Recombinant TIM-4 proteins, chimeric antigen receptor (CAR) T cell delivery systems, and methods of making and using same

Abstract

The present invention provides recombinant TIM-4 fusion proteins comprising a TIM-4 domain and a CD3-specific scFv, as well as methods of making and methods of using the same. The fusion proteins provided herein can be administered in combination with CAR T cells. In addition, CAR T cells engineered to express the TIM-4 fusion protein described herein are also provided, which can be used in the methods described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 329,551, filed on April 11, 2022, the entire contents of which are incorporated herein by reference.

[0003] References to electronic sequence listings

[0004] This application includes a sequence listing in XML format, entitled "155554.00691.xml", 36,976 bytes in size, created on April 10, 2023. The sequence listing is submitted electronically with this application through Patent Center, and its entire contents are incorporated herein by reference. Background Art

[0005] Over the past few decades, immune-based therapies such as immune checkpoint blockade (ICB) and T cell redirection strategies (e.g., chimeric antigen receptor (CAR) T cells) have achieved exciting success in certain cancer subsets. However, success in solid tumors has been limited, a shortcoming that is often attributed to the antigenic heterogeneity of solid tumors. Expanding the repertoire of cancer types that can be effectively treated with immunotherapy remains a major goal of cancer immunology groups worldwide. Multispecific fusion proteins consist of two domains: one domain confers specificity for tumor antigens, while the other domain consists of an anti-CD3 single-chain variable fragment (scFv) that activates T cells through their endogenous T cell receptor. Thus, multispecific fusion proteins redirect T cells to selected tumor antigens independent of T cell receptor specificity. T cell redirection therapy will be successful when all tumor cells express the same targetable antigen. For example, CAR T cells and multispecific fusion proteins have revolutionized the treatment of B cell cancers due to the almost universal expression of CD19 by malignant cells. Unfortunately, T cell redirection therapy performs poorly in solid tumors. These failures are often attributed to the extensive antigenic heterogeneity exhibited by solid tumors.

[0006] Therefore, there is still a need in the art for a new means of identifying and targeting tumor cells. As described in this application, the inventors were able to break conventional thinking and rely on unconventional non-protein targets to circumvent the challenges posed by tumor heterogeneity. Summary of the invention

[0007] TIM-4 fusion proteins and methods for preparing and using the same are described herein, wherein the TIM-4 fusion proteins are composed of a TIM-4 domain connected to a human anti-CD3 scFv. One aspect of the present disclosure provides a TIM-4 fusion protein comprising a TIM-4 domain connected to a CD3-specific single-chain variable fragment (scFv), wherein the TIM-4 domain comprises SEQ ID NO: 3 or a sequence with 95% identity to SEQ ID NO: 3, and wherein the CD3-specific scFv comprises a complementary determining region (CDR) of SEQ ID NO: 22-27 or a sequence with at least 95% identity to SEQ ID NO: 22-27. In some embodiments, the CD3-specific scFv comprises SEQ ID NO: 5 or a sequence with at least 95% identity to SEQ ID NO: 5. Some embodiments provide a pharmaceutical composition comprising a recombinant TIM-4 fusion protein as described herein and a pharmaceutically acceptable excipient, carrier and / or diluent. Some embodiments provide a construct comprising a polynucleotide sequence encoding a TIM-4 fusion protein as described herein. Some embodiments provide a chimeric antigen receptor (CAR)-T cell comprising a construct provided herein.

[0008] Another aspect of the present disclosure provides a method for treating cancer, comprising: administering a therapeutically effective amount of a recombinant TIM-4 fusion protein as described herein and a pharmaceutically acceptable excipient, carrier and / or diluent. In some embodiments, the method further comprises: administering a T lymphocyte having a TIM4 fusion protein, wherein the T lymphocyte is activated when bound to the TIM-4 fusion protein. In some embodiments, the T lymphocyte is a CAR T cell. In yet another embodiment, the CAR T cell can be engineered to express the TIM4 fusion protein and the CAR. In some embodiments, the cancer is a PS-related cancer or an EGFR-related cancer.

[0009] Another aspect of the present disclosure provides a method for diagnosing cancer, comprising: administering the fusion protein described herein; and detecting the presence or accumulation of a tag in a subject suspected of having cancer. The tag allows for in vivo detection of cancer. The TIM4 fusion protein can also be used to detect cancer in a biopsy or other sample from a subject suspected of having cancer in vitro by detecting the binding of TIM4 to cells using the tag.

[0010] Another aspect of the present disclosure provides a method of inducing a T cell response in a subject having cancer, comprising: administering to the subject a therapeutically effective amount of the fusion protein or pharmaceutical composition described herein, wherein an anti-tumor response to the cancer is induced. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 TIM-4 binds to glioma and non-glioma tumor cells. Murine glioma (CT-2A, SMA-560), murine non-glioma (B16F10, Lewis lung carcinoma) and human glioma (U87) cells were stained with APC-conjugated recombinant TIM-4.

[0012] Figure 2 . Irradiation enhances TIM-4 binding to CT-2A glioma cells. A. CT-2A cells cultured in vitro were exposed to 10 Gy of radiation, and TIM-4 binding assays were performed on these cells at 0 h and 24 h after irradiation. B, C. 10 4 CT-2A glioma cells were transplanted into the brain of C57BL / 6 mice. After 14 days, mice received 10Gy whole body irradiation or no irradiation. After 48 hours, tumors were transplanted and stained with TIM-4-APC. Representative overlapping histograms of TIM-4-APC binding to CT-2A are shown in B, and the mean fluorescence intensity (MFI) of TIM-4-APC is shown in C.

[0013] Figure 3 . Schematic diagram and sequence of recombinant TIM-4 fusion protein. A. Model depicting the tumor and T cell binding domains of the recombinant TIM-4 fusion protein. B. Amino acid sequence of the recombinant TIM-4 fusion protein. The fusion protein consists of a signal sequence (SEQ ID NO: 2), an extracellular domain of murine TIM-4 (SEQ ID NO: 7) linked to a scFv derived from the anti-human CD3 antibody OKT3 (SEQ ID NO: 5) (SEQ ID NO: 3), and a tag (SEQ ID NO: 28).

[0014] Figure 4 . Recombinant TIM-4 fusion protein binds to tumor cells and human T cells. A. U87 tumor cells were incubated with recombinant TIM-4 fusion protein expression supernatant or Mock expression supernatant for 30 minutes. Cells were washed thoroughly before incubation with biotinylated protein L. After further extensive washing, cells were incubated with PE-labeled streptavidin and then analyzed by flow cytometry. B, C. Human (B) or mouse (C) CD3 + T cells were incubated with recombinant TIM-4 fusion protein expression supernatant or Mock expression supernatant for 30 min and then incubated with anti-mouse TIM-4-PE.

[0015] Figure 5.Recombinant TIM-4 fusion protein mediates potent tumor cell cytotoxicity in vitro. 100,000 human T cells were co-cultured with 10,000 CellTrace Violet-labeled U87 tumor cells (A) or a 50:50 mixture of 5000 CTV-labeled U87 tumor cells and 5000 CTV-labeled U87vIII tumor cells (B). Individual recombinant TIM-4 fusion proteins or EGFRvIII-anti-CD3 scFv were titrated into the co-cultures. After 24 hours, surviving tumor cells were counted and normalized to the "no fusion protein" control to give tumor survival rates.

[0016] Figure 6 The combination of recombinant TIM-4 fusion protein and EGFRvIIICAR T cells prolonged the survival of mice bearing heterogeneous tumors. 25,000 cells of an 80:20 mixture of U87 and U87vIII tumor cells were intracranially implanted into NOD-SCID-gamma (NSG) mice. Three days later, 2x10 VIIICAR or EGFRvIII secreting TIM-4 fusion protein were intracranially administered. 6 Mice were treated with VIIICAR or not. The survival of mice was followed. *p<0.05.

[0017] Figure 7 .EGFRvIIICAR modified to secrete recombinant TIM-4 fusion protein prolonged the survival of mice bearing EGFRvIII heterogeneous tumors. A 50:50 mixture of CT2A:CT2A vIII was intracranially administered to C57BL / 6 mice. After 7 days, 2x10 6 Important: No lymphodepletion pretreatment was performed before CAR administration. DETAILED DESCRIPTION

[0018] Over the past few decades, immune-based therapies such as immune checkpoint blockade and T cell redirection strategies (e.g., chimeric antigen receptor (CAR) T cells) have achieved exciting success in certain cancer subsets. However, success in solid tumors has been limited, a shortcoming that is often attributed to the antigenic heterogeneity of solid tumors. New means for identifying and targeting tumor cells are needed that break with conventional thinking and rely on unconventional non-protein targets to circumvent the challenges posed by tumor heterogeneity. This article describes a TIM-4 fusion protein, and methods for making and using the same, which is composed of a TIM-4 domain linked to a human anti-CD3 scFv.

[0019] Fusion proteins and constructs:

[0020] One aspect of the present invention provides a recombinant TIM-4 fusion protein comprising a TIM-4 domain optionally connected to a CD3-specific single-chain variable fragment (scFv) via a linker. As used herein, "fusion protein" may also be referred to as a chimeric protein, which is a protein produced by combining two or more genes or portions thereof that originally encode different proteins. The translation of the fusion gene produces one or more polypeptides that have functional properties derived from each original protein. Fusion proteins can be produced in vivo by DNA transfer between chromosomes, or by combining genes or portions of genes to form a single transcript encoding two polypeptides from the same or different organisms in a frame to prepare in a recombinant manner.

[0021] T cell membrane protein 4 or TIM-4 (also known as T cell membrane protein 4 or T cell immunoglobulin and mucin domain-containing protein 4) is a protein encoded by the TIMD4 gene in humans. Tim-4 is a phosphatidylserine (PS) receptor expressed on a variety of immune cells and macrophage subsets. PS is a phospholipid normally found on the side of the plasma membrane facing the cytoplasm, where it is always invisible to the immune system. Cellular stress can lead to dysregulation of the processes that keep PS facing inward, which in turn promotes exposure of PS to the cell surface. Tumor cells often lose the ability to regulate their plasma membrane and present detectable levels of PS on their surface, as normal cells undergoing apoptosis might. When exposed to the cell surface, PS can have a tolerizing effect on the immune system. Although PS exposure can therefore be tumor-adaptive, it also leaves the tumor with an "Achilles' heel" that can be targeted immunologically or otherwise. For example, TIM family proteins bind PS with different affinities, with the strongest binder being TIM-4.

[0022] The recombinant TIM-4 fusion protein disclosed herein comprises a TIM-4 domain comprising SEQ ID NO:3 or a sequence having 95% identity to SEQ ID NO:3 and capable of binding to PS. The recombinant TIM-4 fusion protein can be connected to a CD3-specific scFv via a linker. A peptide linker is a short peptide sequence that can be added to connect protein domains. Linkers are typically composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. The length and content of the linker can vary as needed. In some embodiments, the linker may comprise a combination of glycine and / or serine amino acids. The linker may comprise SEQ ID NO:4 or any number of consecutive repetitions of SEQ ID NO:4 or portions thereof. In the examples, a linker of SEQ ID NO:4 was used to produce Figure 3The TIM4 fusion protein shown in B. Therefore, SEQ ID NO: 4 can be used to connect the TIM4 domain of SEQ ID NO: 3 to the CD3 scFv to produce a TIM4 fusion protein specifically for humans.

[0023] CD3 (differentiation cluster 3) is a protein complex and T cell co-receptor that is involved in activating cytotoxic T cells (CD8+ naive T cells) and helper T cells (CD4+ naive T cells). It consists of four different chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and CD3-ζ (ζ chain) to produce activation signals in T lymphocytes. The recombinant TIM-4 fusion protein disclosed herein can be connected to a CD3-specific single-chain variable fragment (scFv) via a linker. ScFv is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are linked together with a short linker. The CD3-specific scFv may comprise the complementarity determining regions (CDRs) of SEQ ID NO: 22 (VH CDR1), SEQ ID NO: 23 (VH CDR2), SEQ ID NO: 24 (VH CDR3), SEQ ID NO: 25 (VL CDR1), SEQ ID NO: 26 (VL CDR2), and SEQ ID NO: 27 (VL CDR3), or a sequence having at least 95% identity to SEQ ID NO: 22-27. The anti-CD3 scFv of the present disclosure may comprise SEQ ID NO: 5, or a sequence having at least 95% identity to SEQ ID NO: 5. The CD3-specific scFv is capable of binding to and activating T cells through CD3.

[0024] The TIM-4 fusion protein may further comprise an N-terminal signal sequence. The N-terminal signal sequence may comprise a secretion signal sequence. The secretion signal sequence or peptide comprises a short peptide present at the N-terminus of the protein, and these proteins are directed to the secretion pathway. These proteins include proteins that reside in certain organelles, are secreted from the cell, or are inserted into the cell membrane. The signal sequence can be removed or excised from the fusion protein. Therefore, when administered as a protein, the TIM4 fusion protein will generally lack a signal sequence, but a signal sequence may be required to achieve full expression and secretion of the protein. The nucleotide sequence encoding the protein will generally encode a signal sequence. In some embodiments, the secretion signal sequence comprises a signal sequence of SEQ ID NO: 2 or a sequence having at least 90% identity with SEQ ID NO: 2. The length of the signal sequence is generally 15-22 amino acids, and those skilled in the art will recognize that a signal sequence can be removed and replaced with another signal sequence.

[0025] The fusion protein provided herein can additionally include a label. For various purposes, the label can be attached to the protein. The label can include any label known in the art, and can be used for purification, tracking or imaging purposes of the fusion protein. The type of label can include affinity tags, solubilization tags, chromatography tags, epitope tags, fluorescent tags or enzymatic modification tags. In some embodiments, the label can include His tags, FLAG tags, fluorescent tags and Myc tags. In an embodiment, a His tag is used.

[0026] The fusion protein may comprise a TIM4 domain, a linker, and a CD3 scFv. The TIM4 domain and the CD3 scFv may be present in any order relative to each other. For example, the TIM4 domain may be at the N-terminus of the fusion protein, and the CD3 scFv may be at the C-terminus. Alternatively, the CD3 scFv may be at the N-terminus, and the TIM4 domain may be at the C-terminus. For example, the fusion protein may comprise SEQ ID NOs: 3, 4, and 5, or a sequence having at least 95% identity to SEQ ID NOs: 3, 4, and 5, wherein SEQ ID NOs: 3 and 5 are connected by SEQ ID NO: 4 in any order relative to each other. The fusion protein may comprise SEQ ID NOs: 2, 3, 4, and 5, or a sequence having at least 95% identity to SEQ ID NOs: 2, 3, 4, and 5. The fusion protein may comprise SEQ ID NO: 1. The fusion protein may comprise a sequence at least 95% identical to SEQ ID NOs: 3, 4 and 5 and a tag, or a sequence at least 95% identical to SEQ ID NOs: 2, 3, 4 and 5 and a tag, or a sequence at least 95% identical to SEQ ID NO: 1 and a tag.

[0027] In alternative embodiments, the recombinant TIM-4 fusion protein may include a PS-specific antibody portion or scFv. In some embodiments, the antibody portion specifically binds to PS present on the cell surface. When PS is present on the cell surface, TIM4 or other regions capable of binding to PS will specifically bind to PS. The TIM4 domain or region capable of binding to PS will not significantly bind to normal healthy cells that usually do not express PS on their surface. When the cell is a cancer cell in which PS is not properly regulated but is present on the extracellular side of the cell membrane, the TIM4 region of the fusion protein can bind to the cell. Cancer cells with dysregulated PS localization may be present in solid tumors, such as metastatic cancer cells.

[0028] The TIM-4 fusion protein may include a CD3-specific scFv or another CD3-specific antigen-binding fragment. For example, an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv) and single-chain antibody molecules (scFv). In some embodiments, the recombinant TIM-4 fusion protein includes a CD3-specific scFv. In some embodiments, the CD3-specific scFv includes SEQ ID NO:22 (VH CDR1), SEQ ID NO:23 (VH CDR2), SEQ ID NO:24 (VH CDR3), SEQ ID NO:25 (VL CDR1), SEQ ID NO:26 (VL CDR2) and SEQ ID NO:27 (VL CDR3) complementary determining regions (CDRs). In some embodiments, the CD3-specific scFV includes SEQ ID NO:5. The recombinant TIM-4 fusion protein may be a human, humanized or semi-synthetic recombinant TIM-4 fusion protein.

[0029] The present disclosure provides pharmaceutical compositions comprising one or more compositions or fusion proteins as described herein and suitable carriers, excipients or diluents. The exact nature of the carrier, excipient or diluent will depend on the intended application of the composition, and may be suitable for or acceptable for veterinary applications, suitable for or acceptable for human applications. The composition may optionally comprise one or more additional compounds.

[0030] When used to treat or prevent diseases (such as cancer) or disease symptoms, the compositions described herein can be administered alone, as a mixture of one or more compounds, or mixed or combined with other agents (e.g., therapeutic agents) useful for treating these diseases and / or symptoms associated with these diseases. The compounds can be administered in the form of the compound (i.e., protein) itself or in the form of a pharmaceutical composition comprising the compound.

[0031] The pharmaceutical composition comprising the composition can be prepared by conventional mixing, dissolving, granulating, preparing dragees, grinding, emulsifying, preparing capsules, embedding or freeze-drying processes. The composition can be prepared in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that help process the composition into a pharmaceutically acceptable formulation. The pharmaceutical composition can take the form suitable for almost any mode of administration (e.g., including topical administration, eye administration, oral administration, oral buccal administration, systemic administration, nasal administration, injection, transdermal administration, rectal administration, vaginal administration, etc.) or the form suitable for inhalation or insufflation administration.

[0032] The compositions described herein will generally be used in an amount effective to achieve the desired result (e.g., in an amount effective to treat or prevent cancer). Therapeutic benefit refers to the eradication or improvement of the underlying disease being treated and / or the eradication or improvement of one or more symptoms associated with the underlying disease, so that the patient reports feeling or condition improved, although the patient may still suffer from the underlying disease. Therapeutic benefit also generally includes interrupting or slowing the development of the disease, regardless of whether improvement is achieved. Cancer treatment includes, but is not limited to, reducing the number of cancer cells or the size of tumors in the subject, slowing the development of cancer to a more aggressive form, reducing the proliferation of cancer cells or reducing the rate of tumor growth, killing cancer cells, reducing the metastasis of cancer cells, or reducing the possibility of cancer recurrence in the subject. As used herein, treatment of a subject refers to any type of treatment that brings benefits to a subject suffering from a disease or at risk of disease, including improving the condition of the subject (e.g., one or more symptoms), delaying the development of the disease, delaying the onset of symptoms, or slowing the development of symptoms, etc.

[0033] The amount of the composition administered will depend on a variety of factors including, for example, the specific indication being treated, the mode of administration, whether the desired benefit is preventive or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular composition, the conversion rate and delivery efficiency under the selected route of administration, etc.

[0034] Those skilled in the art are well capable of determining effective doses for specific applications and modes of administration. Effective doses can be preliminarily estimated based on in vitro activity and metabolism assays. For example, an initial dose for an animal can be formulated so that the circulating blood or serum concentration of the composition reaches or exceeds the IC value of the specific composition measured in an in vitro assay. 50 The skilled artisan is well-equipped to calculate the dose that achieves such circulating blood or serum concentrations, taking into account the bioavailability of a particular composition by the desired route of administration. The initial dose may also be estimated based on in vivo data, such as animal models. Animal models for testing the efficacy of active metabolites in treating or preventing the various diseases described above are well known in the art. Animal models suitable for testing the bioavailability and / or metabolism of a composition are also well known. A person of ordinary skill can routinely modify this information to determine a dose suitable for administration to humans.

[0035] The dosage is usually in the range of about 0.0001 mg / kg / day, 0.001 mg / kg / day or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower, depending on the activity of the active composition, the bioavailability of the composition, its metabolic kinetics and other pharmacokinetic properties, the mode of administration and various other factors discussed above, etc. The dosage and dosing interval can be adjusted individually to provide a plasma level sufficient to maintain a therapeutic or preventive effect. For example, the composition can be administered once a week, several times a week (e.g., every other day), once a day, or multiple times a day, depending on the mode of administration, the specific indication being treated, and the judgment of the prescribing physician, etc. In the case of local administration or selective uptake (such as topical administration), the effective local concentration of the composition may be independent of the plasma concentration. The technician can optimize the effective dose without excessive experiments.

[0036] The dosage of the recombinant TIM-4 fusion protein administered to an individual (such as a human) may vary with the specific composition, mode of administration, and type of disease being treated. In some embodiments, the amount of the composition effectively produces an objective response (such as a partial response or a complete response). In some embodiments, the amount of the recombinant TIM-4 fusion protein in the composition is included in the range of, for example, about 0.001 pg to about 1000 pg. In some embodiments of any of the above aspects, the effective amount of the recombinant TIM-4 fusion protein in the composition is in the range of about 0.1 pg / kg total body weight to about 100 mg / kg total body weight.

[0037] Recombinant TIM-4 fusion proteins can be administered to individuals (such as humans) by a variety of routes, including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhaled, intracapsular, intramuscular, intratracheal, subcutaneous, intradermal, intraocular, intrathecal, transmucosal and transdermal routes. In some embodiments, sustained release formulations of the composition can be used.

[0038] In some embodiments, a construct is provided, which comprises a polynucleotide sequence encoding any recombinant TIM-4 fusion protein described herein. The term "construct" or "polynucleotide construct" is a polynucleotide (DNA or RNA) that allows the coding sequence to be replicated and / or expressed in a target cell. The construct may contain an exogenous promoter that is operably linked to any polynucleotide described herein. As used herein, when a polynucleotide is in a functional relationship with a second polynucleotide sequence, the polynucleotide is "operably linked" or "operably linked". The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that causes the latter to be expressed. For example, when a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA sequences are continuous and are located in the same reading frame when two protein coding regions need to be combined.

[0039] As used herein, the terms "heterologous promoter", "promoter", "promoter region" or "promoter sequence" generally refer to the transcriptional regulatory region of a gene, which can be present on the 5' or 3' side of the polynucleotides described herein or within the coding region of the polynucleotides. Generally, a promoter is a DNA regulatory region that is capable of binding to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A typical 5' promoter sequence is bounded at its 3' end by a transcription start site and extends upstream (5' direction), thereby containing the minimum number of bases or elements required to initiate transcription above a detectable level of background. Within the promoter sequence is a transcription start site (which can be conveniently defined by mapping with nuclease S1) and a protein binding domain (consensus sequence) responsible for binding RNA polymerase.

[0040] In some embodiments, the construct is in an expression construct, a vector or a viral vector. A vector is any particle used as a medium for artificially carrying an external nucleic acid sequence (usually DNA) into another cell, in which it can be replicated and / or expressed. A vector containing external DNA is called recombinant DNA. Four main vector types are plasmids, viral vectors, cosmids and artificial chromosomes. An expression construct comprises a heterologous promoter and a nucleic acid sequence encoding a target protein (e.g., recombinant TIM-4, CD3 scFv), which can be expressed in an introduced cell. An expression construct includes a vector capable of directing the expression of an exogenous gene operably connected thereto. Such vectors are referred to herein as "recombinant constructs", "expression constructs" and "recombinant expression vectors" (or simply "expression vectors" or "vectors"), and can be used interchangeably. Suitable vectors are known in the art and contain necessary elements so that the genes encoded in the vector are expressed as proteins in host cells. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments (especially exogenous DNA segments encoding mutant α-gal proteins) can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. The viral vector is incorporated into viral particles, and then these viral particles are used to transport the viral polynucleotide encoding the target protein into the target cell. Some vectors can replicate autonomously in the host cell into which they are introduced. Other vectors (e.g., lentiviral vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of exogenous genes operably connected thereto. Generally, vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, "vector" includes expression vectors, such as viral vectors (e.g., replication-defective retroviruses (including lentiviruses), adenoviruses, and adeno-associated viruses (AAV)), which have equivalent functions.

[0041] A vector is a heterogeneous exogenous construct containing sequences from two or more different sources. Suitable vectors include, but are not limited to, plasmids, expression vectors, slow viruses (slow virus vectors), adeno-associated virus vectors (rAAV), etc., and include constructs capable of expressing target proteins in cells. Preferred vectors are slow virus vectors or adeno-associated vectors. Suitable viral particle preparation methods are known in the art to be able to transform cells so as to express target proteins in cells. These cells may be T cells, and may be autologous T cells or CAR T cells, which will be described more fully below. CAR T cells may be CAR T cells capable of targeting cancer, such as EGFR vIII CAR T cells that may include SEQ ID NO: 10 or D2C7 CAR T cells that may include SEQ ID NO: 17. Those skilled in the art may determine other CAR T cells that may be used in combination with the TIM4 fusion protein and the construct encoding the TIM4 fusion protein provided herein.

[0042] Heterologous promoters that can be used to practice the present invention include, but are not limited to, constitutive promoters, inducible promoters, temporally regulated promoters, developmentally regulated promoters, chemically regulated promoters, tissue-preferred promoters, tissue-specific promoters, and cell-type-specific promoters. The heterologous promoter can be a plant promoter, an animal promoter, a bacterial promoter, a fungal promoter, or a synthetic promoter. Suitable promoters are known and described in the art. In mammalian cells, typical promoters include, but are not limited to, promoters of Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, and the translation elongation factor EF-1α promoter or the ubiquitin promoter.

[0043] In some embodiments, the construct comprising a polynucleotide sequence encoding any recombinant TIM-4 fusion protein described herein may further include a sequence encoding a chimeric antigen receptor (CAR). CAR and TIM4 fusion proteins can be expressed using different promoters, or can be expressed using a single promoter as described below, and can be in a single construct or expression vector or in separate constructs or expression vectors, to transfect or be introduced into T cells. The term "chimeric antigen receptor" or "chimeric receptor" or "CAR" or "CARs" used herein refers to such a polypeptide: it has a predetermined binding specificity to a desired target, and is operably connected (for example, as a fusion or as a separate chain connected by one or more disulfide bonds, etc.) to the intracellular portion of the T cell activation domain. More specifically, CAR is an engineered receptor that transplants antigen specificity to cytotoxic cells (for example, T cells, NK cells or macrophages) when expressed. For example, CAR protein is engineered to give T cells a new ability to target specific proteins. The CAR of the present invention may include an extracellular domain with at least one antigen-specific targeting region, a transmembrane domain (TM), and a combination of an intracellular domain (ID) comprising one or more costimulatory domains (CSD), which is not naturally present together on a single protein. This particularly includes receptors in which the extracellular domain and the cytoplasmic domain are not naturally present together on a single receptor protein. In addition, chimeric receptors are different from TCRs expressed in natural T cell lymphocytes.

[0044] The term "CAR-T cell" as used herein refers to a T cell or a population thereof that has been modified by molecular biology methods to express a chimeric antigen receptor (CAR) on the surface of the T cell. CAR is a polypeptide that has a predetermined binding specificity for the desired target being expressed and is operably linked (e.g., as a fusion or as a separate chain linked by one or more disulfide bonds, etc.) to the intracellular portion of the T cell activation domain. By bypassing MHC class I and class II restrictions, CD8 + and CD4 +The CAR engineered T cells of both subgroups can be recruited for redirected target cell recognition. The most common CAR is a fusion of immunoglobulin binding functionality (e.g., as a single-chain variable fragment (scFv) derived from a monoclonal antibody) and CD3-ζ (CD3ζ) transmembrane and intracellular domains. Such molecules will cause the transmission of ζ signals in response to target recognition by immunoglobulin binding functionality. However, there are many alternatives. For example, antigen recognition domains from natural T cell receptor (TCR) α and β single chains can be used as binding functionality. Alternatively, receptor extracellular domains (e.g., CD4 extracellular domains) or cytokines (which can lead to recognition of cells with homologous cytokine receptors) can be used. All requirements for binding functionality are that it binds to a given target in a specific manner and with high affinity.

[0045] The membrane-spanning domain can be derived from a natural source or a synthetic source. If the source is natural, the domain can be derived from any membrane-bound protein or membrane-spanning protein. The membrane-spanning region used in the present invention can be derived from a, b, d or g chains of a T cell receptor, CD28, CD3ε, CD3z, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 (i.e., at least comprising one or more membrane-spanning regions thereof). In some embodiments, the membrane-spanning domain can be synthetic, in which case the membrane-spanning domain can mainly comprise hydrophobic residues, such as leucine and valine. In some embodiments, the triplets of phenylalanine, tryptophan and valine can be present at each end of the synthetic membrane-spanning domain. In some embodiments, a short oligopeptide or polypeptide linker of, for example, about 2 to about 10 (such as about 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids in length can form a connection between the transmembrane domain of the CAR provided herein and the intracellular signaling domain. In some embodiments, the linker is a glycine-serine doublet.

[0046] The intracellular signaling domain of the CAR-T cells provided herein is responsible for activating at least one normal effector function of an immune cell (in which the CAR-T cells provided herein have been placed). For example, the effector function of a T cell may be a cytolytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and instructs cells to perform specialized functions. Although the entire intracellular signaling domain can generally be used, it is not necessary to use the entire chain in many cases. In terms of using a truncated portion of an intracellular signaling domain, this truncated portion can be used to replace the complete chain as long as it can transduce an effector function signal. Therefore, the term "intracellular signaling sequence" means any truncated portion that contains an intracellular signaling domain sufficient to transduce an effector function signal.

[0047] Examples of intracellular signaling domains for use in CAR-T cells according to the present disclosure include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0048] The present disclosure may include a CAR-T cell comprising any recombinant TIM-4 fusion protein described herein and a construct of a CAR with any specificity. Therefore, the TIM-4 recombinant fusion protein can be used in combination with any CAR that is desired to target PS. Any recombinant TIM-4 fusion protein described herein can be combined with CAR in one or more constructs. When the recombinant TIM-4 fusion protein is contained in a single construct with CAR, the recombinant TIM-4 fusion protein may include a self-cleavage site, such as a 2A self-cleavage peptide. The CAR bound to the TIM-4 fusion protein may include an extracellular domain that includes an antigen binding region that binds to one or both of the wild-type EGFR and EGFR VIII variants. The extracellular antigen binding region may include a scFv of SEQ ID NO: 10 or a sequence having at least 95% identity to SEQ ID NO: 10 or a scFv of SEQ ID NO: 17 or a sequence having at least 95% identity to SEQ ID NO: 17. In some embodiments, the CAR and TIM-4 fusion protein are connected by a self-cleavage site. In some embodiments, the self-cleaving sequence may comprise a 2A self-cleaving peptide. In one embodiment, the 2A self-cleaving peptide is SEQ ID NO:11 or a sequence having at least 95% identity to SEQ ID NO:11.

[0049] The present disclosure may include a CAR-T cell comprising any recombinant TIM-4 fusion protein described herein and any CAR construct described herein or elsewhere. The CAR-T may include a construct comprising a polynucleotide encoding an EGFR VIII CAR of SEQ ID NO: 10, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 22-27, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity with SEQ ID NO: 10, 3, 22, 24, 25, 26, and 27. The CAR-T may include a construct comprising a polynucleotide encoding an EGFR VIII CAR of SEQ ID NO: 10, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 5, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity with SEQ ID NO: 10, 3, and 5. In some embodiments, the CAR-T may comprise a construct comprising a polynucleotide encoding a D2C7 CAR of SEQ ID NO: 17, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 22-27, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO: 17, 3, 22, 23, 24, 25, 26, and 27. In some embodiments, the CAR-T may comprise a construct comprising a polynucleotide encoding a D2C7 CAR of SEQ ID NO: 17, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 5, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO: 17, 3, and 5. The constructs may comprise a self-cleavage site comprising SEQ ID NO: 11 or a sequence having at least 95% sequence identity to SEQ ID NO: 11. The constructs may comprise a signal sequence comprising SEQ ID NO: 2 or a sequence having at least 95% sequence identity to SEQ ID NO: 2. The constructs may comprise a linker of SEQ ID NO:4.

[0050] The present disclosure provides a CAR-T cell, comprising a construct encoding a CAR of SEQ ID NO: 10, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NO: 22-27. The present disclosure provides a CAR-T cell, comprising a construct encoding a CAR of SEQ ID NO: 10, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NO: 5. The present disclosure provides a CAR-T cell, comprising a construct encoding a CAR of SEQ ID NO: 17, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NO: 22-27. The present disclosure provides a CAR-T cell comprising a construct encoding a CAR of SEQ ID NO: 17, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NO: 5. A CAR-T cell is provided, comprising a construct encoding SEQ ID NO: 20 or a sequence having at least 95% sequence identity with SEQ ID NO: 20. A CAR-T cell is also provided, comprising a construct encoding SEQ ID NO: 21 or a sequence having at least 95% sequence identity with SEQ ID NO: 21.

[0051] In order to assess the expression of polypeptide or its part, the expression vector to be introduced into the cell can also contain selectable marker gene or reporter gene or both, so as to identify and select expressive cells from the cell colony attempted to be transfected or infected by viral vector. In other aspects, selectable marker can be carried on a separate DNA and used for cotransfection procedure. Both the side of selectable marker and reporter gene can have suitable regulatory sequences to realize expression in host cells. Available selectable markers for example include antibiotic resistance genes such as neo.

[0052] Reporter gene is used to identify the function of potential transfected cells and evaluation regulatory sequences. Generally, reporter gene is a gene that is not present in the receiving organism or tissue or is not expressed by the receiving organism or tissue, and the expression of its encoded polypeptide can be confirmed by some easily detectable characteristics (for example, enzyme activity). After DNA is introduced into the receiving cell, the expression of reporter gene is measured at the appropriate time. These reporter genes can also be used as labels and can be encoded as a part of TIM4 fusion protein, and can also be used as a means to ensure that TIM4 protein is expressed. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase and secretory alkaline phosphatase or green fluorescent protein genes (for example, Ui-Tel, FEBS Letters, 479, 2000). Several reporter genes in these reporter genes have split reporter gene function (split reporter function), wherein a part of the reporter gene can be provided by trans (intrans), to significantly reduce the size of the label, so as to achieve the labeling of the fusion protein. Such split reporter genes can be purchased from the market, and are well known to those skilled in the art. Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, constructs with minimal 5' flanking regions that show the highest levels of reporter gene expression are identified as promoters. These promoter regions can be linked to reporter genes and can be used to assess the ability of agents to regulate promoter-driven transcription.

[0053] Methods for introducing genes into cells and expressing genes are known in the art. In the case of expression vectors, the vectors can be easily introduced into host cells (e.g., mammalian cells, bacterial cells, yeast cells, or insect cells) by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0054] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, gene gun, microinjection, electroporation, etc. Methods for making cells containing vectors and / or exogenous nucleic acids are well known in the art. For example, see Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0055] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors (especially retroviral vectors) have become the most widely used methods for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from slow viruses, poxviruses, herpes simplex virus 1, adenoviruses, and adeno-associated viruses, etc. For example, see U.S. Patents Nos. 5,350,674 and 5,585,362.

[0056] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0057] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is envisioned that (in vitro, in vitro or in vivo) a lipid formulation is used to introduce nucleic acid into a host cell. In another aspect, nucleic acid can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome by a linker molecule that is associated with both the liposome and the oligonucleotide, embedded in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles or compounded with micelles or otherwise associated with lipids. Lipid, lipid / DNA or lipid / expression vector association compositions are not limited to any specific structure in the solution. For example, they can be present in a double-layer structure, as micelles or in a "collapsed (collapsed)" structure. They can also be simply dispersed in the solution, thereby possibly forming aggregates of inconsistent size or shape. Lipids are fatty substances, and they can be naturally occurring lipids or synthetic lipids. For example, lipids include naturally occurring fat droplets in the cytoplasm as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0058] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to the inhibitors of the invention, a variety of assays can be performed to confirm the presence of recombinant DNA sequences in host cells. For example, such assays include "molecular biology" assays well known to those skilled in the art (such as Southern and Northern blotting, RT-PCR and PCR); "biochemical" assays (such as, for example, by immunological means (ELISA and Western blotting) or by assays to detect the presence of specific peptides).

[0059] In some embodiments, sequence variants provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody portion. Amino acid sequence variants of fusion proteins or constructs can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the fusion protein or by peptide synthesis. For example, such modifications include deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody portion. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, as long as the final construct has the desired properties (e.g., antigen binding). The compositions and sequences provided herein (including recombinant TIM-4 fusion proteins) can also include species-specific variations. For example, SEQ ID NO: 1, 2, 3, 4, 5, 10 and 17 are human sequences, while SEQ ID NO: 7, 8, 14 and 17 are mouse sequences. Species-specific sequences and variants can be used according to the desired subject species and results.

[0060] method:

[0061] A second aspect of the present invention provides a method for treating cancer, comprising: administering a therapeutically effective amount of any recombinant TIM-4 fusion protein described herein and a pharmaceutically acceptable excipient, carrier and / or diluent.

[0062] Treating cancer in a subject includes reducing, inhibiting, slowing or preventing cancer growth, reducing tumor volume and / or preventing, inhibiting, slowing or reducing tumor metastasis. Treating cancer in a subject also includes reducing the number of tumor cells in the subject. The term "treatment" can be characterized by at least one of the following: (a) reducing, slowing or inhibiting the growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing the metastasis of cancer cells in the subject; and (d) reducing or improving at least one symptom of cancer. In some embodiments, a person skilled in the art can easily determine the optimal effective amount using routine experiments. The purpose of treatment includes alleviating or preventing symptoms, slowing or preventing the development or deterioration of a disease, disorder or condition, and / or alleviating a disease, disorder or condition. The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result.

[0063] As used herein, the term "administering" an agent (such as a therapeutic entity of a composition described herein) to an animal or cell means distributing, delivering or administering a substance to an intended target. With respect to a therapeutic composition, the term "administering" means contacting a therapeutic agent with a subject or distributing, delivering or administering the therapeutic agent to a subject by any suitable route to deliver the therapeutic agent to a desired location in an animal, including delivery by parenteral or oral routes, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by intranasal or respiratory routes.

[0064] The methods disclosed herein can be used to treat any cancer and any metastasis thereof, including but not limited to carcinoma, lymphoma, blastoma, sarcoma and leukemia. In some embodiments, the cancer is characterized by or associated with phosphatidylserine (PS) expression (referred to herein as "PS-related cancer"). PS is a phospholipid that is typically present on the inner leaflet of normal cells. However, apoptotic cells and non-apoptotic cancer cells (such as malignant melanoma, leukemia, neuroblastoma and gastric cancer) have been shown to express PS extensively on their surfaces. PS exposed on the surface of tumor cells helps to inhibit T cell activity and block tumor clearance. In some embodiments, cancer includes cancer having PS on the cell surface. In some embodiments, cancer includes EGFR-related cancer, i.e., cancer associated with EGFR expression. Suitable examples of such cancers include, but are not limited to, breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, cervical cancer, endometrial cancer, salivary gland cancer, mesothelioma, kidney cancer, vulvar cancer, pancreatic cancer, thyroid cancer, liver cancer, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma, and peripheral neuroepithelioma. In some embodiments, the cancer comprises glioblastoma.

[0065] In some embodiments, the method further includes administering effector cells (especially T lymphocytes), wherein T lymphocytes are activated when combined with TIM-4 fusion proteins. T lymphocytes can be autologous or allogeneic T lymphocytes, or differentiated T cells from stem cells or pluripotent cells. T lymphocytes may be non-specific to targets or antigens. Exemplary effector cells that can be used for the present disclosure include but are not limited to dendritic cells (including immature dendritic cells and mature dendritic cells), T lymphocytes (such as initial T cells, effector T cells, memory T cells, cytotoxic T lymphocytes, T helper cells, natural killer T cells, regulatory T cells, tumor infiltrating lymphocytes (TIL) and lymphokine-activated killer (LAK) cells), B cells, natural killer (NK) cells, monocytes, macrophages, neutrophils, granulocytes and combinations thereof. Effector cell subsets can be defined by the presence or absence of one or more cell surface markers known in the art (e.g., CD3, CD4, CD8, CD19, CD20, CD11c, CD123, CD56, CD34, CD14, CD33, etc.).

[0066] T cell activation refers to the process in which mature T cells can express antigen-specific T cell receptors on their surfaces to recognize their cognate antigens and react by entering the cell cycle, clonal expansion and differentiation, secreting cytokines or lytic enzymes, and starting the immune system based on the function of cells. The release of cytokines is the result of T cell activation and efficacy. Preferably, at least a portion of activated T cells produces one or more cytokines, such as cytokines selected from IL-1, IL-1, IL-2, IL-4, IFN-, IL-10, IL-12, TNF- and GM-CSF. In addition, at least a portion of activated T cells preferably expresses one or more surface markers selected from CD2, CD28, CTLA4, CD40 ligands (gp39), CD18, CD25, CD69, CD16 / CD56, MHC class I, MHC class II, CD8, CD4, CD3 / TcR, CD54, LFA-1 and VLA-4. T cells can be activated in any manner known in the art before administration. T lymphocytes can also become activated or specifically activated when bound to TIM-4 fusion proteins. T lymphocytes can bind to the scFv anti-CD3 domain of TIM-4 fusion proteins. T lymphocytes can be CAR T cells. CAR T cells can include EGFR VIIICAR of SEQ ID NO: 10 or D2C7 CAR of SEQ ID NO: 17 or a sequence having at least 95% identity with SEQ ID NO: 10 or SEQ ID NO: 17.

[0067] In some embodiments, the recombinant TIM-4 fusion protein can be administered separately from T lymphocytes or CAR-T cells. In some embodiments, the recombinant TIM-4 fusion protein can be administered together with T lymphocytes and / or include CAR-T cells when administered. The recombinant TIM-4 fusion protein of the present disclosure can be administered before, simultaneously with, or after administering T lymphocytes, CAR-T cells, or any other cancer therapy (including other immunotherapies, chemotherapy, radiotherapy, surgery, or any other standard treatment cancer therapy). The recombinant TIM-4 fusion protein of the present disclosure can be administered together with CAR-T cells having any specificity.

[0068] Some embodiments of the present disclosure provide a method for treating cancer, comprising: administering a therapeutically effective amount of any CAR-T cell described herein and a pharmaceutically acceptable excipient, carrier and / or diluent. In some embodiments, the cancer is a PS-related cancer or an EGFR-related cancer, including those described herein. Some embodiments of the present disclosure provide a method for treating cancer, comprising: administering a cell comprising any construct described herein. In some embodiments, the cancer is a PS-related cancer or an EGFR-related cancer, including those described herein.

[0069] Another aspect of the present disclosure provides a method for diagnosing cancer, comprising any TIM-4 fusion protein provided herein, detecting the presence or accumulation of a label in a subject suspected of having cancer, wherein the label allows for in vivo or in vitro detection of cancer by contacting cells from the subject. Medical diagnosis is the process of determining which disease or condition can explain a person's symptoms and signs. Cancer can be a PS-related cancer or an EGFR-related cancer, including those described herein.

[0070] In some embodiments, the present disclosure demonstrates a method of inducing a T cell response in a subject having cancer, comprising: administering to the subject a therapeutically effective amount of any fusion protein or pharmaceutical composition described herein, wherein an anti-tumor response to the cancer is induced.

[0071] Another embodiment of the present disclosure provides a method of inducing a T cell response in a subject having cancer, comprising: administering to the subject a therapeutically effective amount of any CAR-T cell described herein, wherein an anti-tumor response to the cancer is induced.

[0072] Additional Definitions

[0073] Unless the context requires or indicates otherwise, a noun without a quantifier means "one or more." For example, "molecule" should be understood to mean "one or more molecules."

[0074] As used herein, "about", "approximately", "substantially" and "significantly" should be understood by one of ordinary skill in the art and should vary to some extent depending on the context in which they are used. If the use of a term is unclear to one of ordinary skill in the art in the context in which it is used, "about" and "approximately" will mean plus or minus ≤10% of the particular term, and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0075] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising". The terms "comprise" and "comprising" should be understood as "open" transition terms, which allow the inclusion of additional components in addition to those components listed in the claims. The terms "consist" and "consisting of" should be understood as "closed" transition terms, which do not allow the inclusion of additional components in addition to those components listed in the claims. The term "consisting essentially of" should be understood as being partially closed and only allowing the inclusion of additional components that do not fundamentally change the nature of the claimed subject matter.

[0076] As used herein, the term "specifically" or "selectively" binds when referring to a ligand / receptor, nucleic acid / complementary nucleic acid, antibody / antigen or other binding pair (e.g., cytokine to cytokine receptor) indicating a binding reaction that determines the presence of the protein in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions, the specified ligand binds to a specific receptor and does not bind to other proteins present in the sample in a significant amount. For example, specific binding can also mean that the binding affinity of the binding compound, nucleic acid ligand, antibody or binding composition derived from the antigen binding site of an antibody in the contemplated method to its target is generally at least 25% greater (more generally at least 50% greater, most generally at least 100% greater (2-fold), generally at least 10-fold greater, more generally at least 20-fold greater, and most generally at least 100-fold greater) than the affinity to any other binding compound.

[0077] "Percentage of sequence identity" or "percentage of similarity" is determined by comparing two optimally aligned sequences within a comparison window, wherein a portion of the polynucleotide or peptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (not containing additions or deletions) to achieve optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to obtain the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window; and multiplying the result by 100 to obtain the percentage of sequence identity.

[0078] The term "substantial identity" or "substantial similarity" of a polynucleotide or peptide sequence refers to a polynucleotide or peptide comprising a sequence having at least 75% sequence identity. Alternatively, the percent identity can be any integer between 75% and 100%. More preferred embodiments include at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein (preferably BLAST using the standard parameters described). These values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0079] Unless otherwise specified herein or there is a clear contradiction with the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is only to better illustrate the present invention, rather than to limit the scope of the present invention. Any language in the specification should not be understood to indicate that any unclaimed element is necessary for the practice of the present invention. The method for treating the human body can be modified to a second medical use or other related claim format, and includes the use of the composition provided herein in the preparation of a drug for treating cancer or diagnosing cancer.

[0080] The present disclosure is not limited to the specific details of the construction, component arrangement or method steps set forth herein. The compositions and methods disclosed herein can be prepared, practiced, used, implemented and / or formed in various ways (based on the following disclosure, these ways will be obvious to those skilled in the art). The wording and terminology used herein are only for descriptive purposes and should not be considered as limiting the scope of the claims. The order indicators (such as the first, second and third) used to indicate various structures or method steps in the specification and claims are not meant to be interpreted as indicating any specific structure or step or any specific order or configuration of these structures or steps. Unless otherwise specified herein or there is a clear contradiction with the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (such as "such as") provided herein is only for the convenience of disclosure and does not mean any limitation to the scope of the present disclosure. Any language in the specification and any structure shown in the drawings should not be interpreted as indicating that any unclaimed element is necessary for implementing the disclosed subject matter. The use of "including," "comprising," or "having," and variations thereof herein, is intended to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including," "comprising," or "having" certain elements are also contemplated as "consisting essentially of" and "consisting of" those certain elements.

[0081] Unless otherwise specified herein, the description of the numerical range herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually stated herein. For example, if the concentration range is stated as 1% to 50%, it means that values ​​such as 2% to 40%, 10% to 30% or 1% to 3% are explicitly listed in this specification. These are merely examples of specific expected values, and all possible numerical combinations between and including the lowest and highest values ​​listed are considered to be explicitly stated in this disclosure. The use of the word "about" to describe the specific amount or range of amounts recorded is intended to indicate that values ​​very close to the recorded amount (such as values ​​that may or naturally be considered due to manufacturing tolerances, instrument errors and human errors generated when forming measurement results, etc.) are included in the amount. Unless otherwise specified, all percentages involving amounts are weight percentages.

[0082] It is not acknowledged that any reference (including any non-patent or patent document cited in this specification) constitutes prior art. In particular, it should be understood that, unless otherwise stated, the reference to any document herein does not constitute an admission that any of these documents constitute part of the common general knowledge in the art in the United States or any other country. Any discussion of the references states the claims of their authors, and the applicant reserves the right to question the accuracy and relevance of any document cited herein. Unless otherwise expressly stated, all references cited herein are fully incorporated herein by reference. If there is any difference between any definition and / or description appearing in the cited references, the present disclosure shall prevail.

[0083] The following examples are merely illustrative and are not meant to limit the scope of the invention or the appended claims.

[0084] Example

[0085] In the following examples, the inventors describe recombinant TIM-4 fusion proteins and methods of use thereof.

[0086] To validate the strategy of targeting PS with TIM-4, we initially sought to determine the extent to which TIM-4 binds to a variety of murine and human tumor cell lines. For these studies, recombinant TIM-4 with a fluorescent label of allophycocyanin (APC) was used to detect TIM-4 binding by flow cytometry. TIM-4 bound to virtually all tumor cell lines tested ( Figure 1). These tumor cell lines included mouse CT-2A and SMA-560 gliomas, mouse B16F10 melanoma, mouse Lewis lung carcinoma (LLC), and human U87 glioma. Notably, TIM-4 binding overlapped with Annexin V, a known PS binder, indicating that TIM-4 is indeed binding to PS on tumor cells (data not shown). These data demonstrate that TIM-4 is a viable targeting moiety for multiple cancer types.

[0087] One risk associated with any targeted therapy is toxicity. PS exposure is not limited to tumor cells, but can also be present in platelets, activated T cells, and apoptotic cells. Therefore, recombinant TIM-4 may potentially induce off-tumor on-target toxicity. To limit these toxicities, radiation is used to increase PS exposure on the surface of tumor cells. This increases the target load, ideally allowing the recombinant TIM-4 fusion protein to achieve more tumor-specific killing. To test this, CT-2A tumor cells were first irradiated in vitro at a dose of 10 Gy and then seeded into flasks. After 24 hours, the cells were removed from the culture and stained with APC-conjugated TIM-4. The mean fluorescence intensity (MFI) of TIM-4 binding in irradiated tumor cells was significantly higher than that in unirradiated tumor cells, indicating that PS exposure after irradiation was enhanced ( Figure 2 A). Next, we sought to determine whether PS exposure and TIM-4 binding were increased after tumor irradiation in vivo. To test this, CT-2A were transplanted into the cranium of C57BL / 6 mice. Fourteen days later, the mice received either 10 Gy whole-body irradiation or no irradiation. After 48 hours, the tumors were harvested and isolated, and these tumors were stained with APC-conjugated TIM-4. Similar to the in vitro observations, TIM-4 binding and PS exposure on tumor cells increased after irradiation ( Figure 2 B, C). These data demonstrate that radiation, as a mainstay of cancer treatment, enhances PS exposure and should therefore work synergistically with the TIM-4 targeting strategy according to the present disclosure.

[0088] In the case where tumor antigens are uniformly expressed, multispecific antibodies exhibit powerful tumor cell toxicity. Unfortunately, uniform antigen expression is rare, especially in solid tumors. However, based on the data provided herein, PS represents a universal targetable entity. Therefore, the inventors attempted to develop a novel T cell redirection therapy based on multispecific antibody technology, which targets PS exposed to the surface. To this end, a multispecific antibody molecule was designed and expressed, which comprises a TIM-4 extracellular domain covalently linked to an agonist anti-human CD3 scFv. Figure 3B shows a schematic diagram of the structure of a recombinant TIM-4 fusion protein and the amino acid sequence of the recombinant TIM-4 fusion protein according to one embodiment of the present disclosure.

[0089] We first sought to determine whether each domain of the recombinant TIM-4 fusion protein maintained binding to its respective target. Recombinant TIM-4 fusion protein (human) was incubated with U87 tumor cells and subsequently probed with protein-L (which binds to the scFv) ( Figure 4 A). Recombinant TIM-4 was detected on U87 cells, indicating that the recombinant TIM-4 fusion protein was able to bind to tumor cells via PS. Next, the recombinant TIM-4 fusion protein was incubated with human T cells and then detected with a fluorescently labeled anti-mouse TIM-4 antibody ( Figure 4 B). Recombinant TIM-4 was detected on human T cells, indicating that the recombinant TIM-4 fusion protein was able to redirect T cells to PS on tumor cells. This T cell binding was specific to human T cells, as recombinant TIM-4 was undetectable after incubation with mouse T cells ( Figure 4 C).

[0090] With the recombinant TIM-4 fusion protein in hand, the functionality was next tested by determining whether recombinant TIM-4 could efficiently mediate tumor cell killing in in vitro cytotoxicity assays. In these assays, increasing concentrations of human recombinant TIM-4 fusion protein were titrated into co-cultures of human T cells and tumor cells. Tumor cells were labeled with a fluorescent dye for quantification by flow cytometry after 24 hours. The percent survival was calculated by normalizing the number of tumor cells maintained under each condition relative to the number of tumor cells in the absence of recombinant TIM-4. Recombinant TIM-4 demonstrated potent dose-dependent killing of U87 ( Figure 5 A). Importantly, in a heterogeneous tumor population consisting of U87 and U87 transfected with the tumor-specific antigen EGFRvIII (U87vIII), recombinant TIM-4 was more effective than EGFRvIII-targeted recombinant TIM-4 ( Figure 5 B). These data demonstrate that recombinant TIM-4 mediates potent tumor cytotoxicity even when tumors exhibit antigenic heterogeneity. Likewise, this suggests that recombinant TIM-4 is capable of achieving the goal of sustained potent cytotoxicity even in the face of protein antigenic heterogeneity—and that their ability to do so is superior to current platforms.

[0091] The half-life of multispecific antibodies in vivo is relatively short, which means that long-term administration of multispecific antibodies is required to maintain the systemic levels required for efficacy. In order to overcome this problem and reduce the possibility of non-tumor targeted toxicity produced by recombinant TIM-4, the inventors engineered tumor antigen (EGFRvIII)-specific CAR T cells as tumor localization vehicles, which secrete recombinant TIM-4 fusion proteins at the tumor site in a renewable fashion. Therefore, the recombinant TIM-4 fusion protein is locally delivered by CAR T cells that also have their own anti-tumor efficacy, thereby providing a combination therapy. To verify the concept, VIII-targeted CARs that secrete recombinant TIM-4 were engineered. These CAR T cells were then administered to the intracranial ( Figure 6 ). Compared with VIIICAR alone, VIIICAR secreting recombinant TIM-4 significantly prolonged the survival of mice. These data demonstrate the in vivo efficacy of recombinant TIM-4 and demonstrate that CAR T cells secreting recombinant TIM-4 can be used as an effective delivery vehicle for treatment.

[0092] The data presented herein provide examples of heterologous species combinations comprising recombinant mouse sequences as well as human T cells. Figure 7 The fully mouse recombinant TIM-4VIIICAR vector in mouse T cells is shown. This in vivo experiment shows that recombinant TIM-4 secreted by VIIICAR prolongs the survival of mice bearing heterologous tumors without lymphocyte depletion pretreatment. Figure 7 This indicates that the same results can be obtained when the recombinant TIM-4 fusion protein and / or CAR are the same species as the one being treated. That is, mouse recombinant TIM-4 together with mouse VIIICAR and mouse T cells (including human T cells with mouse recombinant TIM-4 and VIIICAR) improves survival (compared to Figure 6 Together, these data suggest that similar results can be expected in fully human systems, including those comprising SEQ ID NOs: 3, 5, 17, and 10.

[0093] Those skilled in the art will readily appreciate that the present disclosure is well adapted to achieve these objects and obtain the objects and advantages mentioned as well as those inherent therein. The present disclosure described herein presently represents preferred embodiments, which are exemplary and not intended to limit the scope of the present disclosure. Those skilled in the art will be able to envision changes therein and other applications, which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

[0094] It is not acknowledged that any reference (including any non-patent or patent document cited in this specification) constitutes prior art. In particular, it should be understood that, unless otherwise stated, the reference to any document herein does not represent an admission that any of these documents constitute part of the common general knowledge in the art in the United States or any other country. Any discussion of the references states the claims of their authors, and the applicant reserves the right to question the accuracy and relevance of any document cited herein. Unless otherwise expressly stated, all references cited herein are incorporated herein in their entirety by reference. If there is any difference between any definition and / or description appearing in the cited references, the present disclosure shall prevail.

[0095]

Claims

1. A TIM-4 fusion protein comprising a TIM-4 domain connected to a CD3-specific single-chain variable fragment (scFv), wherein the TIM-4 domain comprises SEQ ID NO: 3 or a sequence with 95% identity to SEQ ID NO: 3, and wherein the CD3-specific scFv comprises a complementarity determining region (CDR) of SEQ ID NO: 22-27 or a sequence with at least 95% identity to SEQ ID NO: 22-27. 2 . The fusion protein of claim 1 , wherein the CD3-specific scFV comprises SEQ ID NO: 5 or a sequence having at least 95% identity to SEQ ID NO:

5.

3. The fusion protein according to claim 1 or 2, further comprising an N-terminal signal sequence, wherein the N-terminal signal sequence comprises a secretion signal sequence. The fusion protein of claim 3 , wherein the secretory signal sequence comprises the signal sequence of SEQ ID NO: 2 or a sequence having at least 95% identity to SEQ ID NO:

2.

5. The fusion protein according to any one of claims 1 to 4, wherein the TIM-4 domain is linked to the CD3-specific scFv via a linker peptide, and optionally wherein the linker is a glycine-serine linker. The fusion protein of claim 5 , wherein the linker comprises at least one copy of SEQ ID NO:

4.

7. The fusion protein according to any one of claims 1 to 6, further comprising a tag. The fusion protein according to claim 7 , wherein the tag can be used for purification, tracking or imaging of the fusion protein. 9 . The fusion protein of claim 1 , comprising SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO:

1.

10. The fusion protein according to any one of claims 1 to 8, comprising the TIM-4 domain of SEQ ID NO: 3, the linker of SEQ ID NO: 4, and the CD3-specific scFv of SEQ ID NO: 22-27.

11. The fusion protein according to any one of claims 1 to 8, comprising the TIM-4 domain of SEQ ID NO: 3, the linker of SEQ ID NO: 4, and the CD3-specific scFv of SEQ ID NO:

5. 12 . The fusion protein according to claim 10 , further comprising a tag selected from the group consisting of a His tag, a FLAG tag and a fluorescent tag. The fusion protein according to claim 10 , further comprising a signal sequence of SEQ ID NO:

2.

14. A pharmaceutical composition comprising a recombinant TIM-4 fusion protein according to any one of the preceding claims and a pharmaceutically acceptable excipient, carrier and / or diluent.

15. A construct comprising a polynucleotide sequence encoding the TIM-4 fusion protein according to any one of the preceding claims.

16. The construct according to claim 15, further comprising a promoter operably linked to a polynucleotide sequence encoding the TIM-4 fusion protein.

17. The construct of claim 15 or 16, further comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

18. The construct of claim 17, wherein the CAR comprises an extracellular domain comprising an antigen binding region that binds to wild-type EGFR and EGFR VIII variant.

19. The construct of claim 18, wherein the antigen binding region comprises the scFv of SEQ ID NO: 10 or a sequence having at least 95% identity to SEQ ID NO:

10.

20. The construct of claim 18, wherein the antigen binding region comprises the scFv of SEQ ID NO: 17 or a sequence having at least 95% identity to SEQ ID NO:

17.

21. The construct of any one of claims 17 to 20, further comprising a heterologous promoter operably linked to a sequence encoding the CAR.

22. The construct of any one of claims 17 to 21, wherein the CAR and the TIM-4 fusion protein are connected by a self-cleavage site.

23. The construct of any one of claims 15 to 22, wherein the construct is contained in a lentiviral, retroviral or AAV vector.

24. A chimeric antigen receptor (CAR)-T cell comprising a construct according to any one of claims 15 to 23.

25. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding an EGFRVIIICAR of SEQ ID NO: 10, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 22-27, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO: 10, 3, 22, 24, 25, 26, and 27.

26. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding an EGFRVIIICAR of SEQ ID NO: 10, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 5, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NOs: 10, 3, and 5.

27. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding a D2C7CAR of SEQ ID NO: 17, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 22-27, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO: 17, 3, 22, 23, 24, 25, 26, and 27.

28. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding a D2C7CAR of SEQ ID NO: 17, a self-cleavage site, a signal sequence, a TIM-4 domain of SEQ ID NO: 3, a linker, and a CD3-specific scFv of SEQ ID NO: 5, or wherein the construct comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NOs: 17, 3, and 5.

29. The CAR-T cell according to any one of claims 24 to 28, wherein the self-cleavage site comprises SEQ ID NO: 11 or a sequence having at least 95% sequence identity with SEQ ID NO:

11.

30. The CAR-T cell according to any one of claims 24 to 29, wherein the signal sequence comprises SEQ ID NO: 2 or a sequence having at least 95% sequence identity with SEQ ID NO:

2.

31. The CAR-T cell of any one of claims 24 to 30, wherein the linker comprises SEQ ID NO:

4.

32. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding a CAR of SEQ ID NO: 10, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NOs: 22-27.

33. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding a CAR of SEQ ID NO: 10, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NO:

5.

34. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding a CAR of SEQ ID NO: 17, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NOs: 22-27.

35. The CAR-T cell of claim 24, wherein the construct comprises a polynucleotide encoding a CAR of SEQ ID NO: 17, a self-cleavage site of SEQ ID NO: 11, a signal sequence of SEQ ID NO: 2, a TIM-4 domain of SEQ ID NO: 3, a linker of SEQ ID NO: 4, and a CD3-specific scFv of SEQ ID NO:

5.

36. A CAR-T cell comprising a polynucleotide encoding SEQ ID NO: 20 or a sequence having at least 95% sequence identity with SEQ ID NO:

20.

37. A CAR-T cell comprising a polynucleotide encoding SEQ ID NO: 21 or a sequence having at least 95% sequence identity with SEQ ID NO:

21.

38. A method for treating cancer, the method comprising: Administering a therapeutically effective amount of a recombinant TIM-4 fusion protein according to any one of claims 1 to 13 and a pharmaceutically acceptable excipient, carrier and / or diluent.

39. The method of claim 38, further comprising: T lymphocytes are administered, wherein the T lymphocytes are activated when bound to the TIM-4 fusion protein.

40. The method of claim 39, wherein the T lymphocytes are CAR T cells.

41. The method of claim 40, wherein the CAR T cell comprises the EGFRVIII CAR of SEQ ID NO: 10 or the D2C7 CAR of SEQ ID NO: 17 or a sequence having at least 95% identity to SEQ ID NO: 10 or SEQ ID NO:

17.

42. A method for treating cancer, the method comprising: Administering a therapeutically effective amount of the CAR-T cells according to any one of claims 24 to 37 and a pharmaceutically acceptable excipient, carrier and / or diluent.

43. A method for treating cancer, the method comprising: Administration of a cell comprising the construct according to any one of claims 15 to 23.

44. The method of any one of claims 38 to 43, wherein the cancer is an EGFR-related cancer.

45. The method of any one of claims 38 to 43, wherein the cancer is a PS-related cancer.

46. ​​The method of claim 44, wherein the EGFR-related cancer comprises glioma, glioblastoma, medulloblastoma, ependymoma, diffuse intrinsic pontine glioma (DIPG), brain metastasis, head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, or esophageal cancer.

47. The method of claim 44 or 46, wherein the treatment of the cancer results in the induction of an anti-tumor response to the EGFR-related cancer.

48. A method for diagnosing cancer, the method comprising: Administering a fusion protein according to claim 7 to 8 or 12 to detect the presence or accumulation of a tag in a subject suspected of having cancer, wherein the tag allows for in vivo detection of the cancer.

49. A method for diagnosing cancer, the method comprising: A sample containing cells or tissues is contacted with the fusion protein according to any one of claims 7 to 8 or 12 or the TIM-4 domain of SEQ ID NO: 3 connected to a tag, and the binding of the fusion protein or the TIM-4 domain to the sample is detected by detecting the tag, wherein the binding of the TIM-4 to the sample indicates that the sample contains cancer cells.

50. The method of claim 48 or 49, wherein the diagnosed cancer is glioma, glioblastoma, medulloblastoma, ependymoma, diffuse intrinsic pontine glioma (DIPG), brain metastasis, head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, or esophageal cancer.

51. A method of inducing a T cell response in a subject having cancer, the method comprising: A therapeutically effective amount of the fusion protein according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 14 is administered to the subject, wherein an anti-tumor response to the cancer is induced.

52. A method of inducing a T cell response in a subject suffering from cancer, the method comprising: A therapeutically effective amount of the CAR-T cells according to any one of claims 24 to 37 is administered to the subject, wherein an anti-tumor response to the cancer is induced.

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