techniques), 1998). See also current immunological protocols (Current Protocols in Immunology); weir, manual for laboratory immunology (Handbook of Experimental Immunology), blacker science publication (Blackwell Scientific), boston, mass (1986); mishell and Shikii (editions), "selected methods in cellular immunology (Selected Methods in Cellular Immunology), frieman Press (Freeman Publishing), san Francisco, california (1979); green and 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 (e.g., fineA combination of intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from antigen-specific priming or stimulation of an immune response can be determined by isolating lymphocytes, such as circulating lymphocytes in a sample of peripheral blood cells or cells from lymph nodes, stimulating cells with antigen, and measuring cytokine production, cell proliferation, and/or cell viability, such as by incorporating tritiated thymidine or a non-radioactive assay, such as an MTT assay, and the like.

In certain embodiments, the host cell modified with the chimeric Tim receptor has a phagocytic index of about 20 to about 1,500 for the target cell. The "phagocytic index" is a measure of the phagocytic activity of a transduced host cell, as determined by counting the number of target cells or particles taken up by each chimeric Tim receptor-modified host cell during a period of time in which a suspension of target cells or particles and chimeric Tim receptor-modified host cells is incubated in a medium. The phagocytic index may be calculated by multiplying [ total number of target cells phagocytosed/total number of chimeric Tim receptor modified cells counted (e.g., phagocytic frequency) ]x [ average area per chimeric Tim receptor + target cells or particle staining of host cells x 100 (e.g., hybridization capture) ] or by [ total number of particles phagocytosed/total number of chimeric Tim receptor modified host cells counted ] × [ number of chimeric Tim receptor modified host cells containing particles phagocytosed/total number of chimeric Tim receptor cells counted ] x100. In certain embodiments, the chimeric Tim receptor-modified cells have a molecular weight of about 30 to about 1,500; about 40 to about 1,500; about 50 to about 1,500; about 75 to about 1,500; about 100 to about 1,500; about 200 to about 1,500; about 300 to about 1,500; about 400 to about 1,500; about 500 to about 1,500; about 20 to about 1,400; about 30 to about 1,400; about 40 to about 1,400; about 50 to about 1,400; about 100 to about 1,400; about 200 to about 1,400; about 300 to about 1,400; about 400 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 100 to about 1,300; about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300; about 500 to about 1,300; about 20 to about 1,200; about 30 to about 1,200; about 40 to about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200; about 300 to about 1,200; about 400 to about 1,200; about 500 to about 1,200; about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 200 to about 1,100; about 300 to about 1,100; about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about 30 to about 1,000; about 40 to about 1,000; about 50 to about 1,000; about 100 to about 1,000; about 200 to about 1,000; about 300 to about 1,000; about 400 to about 1,000; or about 500 to about 1,000; about 20 to about 750; about 30 to about 750; about 40 to about 750; about 50 to about 750; about 100 to about 750; about 200 to about 750; about 300 to about 750; about 400 to about 750; or about 500 to about 750; about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; about 200 to about 500; or a phagocytic index of about 300 to about 500. 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 cells modified with the chimeric Tim receptor exhibit a phagocytic index that is statistically significantly higher than cells transduced with a truncated EGFR control. Phagocytosis index can be calculated using methods known in the art, and as further described in the examples and PCT application No. PCT/US2017/053553 (incorporated herein by reference in its entirety), including quantification by flow cytometry or fluorescence microscopy.

The host cell may be from an animal, such as a human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, or a combination thereof. In a preferred embodiment, the animal is a human. The host cell may be obtained from a healthy subject or a subject suffering from a disease associated with the expression or overexpression of an antigen.

PARP inhibitors

Any suitable PARP inhibitor may be used in the compositions and methods of the present disclosure. Exemplary PARP inhibitors include talazapanib, nilaparib, lu Kapa, olaparib (AZ 2281, KU 59434), veliparib (ABT 888), CEP 9722, E7016, AG014699, MK4827, BMN-673 and pamiparib (BGB-290).

In particular embodiments, the PARP inhibitor comprises nilaparib. In particular embodiments, the PARP inhibitor comprises talazapanib. In a particular embodiment, the PARP inhibitor comprises Lu Kapa ni. In particular embodiments, the PARP inhibitor comprises olaparib. In particular embodiments, the PARP inhibitor comprises veliparib. In particular embodiments, the PARP inhibitor comprises CEP 9722. In particular embodiments, the PARP inhibitor comprises E7016. In particular embodiments, the PARP inhibitor comprises AG014699. In particular embodiments, the PARP inhibitor comprises MK4827. In a specific embodiment, the PARP inhibitor comprises BMN-673. In particular embodiments, the PARP inhibitor comprises pamipril.

Therapeutic method

The chimeric Tim receptor, polynucleotide encoding the chimeric Tim receptor, chimeric Tim receptor vector, or host cell expressing the chimeric Tim receptor, in combination with a PARP inhibitor according to any of the embodiments provided herein, can be used in a method of treating a subject having a disease, disorder, or undesired condition. Embodiments of these methods comprise administering to a subject (i) a therapeutically effective amount of a pharmaceutical composition comprising one or more chimeric Tim receptors, a polynucleotide encoding one or more chimeric Tim receptors, a vector comprising a polynucleotide encoding one or more chimeric Tim receptors, or a population of host cells genetically modified to express one or more chimeric Tim receptors according to the present description; and (ii) a therapeutically effective amount of a pharmaceutical composition comprising a PARP inhibitor.

The chimeric Tim receptor compositions as described herein can be administered prior to PARP inhibitor therapy (e.g., 1 day to 30 days or more prior to PARP inhibitor therapy), concurrently with PARP inhibitor therapy (on the same day) or after PARP inhibitor therapy (e.g., 1 day to 30 days or more after PARP inhibitor therapy). In certain embodiments, the chimeric Tim receptor-modified cells are administered after administration of one or more additional therapies. In further embodiments, the cells modified with the chimeric Tim receptor are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the PARP inhibitor. In still further embodiments, the chimeric Tim receptor-modified cells are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the PARP inhibitor therapy. Where PARP inhibitor therapy involves multiple doses, the chimeric Tim receptor-modified cells can be administered after an initial dose of one or more additional therapies, after a final dose of one or more additional therapies, or between multiple doses of one or more additional therapies.

Diseases that can be treated with cells expressing a chimeric Tim receptor as described in the present disclosure include cancer. Adoptive immunity and gene therapy are promising treatments for various types of cancer (Morgan et al, science 314:126, 2006; schmitt et al, human gene therapy 20:1240, 2009; june, journal of clinical research (J.Clin. Invest.) 117:1466, 2007) and infectious diseases (Kitchen et al, public science library. Complex. 4:38208, 2009; rossi et al, natural biotechnology 25:1444, 2007; zhang et al, PLoS Pathos.) > 6:e1001018, 2010; luo et al, journal of molecular medicine (J.mol. Med.)) 89:903, 2011).

A variety of cancers (including solid tumors and leukemia) are suitable for use in the compositions and methods disclosed herein. Exemplary cancers that can be treated using the receptors, modified host cells, and compositions described herein include adenocarcinomas of the breast, prostate, and colon; all forms of lung bronchogenic carcinoma; myeloid leukemia; melanoma; liver cancer; neuroblastoma; papillomas; amine precursor uptake and decarboxylation of the cell tumor; a vaginosis tumor; gill tumor; malignant carcinoid syndrome; carcinoid heart disease; and cancers (e.g., walker cancer, basal cell carcinoma, basal squamous cell carcinoma, brown-Pearce cancer, ductal carcinoma, ehrlich tumor, krebs2 cancer, merkel cell carcinoma, mucinous cancer, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, hard carcinoma, bronchiolar carcinoma, phosphocellular carcinoma, and transitional cell carcinoma). Other cancer types that can be treated using the receptors, modified host cells, and compositions described herein include histiocyte disorders; malignant tissue cytopathy; leukemia; hodgkin's disease; immunoproliferative small bowel disease; non-hodgkin's lymphoma; plasmacytoma; multiple myeloma; chronic Myelogenous Leukemia (CML); acute Myelogenous Leukemia (AML); plasmacytoma; reticuloendothelial tissue proliferation; melanoma; chondroblastoma; cartilage tumor; chondrosarcoma; fibroids; fibrosarcoma; giant cell tumor; histiocytoma; a fatty tumor; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngeal pipe tumor; a vegetative cell tumor; hamartoma; a stromal tumor; mesonephroma; myosarcoma; enameloblastoma; cementoma; dental tumor; teratoma; thymoma; nourishing cell tumor. Furthermore, the following types of cancers are also contemplated as suitable for treatment using the receptors, modified host cells, and compositions described herein: adenoma; gall bladder tumor; cholesteatoma; cylindrical tumors; cystic adenocarcinoma; cystic adenoma; granulosa cell tumors; ampholytic embryonal cytoma; liver cancer; sweat gland tumor; islet cell tumor; leydig cell tumor; papillomas; support cell tumor; follicular membrane cytoma; smooth myoma; leiomyosarcoma; myoblasts; myomas; myosarcoma; rhabdomyomas; rhabdomyosarcoma; ventricular tube membranoma; gangliocytoma; glioma; medulloblastoma; meningioma; a schwannoma; neuroblastoma; neuroepithelial tumors; neurofibromatosis; neuroma; paraganglioma; non-chromaphilic paragangliomas. The types of cancers that can be treated also include angiokeratomas; vascular lymphoid hyperplasia is accompanied by eosinophilia; sclerosing hemangioma; hemangiomatosis; glomeroclavicular tumor; vascular endothelial tumors; hemangioma; vascular endothelial cell tumor; hemangiosarcoma; lymphangioma; lymphangiomyomas; lymphangiosarcoma; pineal tumor; carcinoma sarcoma; chondrosarcoma; she Zhuangnang sarcoma; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukemia sarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasms; neurofibromatosis; cervical dysplasia; and peritoneal cancer.

In some embodiments, the compositions and methods of the present disclosure may be used to treat 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 a triple negative breast cancer. In some embodiments, the ovarian cancer is advanced ovarian cancer. In some embodiments, the prostate cancer is 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 further embodiments, the cancer is a breast cancer (BRCA) mutated cancer. In particular embodiments, the cancer is BRCA1 mutated cancer, BRCA2 mutated cancer, or both.

The chimeric Tim receptors of the disclosure can be administered to a subject in a cell-bound form (e.g., gene therapy of a target cell population). Thus, for example, the chimeric Tim receptors of the present disclosure can be administered to a subject expressed on the surface of T cells, natural killer T cells, B cells, lymphoid precursor cells, antigen presenting cells, dendritic cells, langerhans cells, bone marrow precursor cells, mature bone marrow cells (including subpopulations thereof), or any combination thereof. In certain embodiments, the method of treating a subject comprises administering an effective amount of a chimeric Tim receptor-modified cell (i.e., a recombinant cell expressing one or more chimeric Tim receptors). The cells modified with the chimeric Tim receptor can be allogeneic, syngeneic, allogeneic or autologous to the subject.

The chimeric Tim receptor modified cells and PARP inhibitors can be administered in a manner appropriate for the disease or condition to be treated (or prevented), as determined by one of skill in the medical arts. The appropriate dosage, appropriate duration and frequency of administration of the composition will be determined by factors such as the condition, body type, body weight, body surface area, age, sex, type and severity of the disease of the patient, the particular therapy to be administered, the particular form of the active ingredient, the time and method of administration, and other drugs being administered simultaneously. The present disclosure provides pharmaceutical compositions comprising cells modified with a chimeric Tim receptor and a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients include water, saline, dextrose, glycerol, and the like, as well as combinations thereof. Other suitable infusion media may be any isotonic media formulation, including saline, normosol R (Abbott), plasma-Lyte A (Baxter), 5% dextrose in water, or lactated ringer's solution.

A therapeutically effective amount of the cells in the pharmaceutical composition is at least one cell (e.g., a T cell modified by a chimeric Tim receptor) or more typically greater than 10 ² Individual cells, e.g. up to 10 ⁶ Up to 10 ⁷ Up to 10 ⁸ Individual cells, up to 10 ⁹ Individual cells, up to 10 ¹⁰ Individual cells or up to 10 ¹¹ Individual cells or more. In some embodiments, at about 10 ⁶ To about 10 ¹⁰ Individual cells/m ² Is preferably about 10 ⁷ To about 10 ⁹ Individual cells/m ² Is administered to the cell. The number of cells will depend on the end use for which the composition is intended and the type of cells contained therein. For example, a composition comprising cells modified to contain a chimeric Tim receptor will comprise a population of cells comprising about 5% to about 95% or more of such cells. In certain embodiments, compositions comprising cells modified with a chimeric Tim receptor include cell populations comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the uses provided herein, the volume of the cells is typically 1 liter or less, 500ml or less, 250ml or less, or 100ml or less. Thus, the desired cell density is typically greater than 10 ⁴ Individual cells/ml and typically greater than 10 ⁷ Individual cells/ml, typically greater than 10 ⁸ Individual cells/ml or greater. The cells may be administered as a single infusion or as multiple infusions over a period of time. Repeated infusions of cells modified with chimeric Tim receptors can be spaced days, weeks, months or even years apart if there is a recurrence of the disease or disease activity. The clinically relevant number of immune cells can be distributed to accumulate at or above 10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ Or 10 ¹¹ Multiple infusions of individual cells. For administration including as hereinPreferred doses of host cells of the recombinant expression vector are about 10 ⁷ Individual cells/m ² About 5X 10 ⁷ Individual cells/m ² About 10 ⁸ Individual cells/m ² About 5X 10 ⁸ Individual cells/m ² About 10 ⁹ Individual cells/m ² About 5X 10 ⁹ Individual cells/m ² About 10 ¹⁰ Individual cells/m ² About 5X 10 ¹⁰ Individual cells/m ² Or about 10 ¹¹ Individual cells/m ² 。

Chimeric Tim receptors and PARP inhibitors as described herein can be administered intravenously, intraperitoneally, intranasally, intratumorally into bone marrow, lymph nodes, and/or cerebrospinal fluid.

In one aspect, the methods of the present disclosure comprise conferring or enhancing phosphatidylserine-specific cytotoxic activity on a cell, comprising 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 expressing at least one chimeric Tim receptor in the host cell, wherein the at least one chimeric Tim receptor enhances phosphatidylserine specific cytotoxic activity of the host cell compared to the host cell prior to modification to express the chimeric Tim receptor. In certain embodiments, the cytotoxic activity of the host cell is increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more compared to the host cell prior to modification with the nucleic acid molecule encoding the chimeric Tim receptor or the chimeric Tim receptor vector. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell or NK cell. The method for measuring the cytotoxic activity of host cells, particularly immune cells such as T cells and NK cells, comprises ⁵¹ Cr), beta-gal or firefly luciferase release assay, flow cytometry methods that mediate target cell death and effector cell activity (see, e.g., vaccine specialist reviews (Expert Rev. Vaccines), 2010, 9:601-616).

In certain embodiments, the methods of the present disclosure comprise conferring or enhancing phosphatidylserine-specific cytotoxic activity on a cell, which further comprises conferring or enhancing phosphatidylserine-specific phagocytic activity on a host cell expressing at least one chimeric Tim receptor. In certain such embodiments, the host cell does not naturally exhibit a phagocytic phenotype prior to modification with the chimeric Tim receptor. For example, in certain such embodiments, phagocytic activity of the host cell is increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more as compared to the host cell prior to modification to express the chimeric Tim receptor vector. In certain embodiments, the host cell does not naturally have phagocytic activity. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell or NK cell. Methods of measuring phagocytic activity of a host cell include methods as described in PCT/US2017/053553 (incorporated herein by reference in its entirety).

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

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

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

In yet other aspects, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell expressing a chimeric Tim receptor according to any of the embodiments provided herein can be used in a method of eliminating a target cell with surface-exposed phosphatidylserine, e.g., for eliminating a cancer cell with surface-presented phosphatidylserine. In certain embodiments, the target cell is a damaged, stressed, apoptotic, necrotic cell (e.g., a tumor cell) with surface exposed phosphatidylserine. In certain embodiments, host cells expressing the chimeric Tim receptor clear damaged, stressed, apoptotic, or necrotic target cells with surface exposed phosphatidylserine via induction of apoptosis or induction of both apoptosis and phagocytosis. Host cells expressing the chimeric Tim receptor can be administered to a subject alone or in combination with other therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiation therapies, chemotherapy, small molecules, oncolytic viruses, electric pulse therapies, and the like.

In another aspect, a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim receptor, a chimeric Tim receptor vector, or a host cell expressing a chimeric Tim receptor according to any of the embodiments provided herein can be used in a method of enhancing the effect of a therapeutic agent that induces stress, injury, necrosis, or apoptosis in a cell. Certain therapies, such as chemotherapy, radiation therapy, UV light therapy, electrical pulse therapy, adoptive cell immunotherapy (e.g., CAR-T cells, TCRs) and oncolytic virus therapy, can induce cell damage or death of tumor cells, diseased cells and cells in their surroundings. Cells expressing the chimeric Tim receptor can be administered in combination with cell injury/cytotoxicity therapy to bind to phosphatidylserine moieties exposed on the outer leaflet of the targeted cells and to clear stressed, injured, diseased, apoptotic, necrotic cells.

The 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 may be administered in combination with a chimeric Tim composition according to the present description include radiation therapies, adoptive cellular immunotherapeutic agents (e.g., recombinant TCRs, affinity-enhanced TCR, CAR, TCR-CARs, scTCR fusion proteins, dendritic cell vaccines), antibody therapies, immune checkpoint molecule inhibitor therapies, UV light therapies, electrical pulse therapies, high intensity focused ultrasound therapies, oncolytic virus therapies, or drug therapies, such as chemotherapeutic agents, therapeutic peptides, hormones, aptamers, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, small molecule therapies, or any combination thereof. In certain embodiments, host cells modified with chimeric Tim receptors can clear stressed, damaged, apoptotic, necrotic, infected, dead cells exhibiting surface phosphatidylserine induced by one or more additional therapeutic agents.

In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapeutic (e.g., chimeric antigen receptor, recombinant TCR, etc.). In certain embodiments, a single polynucleotide encoding a chimeric Tim receptor and a cellular immunotherapeutic (e.g., CAR) is cloned into a cloning site and expressed from a single promoter, wherein the chimeric Tim receptor sequence and the cellular immunotherapeutic (e.g., CAR) sequence are separated from each other by an Internal Ribosome Entry Site (IRES), a furin cleavage site, or a viral 2A peptide, to allow co-expression of multiple genes from a single open reading frame (e.g., a polycistronic vector). In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), thosea asigna virus (T2A), equine rhinitis a virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. Exemplary T2A peptides include the amino acid sequences of SEQ ID NO. 12, 28, 29 or 30. Exemplary P2A peptides include the amino acid sequence of SEQ ID NO. 13 or 31. Exemplary E2A peptide sequences include the amino acid sequence of SEQ ID NO. 14. Exemplary F2A peptide sequences include 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 (e.g., CAR) binding protein are ligated together into a single polynucleotide, and then inserted into a vector. In other embodiments, the polynucleotide encoding the CER and the polynucleotide encoding the CAR or TCR binding protein, respectively, may be inserted into the vector at the same or different cloning site such that the expressed amino acid sequence produces a functional CER and CAR/or TCR. Vectors encoding tandem expression cassettes are referred to herein as "tandem expression vectors".

In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim receptor and a polynucleotide encoding a cellular immunotherapeutic agent (e.g., CAR). Polynucleotides encoding the chimeric Tim receptor and the cellular immunotherapeutic (e.g., CAR) may be sequentially cloned into vectors at different cloning sites, with the chimeric Tim receptor and the cellular immunotherapeutic (e.g., CAR) expressed under the control of different promoters.

In certain embodiments, host cells modified with chimeric Tim receptors may also be modified to co-express one or more small gtpases. Rho GTPase, a small (about 21k Da) subfamily of signaling G proteins and Ras superfamily, regulates actin cytoskeletal organization in various cell types and promotes pseudopodia extension and phagosome closure during phagocytosis (see, e.g., castellano et al, 2000, journal of cytosciences (j. Cell sci.) 113:2955-2961). Phagocytosis requires the recruitment of F-actin under tethered cells or particles, and F-actin rearrangement to allow membrane extension, leading to cell or particle internalization. RhoGTPase comprises RhoA, rac1, rac2, rhoG and CDC42. Other small gtpases, such as Rap1, are involved in regulating complement-mediated phagocytosis. Co-expression of small GTPases with chimeric Tim receptors may promote internalization and/or phagosome formation of target cells or particles by host cells. In some embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on a different vector than the vector containing the chimeric Tim receptor. In other embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on the same vector as the chimeric Tim receptor. The GTPase and chimeric Tim receptor can be expressed on the same vector under the control of different promoters (e.g., at different multiple cloning sites). Alternatively, the chimeric Tim receptor and GTPase may be expressed in a polycistronic vector under the control of one promoter. The polynucleotide sequence encoding the chimeric Tim receptor and the polynucleotide sequence encoding the small GTPase may be isolated from each other by IRES or viral 2A peptide in a polycistronic 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 may be co-expressed with the chimeric Tim receptor include Rac1, rac2, rab5 (also known as Rab5 a), rab7, rap1, rhoA, rhoG, CDC42, or any combination thereof. In particular embodiments, the GTPase comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the Rac1 amino acid sequence of SEQ ID NO:17, the Rab5 amino acid sequence of SEQ ID NO:18, the Rab7 amino acid sequence of SEQ ID NO:19, the Rap1 amino acid sequence of SEQ ID NO:20, the RhoA amino acid sequence of SEQ ID NO:21, the CDC42 amino acid sequence of SEQ ID NO:22, or any combination thereof.

In certain embodiments, host cells modified with chimeric Tim receptors can also be modified to co-express cellular immunotherapeutic agents (e.g., CARs, TCRs, etc.). In some embodiments, the cellular immunotherapeutic agent comprises a Chimeric Antigen Receptor (CAR). CARs are recombinant receptors, which generally include: an extracellular domain comprising a binding domain that binds to a target antigen; an intracellular signaling domain (e.g., including an intracellular signaling domain comprising ITAM and optionally an intracellular co-stimulatory domain), and a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

Binding domains suitable for use in the CARs of the present disclosure comprise any antigen binding polypeptide. The binding domain may comprise an antibody or an anti-antibody thereofThe pro-binding fragments comprise, for example, full-length heavy chains, fab fragments, fab ', F (ab') ₂ sFv, VH domain, VL domain, dAb, VHH, CDR and scFv. In certain embodiments, the CAR binding domain is murine, chimeric, human, or humanized.

In certain embodiments, the binding domain of the CAR targets a cancer or tumor antigen. Exemplary antigens that a CAR may 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-13 Ralpha 2, IL-11 Ralpha, PSA, fcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, hepcidin A2, hepcidin B2, lewis A antigen, MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, SSX-4, LGT 3, acetyl sugar-2, acetyl sugar G2, and GD-2.

In certain embodiments, the extracellular domain of a CAR provided in the present disclosure optionally includes an extracellular non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain at a location remote from the surface of the host cell to further allow for proper cell-to-cell contact, binding and activation. The extracellular spacer domain is typically 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, the selected binding epitope, the binding domain size, and affinity (see, e.g., guest et al, journal of immunotherapy, 28:203-11, 2005; pct publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., igG1, igG2, igG3, igG4, igA, igD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Altered IgG is described in PCT publication No. WO 2014/031687 ₄ A hinge region incorporated in its entirety by referenceHerein, a method for manufacturing a semiconductor device is provided. In a particular embodiment, the extracellular spacer domain comprises a modified IgG having the amino acid sequence of SEQ ID NO. 3 ₄ A hinge region.

Other examples of hinge regions that can be used in the CARs described herein include hinge regions from extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28, and CD7, which can be wild-type or variants thereof. In a particular embodiment, 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 further embodiments, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT publication WO2014/031687, which is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may include a stem region of a type II C-lectin (an extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins comprise CD23, CD69, CD72, CD94, NKG2A and NKG2D.

The CARs of the disclosure include a transmembrane domain connecting and between an extracellular domain and an intracellular signaling domain. The transmembrane domain ranges from about 15 amino acids to about 30 amino acids in length. The transmembrane domain is a hydrophobic alpha helix that passes through the host cell membrane and anchors the CAR in the host cell membrane. The transmembrane domain may be fused directly to the binding domain or extracellular spacer domain, if present. In certain embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., 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 intracellular signaling domain (e.g., the CAR comprises a CD28 costimulatory signaling domain and a 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 yet other embodiments, the transmembrane domain, extracellular domain and intracellular signaling domain are each selected from different molecules.

Exemplary transmembrane domains for the CARs of the present disclosure include the CD28, CD2, CD4, CD8a, CD5, CD3 epsilon, CD3 delta, CD3 zeta, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD, B, CD, CD86, CD95 (Fas), CD134 (OX 40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD154 (CD 40L), CD200R, CD223 (LAG 3), 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, ch3, ch4, tcrp 2, PTCH2, ptk, ltrp α, and the transmembrane domain Zap domain. Exemplary CD28 transmembrane domains include the amino acid sequence of SEQ ID NO. 7. In a particular embodiment, 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 and is capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the CAR to a target molecule (e.g., a cancer antigen) and activating at least one of a normal effector function or response of an immune cell (e.g., a T cell engineered to express the CAR). In some embodiments, the CAR induces a function of the T cell, such as cytolytic activity or T helper cell activity, such as secretion of cytokines or other factors. An intracellular signaling domain may be any portion of an intracellular signaling molecule that retains sufficient signaling activity. In some embodiments, the intracellular signaling domain is obtained from an antigen receptor component (e.g., TCR) or a co-stimulatory molecule. In some embodiments, the full-length intracellular signaling domain of an antigen receptor or co-stimulatory molecule is used. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor or co-stimulatory molecule is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the intracellular signaling domain is a variant of the full length or truncated portion 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 a signaling domain comprising an immune receptor tyrosine activation motif (ITAM). The signaling domain containing ITAM typically contains at least one (one, two, three, four or more) ITAM, which refers to YXXL/I-X _6-8 Conserved motifs of YXXL/I. The signaling domain containing ITAM can initiate T cell activation signaling following antigen binding or ligand binding. The ITAM signaling domain comprises intracellular signaling domains such as cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP12, fcrγ, and CD66 d. Exemplary CD3 zeta signaling domains that can be used in the CARs of the present disclosure include the amino acid sequences of SEQ ID NO 27 or 5.

The CAR intracellular signaling domain optionally includes a co-stimulatory signaling domain that, when activated with a primary or classical (e.g., ITAM-driven) activation signal, promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. Co-stimulatory signaling domains for the CAR include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1 BB), CD150 (SLAMF 1), CD152 (CTLA 4), CD223 (LAG 3), 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 particular embodiments, the costimulatory signaling domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), or 4-1BB (CD 137) signaling domain. Exemplary CD28 costimulatory signaling domains that can be used in the CARs of the present disclosure include the amino acid sequences of SEQ ID NO 26 or 4. An exemplary 4-1BB costimulatory 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 scFv binding domain derived from an antibody, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is from a TCR.

In certain embodiments, the chimeric antigen receptor comprises an amino acid sequence from any mammalian species, including human, primate, cow, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, transgenic species thereof, or any combination thereof. In certain embodiments, the chimeric antigen receptor is murine, chimeric, human or humanized.

In certain embodiments, the CAR is a first generation CAR, a second generation CAR, or a third generation CAR. First generation CARs typically have an intracellular signaling domain comprising cd3ζ, fcγri, or other intracellular signaling domain containing an activation domain of ITAM to provide a T cell activation signal. The second generation CAR further comprises a costimulatory signaling domain (e.g., from an endogenous T cell costimulatory receptor, such as CD28, 4-1BB, or ICOS). The third generation CAR includes an activation domain comprising ITAM, a first costimulatory signaling domain, and a second costimulatory signaling domain.

In some embodiments, one or more of the extracellular domain, the binding domain, the linker, the transmembrane domain, the intracellular signaling domain, or the costimulatory domain comprises a linking amino acid. "connecting amino acid" or "connecting amino acid residues" refers to one or more (e.g., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules or fragments of a protein, such as between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or at one or both ends of a linker connecting the 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). The linking amino acids may be created by the construct design of the fusion protein (e.g., amino acid residues generated during construction of the polynucleotide encoding the fusion protein by use of restriction sites or self-cleaving peptide sequences). For example, the transmembrane domain of the fusion protein may have one or more linking amino acids at the amino terminus, the carboxy terminus, or both.

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

Table 11.

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In certain embodiments, the host cell modified with the chimeric Tim receptor co-expresses a recombinant TCR. Recombinant TCR proteins comprise "traditional" TCRs consisting of heterodimers of an alpha chain polypeptide and a beta chain polypeptide or heterodimers of a gamma chain polypeptide and a delta chain polypeptide, binding fragments thereof, and fusion proteins, including, for example, single chain TCRs, single domain TCRs, soluble TCR fusion TCR proteins, and TCR fusion constructs (trucs ^TM ). In certain embodiments, the tandem expression cassette includes a polynucleotide encoding a recombinant tcrp chain comprising a tcrp variable region and a tcrp constant region, and a polynucleotide encoding a recombinant tcra chain comprising a tcra variable region and a tcra constant region. In certain embodiments, the recombinant TCR is an enhanced affinity TCR. In one embodiment, the recombinant TCR is an enhanced affinity TCR.

In certain embodiments, the recombinant TCR-binding protein is a single chain TCR (scTCR) comprising vα linked to vβ via 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, host cells modified with chimeric Tim receptors may also be modified to co-express single chain TCR (scTCR) fusion proteins. The scTCR fusion protein includes a binding domain comprising a scTCR (TCR V.alpha.domain linked to a TCR V.beta.domain), optionally an extracellular spacer, a transmembrane domain, and an intracellular component comprising a single intracellular signaling domain providing T cell activation signals (e.g., an activation domain containing CD3 zeta ITAM) and optionally a costimulatory signaling domain (see, aggen et al 2012, gene therapy 19:365-374; stone et al, cancer immunology and immunotherapy (Cancer immunology), 2014, 63:1163-76).

In certain embodiments, host cells modified with a chimeric Tim receptor can also be modified to co-express a T cell receptor-based chimeric antigen receptor (TCR-CAR). TCR-CARs are heterodimeric fusion proteins, which typically comprise a soluble TCR (a polypeptide chain comprising a vα domain and a cα domain, and a polypeptide chain comprising a vβ domain and a cβ domain), wherein the vβcβ polypeptide chain is linked to a transmembrane domain and an intracellular signaling component (e.g., an ITAM-containing activation domain and optionally a co-stimulatory signaling domain) (see, e.g., walsen et al, 2017, science report (Scientific Reports) & 7: 10713).

In certain embodiments, an engineered host cell that co-expresses a chimeric Tim receptor and a cellular immunotherapeutic (e.g., CAR, TCR, etc.) includes a recombinant nucleic acid encoding the chimeric Tim receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapeutic on different vectors within the engineered host cell.

In some embodiments, an engineered host cell that co-expresses a chimeric Tim receptor and a cellular immunotherapeutic (e.g., CAR, TCR, etc.) includes a recombinant nucleic acid encoding the chimeric Tim receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapeutic on the same vector as the chimeric Tim receptor within the engineered host cell. The chimeric Tim receptor and the cellular immunotherapeutic agent may be expressed under the control of different promoters on the same vector (e.g., at different multiple cloning sites). Alternatively, the chimeric Tim receptor and cellular immunotherapeutic agent may be expressed under the control of one promoter in a polycistronic vector (e.g., a tandem expression vector). The polynucleotide sequence encoding the chimeric Tim receptor and the polynucleotide sequence encoding the cellular immunotherapeutic may be isolated by IRES or viral 2A peptide in a polycistronic vector.

Tandem expression cassettes, tandem expression vectors, and engineered host cells comprising them are described in international application publication No. WO2019/191339, which is incorporated herein by reference in its entirety.

Host cells expressing the chimeric Tim receptor can be administered to a subject alone or in combination with other therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiation therapies, chemotherapy, small molecules, oncolytic viruses, electric pulse therapies, and the like.

In certain embodiments, the chimeric Tim receptor and the adoptive cellular immunotherapeutic (e.g., CAR, TCR-CAR, TCR, etc., as described above) are administered to a subject in the same host cell or in different host cells. In certain embodiments, the chimeric Tim receptor and the adoptive cellular immunotherapeutic are expressed in the same host cell from the same vector or from different vectors. In certain embodiments, the chimeric Tim receptor and the adoptive cellular immunotherapeutic are expressed in the same host cell from a polycistronic vector. In certain embodiments, the chimeric Tim receptor is expressed in the same host cell type as the adoptive cellular immunotherapeutic (e.g., the chimeric Tim receptor is expressed in CD 4T cells and the CAR/or TCR is expressed in CD 4T cells, or the chimeric Tim receptor is expressed in CD8T cells and the CAR/or TCR is expressed in CD8T cells). In other embodiments, the chimeric Tim receptor is expressed in a different host cell type as an adoptive immunotherapeutic (e.g., the chimeric Tim receptor is expressed in CD 4T cells and the CAR/or TCR is expressed in CD8T cells). Cellular immunotherapy compositions, methods of manufacture, and methods of use comprising combinations of immune cells or cell subsets engineered with chimeric Tim receptors and cellular immunotherapeutic agents (e.g., CARs, TCRs, etc.) are described in PCT international publication No. WO2019/191340, which is incorporated herein by reference in its entirety.

Exemplary antigens that recombinant TCR, affinity enhanced TCR, CAR, TCR-CAR or scTCR fusion proteins can target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, survivin, alpha fetoprotein and tumor specific neoantigens.

The CARs of the present disclosure can target a variety of antigens, including viral antigens, bacterial antigens, fungal antigens, parasitic antigens, tumor antigens, autoimmune disease antigens. Exemplary antigens that a CAR may 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-13 Ralpha 2, IL-11 Ralpha, PSA, fcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, hepcidin A2, hepcidin B2, lewis A antigen, MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, SSX-4, LGT 3, acetyl sugar-2, acetyl sugar G2, and GD-2.

Radiation therapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy, intensity modulated radiation therapy, volume modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiation therapy, or any combination thereof.

Exemplary antibodies for use in combination with the chimeric Tim compositions described herein include rituximab (rituximab), pertuzumab (pertuzumab), trastuzumab (trastuzumab), alemtuzumab (alemtuzumab), tiuximab (Ibritumomab tiuxetan), bentuximab (Brentuximab vedotin), cetuximab (cetuximab), bevacizumab (bevacizumab), acimumab (abciximab), adalimumab (adalimumab), alfumagram (alexaprop), barlizumab (basilizimab), belimumab (bezimab), bei Zuoluo mab (bezlotoxumab), cinacazumab (canakiumab), cetuzumab (certolizumab pegol), daclizumab (daclizumab), dienumab (denoxiab), alemtuzumab (bevacizumab), lizumab (alemtuzumab), and adalimumab (paluzumab).

Exemplary inhibitors of immune checkpoint molecules that may be used in conjunction with the chimeric Tim compositions described herein include checkpoint inhibitors that target PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, PVRIG/CD112R, or any combination thereof. In certain embodiments, the immune checkpoint inhibitor may be an antibody, a peptide, an RNAi agent, or a small molecule. Antibodies specific for CTLA-4 may be ipilimumab (ipilimumab) or tremelimumab (tremelimumab). The antibody specific for PD-1 may be pidizumab (pidirizumab), nivolumab (nivolumab) or pembrolizumab (pembrolizumab). The antibody specific for PD-L1 may be Dewaruzumab (durvalumab), ab-zhuzumab (atezolizumab), or Avelumab (avelumab).

Exemplary chemotherapies for use in conjunction with the chimeric Tim receptor compositions described herein can include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule-inhibiting drugs, DNA damaging agents, antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (e.g., intercalating agents), and DNA repair inhibitors.

Chemotherapy includes nonspecific cytotoxic agents that inhibit mitosis or cell division, as well as molecular targeted therapies that prevent the growth and spread of cancer cells by targeting specific molecules (e.g., oncogenes) that are involved in tumor growth, progression, and metastasis. Exemplary non-specific chemotherapeutics for use in conjunction with the expression cassette compositions described herein can include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule-inhibiting drugs, 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), hypomethylation agents, and DNA repair inhibitors.

Examples of chemotherapeutic agents contemplated for use in the combination therapies contemplated herein include vemurafenib,Dabrafenib, trametinib, cobicitinib and anastrozoleBicalutamide->Bleomycin sulfateBusulfan->Busulfan injection>Capecitabine->N4-pentoxycarbonyl-5-deoxy-5-fluorocytosine nucleoside, carboplatin +.>Carmustine>ChlorambucilCisplatin->Cladribine>Cyclophosphamide (/ -s)>Or->) Cytarabine, cytosine-ArabicGlycoside->Cytarabine liposome injection>Dacarbazine->Actinomycin (actinomycin D, cosmegan), daunorubicin hydrochloride +.>Daunorubicin citrate liposome injection>Dexamethasone, docetaxel +.>Doxorubicin hydrochloride->Etoposide->Fludarabine phosphate->5-fluorouracilFluotamide->Tizacitabine, gemcitabine (difluodeoxycytidine), hydroxyureaIdarubicin->Ifosfamide->Irinotecan->L-asparaginase->Calcium leucovorin, melphalan +.>6-mercaptopurine->Methotrexate>Mitoxantrone->Getuzumab, paclitaxel +.>Phoenix (yttrium 90/MX-DTPA), penstatin and polifeprosan 20 +/f for carmustine-containing implants>Tamoxifen citrateTeniposide->6-thioguanine, thiotepa, tirapazamine >Topotecane hydrochloride for injection>Vinblastine->Vincristine->Ibrutinib, vinatoxin, crizotinib, aprepitant, bujitinib, ceritinib and vinorelbine +.>

Exemplary alkylating agents for combination therapies contemplated herein include nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes, uratemustine (amisulci) Uracil nitrogen/>) Mustard of nitrogenCyclophosphamide (/ -s)>Revimmune ^TM ) Ifosfamide->Melamine->Chlorambucil->Pipobromine-> Triethylenemelamine->Triethylenethiophosphamide, temozolomide +.>Thiotepa->Busulfan (Busulfan)Carmustine>Lomustine>StreptozotocinAnd dacarbazine->Other exemplary alkylating agents for combination therapies contemplated herein include, but are not limited to, oxaliplatin +.>Temozolomide (+)>And->) The method comprises the steps of carrying out a first treatment on the surface of the Actinomycetes (actinomycetes)Plain (also called actinomycin D,)>) The method comprises the steps of carrying out a first treatment on the surface of the Melphalan (also known as L-PAM, L-oncolytic toxin and phenylalanine mustard,/->) The method comprises the steps of carrying out a first treatment on the surface of the Altretamine (also known as altretamine (HMM)) ->) The method comprises the steps of carrying out a first treatment on the surface of the Carmustine>BendamustineBusulfan (/ -herba)>And->) The method comprises the steps of carrying out a first treatment on the surface of the Carboplatin->Lomustine (also known as CCNU,) The method comprises the steps of carrying out a first treatment on the surface of the Cisplatin (also known as CDDP,>and->-AQ); chlorambucil->Cyclophosphamide (/ -s)>And->) The method comprises the steps of carrying out a first treatment on the surface of the Dacarbazine (also known as DTIC, DIC and imidazole carboxamide,) >) The method comprises the steps of carrying out a first treatment on the surface of the Altretamine (also known as altretamine (HMM)) ->) The method comprises the steps of carrying out a first treatment on the surface of the Ifosfamide->Prednumustine; methyl benzyl hydrazineDichloromethyldiethylamine (also known as nitrogen mustard, nitrogen medium and mechlorethamine hydrochloride,/-)>) The method comprises the steps of carrying out a first treatment on the surface of the Streptozotocin->Thiotepa (also known as thiophosphamide, TESPA and TSPA,/-for example)>) The method comprises the steps of carrying out a first treatment on the surface of the Cyclophosphamide (cyclophosphamide)And bendamustine hydrochloride

Exemplary platinum-based agents for the combination therapies contemplated herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthrlatin, and triplatin tetranitrate.

Exemplary hypomethylation agents for combination therapy include azacytidine and decitabine.

Exemplary molecular targeted inhibitors for use in conjunction with the chimeric Tim receptor compositions described herein include small molecules that target molecules involved in cancer cell growth and survival, including, 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.

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

Exemplary angiogenesis inhibitors include, but are not limited to, A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), ABT-627 (atrasentan) (Abbott Laboratories/Xinlay), ABT-869 (Abbott laboratories), actimid (CC 4047, pomalidomide) (Celgene Corporation), adGVGPEDF.11D (GenVec), ADH-1 (Exherin) (Adherex Technologies), AEE788 (Novartis), AG-013136 (axitinib) (Pfizer), AG3340 (primenta) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP O2) (Alnylam Pharmaceuticals), AMG 386 (Amgen), AMG706 (Amgen) Apatinib (YN 968D 1) (Jiangsu Hengrui Medicine), AP23573 (ground phosphorus limus/MK 8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197 (ArQule), ASA404 (Novartis/Antisoma), attomode (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), avastin (Bevacizumab) (Genntech), AZD2171 (Sidinib/Recentin) (AstraZeneca), BAY 57-9352 (tiratinib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim Pharmaceuticals), BIBW 2992 (Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-Myers Squibb), BMS-582664 (brinib) (Bristol-Myers Squibb), BMS-690514 (Bristol-Myers Squibb), calcitriol, CCI-779 (torisel) (Wyeth), CDP-791 (Imclone Systems), cephalotaxine (homocephalotaxine/HHT) (ChemGenex Therapeutics), celecoxib (celecoxib) (Pfizer), CEP-7055 (Cephalon/Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Metastat) (Collagenex Pharmaceuticals), combretastatin (Oxigene), CP-751, 871 (Figitumumab) (Pfizer), CP-547, 632 (Pfizer), CS-7017 (Daiichi Sankyo Pharma), CT-322 (Adginex) (Adneus) curcumin, dalteparin (famoxamine) (Pfizer), disulfiram (antabust), E7820 (Eisai Limited), E7080 (Eisai Limited), EMD 121974 (cilengitide) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), endness (simcore), erbitux (ImClone/Bristol-Myers Squibb), EZN-2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals), GC1008 (Genzyme), genistein, GSK1363089 (fortinib) (GlaxoSmithKline), GW786034 (pazosmithkline), GT-111 (Vascular Biogenics ltd), GT-111 (paxosmithkline), IMC-1121B (ramucirumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (Introgen Therapeutics), iressa (ZD 1839/gefitinib), LBH589 (Faridak/Panobinostst) (Novartis), lucentis (ranibizumab) (Genntech/Novartis), LY317615 (Enzastaurin) (Eli Lilly and Company), macugen (piplitani) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (tanditinib) (Millennium), neovastat (AE 941/benifene) (Aetenna Zentaris), nexavar (Bayer/Onyx), NM-3 (Genzymoport), nadine (Cougar Biotechnology), NPI-2358), OSI (Paloma Pharmaceuticals-Paloma Pharmaceuticals (6339), inc.), panzem capsules (2 ME 2) (EntreMed), panzem NCD (2 ME 2) (EntreMed), PF-0234066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen Industries/Medigen Biotechnology), PKC412 (Novartis), tea polyphenol E (green tea extract) (Polypheno E International, inc.), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Watanani) (Novartis), PXD101 (belisestat) (CuraGen Corporation), RAD001 (everolimus) (Novartis), RAF265 (Novartis), ruigafini (BAY 73-4506) (Bayer), revlimid (Celgene), anecortave (Alcon Research), SN38 (liposome) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (Papanicotide) (Novartis), squalamine (Genaera), suramin, soptan (Pfizer), taroKam (Genntech), TB-403 (Thrombogenics), tempostatin (Collard Biopharmaceuticals), tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), thalidomide (Celgene Corporation), tinzaparin sodium, TKI258 (Novartis), TRC093 (Tracon Pharmaceuticals Int.), VEGF Trap (Abelcep) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), veglin (VasGene Therapeutics), bortezomib (lennium), XL (Exelxis), XL (XL 6474), exelxis (TK), xl (XK 6474), exelxix (ZS), XK (ZS 6474) and Exelxix (ZS).

Exemplary B-Raf inhibitors include vemurafenib, dabrafenib and Kang Naifei ni.

Exemplary MEK inhibitors include bimetanib, cobetanib, refatinib, semetanib, and tramatinib.

Exemplary BTK inhibitors include ibrutinib, loxo-305, tiratinib, GDC-0853, acartinib, ONO-4059, capetinib, BGB-3111, HM71224 and M7583.

Exemplary TRK inhibitors include emtrictinib, lartinib, CH7057288, ONO-7579, LOXO-101, letatinib, and LOXO-195.

Exemplary c-KIT inhibitors include imatinib, sunitinib, and bunatinib.

Exemplary c-MET inhibitors include carbamazepine, crizotinib, tivantinib, onapristal bead mab, INCB28060, AMG-458, sivoratib, and terbutanib.

Exemplary CDK4/6 inhibitors include palbociclib, rebabociclib, flumazenil and troraxili.

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

Exemplary FGFR inhibitors include erdasatinib, pemitinib, inflitinib, luo Jiati ni, AZD4547, BGJ398, FP-1039, and ARQ 087.

Exemplary FLT-3 inhibitors include quezatinib, clarithromycin, ji Ruiti, midostaurin, and letatinib.

Exemplary IDH1 inhibitors include Ai Funi cloth, BAY-1436032 and AGI-5198.

Exemplary IDH2 inhibitors include azepine.

Exemplary PDGFRA inhibitors include imatinib, regorafenib, clarithromycin, and olamumab.

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

Exemplary RET inhibitors include lenvatinib, aletinib, vandetanib, cabozitinib, BLU-667, and LOXO-292.

Exemplary ROS1 inhibitors include ceritinib, loratidine, emtrictinib, crizotinib, TPX-0005, and DS-6051b.

Exemplary Vascular Endothelial Growth Factor (VEGF) receptor inhibitors include, but are not limited to bevacizumabAxitinib->Alanine brinib (BMS-582664, (S) - ((R) -1- (4- (4-fluoro-2-methyl-1H-indol-5-yloxy) -5-methylpyrrolo [2, 1-f)][1,2,4]Triazin-6-yloxy) propan-2-yl) 2-aminopropionate); sorafenib->Pazopanib->Sunitinib malate->Sidinib (AZD 2171, CAS 288383-20-1); nidaminib (BIBF 1120, CAS 928326-83-4); foretinib (GSK 1363089); tiratinib (BAY 57-9352, CAS 332012-40-5); apatinib (YN 968D1, CAS 811803-05-1); imatinib- >Panatinib (AP 24534, CAS 943319-70-8); tivozanib (AV 951, CAS 475108-18-0); regorafenib (BAY 73-4506, CAS 755037-03-7); varanib dihydrochloride (PTK 787, CAS 212141-51-0); brinib (BMS-540215, CAS 649735-46-6); vandetanib (>Or AZD 6474); motrasenib diphosphate (AMG 706, CAS 857876-30-3, N- (2, 3-dihydro-3, 3-dimethyl-1H-indol-6-yl) -2- [ (4-pyridylmethyl) amino)]-3-pyridinecarboxamide as described in PCT publication No. WO 02/066470); poly Wei Tini di-lactate (TKI 258, CAS 852433-84-2); linfanib (ABT 869, CAS 796967-16-3); cabotinib(XL 184, CAS 849217-68-1); litatinib (CAS 111358-88-4); n- [5- [ [ [5- (1, 1-dimethylethyl) -2-oxazolyl ]]Methyl group]Thio-]-2-thiazolyl]-4-piperidinecarboxamide (BMS 3803, 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 (BMS 690514); n- (3, 4-dichloro-2-fluorophenyl) -6-methoxy-7- [ [ (3 a alpha, 5 beta, 6a alpha) -octahydro-2-methylcyclopenta [ c ]]Pyrrol-5-yl]Methoxy group]-4-quinazolinamine (XL 647, CAS 781613-23-8); 4-methyl-3- [ [ 1-methyl-6- (3-pyridinyl) -1H-pyrazolo [3,4-d ] ]Pyrimidin-4-yl]Amino group]-N- [3- (trifluoromethyl) phenyl ]]Benzamide (BHG 712, CAS 940310-85-0); and Abelmoschus->

Exemplary EGF pathway inhibitors include, but are not limited to, tyrosine phosphorylation inhibitor 46, EKB-569, erlotinibGefitinib>Erbitux, nimotuzumab, lapatinib +.>Cetuximab (anti-EGFR mAb), ¹⁸⁸ Re-labeled Nituzumab (anti-EGFR mAb), and those compounds generally and specifically disclosed in WO 97/02266, EP 0 564 409, WO 99/03854, EP 0 520 722, EP 0 566 226, EP 0 787 722, EP 0 837 063, U.S. Pat. No. 5,747,498, WO 98/10767, WO 97/30034, WO 97/49688, WO97/38983 and WO 96/33980. Exemplary EGFR antibodies include, but are not limited to, cetuximab +.>Panitumumab->Matuzumab (EM)D-72000); trastuzumab->Nituzumab (hR 3); zatuzumab; theraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). Exemplary Epidermal Growth Factor Receptor (EGFR) inhibitors include, but are not limited to erlotinib hydrochloride->Ceritinib; b, b; ornitinib; icotinib; gefitinib>N- [4- [ (3-chloro-4-fluorobenzene) amino group ]-7- [ [ (3 "S") -tetrahydro-3-furanyl group]Oxy group]-6-quinazolinyl]-4 (dimethylamino) -2-butenamide,) The method comprises the steps of carrying out a first treatment on the surface of the Vandetanib->Lapatinib->(3R, 4R) -4-amino-1- ((4- ((3-methoxyphenyl) amino) pyrrolo [2, 1-f)][1,2,4]Triazin-5-yl) methyl) piperidin-3-ol (BMS 690514); kanetinib dihydrochloride (CI-1033); 6- [4- [ (4-ethyl-1-piperazinyl) methyl]Phenyl group]-N- [ (1R) -1-phenylethyl]-7H-pyrrolo [2,3-d]Pyrimidine-4-amine (AEE 788, CAS 497839-62-0); xylolitinib (TAK 165); pelitinib (EKB 569); afatinib (BIBW 2992); lenatinib (HKI-272); n- [4- [ [1- [ (3-fluorophenyl) methyl)]-1H-indazol-5-yl]Amino group]-5-methylpyrrolo [2,1-f][1,2,4]Triazin-6-yl]-carbamic acid, (3S) -3-morpholinomethyl ester (BMS 599626); n- (3, 4-dichloro-2-fluorophenyl) -6-methoxy-7- [ [ (3 a alpha, 5 beta, 6a alpha) -octahydro-2-methylcyclopenta [ c ]]Pyrrol-5-yl]Methoxy group]-4-quinazolinamine (XL 647, CAS 781613-23-8); 4- [4- [ [ (1R) -1-phenylethyl]Amino group]-7H-pyrrolo [2,3-d]Pyrimidin-6-yl]Phenol (PKI 166, CAS 187724)61-4); luo Xiti Ni.

Exemplary mTOR inhibitors include, but are not limited to, rapamycinAnd analogs and derivatives thereof; SDZ-RAD; temsirolimus (+)>Also known as CCI-779); gespholimus (previously known as deferolimus, dimethyl phosphinic acid (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-pentoxy-11, 36-dioxa-4-azatricyclo [ 30.3.1.0) ^4,9 ]Thirty-sixteen-16,24,26,28-tetraen-12-yl]Propyl group]-2-methoxycyclohexyl, also known as AP23573 and MK8669, and described in PCT publication No. WO 03/064383; everolimus (+)>Or RAD 001); rapamycin (AY 22989,)>) The method comprises the steps of carrying out a first treatment on the surface of the Sima Pi Mord (CAS 164301-51-3); (5- {2, 4-bis [ (3S) -3-methylmorpholin-4-yl)]Pyrido [2,3-d ]]Pyrimidin-7-yl } -2-methoxyphenyl) methanol (AZD 8055); 2-amino-8- [ trans-4- (2-hydroxyethoxy) cyclohexyl]-6- (6-methoxy-3-pyridinyl) -4-methyl-pyrido [2,3-d]Pyrimidin-7 (8H) -one (PF 04691502, CAS 1013101-36-4); and N ² - [1, 4-dioxo- [ [4- (4-oxo-8-phenyl-4H-1-benzopyran-2-yl) morpholin-4-yl ]]Methoxy group]Butyl group]-L-arginyl glycyl-L-alpha-aspartyl-L-serine-inner salt (SF 1126, CAS 936487-67-1).

Exemplary phosphoinositide 3-kinase (PI 3K) inhibitors include, but are not limited to, du Weili sibutra, iderani, 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 publication nos. WO 09/036082 and WO 09/055730); 2-methyl-2- [4- [ 3-methyl-2-oxo-8- (quinolin-3-yl) -2, 3-dihydroimidazo [4,5-c ] quinolin-1-yl ] phenyl ] propionitrile (also known as BEZ 235 or NVP-BEZ 235 and described in PCT publication No. WO 06/122806); 4- (trifluoromethyl) -5- (2, 6-dimorpholin-4-yl) pyridin-2-amine (also known as BKM120 or NVP-BKM120 and described in PCT publication No. WO 2007/084786); cerclage (VX 680 or MK-0457, cas 639089-54-6); (5Z) -5- [ [4- (4-pyridinyl) -6-quinolinyl ] methylene ] -2, 4-thiazolidinedione (GSK 1059615, CAS 958852-01-2); (1E, 4S,4aR,5R,6aS,9 aR) -5- (acetoxy) -1- [ (di-2-propenylamino) methylene ] -4,4a,5, 6a, 8,9 a-octahydro-11-hydroxy-4- (methoxymethyl) -4a, 6 a-dimethyl-cyclopenta [5,6] naphtho [1,2-c ] pyran-2,7,10 (1H) -trione (PX 866, CAS 502632-66-8); and 8-phenyl-2- (morpholin-4-yl) -chromen-4-one (LY 294002, CAS 154447-36-6). Exemplary Protein Kinase B (PKB) or AKT inhibitors include, but are not limited to, 8- [4- (1-aminocyclobutyl) benzene ] -9-phenyl-1, 2, 4-thiazolo [3,4-f ] [1,6] naphthyridin-3 (2H) -one (MK-2206, CAS 1032349-93-1); pirifaxine (KRX 0401); 4-dodecyl-N-1, 3, 4-thiadiazol-2-yl-benzenesulfonamide (PHT-427, CAS 1191951-57-1); 4- [2- (4-amino-1, 2, 5-oxadiazol-3-yl) -1-ethyl-7- [ (3S) -3-piperidinylmethoxy ] -1H-imidazo [4,5-c ] pyridin-4-yl ] -2-methyl-3-butyn-2-ol (GSK 690693, CAS 937174-76-0); 8- (1-hydroxyethyl) -2-methoxy-3- [ (4-methoxyphenyl) methoxy ] -6H-dibenzo [ b, d ] pyran-6-one (palomid 529, P529, or SG-00529); tricirbin (6-amino-4-methyl-8- (. Beta. -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 ] -phenethylamine (a 675563, CAS 552325-73-2); 4- [ (4-chlorophenyl) methyl ] -1- (7H-pyrrolo [2,3-d ] pyrimidin-4-yl) -4-piperidinamine (CCT 128930, CAS 885499-61-6); 4- (4-chlorophenyl) -4- [4- (1H-pyrazol-4-yl) phenyl ] -piperidine (AT 7867, CAS 857531-00-1); and Archexin (RX-0201, CAS 663232-27-7).

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, aletinib, buganidine, dalanatercept, emtrictinib, and loratidine.

In certain embodiments in which the chimeric Tim receptor-modified cells are administered in combination with one or more additional therapies, the one or more additional therapies may be administered at a dose that if administered as monotherapy may be considered to be lower than the therapeutic dose. In such embodiments, the chimeric Tim receptors can provide additive or synergistic effects such that one or more additional therapies can be administered at lower doses. Combination therapy comprises administering a chimeric Tim receptor composition as described herein prior to (e.g., 1 day to 30 days or more prior to) additional therapy, concurrently with (on the same day) or after (e.g., 1 day to 30 days or more after) additional therapy. In certain embodiments, the chimeric Tim receptor-modified cells are administered after administration of one or more additional therapies. In further embodiments, the chimeric Tim receptor modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the one or more additional therapies. In still further embodiments, the cells modified with the chimeric Tim receptor are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the one or more additional therapies. Where the one or more additional therapies involve multiple doses, the chimeric Tim receptor-modified cells can be administered after an initial dose of the one or more additional therapies, after a final dose of the one or more additional therapies, or between multiple doses of the one or more additional therapies.

In certain embodiments, the methods of the present disclosure comprise a depletion step. To mitigate toxicity to the subject, the step of removing the chimeric Tim receptor from the subject can be performed after a sufficiently long period of therapeutic benefit. In such embodiments, the chimeric Tim receptor vector can comprise an inducible suicide gene, such as iCASP9, inducible Fas, or HSV-TK. Similarly, chimeric Tim receptor vectors can be designed to express known cell surface antigens, such as CD20 or truncated EGFR (SEQ ID NO: 16), which facilitate depletion of transduced cells by infusion of related monoclonal antibodies (mabs), such as rituximab against CD20 or cetuximab against EGFR. Alemtuzumab (Alemtuzumab) targeting 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 with 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. The subject 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, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Examples

Example 1

Evaluation of the synergistic effect of TIM-CER-T cells on synthetic lethal PARP inhibitors in ovarian cancer

Kuramochi ovarian cancer cells expressing mCherry-NLS plasmid were incubated with 0.78 to 100uM of PARP inhibitor nilaparib. The number of Kuramochi cells/wells was tracked via imaging. At t=50, the number of Kuramochi cells in the culture monotonically decreased with increasing nilaparib concentration. The results are shown in fig. 1A.

Similar experiments were performed at different doses of the PARP inhibitors nilaparib, olaparib, tazopanib, veliparib and Lu Kapa, and Kuramochi cell numbers were plotted at t=50 h. Taraxazopanib and nilaparib showed the most sensitive EC ₅₀ Values, followed by Lu Kapa and olaparib. Among the five PARP inhibitors tested, veliparib had minimal effect on the number of Kuramochi cells detected. At t=5 At 0h, nilaparib and Lu Kapa rib almost eliminated Kuramochi cultures. The results are shown in fig. 1B.

Example 2

PARP inhibitors up-regulate surface phosphatidylserine and have negligible effect on CD4/CD 8T cells

Kuramochi cells expressing mCherry-NLS plasmid were incubated with different concentrations of nilaparib, tim4-Fc chimera and anti-mouse IgG2a antibody conjugated to Alexa 488. The number of tim4+kuramochi cells (i.e., green subjects) increased significantly and above the IgG only control when it responded to incubation with nilaparib even at a concentration of 1.56 μm (fig. 2A-2B). In response to 1.56-12.5 μm nilaparib (fig. 2C), the number of Kuramochi cells increased, indicating that PARP inhibition induced surface phosphatidylserine without killing Kuramochi cells.

Co-cultures of primary human CD4+ and CD8+ T cells were incubated with different concentrations of Nilapatinib and T cell viability, diameter and density were measured using a Vicell cell counter on days 7, 8 and 9 (corresponding to days 1, 2 and 3 after incubation with Nilapatinib) after activation with TransAct. T cell viability, average diameter and expansion showed negligible changes from day 7 to day 9 as a function of nilaparib concentration (fig. 3).

Example 3

Chimeric TIM receptor/nilaparib co-kills kuramiochi cells at different doses in vitro

Kuramochi cells expressing mCherry-NLS plasmid were incubated with 25, 12.5 or 6.25 μm nilaparib for 20 hours. The next day, the concentration of nilaparib was reduced to 0.52 μm, and a co-culture of primary human cd4+ and cd8+ T cells expressing chimeric Tim receptor construct 13A (Tim 4 binding domain-CD 28 transmembrane domain-CD 28 co-stimulatory domain-CD 3 zeta signaling domain) (SEQ ID NO: 162) was added to Kuramochi culture. According to experimental conditions, T cell cultures expressing chimeric Tim receptor construct 13A significantly reduced the number of Kuramochi cells and were significantly lower than the number of Kuramochi cells in cultures mimicking the transduction control (control-T). The number of Kuramochi cells expressing mCherry-NLS was monitored via IncuCyte. The results are shown in fig. 4. The combination therapy of chimeric Tim receptor construct 13A and nilaparib at different doses synergistically kills Kuramochi cells.

Kuramochi cells expressing mCherry-NLS plasmid were incubated with 1.56. Mu.M nilaparib for about 20 hours. The following day, the medium was removed from the cells and replaced with fresh medium with 0. Mu.M nilaparib, and primary human CD4+ and CD8+ T cells expressing the chimeric Tim receptor CTX140 (Tim 4 binding domain-CD 28 hinge-CD 28 transmembrane-CD 28 costimulatory domain-DAP 12) (SEQ ID NO: 195) or CTX156 (tEGFR control) were added to the Kuramochi culture. The number of Kuramochi cells expressing mCherry-NLS was monitored via IncuCyte. The results are shown in fig. 5. T cell cultures expressing the chimeric Tim receptor construct CTX140 significantly reduced the number of Kuramochi cells and were significantly lower than the number of Kuramochi cells in cultures mimicking the transduction control.

2,500 Kuramochi ovarian cancer cells expressing the mCherry-NLS plasmid were plated on day 0. 25, 12.5 or 6.25. Mu.M nilaparib was added to the cell culture after about 4 hours. About 20 hours after the initial nilaparib dose, primary human cd4+ and cd8+ T cells expressing chimeric Tim receptor CTX137 (Tim 4 binding domain-CD 28 transmembrane-CD 28 costimulatory domain-DAP 12) (SEQ ID NO: 197) or CTX156 (tgfr control) and a maintenance dose of 0.52 μm nilaparib were added. The results of the 25 μm nilaparib pretreatment are shown in fig. 6. The results of the 25, 12.5 or 6.25 μm nilaparib pretreatment with varying effector to target (E: T) ratios are shown in fig. 7. T cell cultures expressing the chimeric Tim receptor construct CTX137 significantly reduced the number of Kuramochi cells and were significantly lower than the number of Kuramochi cells in cultures mimicking the transduction control.

To induce Ptd-Ser exposure, BRCA-2 mutated Kuramochi cell lines were treated with a therapeutic dose of the PARP inhibitor nilaparib. Brief exposure to nilaparib causes changes in membrane phospholipid symmetry in a dose-dependent manner and effectively inhibits Kuramochi cell growth (fig. 8A). The addition of the chimeric Tim4 receptor pCTX133 (Tim 4-TLR2-CD3 z) enhanced the in vitro potency of nilaparib at a low effector to target ratio (1:1) compared to the transduced control, demonstrating the ability of pCTX133 to elicit a direct cytotoxic effect on target cells (fig. 8B).

Example 4

Chimeric TIM receptor/nilaparib synergistic killing of a2780 cells

T cell transduction and culture: CD4 and CD 8T cells were isolated from frozen healthy donor PBMCs using Miltenyi cd4+ and cd8+ isolation kits. CD4 and CD 8T cells were then mixed at a 1:1 ratio and incubated overnight in OpTmizer medium supplemented with cell serum replacement, L-glutamine, glutaMAX, and IL-2, IL-7, and IL-15 cytokines in flat bottom 24-well plates, and the tranact diluted 1:50. T cells were transduced with the appropriate virus with moi=10, 8 μg/mL polyaromatic and 1:50 transactt in flat bottom 96 well plates 16-24 hours after activation with transactt. About 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. T cells were counted approximately every two days thereafter and fresh medium was replenished.

Cell line engineering:a2780 (human ovarian carcinoma cell line) and Kuramochi (BRCA-deficient human ovarian carcinoma cell line) cells were transduced with lentiviral vectors encoding mCherry-Nuclear Localization Sequences (NLS) genes, and then classified according to mCherry expression. Clone sorting, expansion and banking were performed on the A2780-mCherry-NLS cell line, while expansion and extensive banking were performed on the Kuramochi-mCherry-NLS cells. Cell lines were thawed in RPMI 1640 supplemented with 10% FBS and maintained in culture up to about passage 25.

Co-culture assay: on day 0, 2,500A 2780-mCh-NLS or Kuramoch-mCh-NLS cells were placed in NUNC Edge 96 well plates and Nilapatinib (free base) was added at appropriate concentrations after about 4 hours, all in RPMI 1640+10% FBS. After about 20 hours, the medium was removed and replaced with the appropriate concentration of nilaparib (free base) and the appropriate number of T cells, all in the OpTmizer medium supplemented with cell serum replacement, L-glutamine, glutaMAX, and IL-2, IL-7, and IL-15 cytokines. When using A2780-mCh-NLS cells, the supernatant is centrifuged and resuspended in T cell culture medium to preserve any suspended components of A2780 cells. Plates were immediately transferred to IncuCyte as appropriate.

Flow cytometry measurement of surface PS:A2780-mCh-NLS cells were treated with 1.56 or 25. Mu.M nilaparib or with an equivalent volume of DMSO (control). After 48 hours, the samples were trypsinized and stained with Tim4-Fc followed by fluorescent-labeled secondary antibodies to Tim 4-Fc. The samples were immediately analyzed via flow cytometry.

As shown in FIGS. 9A-9B, T cells modified with chimeric Tim4 receptor pCTX247 (Tim 4 binding domain-CD 28 transmembrane-CD 28 signaling domain-CD 3 zeta signaling domain; SEQ ID NO: 257) exhibited anti-tumor activity in vitro in two ovarian cancer cell lines. As shown in FIG. 10, T cells modified with the chimeric Tim4 receptor pCTX797 (Tim 4 binding domain-CD 28 transmembrane-CD 28 signaling domain-CD 3 zeta signaling domain; SEQ ID NO: 258) exhibited in vitro antitumor activity in the A2780 ovarian cancer model.

As shown in fig. 11A-11B, phosphatidylserine was presented on the cell surface in response to nilaparib in both ovarian cancer models.

As shown in FIG. 12, the chimeric Tim4 receptor pCTX797 (Tim 4 binding domain-CD 28 transmembrane-CD 28 signaling domain-CD 3 zeta signaling domain; SEQ ID NO: 258) showed synergy with additional PARP inhibitors (Lu Kapa Ni, olaparib and Nilaparib) compared to the truncated EGFR (EGFRt) control (pCTX 236).

The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in this specification and/or listed in the application data sheet, including, but not limited to, U.S. provisional patent application No. 63/066,150 filed on 8/14/2020, U.S. provisional patent application No. 63/087,049 filed on 10/2020, and U.S. provisional patent application No. 63/226,712 filed on 7/28/2021, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

Sequence listing

<110> Senro treatment Co

<120> compositions and methods for treating cancer with chimeric TIM receptor in combination with poly (ADP-ribose) polymerase inhibitors

<130> 200265.415WO

<150> 63/066,150

<151> 2020-08-14

<150> 63/087,049

<151> 2020-10-02

<150> 63/226,712

<151> 2021-07-28

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Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

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Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu Met Ser Lys Glu Pro Leu

370 375 380

Ile Leu Trp Leu Met Ile Glu Phe Trp Trp Leu Tyr Leu Thr Pro Val

385 390 395 400

Thr Ser Glu Thr Val Val Thr Glu Val Leu Gly His Arg Val Thr Leu

405 410 415

Pro Cys Leu Tyr Ser Ser Trp Ser His Asn Ser Asn Ser Met Cys Trp

420 425 430

Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys Lys Glu Ala Leu Ile Arg

435 440 445

Thr Asp Gly Met Arg Val Thr Ser Arg Lys Ser Ala Lys Tyr Arg Leu

450 455 460

Gln Gly Thr Ile Pro Arg Gly Asp Val Ser Leu Thr Ile Leu Asn Pro

465 470 475 480

Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys Arg Ile Glu Val Pro Gly

485 490 495

Trp Phe Asn Asp Val Lys Ile Asn Val Arg Leu Asn Leu Gln Arg Ala

500 505 510

Ser Thr Thr Thr His Arg Thr Ala Thr Thr Thr Thr Arg Arg Thr Thr

515 520 525

Thr Thr Ser Pro Thr Thr Thr Arg Gln Met Thr Thr Thr Pro Ala Ala

530 535 540

Leu Pro Thr Thr Val Val Thr Thr Pro Asp Leu Thr Thr Gly Thr Pro

545 550 555 560

Leu Gln Met Thr Thr Ile Ala Val Phe Thr Thr Ala Asn Thr Cys Leu

565 570 575

Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu Ala Thr Gly Leu Leu Thr

580 585 590

Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu Thr Ala Glu Ser Glu Thr

595 600 605

Val Leu Pro Ser Asp Ser Trp Ser Ser Val Glu Ser Thr Ser Ala Asp

610 615 620

Thr Val Leu Leu Thr Ser Lys Glu Ser Lys Val Trp Asp Leu Pro Ser

625 630 635 640

Thr Ser His Val Ser Met Trp Lys Thr Ser Asp Ser Val Ser Ser Pro

645 650 655

Gln Pro Gly Ala Ser Asp Thr Ala Val Pro Glu Gln Asn Lys Thr Thr

660 665 670

Lys Thr Gly Gln Met Asp Gly Ile Pro Met Ser Met Lys Asn Glu Met

675 680 685

Pro Ile Ser Gln Leu Leu Met Ile Ile Ala Pro Ser Leu Gly Phe Val

690 695 700

Leu Phe Ala Leu Phe Val Ala Phe Leu Leu Arg Gly Lys Leu Met Glu

705 710 715 720

Thr Tyr Cys Ser Gln Lys His Thr Arg Leu Asp Tyr Ile Gly Asp Ser

725 730 735

Lys Asn Val Leu Asn Asp Val Gln His Gly Arg Glu Asp Glu Asp Gly

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Leu Phe Thr Leu

755

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Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln

305 310

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Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

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Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

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Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 5

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<223> mutant human CD3z contains the activation domain of ITAM

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Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

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Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

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Leu Leu Met Ile Ile Ala Pro Ser Leu Gly Phe Val Leu Phe Ala Leu

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Phe Val Ala Phe Leu

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<210> 7

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Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 8

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Ile Tyr Ala Gly Val Cys Ile Ser Val Leu Val Leu Leu Ala Leu Leu

1 5 10 15

Gly Val Ile Ile Ala

20

<210> 9

<211> 52

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Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala

1 5 10 15

Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu

20 25 30

Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg

35 40 45

Pro Tyr Tyr Lys

50

<210> 10

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Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 11

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Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser

20

<210> 12

<211> 18

<212> PRT

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<220>

<223> T2A self-cleaving peptides

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Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

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Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 14

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Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

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Asn Pro Gly Pro

20

<210> 15

<211> 22

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<220>

<223> F2A self-cleaving peptides

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Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 16

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Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His

275 280 285

<210> 17

<211> 192

<212> PRT

<213> Chile person

<400> 17

Met Gln Ala Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys

1 5 10 15

Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr

20 25 30

Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Val Asp Gly

35 40 45

Lys Pro Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln Glu Asp Tyr

50 55 60

Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Ile

65 70 75 80

Cys Phe Ser Leu Val Ser Pro Ala Ser Phe Glu Asn Val Arg Ala Lys

85 90 95

Trp Tyr Pro Glu Val Arg His His Cys Pro Asn Thr Pro Ile Ile Leu

100 105 110

Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Asp Thr Ile Glu Lys

115 120 125

Leu Lys Glu Lys Lys Leu Thr Pro Ile Thr Tyr Pro Gln Gly Leu Ala

130 135 140

Met Ala Lys Glu Ile Gly Ala Val Lys Tyr Leu Glu Cys Ser Ala Leu

145 150 155 160

Thr Gln Arg Gly Leu Lys Thr Val Phe Asp Glu Ala Ile Arg Ala Val

165 170 175

Leu Cys Pro Pro Pro Val Lys Lys Arg Lys Arg Lys Cys Leu Leu Leu

180 185 190

<210> 18

<211> 215

<212> PRT

<213> Chile person

<400> 18

Met Ala Ser Arg Gly Ala Thr Arg Pro Asn Gly Pro Asn Thr Gly Asn

1 5 10 15

Lys Ile Cys Gln Phe Lys Leu Val Leu Leu Gly Glu Ser Ala Val Gly

20 25 30

Lys Ser Ser Leu Val Leu Arg Phe Val Lys Gly Gln Phe His Glu Phe

35 40 45

Gln Glu Ser Thr Ile Gly Ala Ala Phe Leu Thr Gln Thr Val Cys Leu

50 55 60

Asp Asp Thr Thr Val Lys Phe Glu Ile Trp Asp Thr Ala Gly Gln Glu

65 70 75 80

Arg Tyr His Ser Leu Ala Pro Met Tyr Tyr Arg Gly Ala Gln Ala Ala

85 90 95

Ile Val Val Tyr Asp Ile Thr Asn Glu Glu Ser Phe Ala Arg Ala Lys

100 105 110

Asn Trp Val Lys Glu Leu Gln Arg Gln Ala Ser Pro Asn Ile Val Ile

115 120 125

Ala Leu Ser Gly Asn Lys Ala Asp Leu Ala Asn Lys Arg Ala Val Asp

130 135 140

Phe Gln Glu Ala Gln Ser Tyr Ala Asp Asp Asn Ser Leu Leu Phe Met

145 150 155 160

Glu Thr Ser Ala Lys Thr Ser Met Asn Val Asn Glu Ile Phe Met Ala

165 170 175

Ile Ala Lys Lys Leu Pro Lys Asn Glu Pro Gln Asn Pro Gly Ala Asn

180 185 190

Ser Ala Arg Gly Arg Gly Val Asp Leu Thr Glu Pro Thr Gln Pro Thr

195 200 205

Arg Asn Gln Cys Cys Ser Asn

210 215

<210> 19

<211> 207

<212> PRT

<213> Chile person

<400> 19

Met Thr Ser Arg Lys Lys Val Leu Leu Lys Val Ile Ile Leu Gly Asp

1 5 10 15

Ser Gly Val Gly Lys Thr Ser Leu Met Asn Gln Tyr Val Asn Lys Lys

20 25 30

Phe Ser Asn Gln Tyr Lys Ala Thr Ile Gly Ala Asp Phe Leu Thr Lys

35 40 45

Glu Val Met Val Asp Asp Arg Leu Val Thr Met Gln Ile Trp Asp Thr

50 55 60

Ala Gly Gln Glu Arg Phe Gln Ser Leu Gly Val Ala Phe Tyr Arg Gly

65 70 75 80

Ala Asp Cys Cys Val Leu Val Phe Asp Val Thr Ala Pro Asn Thr Phe

85 90 95

Lys Thr Leu Asp Ser Trp Arg Asp Glu Phe Leu Ile Gln Ala Ser Pro

100 105 110

Arg Asp Pro Glu Asn Phe Pro Phe Val Val Leu Gly Asn Lys Ile Asp

115 120 125

Leu Glu Asn Arg Gln Val Ala Thr Lys Arg Ala Gln Ala Trp Cys Tyr

130 135 140

Ser Lys Asn Asn Ile Pro Tyr Phe Glu Thr Ser Ala Lys Glu Ala Ile

145 150 155 160

Asn Val Glu Gln Ala Phe Gln Thr Ile Ala Arg Asn Ala Leu Lys Gln

165 170 175

Glu Thr Glu Val Glu Leu Tyr Asn Glu Phe Pro Glu Pro Ile Lys Leu

180 185 190

Asp Lys Asn Asp Arg Ala Lys Ala Ser Ala Glu Ser Cys Ser Cys

195 200 205

<210> 20

<211> 184

<212> PRT

<213> Chile person

<400> 20

Met Arg Glu Tyr Lys Leu Val Val Leu Gly Ser Gly Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Val Gln Phe Val Gln Gly Ile Phe Val Glu Lys Tyr

20 25 30

Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp Cys

35 40 45

Gln Gln Cys Met Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe

50 55 60

Thr Ala Met Arg Asp Leu Tyr Met Lys Asn Gly Gln Gly Phe Ala Leu

65 70 75 80

Val Tyr Ser Ile Thr Ala Gln Ser Thr Phe Asn Asp Leu Gln Asp Leu

85 90 95

Arg Glu Gln Ile Leu Arg Val Lys Asp Thr Glu Asp Val Pro Met Ile

100 105 110

Leu Val Gly Asn Lys Cys Asp Leu Glu Asp Glu Arg Val Val Gly Lys

115 120 125

Glu Gln Gly Gln Asn Leu Ala Arg Gln Trp Cys Asn Cys Ala Phe Leu

130 135 140

Glu Ser Ser Ala Lys Ser Lys Ile Asn Val Asn Glu Ile Phe Tyr Asp

145 150 155 160

Leu Val Arg Gln Ile Asn Arg Lys Thr Pro Val Glu Lys Lys Lys Pro

165 170 175

Lys Lys Lys Ser Cys Leu Leu Leu

180

<210> 21

<211> 193

<212> PRT

<213> Chile person

<400> 21

Met Ala Ala Ile Arg Lys Lys Leu Val Ile Val Gly Asp Gly Ala Cys

1 5 10 15

Gly Lys Thr Cys Leu Leu Ile Val Phe Ser Lys Asp Gln Phe Pro Glu

20 25 30

Val Tyr Val Pro Thr Val Phe Glu Asn Tyr Val Ala Asp Ile Glu Val

35 40 45

Asp Gly Lys Gln Val Glu Leu Ala Leu Trp Asp Thr Ala Gly Gln Glu

50 55 60

Asp Tyr Asp Arg Leu Arg Pro Leu Ser Tyr Pro Asp Thr Asp Val Ile

65 70 75 80

Leu Met Cys Phe Ser Ile Asp Ser Pro Asp Ser Leu Glu Asn Ile Pro

85 90 95

Glu Lys Trp Thr Pro Glu Val Lys His Phe Cys Pro Asn Val Pro Ile

100 105 110

Ile Leu Val Gly Asn Lys Lys Asp Leu Arg Asn Asp Glu His Thr Arg

115 120 125

Arg Glu Leu Ala Lys Met Lys Gln Glu Pro Val Lys Pro Glu Glu Gly

130 135 140

Arg Asp Met Ala Asn Arg Ile Gly Ala Phe Gly Tyr Met Glu Cys Ser

145 150 155 160

Ala Lys Thr Lys Asp Gly Val Arg Glu Val Phe Glu Met Ala Thr Arg

165 170 175

Ala Ala Leu Gln Ala Arg Arg Gly Lys Lys Lys Ser Gly Cys Leu Val

180 185 190

Leu

<210> 22

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<212> PRT

<213> Chile person

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Met Gln Thr Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys

1 5 10 15

Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Lys Phe Pro Ser Glu Tyr

20 25 30

Val Pro Thr Val Phe Asp Asn Tyr Ala Val Thr Val Met Ile Gly Gly

35 40 45

Glu Pro Tyr Thr Leu Gly Leu Phe Asp Thr Ala Gly Gln Glu Asp Tyr

50 55 60

Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Val

65 70 75 80

Cys Phe Ser Val Val Ser Pro Ser Ser Phe Glu Asn Val Lys Glu Lys

85 90 95

Trp Val Pro Glu Ile Thr His His Cys Pro Lys Thr Pro Phe Leu Leu

100 105 110

Val Gly Thr Gln Ile Asp Leu Arg Asp Asp Pro Ser Thr Ile Glu Lys

115 120 125

Leu Ala Lys Asn Lys Gln Lys Pro Ile Thr Pro Glu Thr Ala Glu Lys

130 135 140

Leu Ala Arg Asp Leu Lys Ala Val Lys Tyr Val Glu Cys Ser Ala Leu

145 150 155 160

Thr Gln Lys Gly Leu Lys Asn Val Phe Asp Glu Ala Ile Leu Ala Ala

165 170 175

Leu Glu Pro Pro Glu Pro Lys Lys Ser Arg Arg Cys Val Leu Leu

180 185 190

<210> 23

<211> 21

<212> PRT

<213> mice

<400> 23

Ile Leu Ile Ile Ala Cys Cys Val Gly Phe Val Leu Met Val Leu Leu

1 5 10 15

Phe Leu Ala Phe Leu

20

<210> 24

<211> 279

<212> PRT

<213> mice

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 24

Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu

20 25 30

Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser

35 40 45

Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys

50 55 60

Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys

65 70 75 80

Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser

85 90 95

Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg

115 120 125

Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr

130 135 140

Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu

145 150 155 160

Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro

165 170 175

Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro

180 185 190

Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala

195 200 205

Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg

210 215 220

Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly

225 230 235 240

Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys

245 250 255

Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His

260 265 270

Gln Ile Asn Ser Arg Gln Thr

275

<210> 25

<211> 22

<212> PRT

<213> mice

<400> 25

Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Ala

20

<210> 26

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> human CD28 costimulatory signaling domain with L186G/L187G substitution (position reference full-length protein)

<400> 26

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 27

<211> 113

<212> PRT

<213> Chile person

<400> 27

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 28

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> T2A self-cleaving peptide variants

<400> 28

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro

20

<210> 29

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> T2A self-cleaving peptide variants

<400> 29

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro Arg

<210> 30

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> T2A self-cleaving peptide variants

<400> 30

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro Arg

20

<210> 31

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> P2A self-cleaving peptide variants

<400> 31

Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys

1 5 10 15

Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 32

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> CD28 hinge region

<400> 32

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 33

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> CD8a transmembrane domain

<400> 33

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 34

<211> 111

<212> PRT

<213> Chile person

<400> 34

Glu Thr Val Val Thr Glu Val Leu Gly His Arg Val Thr Leu Pro Cys

1 5 10 15

Leu Tyr Ser Ser Trp Ser His Asn Ser Asn Ser Met Cys Trp Gly Lys

20 25 30

Asp Gln Cys Pro Tyr Ser Gly Cys Lys Glu Ala Leu Ile Arg Thr Asp

35 40 45

Gly Met Arg Val Thr Ser Arg Lys Ser Ala Lys Tyr Arg Leu Gln Gly

50 55 60

Thr Ile Pro Arg Gly Asp Val Ser Leu Thr Ile Leu Asn Pro Ser Glu

65 70 75 80

Ser Asp Ser Gly Val Tyr Cys Cys Arg Ile Glu Val Pro Gly Trp Phe

85 90 95

Asn Asp Val Lys Ile Asn Val Arg Leu Asn Leu Gln Arg Ala Ser

100 105 110

<210> 35

<211> 179

<212> PRT

<213> Chile person

<400> 35

Thr Thr Thr His Arg Thr Ala Thr Thr Thr Thr Arg Arg Thr Thr Thr

1 5 10 15

Thr Ser Pro Thr Thr Thr Arg Gln Met Thr Thr Thr Pro Ala Ala Leu

20 25 30

Pro Thr Thr Val Val Thr Thr Pro Asp Leu Thr Thr Gly Thr Pro Leu

35 40 45

Gln Met Thr Thr Ile Ala Val Phe Thr Thr Ala Asn Thr Cys Leu Ser

50 55 60

Leu Thr Pro Ser Thr Leu Pro Glu Glu Ala Thr Gly Leu Leu Thr Pro

65 70 75 80

Glu Pro Ser Lys Glu Gly Pro Ile Leu Thr Ala Glu Ser Glu Thr Val

85 90 95

Leu Pro Ser Asp Ser Trp Ser Ser Val Glu Ser Thr Ser Ala Asp Thr

100 105 110

Val Leu Leu Thr Ser Lys Glu Ser Lys Val Trp Asp Leu Pro Ser Thr

115 120 125

Ser His Val Ser Met Trp Lys Thr Ser Asp Ser Val Ser Ser Pro Gln

130 135 140

Pro Gly Ala Ser Asp Thr Ala Val Pro Glu Gln Asn Lys Thr Thr Lys

145 150 155 160

Thr Gly Gln Met Asp Gly Ile Pro Met Ser Met Lys Asn Glu Met Pro

165 170 175

Ile Ser Gln

<210> 36

<211> 364

<212> PRT

<213> Chile person

<400> 36

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala Lys Lys Tyr Phe

305 310 315 320

Phe Lys Lys Glu Val Gln Gln Leu Ser Val Ser Phe Ser Ser Leu Gln

325 330 335

Ile Lys Ala Leu Gln Asn Ala Val Glu Lys Glu Val Gln Ala Glu Asp

340 345 350

Asn Ile Tyr Ile Glu Asn Ser Leu Tyr Ala Thr Asp

355 360

<210> 37

<211> 295

<212> PRT

<213> Chile person

<220>

<221> SIGNAL

<222> (1)..(20)

<400> 37

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly

290 295

<210> 38

<211> 108

<212> PRT

<213> Chile person

<400> 38

Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val Thr Leu Pro Cys

1 5 10 15

His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn Arg Gly Ser Cys

20 25 30

Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr Asn Gly Thr His

35 40 45

Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu Gly Asp Leu Ser

50 55 60

Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala Val Ser Asp Ser

65 70 75 80

Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp Phe Asn Asp Met

85 90 95

Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

100 105

<210> 39

<211> 167

<212> PRT

<213> Chile person

<400> 39

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

1 5 10 15

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

20 25 30

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

35 40 45

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

50 55 60

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

65 70 75 80

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

85 90 95

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

100 105 110

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

115 120 125

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

130 135 140

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

145 150 155 160

Thr Ala Asn Thr Thr Lys Gly

165

<210> 40

<211> 20

<212> PRT

<213> Chile person

<400> 40

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly

20

<210> 41

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> modified Tim1IgV Domain

<400> 41

Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val Thr Leu Pro Cys

1 5 10 15

His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn Arg Gly Ser Cys

20 25 30

Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr Asn Gly Thr His

35 40 45

Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu Gly Asp Leu Ser

50 55 60

Arg Gly Asp Val Ser Leu Thr Ile Glu Asn Thr Ala Val Ser Asp Ser

65 70 75 80

Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp Phe Asn Asp Met

85 90 95

Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

100 105

<210> 42

<211> 290

<212> PRT

<213> Chile person

<400> 42

Glu Thr Val Val Thr Glu Val Leu Gly His Arg Val Thr Leu Pro Cys

1 5 10 15

Leu Tyr Ser Ser Trp Ser His Asn Ser Asn Ser Met Cys Trp Gly Lys

20 25 30

Asp Gln Cys Pro Tyr Ser Gly Cys Lys Glu Ala Leu Ile Arg Thr Asp

35 40 45

Gly Met Arg Val Thr Ser Arg Lys Ser Ala Lys Tyr Arg Leu Gln Gly

50 55 60

Thr Ile Pro Arg Gly Asp Val Ser Leu Thr Ile Leu Asn Pro Ser Glu

65 70 75 80

Ser Asp Ser Gly Val Tyr Cys Cys Arg Ile Glu Val Pro Gly Trp Phe

85 90 95

Asn Asp Val Lys Ile Asn Val Arg Leu Asn Leu Gln Arg Ala Ser Thr

100 105 110

Thr Thr His Arg Thr Ala Thr Thr Thr Thr Arg Arg Thr Thr Thr Thr

115 120 125

Ser Pro Thr Thr Thr Arg Gln Met Thr Thr Thr Pro Ala Ala Leu Pro

130 135 140

Thr Thr Val Val Thr Thr Pro Asp Leu Thr Thr Gly Thr Pro Leu Gln

145 150 155 160

Met Thr Thr Ile Ala Val Phe Thr Thr Ala Asn Thr Cys Leu Ser Leu

165 170 175

Thr Pro Ser Thr Leu Pro Glu Glu Ala Thr Gly Leu Leu Thr Pro Glu

180 185 190

Pro Ser Lys Glu Gly Pro Ile Leu Thr Ala Glu Ser Glu Thr Val Leu

195 200 205

Pro Ser Asp Ser Trp Ser Ser Val Glu Ser Thr Ser Ala Asp Thr Val

210 215 220

Leu Leu Thr Ser Lys Glu Ser Lys Val Trp Asp Leu Pro Ser Thr Ser

225 230 235 240

His Val Ser Met Trp Lys Thr Ser Asp Ser Val Ser Ser Pro Gln Pro

245 250 255

Gly Ala Ser Asp Thr Ala Val Pro Glu Gln Asn Lys Thr Thr Lys Thr

260 265 270

Gly Gln Met Asp Gly Ile Pro Met Ser Met Lys Asn Glu Met Pro Ile

275 280 285

Ser Gln

290

<210> 43

<211> 275

<212> PRT

<213> Chile person

<400> 43

Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val Thr Leu Pro Cys

1 5 10 15

His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn Arg Gly Ser Cys

20 25 30

Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr Asn Gly Thr His

35 40 45

Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu Gly Asp Leu Ser

50 55 60

Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala Val Ser Asp Ser

65 70 75 80

Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp Phe Asn Asp Met

85 90 95

Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys Val Thr Thr Thr

100 105 110

Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val Arg Thr Ser Thr

115 120 125

Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr Val Pro Thr Thr

130 135 140

Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr Thr Val Leu Thr

145 150 155 160

Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr Thr Thr Ser Ile

165 170 175

Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val Ser Thr Phe Val

180 185 190

Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro Val Ala Thr Ser

195 200 205

Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr Thr Leu Gln Gly

210 215 220

Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr Ser Tyr Thr Thr

225 230 235 240

Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly Leu Trp Asn Asn

245 250 255

Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu Thr Ala Asn Thr

260 265 270

Thr Lys Gly

275

<210> 44

<211> 48

<212> PRT

<213> Chile person

<400> 44

Lys Lys Tyr Phe Phe Lys Lys Glu Val Gln Gln Leu Ser Val Ser Phe

1 5 10 15

Ser Ser Leu Gln Ile Lys Ala Leu Gln Asn Ala Val Glu Lys Glu Val

20 25 30

Gln Ala Glu Asp Asn Ile Tyr Ile Glu Asn Ser Leu Tyr Ala Thr Asp

35 40 45

<210> 45

<211> 43

<212> PRT

<213> Chile person

<400> 45

Leu Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg

1 5 10 15

Leu Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His

20 25 30

Gly Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu

35 40

<210> 46

<211> 274

<212> PRT

<213> artificial sequence

<220>

<223> TRAF6 Signal transduction Domain

<400> 46

Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu

1 5 10 15

Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys

20 25 30

Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser

35 40 45

Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu

50 55 60

Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala

65 70 75 80

Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys

85 90 95

Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu

100 105 110

Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu

115 120 125

Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu

130 135 140

Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met

145 150 155 160

Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile

165 170 175

His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys

180 185 190

Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys

195 200 205

Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg

210 215 220

Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile

225 230 235 240

Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn

245 250 255

His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met

260 265 270

Leu Ala

<210> 47

<211> 193

<212> PRT

<213> artificial sequence

<220>

<223> TLR8 signaling domain

<400> 47

His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu

1 5 10 15

Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr

20 25 30

Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp

35 40 45

Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn

50 55 60

Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile

65 70 75 80

Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe

85 90 95

Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe

100 105 110

Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile

115 120 125

Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu

130 135 140

Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro

145 150 155 160

Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr

165 170 175

Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln

180 185 190

Tyr

<210> 48

<211> 303

<212> PRT

<213> artificial sequence

<220>

<223> TRAF2 Signal transduction Domain

<400> 48

Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser Leu Glu Leu Leu Gln

1 5 10 15

Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala Lys Tyr

20 25 30

Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln

35 40 45

Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser

50 55 60

Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu

65 70 75 80

Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn Ala Ala

85 90 95

Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys

100 105 110

Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg

115 120 125

Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg

130 135 140

Leu Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser

145 150 155 160

Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys

165 170 175

Ala His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys

180 185 190

Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr

195 200 205

Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu

210 215 220

Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu

225 230 235 240

Arg Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro

245 250 255

Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys

260 265 270

Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val

275 280 285

Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys

290 295 300

<210> 49

<211> 476

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-Tim 1TM-Tim1SD-CD3zSD

<400> 49

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala Lys Lys Tyr Phe

305 310 315 320

Phe Lys Lys Glu Val Gln Gln Leu Ser Val Ser Phe Ser Ser Leu Gln

325 330 335

Ile Lys Ala Leu Gln Asn Ala Val Glu Lys Glu Val Gln Ala Glu Asp

340 345 350

Asn Ile Tyr Ile Glu Asn Ser Leu Tyr Ala Thr Asp Arg Val Lys Phe

355 360 365

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

370 375 380

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

385 390 395 400

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

405 410 415

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

420 425 430

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

435 440 445

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

450 455 460

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470 475

<210> 50

<211> 471

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-Tim 1TM-Tim4SD-CD3zSD

<400> 50

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala Leu Arg Gly Lys

305 310 315 320

Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu Asp Tyr Ile

325 330 335

Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly Arg Glu Asp

340 345 350

Glu Asp Gly Leu Phe Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 51

<211> 363

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-CD 28TM-CD28SD

<400> 51

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Phe Trp Val Leu Val Val Val Gly Gly

290 295 300

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

305 310 315 320

Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn

325 330 335

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

340 345 350

Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

355 360

<210> 52

<211> 590

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-Tim 1TM-TRAF6SD

<400> 52

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala Met Ser Leu Leu

305 310 315 320

Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu Ser Asp Cys Cys

325 330 335

Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys Asp Asp Ser Val

340 345 350

Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser Phe Met Glu Glu

355 360 365

Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu Glu Ser Lys Tyr

370 375 380

Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala Val Gln Thr Pro

385 390 395 400

Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys Ser Ile Arg Asp

405 410 415

Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu Leu Glu Asn Gln

420 425 430

Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu Ser Leu Met Val

435 440 445

Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu Leu Arg His Leu

450 455 460

Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met Asp Cys Pro Gln

465 470 475 480

Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile His Ile Leu Lys

485 490 495

Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys Ala Ala Ser Met

500 505 510

Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys Pro Leu Ala Asn

515 520 525

Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg Glu Gln Met Pro

530 535 540

Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile Pro Cys Thr Phe

545 550 555 560

Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn His Leu Ala Arg

565 570 575

His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met Leu Ala

580 585 590

<210> 53

<211> 596

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-CD 28TM-TRAF6SD

<400> 53

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Phe Trp Val Leu Val Val Val Gly Gly

290 295 300

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

305 310 315 320

Trp Val Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln

325 330 335

Ser Glu Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val

340 345 350

Thr Lys Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser

355 360 365

Ser Ser Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro

370 375 380

Pro Leu Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg

385 390 395 400

Glu Ala Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile

405 410 415

Ile Lys Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu

420 425 430

Ile Leu Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu

435 440 445

Ile Leu Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys

450 455 460

Met Glu Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala

465 470 475 480

Leu Met Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile

485 490 495

Asn Ile His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp

500 505 510

Asn Cys Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln

515 520 525

Asn Cys Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu

530 535 540

Ile Arg Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala

545 550 555 560

Pro Ile Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln

565 570 575

Arg Asn His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met

580 585 590

Arg Met Leu Ala

595

<210> 54

<211> 619

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-Tim 1TM-TRAF2SD

<400> 54

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala Met Ala Ala Ala

305 310 315 320

Ser Val Thr Pro Pro Gly Ser Leu Glu Leu Leu Gln Pro Gly Phe Ser

325 330 335

Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala Lys Tyr Leu Cys Ser Ala

340 345 350

Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln Cys Gly His Arg

355 360 365

Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser Gly Pro Gln Asn

370 375 380

Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu Gly Ile Ser Ile

385 390 395 400

Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn Ala Ala Arg Arg Glu Val

405 410 415

Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys Thr Trp Lys Gly

420 425 430

Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg Cys Pro Leu Met

435 440 445

Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg Leu Gly Glu Lys

450 455 460

Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser Leu Ser Cys Arg

465 470 475 480

His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys Ala His His Glu

485 490 495

Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys Gly Lys Lys Lys

500 505 510

Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr Cys Gly Lys Cys

515 520 525

Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu Glu Thr Val Glu

530 535 540

Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu Arg Glu His Leu

545 550 555 560

Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro Leu Leu Gly Asp

565 570 575

Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys Glu Ser Leu Glu

580 585 590

Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val Leu Asn Arg Glu

595 600 605

Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys

610 615

<210> 55

<211> 625

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-CD 28TM-TRAF2SD

<400> 55

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Phe Trp Val Leu Val Val Val Gly Gly

290 295 300

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

305 310 315 320

Trp Val Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser Leu Glu Leu

325 330 335

Leu Gln Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala

340 345 350

Lys Tyr Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln

355 360 365

Ala Gln Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu

370 375 380

Ser Ser Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr

385 390 395 400

Glu Glu Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn

405 410 415

Ala Ala Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp

420 425 430

Gly Cys Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu

435 440 445

Gly Arg Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu

450 455 460

Val Arg Leu Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu

465 470 475 480

Arg Ser Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp

485 490 495

Val Lys Ala His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp

500 505 510

Gly Cys Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val

515 520 525

Lys Thr Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly

530 535 540

Cys Leu Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln

545 550 555 560

Trp Leu Arg Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala

565 570 575

Lys Pro Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln

580 585 590

Arg Cys Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val

595 600 605

Cys Val Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala

610 615 620

Cys

625

<210> 56

<211> 621

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-Tim 1TM-TLR8SD-CD3zSD

<400> 56

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala His His Leu Phe

305 310 315 320

Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys

325 330 335

Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile

340 345 350

Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu

355 360 365

Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys

370 375 380

Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu

385 390 395 400

Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys

405 410 415

Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu

420 425 430

Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu

435 440 445

Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile

450 455 460

Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly

465 470 475 480

Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser

485 490 495

Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr Arg Val Lys

500 505 510

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

515 520 525

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

530 535 540

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

545 550 555 560

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

565 570 575

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

580 585 590

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

595 600 605

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

610 615 620

<210> 57

<211> 415

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-CD 28TM-CD28SD-DAP12SD

<400> 57

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Phe Trp Val Leu Val Val Val Gly Gly

290 295 300

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

305 310 315 320

Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn

325 330 335

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

340 345 350

Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Tyr Phe Leu Gly Arg

355 360 365

Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln

370 375 380

Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg

385 390 395 400

Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

405 410 415

<210> 58

<211> 409

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim1IgV-Tim1 mucin-Tim 1TM-CD28SD-DAP12SD

<400> 58

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala Arg Ser Lys Arg

305 310 315 320

Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro

325 330 335

Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe

340 345 350

Ala Ala Tyr Arg Ser Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg

355 360 365

Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu

370 375 380

Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp

385 390 395 400

Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

405

<210> 59

<211> 490

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-Tim 4TM-Tim4SD-CD3zSD

<400> 59

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 60

<211> 495

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-Tim 4TM-Tim1SD-CD3zSD

<400> 60

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Lys

325 330 335

Lys Tyr Phe Phe Lys Lys Glu Val Gln Gln Leu Ser Val Ser Phe Ser

340 345 350

Ser Leu Gln Ile Lys Ala Leu Gln Asn Ala Val Glu Lys Glu Val Gln

355 360 365

Ala Glu Asp Asn Ile Tyr Ile Glu Asn Ser Leu Tyr Ala Thr Asp Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 61

<211> 382

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-CD 28TM-CD28SD

<400> 61

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

370 375 380

<210> 62

<211> 609

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-Tim 4TM-TRAF6SD

<400> 62

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Met

325 330 335

Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu Ser

340 345 350

Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys Asp

355 360 365

Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser Phe

370 375 380

Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu Glu

385 390 395 400

Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala Val

405 410 415

Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys Ser

420 425 430

Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu Leu

435 440 445

Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu Ser

450 455 460

Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu Leu

465 470 475 480

Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met Asp

485 490 495

Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile His

500 505 510

Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys Ala

515 520 525

Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys Pro

530 535 540

Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg Glu

545 550 555 560

Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile Pro

565 570 575

Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn His

580 585 590

Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met Leu

595 600 605

Ala

<210> 63

<211> 615

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-CD 28TM-TRAF6SD

<400> 63

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly

340 345 350

Ser Ser Gln Ser Glu Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys

355 360 365

Ser Ala Val Thr Lys Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly

370 375 380

Asn Leu Ser Ser Ser Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu

385 390 395 400

Phe Asp Pro Pro Leu Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met

405 410 415

Ala Leu Arg Glu Ala Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys

420 425 430

Ala Cys Ile Ile Lys Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val

435 440 445

Asp Asn Glu Ile Leu Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala

450 455 460

Lys Arg Glu Ile Leu Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys

465 470 475 480

Leu His Lys Met Glu Leu Arg His Leu Glu Asp His Gln Ala His Cys

485 490 495

Glu Phe Ala Leu Met Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys

500 505 510

Phe His Ile Asn Ile His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val

515 520 525

Ser Cys Asp Asn Cys Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile

530 535 540

His Asp Gln Asn Cys Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn

545 550 555 560

Thr Ile Leu Ile Arg Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys

565 570 575

Pro Thr Ala Pro Ile Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu

580 585 590

Lys Met Gln Arg Asn His Leu Ala Arg His Leu Gln Glu Asn Thr Gln

595 600 605

Ser His Met Arg Met Leu Ala

610 615

<210> 64

<211> 638

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-Tim 4TM-TRAF2SD

<400> 64

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Met

325 330 335

Ala Ala Ala Ser Val Thr Pro Pro Gly Ser Leu Glu Leu Leu Gln Pro

340 345 350

Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala Lys Tyr Leu

355 360 365

Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln Cys

370 375 380

Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser Gly

385 390 395 400

Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu Gly

405 410 415

Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn Ala Ala Arg

420 425 430

Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys Thr

435 440 445

Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg Cys

450 455 460

Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg Leu

465 470 475 480

Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser Leu

485 490 495

Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys Ala

500 505 510

His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys Gly

515 520 525

Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr Cys

530 535 540

Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu Glu

545 550 555 560

Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu Arg

565 570 575

Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro Leu

580 585 590

Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys Glu

595 600 605

Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val Leu

610 615 620

Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys

625 630 635

<210> 65

<211> 644

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-CD 28TM-TRAF2SD

<400> 65

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser

340 345 350

Leu Glu Leu Leu Gln Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys

355 360 365

Leu Glu Ala Lys Tyr Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg

370 375 380

Pro Phe Gln Ala Gln Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala

385 390 395 400

Ser Ile Leu Ser Ser Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu

405 410 415

Gly Ile Tyr Glu Glu Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe

420 425 430

Pro Asp Asn Ala Ala Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys

435 440 445

Pro Ser Asp Gly Cys Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser

450 455 460

Cys His Glu Gly Arg Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys

465 470 475 480

Lys Gly Leu Val Arg Leu Gly Glu Lys Glu Arg His Leu Glu His Glu

485 490 495

Cys Pro Glu Arg Ser Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys

500 505 510

Gly Ala Asp Val Lys Ala His His Glu Val Cys Pro Lys Phe Pro Leu

515 520 525

Thr Cys Asp Gly Cys Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln

530 535 540

Asp His Val Lys Thr Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His

545 550 555 560

Ala Ile Gly Cys Leu Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His

565 570 575

Glu Val Gln Trp Leu Arg Glu His Leu Ala Met Leu Leu Ser Ser Val

580 585 590

Leu Glu Ala Lys Pro Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu

595 600 605

Leu Leu Gln Arg Cys Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu

610 615 620

Asn Ile Val Cys Val Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr

625 630 635 640

Ala Glu Ala Cys

<210> 66

<211> 644

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-Tim 1TM-TLR8SD-CD3zSD

<400> 66

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser

340 345 350

Leu Glu Leu Leu Gln Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys

355 360 365

Leu Glu Ala Lys Tyr Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg

370 375 380

Pro Phe Gln Ala Gln Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala

385 390 395 400

Ser Ile Leu Ser Ser Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu

405 410 415

Gly Ile Tyr Glu Glu Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe

420 425 430

Pro Asp Asn Ala Ala Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys

435 440 445

Pro Ser Asp Gly Cys Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser

450 455 460

Cys His Glu Gly Arg Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys

465 470 475 480

Lys Gly Leu Val Arg Leu Gly Glu Lys Glu Arg His Leu Glu His Glu

485 490 495

Cys Pro Glu Arg Ser Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys

500 505 510

Gly Ala Asp Val Lys Ala His His Glu Val Cys Pro Lys Phe Pro Leu

515 520 525

Thr Cys Asp Gly Cys Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln

530 535 540

Asp His Val Lys Thr Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His

545 550 555 560

Ala Ile Gly Cys Leu Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His

565 570 575

Glu Val Gln Trp Leu Arg Glu His Leu Ala Met Leu Leu Ser Ser Val

580 585 590

Leu Glu Ala Lys Pro Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu

595 600 605

Leu Leu Gln Arg Cys Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu

610 615 620

Asn Ile Val Cys Val Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr

625 630 635 640

Ala Glu Ala Cys

<210> 67

<211> 628

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim4 mucin-Tim 1TM-TLR8SD-CD3zSD

<400> 67

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Val Thr Thr Thr Pro Ile Val Thr Thr

130 135 140

Val Pro Thr Val Thr Thr Val Arg Thr Ser Thr Thr Val Pro Thr Thr

145 150 155 160

Thr Thr Val Pro Met Thr Thr Val Pro Thr Thr Thr Val Pro Thr Thr

165 170 175

Met Ser Ile Pro Thr Thr Thr Thr Val Leu Thr Thr Met Thr Val Ser

180 185 190

Thr Thr Thr Ser Val Pro Thr Thr Thr Ser Ile Pro Thr Thr Thr Ser

195 200 205

Val Pro Val Thr Thr Thr Val Ser Thr Phe Val Pro Pro Met Pro Leu

210 215 220

Pro Arg Gln Asn His Glu Pro Val Ala Thr Ser Pro Ser Ser Pro Gln

225 230 235 240

Pro Ala Glu Thr His Pro Thr Thr Leu Gln Gly Ala Ile Arg Arg Glu

245 250 255

Pro Thr Ser Ser Pro Leu Tyr Ser Tyr Thr Thr Asp Gly Asn Asp Thr

260 265 270

Val Thr Glu Ser Ser Asp Gly Leu Trp Asn Asn Asn Gln Thr Gln Leu

275 280 285

Phe Leu Glu His Ser Leu Leu Thr Ala Asn Thr Thr Lys Gly Ile Tyr

290 295 300

Ala Gly Val Cys Ile Ser Val Leu Val Leu Leu Ala Leu Leu Gly Val

305 310 315 320

Ile Ile Ala His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn

325 330 335

Val Cys Leu Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln

340 345 350

Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val

355 360 365

Thr Asp Trp Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg

370 375 380

Asp Lys Asn Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly

385 390 395 400

Leu Ala Ile Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys

405 410 415

Thr Val Phe Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys

420 425 430

Thr Ala Phe Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp

435 440 445

Val Ile Ile Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr

450 455 460

Leu Arg Leu Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro

465 470 475 480

Asp Asn Pro Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val

485 490 495

Val Leu Thr Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser

500 505 510

Ile Lys Gln Tyr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

515 520 525

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

530 535 540

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

545 550 555 560

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

565 570 575

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

580 585 590

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

595 600 605

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

610 615 620

Leu Pro Pro Arg

625

<210> 68

<211> 416

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim1 mucin-Tim 1TM-CD28SD-DAP12SD

<400> 68

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Val Thr Thr Thr Pro Ile Val Thr Thr

130 135 140

Val Pro Thr Val Thr Thr Val Arg Thr Ser Thr Thr Val Pro Thr Thr

145 150 155 160

Thr Thr Val Pro Met Thr Thr Val Pro Thr Thr Thr Val Pro Thr Thr

165 170 175

Met Ser Ile Pro Thr Thr Thr Thr Val Leu Thr Thr Met Thr Val Ser

180 185 190

Thr Thr Thr Ser Val Pro Thr Thr Thr Ser Ile Pro Thr Thr Thr Ser

195 200 205

Val Pro Val Thr Thr Thr Val Ser Thr Phe Val Pro Pro Met Pro Leu

210 215 220

Pro Arg Gln Asn His Glu Pro Val Ala Thr Ser Pro Ser Ser Pro Gln

225 230 235 240

Pro Ala Glu Thr His Pro Thr Thr Leu Gln Gly Ala Ile Arg Arg Glu

245 250 255

Pro Thr Ser Ser Pro Leu Tyr Ser Tyr Thr Thr Asp Gly Asn Asp Thr

260 265 270

Val Thr Glu Ser Ser Asp Gly Leu Trp Asn Asn Asn Gln Thr Gln Leu

275 280 285

Phe Leu Glu His Ser Leu Leu Thr Ala Asn Thr Thr Lys Gly Ile Tyr

290 295 300

Ala Gly Val Cys Ile Ser Val Leu Val Leu Leu Ala Leu Leu Gly Val

305 310 315 320

Ile Ile Ala Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

325 330 335

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

340 345 350

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Tyr Phe Leu Gly

355 360 365

Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys

370 375 380

Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln

385 390 395 400

Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

405 410 415

<210> 69

<211> 422

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-Tim4IgV-Tim1 mucin-CD 28TM-CD28SD-DAP12SD

<400> 69

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Val Thr Thr Thr Pro Ile Val Thr Thr

130 135 140

Val Pro Thr Val Thr Thr Val Arg Thr Ser Thr Thr Val Pro Thr Thr

145 150 155 160

Thr Thr Val Pro Met Thr Thr Val Pro Thr Thr Thr Val Pro Thr Thr

165 170 175

Met Ser Ile Pro Thr Thr Thr Thr Val Leu Thr Thr Met Thr Val Ser

180 185 190

Thr Thr Thr Ser Val Pro Thr Thr Thr Ser Ile Pro Thr Thr Thr Ser

195 200 205

Val Pro Val Thr Thr Thr Val Ser Thr Phe Val Pro Pro Met Pro Leu

210 215 220

Pro Arg Gln Asn His Glu Pro Val Ala Thr Ser Pro Ser Ser Pro Gln

225 230 235 240

Pro Ala Glu Thr His Pro Thr Thr Leu Gln Gly Ala Ile Arg Arg Glu

245 250 255

Pro Thr Ser Ser Pro Leu Tyr Ser Tyr Thr Thr Asp Gly Asn Asp Thr

260 265 270

Val Thr Glu Ser Ser Asp Gly Leu Trp Asn Asn Asn Gln Thr Gln Leu

275 280 285

Phe Leu Glu His Ser Leu Leu Thr Ala Asn Thr Thr Lys Gly Phe Trp

290 295 300

Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val

305 310 315 320

Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu

325 330 335

Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr

340 345 350

Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr

355 360 365

Arg Ser Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala

370 375 380

Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr

385 390 395 400

Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr

405 410 415

Gln Arg Pro Tyr Tyr Lys

420

<210> 70

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> CD8a hinge region

<400> 70

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 71

<211> 496

<212> PRT

<213> artificial sequence

<220>

<223> chimeric Tim-TimIgV-Tim4 mucin-CD 28TM-CD28SD-CD3zSD

<400> 71

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ser Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 72

<211> 510

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TLR2ICD

<220>

<221> SIGNAL

<222> (1)..(24)

<400> 72

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu His

325 330 335

Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu Gln

340 345 350

Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp

355 360 365

Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu

370 375 380

Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu

385 390 395 400

His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile

405 410 415

Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu Asn

420 425 430

Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe

435 440 445

Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu

450 455 460

Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys

465 470 475 480

Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln

485 490 495

Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser

500 505 510

<210> 73

<211> 528

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TLR8ICD

<220>

<221> SIGNAL

<222> (1)..(24)

<400> 73

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu His

325 330 335

His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala

340 345 350

Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp

355 360 365

Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val

370 375 380

Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val

385 390 395 400

Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile

405 410 415

Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val

420 425 430

Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr

435 440 445

Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe

450 455 460

Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg

465 470 475 480

Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys

485 490 495

Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu

500 505 510

Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr

515 520 525

<210> 74

<400> 74

000

<210> 75

<400> 75

000

<210> 76

<400> 76

000

<210> 77

<400> 77

000

<210> 78

<400> 78

000

<210> 79

<400> 79

000

<210> 80

<400> 80

000

<210> 81

<400> 81

000

<210> 82

<400> 82

000

<210> 83

<400> 83

000

<210> 84

<400> 84

000

<210> 85

<400> 85

000

<210> 86

<400> 86

000

<210> 87

<400> 87

000

<210> 88

<400> 88

000

<210> 89

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible Joint

<400> 89

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 90

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible Joint

<400> 90

Gly Thr Gly Gly Ser Gly Gly Thr Gly Ser Gly Thr Gly Thr Ser

1 5 10 15

<210> 91

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> IgG4 hinge region (short)

<400> 91

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 92

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> CD8a hinge region

<400> 92

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 93

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> CD28 hinge region

<400> 93

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 94

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> CD8a transmembrane region

<400> 94

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 95

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> CD28 transmembrane region

<400> 95

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 96

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> wild-type CD3z signaling domain

<400> 96

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 97

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> variant CD3z signaling domain

<400> 97

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 98

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> wild-type CD28 costimulatory signaling domain

<400> 98

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 99

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> variant CD28 Co-stimulatory signaling domain with L186G/L187G substitution, position reference full-length protein

<400> 99

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 100

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> 4-1BB costimulatory signaling domain

<400> 100

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 101

<400> 101

000

<210> 102

<400> 102

000

<210> 103

<400> 103

000

<210> 104

<400> 104

000

<210> 105

<400> 105

000

<210> 106

<400> 106

000

<210> 107

<400> 107

000

<210> 108

<400> 108

000

<210> 109

<400> 109

000

<210> 110

<400> 110

000

<210> 111

<400> 111

000

<210> 112

<400> 112

000

<210> 113

<400> 113

000

<210> 114

<400> 114

000

<210> 115

<400> 115

000

<210> 116

<400> 116

000

<210> 117

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> GMCSFR_signal peptide

<400> 117

Met Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala

1 5 10 15

Phe Leu Leu Ile Pro

20

<210> 118

<211> 41

<212> PRT

<213> Chile person

<400> 118

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 119

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> CD28 costimulatory signaling domain with substitution of L186G/L187G (position reference full-length protein)

<400> 119

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 120

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> OX40 costimulatory signaling Domain

<400> 120

Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His

1 5 10 15

Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln

20 25 30

Ala Asp Ala His Ser Thr Leu Ala Lys Ile

35 40

<210> 121

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> CD2 costimulatory signaling domain

<400> 121

Lys Arg Lys Lys Gln Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr

1 5 10 15

Arg Ala His Arg Val Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln

20 25 30

Ile Pro Ala Ser Thr Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro

35 40 45

Pro Pro Pro Gly His Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro

50 55 60

Pro Gly His Arg Val Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro

65 70 75 80

Ser Gly Thr Gln Val His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro

85 90 95

Arg Val Gln Pro Lys Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser

100 105 110

Pro Ser Ser Asn

115

<210> 122

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> 4-1BB costimulatory signaling domain

<400> 122

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 123

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> CD27 costimulatory signaling domain

<400> 123

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 124

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> ICAM-1 costimulatory signaling Domain

<400> 124

Asn Arg Gln Arg Lys Ile Lys Lys Tyr Arg Leu Gln Gln Ala Gln Lys

1 5 10 15

Gly Thr Pro Met Lys Pro Asn Thr Gln Ala Thr Pro Pro

20 25

<210> 125

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> LFA-1 costimulatory signaling domain

<400> 125

Lys Ala Leu Ile His Leu Ser Asp Leu Arg Glu Tyr Arg Arg Phe Glu

1 5 10 15

Lys Glu Lys Leu Lys Ser Gln Trp Asn Asn Asp Asn Pro Leu Phe Lys

20 25 30

Ser Ala Thr Thr Thr Val Met Asn Pro Lys Phe Ala Glu Ser

35 40 45

<210> 126

<211> 38

<212> PRT

<213> artificial sequence

<220>

<223> ICOS costimulatory signaling Domain

<400> 126

Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn

1 5 10 15

Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg

20 25 30

Leu Thr Asp Val Thr Leu

35

<210> 127

<211> 188

<212> PRT

<213> artificial sequence

<220>

<223> CD30 costimulatory signaling domain

<400> 127

His Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys

1 5 10 15

Tyr Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg

20 25 30

Pro Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu

35 40 45

Pro Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr

50 55 60

Cys His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp

65 70 75 80

Ala Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro

85 90 95

Arg Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile

100 105 110

Met Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro

115 120 125

Glu Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu

130 135 140

Leu Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro

145 150 155 160

Pro Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly

165 170 175

Lys Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys

180 185

<210> 128

<211> 62

<212> PRT

<213> artificial sequence

<220>

<223> CD40 costimulatory signaling domain

<400> 128

Lys Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His Pro Lys Gln

1 5 10 15

Glu Pro Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro Gly Ser Asn Thr

20 25 30

Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln

35 40 45

Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

50 55 60

<210> 129

<211> 97

<212> PRT

<213> artificial sequence

<220>

<223> PD-1 costimulatory signaling domain

<400> 129

Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln

1 5 10 15

Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr

20 25 30

Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val

35 40 45

Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser

50 55 60

Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro

65 70 75 80

Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro

85 90 95

Leu

<210> 130

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> CD7 costimulatory signaling domain

<400> 130

Arg Thr Gln Ile Lys Lys Leu Cys Ser Trp Arg Asp Lys Asn Ser Ala

1 5 10 15

Ala Cys Val Val Tyr Glu Asp Met Ser His Ser Arg Cys Asn Thr Leu

20 25 30

Ser Ser Pro Asn Gln Tyr Gln

35

<210> 131

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> LIGHT co-stimulatory signaling domain

<400> 131

Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val Asp Gly Gln

1 5 10 15

Thr Asp Ile Pro Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser

20 25 30

Cys Ser Val Ala Arg

35

<210> 132

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> NKG2C costimulatory signaling domain

<400> 132

Met Ser Lys Gln Arg Gly Thr Phe Ser Glu Val Ser Leu Ala Gln Asp

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Ser

20 25 30

Gly Thr Glu Gln Glu Ile Phe Gln Val Glu Leu Asn Leu Gln Asn Pro

35 40 45

Ser Leu Asn His Gln Gly Ile Asp Lys Ile Tyr Asp Cys Gln Gly Leu

50 55 60

Leu Pro Pro Pro Glu Lys

65 70

<210> 133

<211> 47

<212> PRT

<213> artificial sequence

<220>

<223> B7-H3 costimulatory signaling domain

<400> 133

Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala

1 5 10 15

Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro

20 25 30

Leu Lys His Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala

35 40 45

<210> 134

<211> 113

<212> PRT

<213> Chile person

<400> 134

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 135

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> mutant human CD3z contains the activation domain of ITAM

<400> 135

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 136

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> CD3g contains the activation domain of ITAM

<400> 136

Gly Gln Asp Gly Val Arg Gln Ser Arg Ala Ser Asp Lys Gln Thr Leu

1 5 10 15

Leu Pro Asn Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg Glu Asp Asp

20 25 30

Gln Tyr Ser His Leu Gln Gly Asn Gln Leu Arg Arg Asn

35 40 45

<210> 137

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> CD3d contains the activation domain of ITAM

<400> 137

Gly His Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu

1 5 10 15

Leu Arg Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala

20 25 30

Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

35 40 45

<210> 138

<211> 55

<212> PRT

<213> artificial sequence

<220>

<223> CD3e contains the activation domain of ITAM

<400> 138

Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala

1 5 10 15

Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro

20 25 30

Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser

35 40 45

Gly Leu Asn Gln Arg Arg Ile

50 55

<210> 139

<211> 93

<212> PRT

<213> artificial sequence

<220>

<223> CD5 contains the activation domain of ITAM

<400> 139

Lys Lys Leu Val Lys Lys Phe Arg Gln Lys Lys Gln Arg Gln Trp Ile

1 5 10 15

Gly Pro Thr Gly Met Asn Gln Asn Met Ser Phe His Arg Asn His Thr

20 25 30

Ala Thr Val Arg Ser His Ala Glu Asn Pro Thr Ala Ser His Val Asp

35 40 45

Asn Glu Tyr Ser Gln Pro Pro Arg Asn Ser His Leu Ser Ala Tyr Pro

50 55 60

Ala Leu Glu Gly Ala Leu His Arg Ser Ser Met Gln Pro Asp Asn Ser

65 70 75 80

Ser Asp Ser Asp Tyr Asp Leu His Gly Ala Gln Arg Leu

85 90

<210> 140

<211> 141

<212> PRT

<213> artificial sequence

<220>

<223> CD22 contains the activation domain of ITAM

<400> 140

Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly Leu Gln

1 5 10 15

Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn Lys Lys Val Arg

20 25 30

Arg Ala Pro Leu Ser Glu Gly Pro His Ser Leu Gly Cys Tyr Asn Pro

35 40 45

Met Met Glu Asp Gly Ile Ser Tyr Thr Thr Leu Arg Phe Pro Glu Met

50 55 60

Asn Ile Pro Arg Thr Gly Asp Ala Glu Ser Ser Glu Met Gln Arg Pro

65 70 75 80

Pro Pro Asp Cys Asp Asp Thr Val Thr Tyr Ser Ala Leu His Lys Arg

85 90 95

Gln Val Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu Asp Glu

100 105 110

Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val Gly Glu Arg Pro

115 120 125

Gln Ala Gln Glu Asn Val Asp Tyr Val Ile Leu Lys His

130 135 140

<210> 141

<211> 61

<212> PRT

<213> artificial sequence

<220>

<223> CD79a contains the activation domain of ITAM

<400> 141

Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp Glu

1 5 10 15

Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser

20 25 30

Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr Gln Asp Val

35 40 45

Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys Pro

50 55 60

<210> 142

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> DAP10 contains the activation Domain of ITAM

<400> 142

Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val

1 5 10 15

Tyr Ile Asn Met Pro Gly Arg Gly

20

<210> 143

<211> 76

<212> PRT

<213> artificial sequence

<220>

<223> CD66d contains the activation domain of ITAM

<400> 143

Ala Lys Thr Gly Arg Thr Ser Ile Gln Arg Asp Leu Lys Glu Gln Gln

1 5 10 15

Pro Gln Ala Leu Ala Pro Gly Arg Gly Pro Ser His Ser Ser Ala Phe

20 25 30

Ser Met Ser Pro Leu Ser Thr Ala Gln Ala Pro Leu Pro Asn Pro Arg

35 40 45

Thr Ala Ala Ser Ile Tyr Glu Glu Leu Leu Lys His Asp Thr Asn Ile

50 55 60

Tyr Cys Arg Met Asp His Lys Ala Glu Val Ala Ser

65 70 75

<210> 144

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 transmembrane Domain

<400> 144

Leu Leu Met Ile Ile Ala Pro Ser Leu Gly Phe Val Leu Phe Ala Leu

1 5 10 15

Phe Val Ala Phe Leu

20

<210> 145

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> CD28 transmembrane Domain

<400> 145

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 146

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> 4-1BB transmembrane Domain

<400> 146

Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu

1 5 10 15

Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val

20 25

<210> 147

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> OX40 transmembrane Domain

<400> 147

Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro

1 5 10 15

Leu Ala Ile Leu Leu

20

<210> 148

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> CD27 transmembrane Domain

<400> 148

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

1 5 10 15

Ala Leu Phe Leu His

20

<210> 149

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> ICOS transmembrane Domain

<400> 149

Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu

1 5 10 15

Gly Cys Ile Leu Ile

20

<210> 150

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> CD2 transmembrane Domain

<400> 150

Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met Val

1 5 10 15

Phe Val Ala Leu Leu Val Phe Tyr Ile Thr

20 25

<210> 151

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> LFA-1 transmembrane Domain

<400> 151

Ile Ala Ala Ile Val Gly Gly Thr Val Ala Gly Ile Val Leu Ile Gly

1 5 10 15

Ile Leu Leu Leu Val Ile Trp

20

<210> 152

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> CD30 transmembrane Domain

<400> 152

Pro Val Leu Asp Ala Gly Pro Val Leu Phe Trp Val Ile Leu Val Leu

1 5 10 15

Val Val Val Val Gly Ser Ser Ala Phe Leu Leu Cys

20 25

<210> 153

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> CD40 transmembrane Domain

<400> 153

Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu

1 5 10 15

Leu Val Leu Val Phe Ile

20

<210> 154

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> PD-1 transmembrane Domain

<400> 154

Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val Trp

1 5 10 15

Val Leu Ala Val Ile

20

<210> 155

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> CD7 transmembrane Domain

<400> 155

Ala Ala Leu Ala Val Ile Ser Phe Leu Leu Gly Leu Gly Leu Gly Val

1 5 10 15

Ala Cys Val Leu Ala

20

<210> 156

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> LIGHT transmembrane domain

<400> 156

Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly Ala Gly Leu Ala Val

1 5 10 15

Gln Gly Trp Phe Leu

20

<210> 157

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> NKG2C transmembrane Domain

<400> 157

Leu Thr Ala Glu Val Leu Gly Ile Ile Cys Ile Val Leu Met Ala Thr

1 5 10 15

Val Leu Lys Thr Ile Val Leu

20

<210> 158

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> OX40 costimulatory signaling Domain

<400> 158

Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu Leu Val

1 5 10 15

Ala Leu Ala Phe Val

20

<210> 159

<211> 347

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 binding Domain-CD 28 transmembrane-CD 28 Co-stimulatory signaling Domain

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 159

Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu

20 25 30

Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser

35 40 45

Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys

50 55 60

Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys

65 70 75 80

Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser

85 90 95

Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg

115 120 125

Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr

130 135 140

Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu

145 150 155 160

Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro

165 170 175

Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro

180 185 190

Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala

195 200 205

Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg

210 215 220

Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly

225 230 235 240

Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys

245 250 255

Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His

260 265 270

Gln Ile Asn Ser Arg Gln Thr Phe Trp Val Leu Val Val Val Gly Gly

275 280 285

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

290 295 300

Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn

305 310 315 320

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

325 330 335

Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

340 345

<210> 160

<211> 341

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 binding Domain-Tim 4 transmembrane-CD 28 Co-stimulatory signaling Domain

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 160

Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu

20 25 30

Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser

35 40 45

Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys

50 55 60

Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys

65 70 75 80

Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser

85 90 95

Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg

115 120 125

Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr

130 135 140

Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu

145 150 155 160

Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro

165 170 175

Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro

180 185 190

Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala

195 200 205

Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg

210 215 220

Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly

225 230 235 240

Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys

245 250 255

Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His

260 265 270

Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly

275 280 285

Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Arg Ser Lys Arg

290 295 300

Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro

305 310 315 320

Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe

325 330 335

Ala Ala Tyr Arg Ser

340

<210> 161

<211> 454

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 binding Domain-Tim 4 transmembrane-4-1 BB costimulatory Signaling Domain-CD 3z contains the activation Domain of ITAM

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 161

Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu

20 25 30

Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser

35 40 45

Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys

50 55 60

Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys

65 70 75 80

Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser

85 90 95

Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg

115 120 125

Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr

130 135 140

Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu

145 150 155 160

Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro

165 170 175

Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro

180 185 190

Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala

195 200 205

Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg

210 215 220

Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly

225 230 235 240

Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys

245 250 255

Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His

260 265 270

Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly

275 280 285

Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Lys Arg Gly Arg

290 295 300

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

305 310 315 320

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

325 330 335

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

340 345 350

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

355 360 365

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

370 375 380

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

385 390 395 400

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

405 410 415

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

420 425 430

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

435 440 445

Gln Ala Leu Pro Pro Arg

450

<210> 162

<211> 496

<212> PRT

<213> artificial sequence

<220>

<223> construct 13ATIM4 binding Domain-CD 28 TM-CD28 costim-CD3z

<220>

<221> SIGNAL

<222> (1)..(24)

<400> 162

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ser Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 163

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> GM-CSF signal peptide

<400> 163

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 164

<211> 41

<212> PRT

<213> Chile person

<400> 164

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 165

<211> 41

<212> PRT

<213> artificial sequence

<220>

<400> 165

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 166

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> OX40 costimulatory signaling Domain

<400> 166

Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His

1 5 10 15

Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln

20 25 30

Ala Asp Ala His Ser Thr Leu Ala Lys Ile

35 40

<210> 167

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> CD2 costimulatory signaling domain

<400> 167

Lys Arg Lys Lys Gln Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr

1 5 10 15

Arg Ala His Arg Val Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln

20 25 30

Ile Pro Ala Ser Thr Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro

35 40 45

Pro Pro Pro Gly His Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro

50 55 60

Pro Gly His Arg Val Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro

65 70 75 80

Ser Gly Thr Gln Val His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro

85 90 95

Arg Val Gln Pro Lys Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser

100 105 110

Pro Ser Ser Asn

115

<210> 168

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> 4-1BB costimulatory signaling domain

<400> 168

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 169

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> CD27 costimulatory signaling domain

<400> 169

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 170

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> ICAM-1 costimulatory signaling Domain

<400> 170

Asn Arg Gln Arg Lys Ile Lys Lys Tyr Arg Leu Gln Gln Ala Gln Lys

1 5 10 15

Gly Thr Pro Met Lys Pro Asn Thr Gln Ala Thr Pro Pro

20 25

<210> 171

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> LFA-1 costimulatory signaling domain

<400> 171

Lys Ala Leu Ile His Leu Ser Asp Leu Arg Glu Tyr Arg Arg Phe Glu

1 5 10 15

Lys Glu Lys Leu Lys Ser Gln Trp Asn Asn Asp Asn Pro Leu Phe Lys

20 25 30

Ser Ala Thr Thr Thr Val Met Asn Pro Lys Phe Ala Glu Ser

35 40 45

<210> 172

<211> 38

<212> PRT

<213> artificial sequence

<220>

<223> ICOS costimulatory signaling Domain

<400> 172

Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn

1 5 10 15

Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg

20 25 30

Leu Thr Asp Val Thr Leu

35

<210> 173

<211> 188

<212> PRT

<213> artificial sequence

<220>

<223> CD30 costimulatory signaling domain

<400> 173

His Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys

1 5 10 15

Tyr Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg

20 25 30

Pro Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu

35 40 45

Pro Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr

50 55 60

Cys His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp

65 70 75 80

Ala Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro

85 90 95

Arg Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile

100 105 110

Met Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro

115 120 125

Glu Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu

130 135 140

Leu Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro

145 150 155 160

Pro Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly

165 170 175

Lys Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys

180 185

<210> 174

<211> 62

<212> PRT

<213> artificial sequence

<220>

<223> CD40 costimulatory signaling domain

<400> 174

Lys Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His Pro Lys Gln

1 5 10 15

Glu Pro Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro Gly Ser Asn Thr

20 25 30

Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr Gln

35 40 45

Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln

50 55 60

<210> 175

<211> 97

<212> PRT

<213> artificial sequence

<220>

<223> PD-1 costimulatory signaling domain

<400> 175

Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln

1 5 10 15

Pro Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr

20 25 30

Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val

35 40 45

Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser

50 55 60

Gly Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro

65 70 75 80

Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro

85 90 95

Leu

<210> 176

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> CD7 costimulatory signaling domain

<400> 176

Arg Thr Gln Ile Lys Lys Leu Cys Ser Trp Arg Asp Lys Asn Ser Ala

1 5 10 15

Ala Cys Val Val Tyr Glu Asp Met Ser His Ser Arg Cys Asn Thr Leu

20 25 30

Ser Ser Pro Asn Gln Tyr Gln

35

<210> 177

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> LIGHT co-stimulatory signaling domain

<400> 177

Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val Asp Gly Gln

1 5 10 15

Thr Asp Ile Pro Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser

20 25 30

Cys Ser Val Ala Arg

35

<210> 178

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> NKG2C costimulatory signaling domain

<400> 178

Met Ser Lys Gln Arg Gly Thr Phe Ser Glu Val Ser Leu Ala Gln Asp

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Ser

20 25 30

Gly Thr Glu Gln Glu Ile Phe Gln Val Glu Leu Asn Leu Gln Asn Pro

35 40 45

Ser Leu Asn His Gln Gly Ile Asp Lys Ile Tyr Asp Cys Gln Gly Leu

50 55 60

Leu Pro Pro Pro Glu Lys

65 70

<210> 179

<211> 47

<212> PRT

<213> artificial sequence

<220>

<223> B7-H3 costimulatory signaling domain

<400> 179

Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala

1 5 10 15

Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro

20 25 30

Leu Lys His Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala

35 40 45

<210> 180

<211> 52

<212> PRT

<213> artificial sequence

<220>

<223> DAP12

<400> 180

Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala

1 5 10 15

Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu

20 25 30

Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg

35 40 45

Pro Tyr Tyr Lys

50

<210> 181

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> CD28 transmembrane Domain

<400> 181

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 182

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> 4-1BB transmembrane Domain

<400> 182

Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu

1 5 10 15

Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val

20 25

<210> 183

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> OX40 transmembrane Domain

<400> 183

Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro

1 5 10 15

Leu Ala Ile Leu Leu

20

<210> 184

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> CD27 transmembrane Domain

<400> 184

Ile Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly

1 5 10 15

Ala Leu Phe Leu His

20

<210> 185

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> ICOS transmembrane Domain

<400> 185

Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu

1 5 10 15

Gly Cys Ile Leu Ile

20

<210> 186

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> CD2 transmembrane Domain

<400> 186

Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met Val

1 5 10 15

Phe Val Ala Leu Leu Val Phe Tyr Ile Thr

20 25

<210> 187

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> LFA-1 transmembrane Domain

<400> 187

Ile Ala Ala Ile Val Gly Gly Thr Val Ala Gly Ile Val Leu Ile Gly

1 5 10 15

Ile Leu Leu Leu Val Ile Trp

20

<210> 188

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> CD30 transmembrane Domain

<400> 188

Pro Val Leu Asp Ala Gly Pro Val Leu Phe Trp Val Ile Leu Val Leu

1 5 10 15

Val Val Val Val Gly Ser Ser Ala Phe Leu Leu Cys

20 25

<210> 189

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> CD40 transmembrane Domain

<400> 189

Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu

1 5 10 15

Leu Val Leu Val Phe Ile

20

<210> 190

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> PD-1 transmembrane Domain

<400> 190

Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val Trp

1 5 10 15

Val Leu Ala Val Ile

20

<210> 191

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> CD7 transmembrane Domain

<400> 191

Ala Ala Leu Ala Val Ile Ser Phe Leu Leu Gly Leu Gly Leu Gly Val

1 5 10 15

Ala Cys Val Leu Ala

20

<210> 192

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> LIGHT transmembrane domain

<400> 192

Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly Ala Gly Leu Ala Val

1 5 10 15

Gln Gly Trp Phe Leu

20

<210> 193

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> NKG2C transmembrane Domain

<400> 193

Leu Thr Ala Glu Val Leu Gly Ile Ile Cys Ile Val Leu Met Ala Thr

1 5 10 15

Val Leu Lys Thr Ile Val Leu

20

<210> 194

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> OX40 costimulatory signaling Domain

<400> 194

Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu Leu Val

1 5 10 15

Ala Leu Ala Phe Val

20

<210> 195

<211> 473

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 binding Domain-CD 28 hinge-CD 28 transmembrane-CD 28 Co-stimulatory signaling

domain-DAP 12 signalling domain, amino acids 1-24 are signal peptides (CTX 140)

<220>

<221> SIGNAL

<222> (1)..(24)

<400> 195

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Ile Glu Val Met Tyr Pro

305 310 315 320

Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val

325 330 335

Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys

340 345 350

Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

355 360 365

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg

370 375 380

Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro

385 390 395 400

Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe

405 410 415

Ala Ala Tyr Arg Ser Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg

420 425 430

Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu

435 440 445

Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp

450 455 460

Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

465 470

<210> 196

<211> 446

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 binding Domain-modified IgG4 hinge-CD 28 transmembrane-CD 28 costimulatory Signaling

domain-DAP 12 signalling domain, amino acids 1-24 are signal peptides (CTX 143)

<220>

<221> SIGNAL

<222> (1)..(24)

<400> 196

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Glu Ser Lys Tyr Gly Pro

305 310 315 320

Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val

325 330 335

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

340 345 350

Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met

355 360 365

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

370 375 380

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Tyr Phe Leu Gly Arg Leu

385 390 395 400

Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg

405 410 415

Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser

420 425 430

Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

435 440 445

<210> 197

<211> 434

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 binding Domain-CD 28 transmembrane-CD 28 Co-stimulatory signaling

domain-DAP 12 signalling domain, amino acids 1-24 are signal peptides (CTX 137)

<220>

<221> SIGNAL

<222> (1)..(24)

<400> 197

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Tyr Phe

370 375 380

Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr

385 390 395 400

Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln

405 410 415

Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr

420 425 430

Tyr Lys

<210> 198

<211> 428

<212> PRT

<213> artificial sequence

<220>

<223> Tim4 binding Domain-Tim 4 TM-CD28-DAP12, amino acids 1-24 are Signal peptides (CTX 256)

<220>

<221> SIGNAL

<222> (1)..(24)

<400> 198

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Arg

325 330 335

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

340 345 350

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

355 360 365

Arg Asp Phe Ala Ala Tyr Arg Ser Tyr Phe Leu Gly Arg Leu Val Pro

370 375 380

Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr

385 390 395 400

Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val

405 410 415

Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

420 425

<210> 199

<400> 199

000

<210> 200

<400> 200

000

<210> 201

<400> 201

000

<210> 202

<400> 202

000

<210> 203

<400> 203

000

<210> 204

<400> 204

000

<210> 205

<400> 205

000

<210> 206

<400> 206

000

<210> 207

<400> 207

000

<210> 208

<400> 208

000

<210> 209

<400> 209

000

<210> 210

<400> 210

000

<210> 211

<400> 211

000

<210> 212

<400> 212

000

<210> 213

<400> 213

000

<210> 214

<400> 214

000

<210> 215

<400> 215

000

<210> 216

<400> 216

000

<210> 217

<400> 217

000

<210> 218

<211> 24

<212> PRT

<213> Chile person

<400> 218

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser

20

<210> 219

<211> 290

<212> PRT

<213> Chile person

<400> 219

Glu Thr Val Val Thr Glu Val Leu Gly His Arg Val Thr Leu Pro Cys

1 5 10 15

Leu Tyr Ser Ser Trp Ser His Asn Ser Asn Ser Met Cys Trp Gly Lys

20 25 30

Asp Gln Cys Pro Tyr Ser Gly Cys Lys Glu Ala Leu Ile Arg Thr Asp

35 40 45

Gly Met Arg Val Thr Ser Arg Lys Ser Ala Lys Tyr Arg Leu Gln Gly

50 55 60

Thr Ile Pro Arg Gly Asp Val Ser Leu Thr Ile Leu Asn Pro Ser Glu

65 70 75 80

Ser Asp Ser Gly Val Tyr Cys Cys Arg Ile Glu Val Pro Gly Trp Phe

85 90 95

Asn Asp Val Lys Ile Asn Val Arg Leu Asn Leu Gln Arg Ala Ser Thr

100 105 110

Thr Thr His Arg Thr Ala Thr Thr Thr Thr Arg Arg Thr Thr Thr Thr

115 120 125

Ser Pro Thr Thr Thr Arg Gln Met Thr Thr Thr Pro Ala Ala Leu Pro

130 135 140

Thr Thr Val Val Thr Thr Pro Asp Leu Thr Thr Gly Thr Pro Leu Gln

145 150 155 160

Met Thr Thr Ile Ala Val Phe Thr Thr Ala Asn Thr Cys Leu Ser Leu

165 170 175

Thr Pro Ser Thr Leu Pro Glu Glu Ala Thr Gly Leu Leu Thr Pro Glu

180 185 190

Pro Ser Lys Glu Gly Pro Ile Leu Thr Ala Glu Ser Glu Thr Val Leu

195 200 205

Pro Ser Asp Ser Trp Ser Ser Val Glu Ser Thr Ser Ala Asp Thr Val

210 215 220

Leu Leu Thr Ser Lys Glu Ser Lys Val Trp Asp Leu Pro Ser Thr Ser

225 230 235 240

His Val Ser Met Trp Lys Thr Ser Asp Ser Val Ser Ser Pro Gln Pro

245 250 255

Gly Ala Ser Asp Thr Ala Val Pro Glu Gln Asn Lys Thr Thr Lys Thr

260 265 270

Gly Gln Met Asp Gly Ile Pro Met Ser Met Lys Asn Glu Met Pro Ile

275 280 285

Ser Gln

290

<210> 220

<211> 27

<212> PRT

<213> Chile person

<400> 220

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 221

<211> 21

<212> PRT

<213> Chile person

<400> 221

Leu Leu Met Ile Ile Ala Pro Ser Leu Gly Phe Val Leu Phe Ala Leu

1 5 10 15

Phe Val Ala Phe Leu

20

<210> 222

<211> 175

<212> PRT

<213> Chile person

<400> 222

His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu

1 5 10 15

Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr

20 25 30

Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn

35 40 45

Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys

50 55 60

Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile

65 70 75 80

Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu

85 90 95

Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His

100 105 110

Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu

115 120 125

Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg

130 135 140

Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala

145 150 155 160

Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser

165 170 175

<210> 223

<211> 193

<212> PRT

<213> Chile person

<400> 223

His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu

1 5 10 15

Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr

20 25 30

Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp

35 40 45

Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn

50 55 60

Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile

65 70 75 80

Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe

85 90 95

Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe

100 105 110

Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile

115 120 125

Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu

130 135 140

Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro

145 150 155 160

Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr

165 170 175

Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln

180 185 190

Tyr

<210> 224

<211> 42

<212> PRT

<213> Chile person

<400> 224

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

1 5 10 15

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

20 25 30

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu

35 40

<210> 225

<211> 43

<212> PRT

<213> Chile person

<400> 225

Met Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg

1 5 10 15

Leu Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His

20 25 30

Gly Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu

35 40

<210> 226

<400> 226

000

<210> 227

<211> 496

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD28icd-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 227

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser

340 345 350

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

355 360 365

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 228

<211> 671

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD28icd-TLR2ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 228

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser

340 345 350

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

355 360 365

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser His

370 375 380

Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu Gln

385 390 395 400

Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp

405 410 415

Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu

420 425 430

Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu

435 440 445

His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile

450 455 460

Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu Asn

465 470 475 480

Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe

485 490 495

Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu

500 505 510

Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys

515 520 525

Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln

530 535 540

Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser Arg Val

545 550 555 560

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

565 570 575

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

580 585 590

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln

595 600 605

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

610 615 620

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

625 630 635 640

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

645 650 655

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 229

<211> 671

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD28icd-CD3zICD-TLR2ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 229

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser

340 345 350

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

355 360 365

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu

500 505 510

Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr

515 520 525

Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn

530 535 540

Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys

545 550 555 560

Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile

565 570 575

Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu

580 585 590

Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His

595 600 605

Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu

610 615 620

Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg

625 630 635 640

Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala

645 650 655

Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser

660 665 670

<210> 230

<211> 689

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD28icd-TLR8ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 230

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser

340 345 350

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

355 360 365

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser His

370 375 380

His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala

385 390 395 400

Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp

405 410 415

Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val

420 425 430

Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val

435 440 445

Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile

450 455 460

Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val

465 470 475 480

Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr

485 490 495

Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe

500 505 510

Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg

515 520 525

Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys

530 535 540

Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu

545 550 555 560

Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr

565 570 575

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

580 585 590

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

595 600 605

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

610 615 620

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

625 630 635 640

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

645 650 655

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

660 665 670

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

675 680 685

Arg

<210> 231

<211> 689

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD28icd-CD3zICD-TLR8ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 231

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser

340 345 350

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

355 360 365

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu

500 505 510

Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr

515 520 525

Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp

530 535 540

Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn

545 550 555 560

Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile

565 570 575

Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe

580 585 590

Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe

595 600 605

Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile

610 615 620

Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu

625 630 635 640

Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro

645 650 655

Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr

660 665 670

Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln

675 680 685

Tyr

<210> 232

<211> 630

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-TLR2ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 232

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val His Arg Phe His Gly Leu Trp Tyr Met Lys

340 345 350

Met Met Trp Ala Trp Leu Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro

355 360 365

Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp

370 375 380

Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu Glu Asn Phe Asn

385 390 395 400

Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe Ile Pro Gly Lys

405 410 415

Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys Ser His Lys Thr

420 425 430

Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr

435 440 445

Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala

450 455 460

Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln

465 470 475 480

Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu

485 490 495

Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp Val Asn Leu Arg

500 505 510

Ala Ala Ile Lys Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

515 520 525

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

530 535 540

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

545 550 555 560

Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu

565 570 575

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

580 585 590

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

595 600 605

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

610 615 620

Gln Ala Leu Pro Pro Arg

625 630

<210> 233

<211> 630

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD3zICD-TLR2ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 233

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

340 345 350

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

355 360 365

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

370 375 380

Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly

385 390 395 400

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

405 410 415

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

420 425 430

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

435 440 445

Met Gln Ala Leu Pro Pro Arg His Arg Phe His Gly Leu Trp Tyr Met

450 455 460

Lys Met Met Trp Ala Trp Leu Gln Ala Lys Arg Lys Pro Arg Lys Ala

465 470 475 480

Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr Ser Glu Arg

485 490 495

Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu Glu Asn Phe

500 505 510

Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe Ile Pro Gly

515 520 525

Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys Ser His Lys

530 535 540

Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu Trp Cys Lys

545 550 555 560

Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu Asn Asn Asp

565 570 575

Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys Ala Ile Pro

580 585 590

Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys Thr Tyr Leu

595 600 605

Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp Val Asn Leu

610 615 620

Arg Ala Ala Ile Lys Ser

625 630

<210> 234

<211> 648

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-TLR8ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 234

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val His His Leu Phe Tyr Trp Asp Val Trp Phe

340 345 350

Ile Tyr Asn Val Cys Leu Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser

355 360 365

Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp

370 375 380

Ala Ser Val Thr Asp Trp Val Ile Asn Glu Leu Arg Tyr His Leu Glu

385 390 395 400

Glu Ser Arg Asp Lys Asn Val Leu Leu Cys Leu Glu Glu Arg Asp Trp

405 410 415

Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu Met Gln Ser Ile Asn Gln

420 425 430

Ser Lys Lys Thr Val Phe Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp

435 440 445

Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu

450 455 460

Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu Pro Val Leu Gln His

465 470 475 480

Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu

485 490 495

Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu

500 505 510

Arg Asn Val Val Leu Thr Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr

515 520 525

Val Asp Ser Ile Lys Gln Tyr Arg Val Lys Phe Ser Arg Ser Ala Asp

530 535 540

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

545 550 555 560

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

565 570 575

Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu

580 585 590

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

595 600 605

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

610 615 620

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

625 630 635 640

His Met Gln Ala Leu Pro Pro Arg

645

<210> 235

<211> 648

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD3zICD-TLR8ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 235

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

340 345 350

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

355 360 365

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

370 375 380

Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly

385 390 395 400

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

405 410 415

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

420 425 430

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

435 440 445

Met Gln Ala Leu Pro Pro Arg His His Leu Phe Tyr Trp Asp Val Trp

450 455 460

Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys Gly Tyr Arg Ser Leu

465 470 475 480

Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr Asp Thr Lys

485 490 495

Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu Leu Arg Tyr His Leu

500 505 510

Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys Leu Glu Glu Arg Asp

515 520 525

Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu Met Gln Ser Ile Asn

530 535 540

Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys Lys Tyr Ala Lys Ser

545 550 555 560

Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln Arg Leu Met Asp

565 570 575

Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu Pro Val Leu Gln

580 585 590

His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys Lys Ser Ser Ile

595 600 605

Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly Leu Phe Trp Gln Thr

610 615 620

Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser Arg Tyr Asn Asn Met

625 630 635 640

Tyr Val Asp Ser Ile Lys Gln Tyr

645

<210> 236

<211> 729

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-TRAF6ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 236

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Met Ser Leu Leu Asn Cys Glu Asn Ser Cys

340 345 350

Gly Ser Ser Gln Ser Glu Ser Asp Cys Cys Val Ala Met Ala Ser Ser

355 360 365

Cys Ser Ala Val Thr Lys Asp Asp Ser Val Gly Gly Thr Ala Ser Thr

370 375 380

Gly Asn Leu Ser Ser Ser Phe Met Glu Glu Ile Gln Gly Tyr Asp Val

385 390 395 400

Glu Phe Asp Pro Pro Leu Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu

405 410 415

Met Ala Leu Arg Glu Ala Val Gln Thr Pro Cys Gly His Arg Phe Cys

420 425 430

Lys Ala Cys Ile Ile Lys Ser Ile Arg Asp Ala Gly His Lys Cys Pro

435 440 445

Val Asp Asn Glu Ile Leu Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe

450 455 460

Ala Lys Arg Glu Ile Leu Ser Leu Met Val Lys Cys Pro Asn Glu Gly

465 470 475 480

Cys Leu His Lys Met Glu Leu Arg His Leu Glu Asp His Gln Ala His

485 490 495

Cys Glu Phe Ala Leu Met Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln

500 505 510

Lys Phe His Ile Asn Ile His Ile Leu Lys Asp Cys Pro Arg Arg Gln

515 520 525

Val Ser Cys Asp Asn Cys Ala Ala Ser Met Ala Phe Glu Asp Lys Glu

530 535 540

Ile His Asp Gln Asn Cys Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys

545 550 555 560

Asn Thr Ile Leu Ile Arg Glu Gln Met Pro Asn His Tyr Asp Leu Asp

565 570 575

Cys Pro Thr Ala Pro Ile Pro Cys Thr Phe Ser Thr Phe Gly Cys His

580 585 590

Glu Lys Met Gln Arg Asn His Leu Ala Arg His Leu Gln Glu Asn Thr

595 600 605

Gln Ser His Met Arg Met Leu Ala Arg Val Lys Phe Ser Arg Ser Ala

610 615 620

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

625 630 635 640

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

645 650 655

Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln

660 665 670

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

675 680 685

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

690 695 700

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

705 710 715 720

Leu His Met Gln Ala Leu Pro Pro Arg

725

<210> 237

<211> 729

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-CD28tm-CD3zICD-TRAF6ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 237

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Gln Phe Trp Val Leu Val

305 310 315 320

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

325 330 335

Phe Ile Ile Phe Trp Val Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

340 345 350

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

355 360 365

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

370 375 380

Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly

385 390 395 400

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

405 410 415

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

420 425 430

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

435 440 445

Met Gln Ala Leu Pro Pro Arg Met Ser Leu Leu Asn Cys Glu Asn Ser

450 455 460

Cys Gly Ser Ser Gln Ser Glu Ser Asp Cys Cys Val Ala Met Ala Ser

465 470 475 480

Ser Cys Ser Ala Val Thr Lys Asp Asp Ser Val Gly Gly Thr Ala Ser

485 490 495

Thr Gly Asn Leu Ser Ser Ser Phe Met Glu Glu Ile Gln Gly Tyr Asp

500 505 510

Val Glu Phe Asp Pro Pro Leu Glu Ser Lys Tyr Glu Cys Pro Ile Cys

515 520 525

Leu Met Ala Leu Arg Glu Ala Val Gln Thr Pro Cys Gly His Arg Phe

530 535 540

Cys Lys Ala Cys Ile Ile Lys Ser Ile Arg Asp Ala Gly His Lys Cys

545 550 555 560

Pro Val Asp Asn Glu Ile Leu Leu Glu Asn Gln Leu Phe Pro Asp Asn

565 570 575

Phe Ala Lys Arg Glu Ile Leu Ser Leu Met Val Lys Cys Pro Asn Glu

580 585 590

Gly Cys Leu His Lys Met Glu Leu Arg His Leu Glu Asp His Gln Ala

595 600 605

His Cys Glu Phe Ala Leu Met Asp Cys Pro Gln Cys Gln Arg Pro Phe

610 615 620

Gln Lys Phe His Ile Asn Ile His Ile Leu Lys Asp Cys Pro Arg Arg

625 630 635 640

Gln Val Ser Cys Asp Asn Cys Ala Ala Ser Met Ala Phe Glu Asp Lys

645 650 655

Glu Ile His Asp Gln Asn Cys Pro Leu Ala Asn Val Ile Cys Glu Tyr

660 665 670

Cys Asn Thr Ile Leu Ile Arg Glu Gln Met Pro Asn His Tyr Asp Leu

675 680 685

Asp Cys Pro Thr Ala Pro Ile Pro Cys Thr Phe Ser Thr Phe Gly Cys

690 695 700

His Glu Lys Met Gln Arg Asn His Leu Ala Arg His Leu Gln Glu Asn

705 710 715 720

Thr Gln Ser His Met Arg Met Leu Ala

725

<210> 238

<211> 490

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD28icd-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 238

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

340 345 350

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

355 360 365

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 239

<211> 665

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD28icd-TLR2ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 239

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

340 345 350

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

355 360 365

Pro Arg Asp Phe Ala Ala Tyr Arg Ser His Arg Phe His Gly Leu Trp

370 375 380

Tyr Met Lys Met Met Trp Ala Trp Leu Gln Ala Lys Arg Lys Pro Arg

385 390 395 400

Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr Ser

405 410 415

Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu Glu

420 425 430

Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe Ile

435 440 445

Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys Ser

450 455 460

His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu Trp

465 470 475 480

Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu Asn

485 490 495

Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys Ala

500 505 510

Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys Thr

515 520 525

Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp Val

530 535 540

Asn Leu Arg Ala Ala Ile Lys Ser Arg Val Lys Phe Ser Arg Ser Ala

545 550 555 560

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

565 570 575

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

580 585 590

Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln

595 600 605

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

610 615 620

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

625 630 635 640

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

645 650 655

Leu His Met Gln Ala Leu Pro Pro Arg

660 665

<210> 240

<211> 665

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD28icd-CD3zICD-TLR2ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 240

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

340 345 350

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

355 360 365

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg His Arg Phe His Gly Leu

485 490 495

Trp Tyr Met Lys Met Met Trp Ala Trp Leu Gln Ala Lys Arg Lys Pro

500 505 510

Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr

515 520 525

Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu

530 535 540

Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe

545 550 555 560

Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys

565 570 575

Ser His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu

580 585 590

Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu

595 600 605

Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys

610 615 620

Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys

625 630 635 640

Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp

645 650 655

Val Asn Leu Arg Ala Ala Ile Lys Ser

660 665

<210> 241

<211> 683

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD28icd-TLR8ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 241

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

340 345 350

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

355 360 365

Pro Arg Asp Phe Ala Ala Tyr Arg Ser His His Leu Phe Tyr Trp Asp

370 375 380

Val Trp Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys Gly Tyr Arg

385 390 395 400

Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr Asp

405 410 415

Thr Lys Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu Leu Arg Tyr

420 425 430

His Leu Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys Leu Glu Glu

435 440 445

Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu Met Gln Ser

450 455 460

Ile Asn Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys Lys Tyr Ala

465 470 475 480

Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln Arg Leu

485 490 495

Met Asp Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu Pro Val

500 505 510

Leu Gln His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys Lys Ser

515 520 525

Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly Leu Phe Trp

530 535 540

Gln Thr Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser Arg Tyr Asn

545 550 555 560

Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr Arg Val Lys Phe Ser Arg

565 570 575

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

580 585 590

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

595 600 605

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

610 615 620

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

625 630 635 640

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

645 650 655

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

660 665 670

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

675 680

<210> 242

<211> 683

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD28icd-CD3zICD-TLR8ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 242

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

340 345 350

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

355 360 365

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg His His Leu Phe Tyr Trp

485 490 495

Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys Gly Tyr

500 505 510

Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr

515 520 525

Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu Leu Arg

530 535 540

Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys Leu Glu

545 550 555 560

Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu Met Gln

565 570 575

Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys Lys Tyr

580 585 590

Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln Arg

595 600 605

Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu Pro

610 615 620

Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys Lys

625 630 635 640

Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly Leu Phe

645 650 655

Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser Arg Tyr

660 665 670

Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr

675 680

<210> 243

<211> 624

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TLR2ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 243

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu

340 345 350

Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr

355 360 365

Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn

370 375 380

Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys

385 390 395 400

Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile

405 410 415

Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu

420 425 430

Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His

435 440 445

Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu

450 455 460

Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg

465 470 475 480

Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala

485 490 495

Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser Arg

500 505 510

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

515 520 525

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

530 535 540

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

545 550 555 560

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

565 570 575

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

580 585 590

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

595 600 605

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

610 615 620

<210> 244

<211> 624

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD3zICD-TLR2ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 244

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

340 345 350

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

355 360 365

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

370 375 380

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

385 390 395 400

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

405 410 415

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

420 425 430

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

435 440 445

Arg His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp

450 455 460

Leu Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys

465 470 475 480

Tyr Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu

485 490 495

Asn Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu

500 505 510

Cys Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn

515 520 525

Ile Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser

530 535 540

Glu Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser

545 550 555 560

His Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu

565 570 575

Leu Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu

580 585 590

Arg Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu

595 600 605

Ala Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser

610 615 620

<210> 245

<211> 642

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TLR8ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 245

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu

340 345 350

Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr

355 360 365

Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp

370 375 380

Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn

385 390 395 400

Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile

405 410 415

Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe

420 425 430

Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe

435 440 445

Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile

450 455 460

Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu

465 470 475 480

Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro

485 490 495

Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr

500 505 510

Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln

515 520 525

Tyr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

530 535 540

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

545 550 555 560

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

565 570 575

Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

580 585 590

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

595 600 605

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

610 615 620

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

625 630 635 640

Pro Arg

<210> 246

<211> 642

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD3zICD-TLR8ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 246

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

340 345 350

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

355 360 365

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

370 375 380

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

385 390 395 400

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

405 410 415

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

420 425 430

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

435 440 445

Arg His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys

450 455 460

Leu Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe

465 470 475 480

Tyr Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp

485 490 495

Trp Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys

500 505 510

Asn Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala

515 520 525

Ile Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val

530 535 540

Phe Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala

545 550 555 560

Phe Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile

565 570 575

Ile Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg

580 585 590

Leu Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn

595 600 605

Pro Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu

610 615 620

Thr Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys

625 630 635 640

Gln Tyr

<210> 247

<211> 723

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TRAF6ICD-CD3zICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 247

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu

340 345 350

Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys

355 360 365

Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser

370 375 380

Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu

385 390 395 400

Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala

405 410 415

Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys

420 425 430

Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu

435 440 445

Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu

450 455 460

Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu

465 470 475 480

Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met

485 490 495

Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile

500 505 510

His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys

515 520 525

Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys

530 535 540

Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg

545 550 555 560

Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile

565 570 575

Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn

580 585 590

His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met

595 600 605

Leu Ala Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

610 615 620

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

625 630 635 640

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

645 650 655

Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

660 665 670

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

675 680 685

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

690 695 700

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

705 710 715 720

Pro Pro Arg

<210> 248

<211> 723

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-CD3zICD-TRAF6ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 248

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

340 345 350

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

355 360 365

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

370 375 380

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

385 390 395 400

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

405 410 415

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

420 425 430

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

435 440 445

Arg Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser

450 455 460

Glu Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr

465 470 475 480

Lys Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser

485 490 495

Ser Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro

500 505 510

Leu Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu

515 520 525

Ala Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile

530 535 540

Lys Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile

545 550 555 560

Leu Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile

565 570 575

Leu Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met

580 585 590

Glu Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu

595 600 605

Met Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn

610 615 620

Ile His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn

625 630 635 640

Cys Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn

645 650 655

Cys Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile

660 665 670

Arg Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro

675 680 685

Ile Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg

690 695 700

Asn His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg

705 710 715 720

Met Leu Ala

<210> 249

<211> 379

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TIM4ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 249

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Met Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg

340 345 350

Leu Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His

355 360 365

Gly Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu

370 375

<210> 250

<211> 666

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TIM4ICD-TLR2-CD3z

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 250

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu His Arg Phe His Gly Leu

370 375 380

Trp Tyr Met Lys Met Met Trp Ala Trp Leu Gln Ala Lys Arg Lys Pro

385 390 395 400

Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr

405 410 415

Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu

420 425 430

Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe

435 440 445

Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys

450 455 460

Ser His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu

465 470 475 480

Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu

485 490 495

Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys

500 505 510

Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys

515 520 525

Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp

530 535 540

Val Asn Leu Arg Ala Ala Ile Lys Ser Arg Val Lys Phe Ser Arg Ser

545 550 555 560

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

565 570 575

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

580 585 590

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro

595 600 605

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

610 615 620

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

625 630 635 640

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

645 650 655

Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665

<210> 251

<211> 666

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TIM4ICD-CD3z-TLR2

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 251

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg His Arg Phe His Gly

485 490 495

Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu Gln Ala Lys Arg Lys

500 505 510

Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser

515 520 525

Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu

530 535 540

Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp

545 550 555 560

Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu

565 570 575

Lys Ser His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser

580 585 590

Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp

595 600 605

Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys

610 615 620

Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr

625 630 635 640

Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe

645 650 655

Trp Val Asn Leu Arg Ala Ala Ile Lys Ser

660 665

<210> 252

<211> 684

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TIM4ICD-TLR8-CD3z

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 252

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu His His Leu Phe Tyr Trp

370 375 380

Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys Gly Tyr

385 390 395 400

Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr

405 410 415

Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu Leu Arg

420 425 430

Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys Leu Glu

435 440 445

Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu Met Gln

450 455 460

Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys Lys Tyr

465 470 475 480

Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln Arg

485 490 495

Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu Pro

500 505 510

Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys Lys

515 520 525

Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly Leu Phe

530 535 540

Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser Arg Tyr

545 550 555 560

Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr Arg Val Lys Phe Ser

565 570 575

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

580 585 590

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

595 600 605

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys

610 615 620

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

625 630 635 640

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

645 650 655

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

660 665 670

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

675 680

<210> 253

<211> 684

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TIM4ICD-CD3z-TLR8

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 253

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg His His Leu Phe Tyr

485 490 495

Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys Gly

500 505 510

Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser

515 520 525

Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu Leu

530 535 540

Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys Leu

545 550 555 560

Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu Met

565 570 575

Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys Lys

580 585 590

Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln

595 600 605

Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu

610 615 620

Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys

625 630 635 640

Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly Leu

645 650 655

Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser Arg

660 665 670

Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr

675 680

<210> 254

<211> 553

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TLR2ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 254

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu His Arg Phe His Gly Leu

370 375 380

Trp Tyr Met Lys Met Met Trp Ala Trp Leu Gln Ala Lys Arg Lys Pro

385 390 395 400

Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr

405 410 415

Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu

420 425 430

Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe

435 440 445

Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys

450 455 460

Ser His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu

465 470 475 480

Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu

485 490 495

Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys

500 505 510

Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys

515 520 525

Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp

530 535 540

Val Asn Leu Arg Ala Ala Ile Lys Ser

545 550

<210> 255

<211> 571

<212> PRT

<213> artificial sequence

<220>

<223> hTIM4-TIM4-TIM4tm-TLR8ICD

<220>

<221> SIGNAL

<222> (1)..(22)

<400> 255

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Leu Leu Met Ile Ile Ala

305 310 315 320

Pro Ser Leu Gly Phe Val Leu Phe Ala Leu Phe Val Ala Phe Leu Leu

325 330 335

Arg Gly Lys Leu Met Glu Thr Tyr Cys Ser Gln Lys His Thr Arg Leu

340 345 350

Asp Tyr Ile Gly Asp Ser Lys Asn Val Leu Asn Asp Val Gln His Gly

355 360 365

Arg Glu Asp Glu Asp Gly Leu Phe Thr Leu His His Leu Phe Tyr Trp

370 375 380

Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys Gly Tyr

385 390 395 400

Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile Ser Tyr

405 410 415

Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu Leu Arg

420 425 430

Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys Leu Glu

435 440 445

Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu Met Gln

450 455 460

Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys Lys Tyr

465 470 475 480

Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu Gln Arg

485 490 495

Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu Glu Pro

500 505 510

Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile Cys Lys

515 520 525

Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly Leu Phe

530 535 540

Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser Arg Tyr

545 550 555 560

Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr

565 570

<210> 256

<211> 598

<212> PRT

<213> artificial sequence

<220>

<223> HPV 16E 7 TCRB chain-P2A-TCRa chain, amino acids 309-335 are P2A sequences

<400> 256

Met Ala Pro Gly Leu Leu Cys Trp Ala Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys

20 25 30

Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His

35 40 45

Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe

50 55 60

Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro

65 70 75 80

Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn

85 90 95

Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Leu Gly Trp Arg Gly Gly Arg Tyr Asn Glu Gln Phe Phe Gly Pro

115 120 125

Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro

130 135 140

Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln

145 150 155 160

Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val

165 170 175

Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys

180 185 190

Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser

195 200 205

Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His

210 215 220

Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp

225 230 235 240

Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala

245 250 255

Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly

260 265 270

Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr

275 280 285

Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys

290 295 300

Arg Lys Asn Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe

305 310 315 320

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met

325 330 335

Trp Gly Val Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr Thr

340 345 350

Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly Ala

355 360 365

Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly Leu

370 375 380

Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser Tyr

385 390 395 400

Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe Leu

405 410 415

Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln Met

420 425 430

Lys Asp Ser Ala Ser Tyr Leu Cys Ala Ser Val Asp Gly Asn Asn Arg

435 440 445

Leu Ala Phe Gly Lys Gly Asn Gln Val Val Val Ile Pro Asn Ile Gln

450 455 460

Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp

465 470 475 480

Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro

485 490 495

Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Cys Val Leu Asp

500 505 510

Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn

515 520 525

Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr

530 535 540

Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser

545 550 555 560

Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val

565 570 575

Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr

580 585 590

Leu Arg Leu Trp Ser Ser

595

<210> 257

<211> 496

<212> PRT

<213> artificial sequence

<220>

<223> pCTX247 Tim4 Signal peptide-Tim 4 ECD-CD28 TM-CD28 ICD-CD3z ICD

<400> 257

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ser Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 258

<211> 496

<212> PRT

<213> artificial sequence

<220>

<223> pCTX797 Tim4 Signal peptide-Tim 4 ECD-CD28 TM-CD28 ICD-CD3z ICD

<400> 258

Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp

1 5 10 15

Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu

20 25 30

Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn

35 40 45

Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys

50 55 60

Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys

65 70 75 80

Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser

85 90 95

Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys

100 105 110

Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg

115 120 125

Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr

130 135 140

Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met

145 150 155 160

Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp

165 170 175

Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr

180 185 190

Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu

195 200 205

Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu

210 215 220

Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val

225 230 235 240

Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys

245 250 255

Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser

260 265 270

Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro

275 280 285

Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met

290 295 300

Ser Met Lys Asn Glu Met Pro Ile Ser Gln Phe Trp Val Leu Val Val

305 310 315 320

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

325 330 335

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

340 345 350

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

355 360 365

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ser Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

Claims

1. A method of 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 administering to the subject an effective amount of:

a chimeric T cell immunoglobulin and mucin (Tim) receptor comprising a single chain chimeric protein comprising:

(a) An extracellular domain comprising a binding domain, the 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, wherein the intracellular signaling domain comprises a primary intracellular signaling domain and optionally a secondary intracellular signaling domain; and

(c) A transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain; and

Inhibitors of poly (ADP-ribose) polymerase (PARP).

2. The method of claim 1, wherein the breast cancer is a triple negative breast cancer.

3. The method of claim 1, wherein the ovarian cancer is advanced ovarian cancer.

4. The method of claim 1, wherein the prostate cancer is advanced prostate cancer.

5. The method of claim 1, wherein the lung cancer is non-small cell lung cancer.

6. The method of any one of claims 1 to 5, wherein the cancer is a breast cancer gene (BRCA) mutated cancer.

7. The method of any one of claims 6, wherein the cancer is BRCA1 mutant cancer, BRCA2 mutant cancer, or both.

8. The method of any one of claims 1 to 7, wherein the PARP inhibitor is talazopanib (tazopanib), nilaparib (ruaparib), lu Kapa ni (ruaparib), olaparib (olaparib), veliparib (veliparib), CEP 9722, E7016, AG014699, MK4827, BMN-673, pamiparib (Pamiparib), or a combination thereof.

9. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises nilaparib.

10. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises talazapanib.

11. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises Lu Kapa ni.

12. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises olaparib.

13. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises veliparib.

14. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises CEP 9722.

15. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises E7016.

16. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises AG014699.

17. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises MK4827.

18. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises BMN-673.

19. The method of any one of claims 1 to 8, wherein the PARP inhibitor comprises pamipril.

20. The method of any one of claims 1 to 19, further comprising administering an additional therapeutic agent.

21. The method of claim 20, wherein the additional therapeutic agent comprises radiation therapy, cellular immunotherapy, antibodies, immune checkpoint molecule inhibitors, chemotherapy, hormonal therapy, peptides, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, UV light therapy, electrical pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, small molecule therapy, or a combination thereof.

22. The method of claim 21, wherein the cellular immunotherapy is a chimeric antigen receptor or a T cell receptor.

23. The method of claim 20 or 21, wherein the additional therapeutic agent comprises an angiogenesis inhibitor (e.g., VEGF pathway inhibitor), a tyrosine kinase inhibitor (e.g., EGF pathway inhibitor), a receptor tyrosine kinase inhibitor, a growth factor inhibitor, a GTPase inhibitor, a serine/threonine kinase inhibitor, a transcription factor inhibitor, a B-Raf inhibitor, a MEK inhibitor, an mTOR inhibitor, an EGFR inhibitor, an ALK inhibitor, a ROS1 inhibitor, a BCL-2 inhibitor, a PI3K inhibitor, a VEGFR inhibitor, a BCR-ABL inhibitor, a MET inhibitor, a MYC inhibitor, an ABL inhibitor, a HER2 inhibitor, a BTK inhibitor, an H-RAS inhibitor, a K-RAS inhibitor, a PDGFR inhibitor, a TRK inhibitor, a c-KIT inhibitor, a c-MET inhibitor, a CDK4/6 inhibitor, a FAK inhibitor, an FGFR inhibitor, an FLT3 inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, a PDGFRA inhibitor, or a RET inhibitor.

24. The method of any one of claims 1-23, wherein the binding domain comprises the Tim1 IgV domain and the Tim1 mucin domain.

25. The method of any one of claims 1-23, wherein the binding domain comprises the Tim4 IgV domain and the Tim4 mucin domain.

26. The method of any one of claims 1-23, wherein the binding domain comprises the Tim1 IgV domain and the Tim4 mucin domain.

27. The method of any one of claims 1-23, wherein the binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain.

28. The method of any one of claims 1 to 24 or 26, wherein the Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID No. 38.

29. The method of any one of claims 1 to 24, 26 or 28, wherein the Tim1 IgV domain is a modified Tim1 IgV domain comprising the R66G substitution in SEQ ID No. 38.

30. The method of claim 29, wherein the modified Tim1 IgV domain comprises the amino acid sequence set forth in SEQ ID No. 41.

31. The method of any one of claims 1 to 25 or 27 to 30, wherein the Tim1 mucin domain comprises the amino acid sequence set forth in SEQ ID NO 39.

32. The method of any one of claims 1 to 23, 25 or 27, wherein the Tim4 IgV domain comprises the amino acid sequence set forth in SEQ ID NO 34.

33. The method of any one of claims 1 to 23, 25 or 26, wherein the Tim4 mucin domain comprises the amino acid sequence set forth in SEQ ID No. 35.

34. The method of any one of claims 1-33, wherein the transmembrane domain comprises a Tim4 transmembrane domain, a Tim1 transmembrane domain, a CD28 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, a CD27 transmembrane domain, an ICOS transmembrane domain, a CD2 transmembrane domain, an LFA-1 transmembrane domain, a CD30 transmembrane domain, a CD40 transmembrane domain, a PD-1 transmembrane domain, a CD7 transmembrane domain, a LIGHT transmembrane domain, a NKG2C transmembrane domain, or a B7-H3 transmembrane domain.

35. The method of claim 34, wherein 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 or consists of the amino acid sequence of SEQ ID NO. 145; the 4-1BB transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO. 146; the OX40 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO 147; the CD27 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 or consists of 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 or consists of 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.

36. The method of any one of claims 1 to 34, wherein the transmembrane domain comprises a Tim1 transmembrane domain, a Tim4 transmembrane domain, or a CD28 transmembrane domain.

37. The method of claim 42, wherein the Tim1 transmembrane domain comprises the amino acid sequence set forth in SEQ ID No. 8, the Tim4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID No. 6 or 23, or the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID No. 7.

38. The method of any one of claims 1 to 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 set forth in SEQ ID NO. 3 or the CD28 hinge region comprises the amino acid sequence set forth in SEQ ID NO. 32.

41. The method of any one of claims 1 to 40, wherein the primary signaling domain comprises a CD28 costimulatory signaling domain; 4-1BB costimulatory signaling domain; a CD27 costimulatory signaling domain; ICOS costimulatory signaling domain; LFA-1 costimulatory signaling domain; OX40 costimulatory signaling domain; a CD2 costimulatory signaling domain; or ICAM-1 costimulatory signaling domain.

42. The method of claim 41, wherein the CD28 costimulatory signaling domain comprises the amino acid sequence of SEQ id No. 118 or 119; the 4-1BB costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO. 122; the CD27 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO. 169; the ICOS costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO. 172; the LFA-1 co-stimulatory signaling domain includes the amino acid sequence of SEQ ID NO. 171; the OX40 co-stimulatory signaling domain includes 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 comprises the amino acid sequence of SEQ ID NO: 170.

43. The method of any one of claims 1-42, wherein the secondary signaling domain comprises a CD3 zeta signaling domain.

44. The method of claim 43, wherein said CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 27.

45. The method of any one of claims 1 to 40, wherein the primary signaling domain comprises a CD28 costimulatory signaling domain; 4-1BB costimulatory signaling domain; a CD27 costimulatory signaling domain; ICOS costimulatory signaling domain; LFA-1 costimulatory signaling domain; OX40 costimulatory signaling domain; a CD2 costimulatory signaling domain; or ICAM-1 costimulatory signaling domain.

46. The method of claim 45, wherein the CD28 costimulatory signaling domain comprises the amino acid sequence of SEQ ID No. 118 or 119; the 4-1BB costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO. 122; the CD27 costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO. 169; the ICOS costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO. 172; the LFA-1 co-stimulatory signaling domain includes the amino acid sequence of SEQ ID NO. 171; the OX40 co-stimulatory signaling domain includes 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.

47. The method of any one of claims 1 to 42, 45 or 46, wherein the secondary signaling domain comprises a DAP12 signaling domain.

48. The method of claim 47, wherein the DAP12 signaling domain comprises the amino acid sequence of SEQ ID NO. 180.

49. The method of any one of claims 1 to 40, wherein the primary intracellular signaling domain comprises a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3 zeta signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

50. The method of claim 49, wherein 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, 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 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, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID No. 5, and the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID No. 222. Or the TLR8 signaling domain comprises the amino acid sequence set forth in SEQ ID No. 47.

51. The method of any one of claims 1 to 42, 49 or 50, wherein the secondary intracellular signaling domain comprises a Tim1 signaling domain, a Tim4 signaling domain, a TRAF2 signaling domain, a TRAF6 signaling domain, a CD28 signaling domain, a DAP12 signaling domain, a CD3 zeta signaling domain, a TLR2 signaling domain, or a TLR8 signaling domain.

52. The method of claim 51, wherein 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, 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 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, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID No. 5, and the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID No. 222. Or the TLR8 signaling domain comprises the amino acid sequence set forth in SEQ ID No. 47.

53. The method of any one of claims 1 to 52, wherein

(i) The binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain, the primary intracellular signaling domain comprises a TLR8 signaling domain, the secondary intracellular signaling domain comprises a CD3 zeta signaling domain, and the transmembrane domain comprises a Tim1 transmembrane domain;

(ii) The binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprises a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmembrane domain comprises a Tim1 transmembrane domain; or alternatively

(iii) The binding domain comprises the Tim4 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprises a CD28 signaling domain, the secondary intracellular signaling domain comprises a DAP12 signaling domain, and the transmembrane domain comprises a CD28 transmembrane domain.

54. The method of claim 53, wherein:

(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 CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO. 5 or 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. 9; and the Tim1 transmembrane domain comprises the amino acid sequence of SEQ ID NO. 8; or alternatively

(iii) 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. 9;

and the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO. 7.

55. The method of claim 53 or 54, wherein:

(i) 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 alternatively

(iii) The single chain chimeric protein comprises amino acids 25-422 of SEQ ID NO. 69.

56. The method of any one of claims 53 to 55, wherein:

(i) 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 alternatively

(iii) The single chain chimeric protein comprises the amino acid sequence of SEQ ID NO. 69.

57. The method of any one of claims 1 to 52, wherein the chimeric receptor comprises:

(i) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a Tim1 signaling domain, the secondary intracellular signaling domain comprising a CD3 zeta signaling domain, and the transmembrane domain comprising a Tim1 transmembrane domain;

(ii) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain, the primary intracellular signaling domain comprising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3 zeta signaling domain; and the transmembrane domain comprising a Tim1 transmembrane domain;

(iii) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a CD28 signaling domain and the transmembrane domain comprising a CD28 transmembrane domain;

(iv) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a TRAF6 signaling domain and the transmembrane domain comprising a Tim1 transmembrane domain;

(v) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a TRAF6 signaling domain and the transmembrane domain comprising a CD28 transmembrane domain;

(vi) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a TRAF2 signaling domain and the transmembrane domain comprising a Tim1 transmembrane domain;

(vii) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a TRAF2 signaling domain and the transmembrane domain comprising a CD28 transmembrane domain;

(viii) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a TLR8 signaling domain, the secondary intracellular signaling domain comprising a CD3 zeta signaling domain, and the transmembrane domain comprising a Tim1 transmembrane domain; (ix) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a CD28 signaling domain, the secondary intracellular signaling domain comprising a DAP12 signaling domain, and the transmembrane domain comprising a CD28 transmembrane domain; or alternatively

(x) The binding domain comprising the Tim1 IgV domain and the Tim1 mucin domain; the primary intracellular signaling domain comprising a CD28 signaling domain, the secondary intracellular signaling domain comprising a DAP12 signaling domain, and the transmembrane domain comprising a Tim1 transmembrane domain.

58. The method of claim 57, wherein the chimeric Tim4 receptor comprises:

(i) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the Tim1 signaling domain comprising the amino acid sequence of SEQ ID No. 44, the CD3 zeta signaling structure comprising the amino acid sequence of SEQ ID No. 4, and the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID No. 8;

(ii) Said Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO. 38, said Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO. 39, said Tim4 signaling domain comprising the amino acid sequence of SEQ ID NO. 45, 224 or 225, said CD3 zeta signaling domain comprising the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 27; said Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID NO. 8;

(iii) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the CD28 signaling domain comprising the amino acid sequence of SEQ ID No. 4, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID No. 7;

(iv) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the TRAF6 signaling domain comprising the amino acid sequence of SEQ ID No. 46, and the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID No. 8;

(v) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the TRAF6 signaling domain comprising the amino acid sequence of SEQ ID No. 46, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID No. 7;

(vi) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the TRAF2 signaling domain comprising the amino acid sequence of SEQ ID No. 48, and the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID No. 8;

(vii) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the TRAF2 signaling domain comprising the amino acid sequence of SEQ ID No. 48, and the CD28 transmembrane domain comprising the amino acid sequence of SEQ ID No. 7;

(viii) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the TLR8 signaling domain comprising the amino acid sequence of SEQ ID No. 47, the CD3 zeta signaling domain comprising the amino acid sequence of SEQ ID No. 5 or SEQ ID No. 27, and the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID No. 8;

(ix) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the 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 CD28 transmembrane domain comprising the amino acid sequence of SEQ ID No. 7; or alternatively

(x) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID NO. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID NO. 39, the 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.

59. The method of any one of claims 1 to 52, wherein the chimeric Tim receptor comprises:

(i) Said single chain chimeric protein comprising amino acids 21-456 of SEQ ID NO. 49;

(ii) Said single chain chimeric protein comprising amino acids 21-471 of SEQ ID NO. 50;

(iii) Said single chain chimeric protein comprising amino acids 21-363 of SEQ ID NO. 51;

(iv) Said single chain chimeric protein comprising amino acids 21-590 of SEQ ID NO. 52;

(v) The single chain chimeric protein comprising amino acids 21-596 of SEQ ID NO. 53;

(vi) The single chain chimeric protein comprising amino acids 21-619 of SEQ ID NO. 54;

(vii) Said single chain chimeric protein comprising amino acids 21-625 of SEQ ID NO. 55;

(viii) Said single chain chimeric protein comprising amino acids 21-621 of SEQ ID NO. 56;

(ix) Said single chain chimeric protein comprising amino acids 21-415 of SEQ ID NO. 57; or alternatively

(x) The single chain chimeric protein comprising amino acids 21-409 of SEQ ID NO. 58.

60. The method of claim 59, wherein the chimeric Tim receptor comprises:

(i) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 49;

(ii) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 50;

(iii) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 51;

(iv) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 52;

(v) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 53;

(vi) Said single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 54;

(vii) Said single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 55;

(viii) Said single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 56;

(ix) Said single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 57; or alternatively

(x) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 58.

61. The method of any one of claims 1 to 52, wherein the chimeric Tim receptor comprises:

(iii) The binding domain comprising the Tim4 IgV domain and the Tim4 mucin domain; the primary intracellular signaling domain comprising a Tim4 signaling domain, the secondary intracellular signaling domain comprising a CD3 zeta signaling domain; and the transmembrane domain comprising a Tim4 transmembrane domain; or alternatively

(iv) The binding domain comprising the Tim4 IgV domain and the Tim4 mucin domain; the primary intracellular signaling domain comprising a Tim1 signaling domain, the secondary intracellular signaling domain comprising a CD3 zeta signaling domain; and the transmembrane domain comprising a Tim4 transmembrane domain.

62. The method of claim 61, wherein the chimeric Tim receptor comprises:

(i) The Tim1 IgV domain comprising the amino acid sequence of SEQ ID No. 38, the Tim1 mucin domain comprising the amino acid sequence of SEQ ID No. 39, the Tim1 signaling domain comprising the amino acid sequence of SEQ ID No. 44, the CD3 zeta signaling domain comprising the amino acid sequence of SEQ ID No. 5 or SEQ ID No. 27, and the Tim1 transmembrane domain comprising the amino acid sequence of SEQ ID No. 8;

(iii) Said Tim4 IgV domain comprising the amino acid sequence of SEQ ID NO. 34, said Tim4 mucin domain comprising the amino acid sequence of SEQ ID NO. 35, said Tim4 signaling domain comprising the amino acid sequence of SEQ ID NO. 45, 224 or 225, said CD3 zeta signaling domain comprising the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 27; said Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO. 6; or alternatively

(iv) The Tim4 IgV domain comprising the amino acid sequence of SEQ ID No. 34, the Tim4 mucin domain comprising the amino acid sequence of SEQ ID No. 35, the Tim1 signaling domain comprising the amino acid sequence of SEQ ID No. 44, the CD3 zeta signaling domain comprising the amino acid sequence of SEQ ID No. 5 or SEQ ID No. 27; and the Tim4 transmembrane domain comprising the amino acid sequence of SEQ ID NO. 6.

63. The method of claim 61 or 62, wherein the chimeric Tim receptor comprises:

(iii) Said single chain chimeric protein comprising amino acids 25-490 of SEQ ID NO. 59; or alternatively

(iv) The single chain chimeric protein comprising amino acids 25-495 of SEQ ID NO. 60.

64. The method of any one of claims 61 to 63, wherein the chimeric Tim receptor comprises:

(iii) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 59; or alternatively

(iv) The single chain chimeric protein comprising the amino acid sequence of SEQ ID NO. 60.

65. The method of any one of claims 1 to 52, wherein the chimeric Tim receptor comprises:

(i) The 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) The 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) The 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 alternatively

(iv) Comprising the amino acid sequence of SEQ ID NO. 198 or amino acids 25-428 of SEQ ID NO. 198.

66. The method of any one of claims 1 to 52, wherein the chimeric Tim receptor comprises:

(a) An extracellular domain comprising a binding domain, the binding domain comprising: (i) a Tim4 IgV domain and a Tim4 mucin domain;

(b) An intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain selected from the group consisting of a CD28 signaling domain, a CD3 zeta signaling domain, and a 4-1BB signaling domain, and a secondary intracellular signaling domain selected from the group consisting of a TLR2 signaling domain or a TLR8 signaling domain; and

(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, or 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 to 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.

73. The method of claim 72, wherein the Tim1 transmembrane domain comprises the amino acid sequence set forth in SEQ ID No. 8, the Tim4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID No. 6 or 23, or the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID No. 7.

74. The method of any one of claims 66-73, wherein the CD28 signaling domain comprises the amino acid sequence set forth in SEQ ID No. 4 or SEQ ID No. 26, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID No. 5 or SEQ ID No. 27, or the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID No. 100.

75. The method of any one of claims 66 to 74, wherein the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID No. 222 or the TLR8 signaling domain comprises the amino acid sequence set forth in SEQ ID No. 47.

76. The method of any one of claims 1 to 52, wherein the chimeric Tim receptor comprises:

(b) An intracellular signaling domain, wherein the intracellular signaling domain comprises a primary intracellular signaling domain comprising a signaling domain comprising an immune receptor tyrosine activation motif (ITAM); a secondary intracellular signaling domain comprising a costimulatory signaling domain, a Tim1 signaling domain, or a Tim4 signaling domain; and a tertiary intracellular signaling domain comprising a TLR signaling domain; and

77. The chimeric Tim receptor of claim 76, wherein the ITAM-containing signaling domain is a CD3 zeta signaling domain or a DAP12 signaling domain.

78. The chimeric Tim receptor of claim 76 or 77, wherein the costimulatory signaling domain is a 4-1BB signaling domain or a CD28 signaling domain.

79. The chimeric Tim receptor of any one of claims 76 to 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 set forth in any one of tables 1, 2, or 4-10.

81. The method of any one of claims 1 to 80, wherein the chimeric Tim receptor is administered as a polynucleotide encoding the single chain protein.

82. The method of claim 81, wherein the chimeric Tim receptor is administered as a vector comprising the polynucleotide.

83. The method of claim 82, wherein the chimeric Tim receptor is administered as an engineered cell comprising the chimeric Tim receptor, the polynucleotide, or the vector.

84. The method of claim 83, wherein the cell is an immune cell.

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 cell is a cd4+ T cell, a cd8+ T cell, or a cd4+/cd8+ T cell.

87. The method of any one of claims 83-85, wherein the cell is a human cell.

88. The method of any one of claims 1 to 87, wherein the chimeric Tim receptor is administered as a composition comprising the chimeric Tim receptor, the polynucleotide, the vector, or the engineered cell, and a pharmaceutically acceptable excipient.