Chimeric antigen receptor system, preparation method and application thereof

Abstract

The present disclosure provides Smart CAR systems in which an exogenous antigen is labeled on a tumor cell, the tumor cell labeled with the exogenous antigen can be recognized by an engineered immune cell expressing a chimeric receptor, the chimeric receptor comprising a binding domain that binds to the exogenous antigen, and the tumor can be at least partially eliminated. The present disclosure also provides methods of preparation, methods of treatment, and uses associated with the Smart CAR system.

Description

Related Applications

[0001] This application claims the priority of International Application No. PCT / CN2022 / 109857, filed on August 3, 2022, the content of which is incorporated herein by reference in its entirety. Sequence Listing

[0002] This application contains a sequence listing, which is submitted electronically as an XML file named "Seq.XML", with a size of 31 KB and created on August 1, 2023. The content contained in the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the treatment of tumors, in which tumor cells are labeled with exogenous antigens and treated with engineered immune cells expressing a chimeric antigen receptor (CAR), and the chimeric antigen receptor comprises a binding domain that recognizes the exogenous antigen. Background Art

[0004] Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable success in clinical trials of B-cell malignancies, their applicability to solid tumors is limited. Multiple factors have led to obstacles in manufacturing effective CAR-T cells for solid tumors, and the lack of tumor-specific antigens is one of the main reasons (1, 2). Identifying antigens that are highly and homogeneously expressed only in solid tumors and not in healthy tissues has proven difficult (2). Current CARs typically contain an extracellular antigen-binding domain, which is usually a single-chain variable fragment (scFv). Screening scFvs with appropriate affinity for tumor antigens requires a great deal of effort. In addition, acquired drug resistance caused by heterogeneity and antigen loss in tumors makes the widespread application of CAR-T cell therapy difficult (3, 4).

[0005] To overcome one or more of these problems, the present disclosure provides a Smart CAR system, in which an exogenous antigen is labeled on the surface of tumor cells, and the tumor cells labeled with the exogenous antigen can be recognized by engineered immune cells expressing a chimeric receptor, and the chimeric receptor comprises a binding domain that binds to the exogenous antigen, and subsequently the tumor can be treated, for example, at least partially eliminated. Summary of the Invention

[0006] In one aspect, the present disclosure provides a method for treating a tumor in a subject in need, comprising: (a) providing a sortase and an exogenous antigen to the tumor, wherein the exogenous antigen comprises an exogenous antigen ligand and a sort tag motif, and wherein the cells in the tumor are sorted and labeled with the exogenous antigen, and (b) Administering to a subject genetically engineered immune cells expressing a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen-binding domain capable of binding the exogenous antigen ligand.

[0007] In some embodiments, the CAR comprising an antigen-binding domain capable of binding the exogenous antigen ligand further comprises a binding domain capable of binding an endogenous antigen in the tumor.

[0008] In some embodiments, the method of treating a tumor further comprises administering to the subject genetically engineered immune cells expressing a second CAR, wherein the second CAR comprises a binding domain that recognizes an endogenous or exogenous antigen in the tumor.

[0009] In one aspect, the present disclosure provides a method of labeling tumor cells with an exogenous antigen, comprising providing sortase and an exogenous antigen to the tumor cells, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein the tumor cells are sorted and labeled with the exogenous antigen.

[0010] In one aspect, the present disclosure provides a tumor cell sorted and labeled with an exogenous antigen, e.g., by the method of labeling tumor cells described herein.

[0011] In one aspect, the present disclosure provides an exogenous antigen comprising a sorting tag motif and an exogenous antigen ligand.

[0012] In one aspect, the present disclosure provides a nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling domains, and a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the antigen-binding domain is capable of binding the exogenous antigen described herein. In some embodiments, the CAR further comprises a binding domain capable of binding an endogenous or exogenous antigen in the tumor.

[0013] In one aspect, the present disclosure provides a vector comprising the nucleic acid described herein.

[0014] In one aspect, the present disclosure provides an engineered immune cell comprising the nucleic acid or vector described herein.

[0015] In one aspect, the present disclosure provides a pharmaceutical composition comprising a population of the engineered immune cells described herein.

[0016] In one aspect, the present disclosure provides a method of modifying an immune cell, comprising delivering to the immune cell the vector or nucleic acid described herein.

[0017] In one aspect, the present disclosure provides a kit comprising a sortase or a nucleic acid or vector encoding the same, and an exogenous antigen. In some embodiments, the kit further comprises a nucleic acid or vector encoding a CAR as described herein.

[0018] In one aspect, the present disclosure provides a genetically engineered immune cell for treating a tumor, wherein cells in the tumor are sorted and labeled with an exogenous antigen mediated by a sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting label motif, and wherein the genetically engineered immune cell expresses a CAR, the CAR comprising an antigen-binding domain capable of binding the exogenous antigen ligand. In some embodiments, the CAR further comprises a binding domain capable of binding an endogenous or exogenous antigen in the tumor.

[0019] In one aspect, the present disclosure provides the use of a genetically engineered immune cell in the preparation of a medicament for treating a tumor, wherein cells in the tumor are sorted and labeled with an exogenous antigen mediated by a sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting label motif, and wherein the genetically engineered immune cell expresses a CAR, the CAR comprising an antigen-binding domain capable of binding the exogenous antigen ligand. In some embodiments, the CAR further comprises a binding domain capable of binding an endogenous or exogenous antigen in the tumor. Brief Description of the Drawings

[0020] Figure 1 Shows the Smart CAR-T system (right), which, compared to the classical CAR-T system (left), comprises a mixture of sortase and exogenous antigen, and Smart CAR-T cells. Figure 1A Displays the Smart CAR-T system (right), which, compared to the classical CAR-T system (left), comprises Smart CAR-T cells having a TNFRSF9 cytoplasmic domain.

[0021] Figure 2 Shows the vector design for monomeric streptavidin (mSA)-CAR.

[0022] Figure 3A Shows the transduction efficiency and mSA-CAR expression on the surface of T cells 2 days, 8 days, and 11 days after transduction. The transduction efficiency is expressed as the percentage of tagBFP+ cells. Detection of mSA-CAR expression on the surface of T cells is performed via the binding of FITC-AhX-GGGGK (biotin)-NH2 (SEQ ID NO:9) through mSA and biotin.

[0023] Figure 3B andFigure 3C showed the proliferation of mSA-CAR-T cells at 0 days (D0), 2 days (D2), 4 days (D4), 8 days (D8), and 11 days (D11) after lentiviral transduction ( Figure 3B ) and viability ( Figure 3C ). mSA-CAR-T: primary T cells with mSA-CAR expression; UN-T: primary T cells without mSA-CAR expression.

[0024] Figure 4A showed the quantification of biotin-AALPETG*G (SEQ ID NO:14) sorted and labeled on various solid tumor cell lines. SORT: tumor cells sorted and labeled with biotin-AALPETG*G (SEQ ID NO:14); Blank: tumor cells without sorting and labeling. The sorting and labeling efficiency was indicated by the intensity of phycoerythrin (PE)-streptavidin.

[0025] Figure 4B showed the quantification of biotin-AALPETG*G (SEQ ID NO:14) on (left) Hela and (right) MDA-MB-231 cells at time points after intratumoral injection in vivo. SORT: tumor cells sorted and labeled with biotin-AALPETG*G (SEQ ID NO:14); Blank: tumor cells without sorting and labeling. The sorting and labeling efficiency was indicated by PE-streptavidin.

[0026] Figure 5A showed the expression of CD25 and CD69 on the surface of T cells when co-cultured with tumor cells. mSA-CAR-T: primary T cells expressing mSA-CAR; UN-T: primary T cells without mSA-CAR expression; SORT: tumor cells sorted and labeled with biotin-AALPETG*G (SEQ ID NO:14); Blank: tumor cells without sorting and labeling.

[0027] Figure 5B showed the cytokine release of T cells against tumor cells. mSA-CAR-T: primary T cells expressing mSA-CAR; UN-T: primary T cells without mSA-CAR expression; The terms ending with "Blank" in the x-axis label indicate tumor cells without sorting and labeling; The terms ending with "Sort" in the x-axis label indicate tumor cells sorted and labeled with biotin-AALPETG*G (SEQ ID NO:14).

[0028] Figure 5CShows the cytotoxicity of mSA-CAR-T cells against Hela, MDA-MB-231, or HepG2 solid tumor cell lines with stable luciferase expression. Blank: Tumor cells without sorting label; SORT: Tumor cells sorted and labeled with biotin-AALPETG*G (SEQ ID NO:14); -luc: Luciferase with stable expression.

[0029] Figure 6 Shows the tumor size curves of mice receiving intratumoral injection of mSA-CAR-T cells and sorting labels. The vertical arrows indicate the time points of injecting a dose of the respective sorting label mixture into the mice, for a total of 6 doses. mSA-CAR-T + biotin-motif: Mice received intratumoral injection of mSA-CAR-T cells and sorting labels; UN-T + biotin-motif: Mice received intratumoral injection of primary T cells and sorting labels; Blank: Mice did not receive primary T cells and did not receive intratumoral injection.

[0030] Figure 7 Shows the Smart CAR vector design for PNEscFv-CAR, which recognizes PNE via anti-PNE scFv. The Smart CAR module design includes PNE scFv, hinge domain, transmembrane domain, co-stimulatory domain, and CD247.

[0031] Figure 8A Shows the transduction efficiency and PNEscFv-CAR expression on the surface of T cells at different days after transduction. The transduction efficiency is indicated by tagBFP. The PNEscFv-CAR expression on the surface of T cells is indicated by PE-streptavidin.

[0032] Figure 8B Shows the identification of biotin-PNE sorted and labeled on 293T cells. SORT: Tumor cells sorted and labeled; Blank: Tumor cells without sorting label. The sorting label efficiency is indicated by anti-biotin-PE.

[0033] Figure 8C Shows the expression of CD25 and CD69 on the surface of T cells when co-cultured with tumor cells. PNEscFv-CAR-T: Primary T cells expressing PNEscFv-CAR; UN-T: Primary T cells without PNEscFv-CAR expression; SORT: Tumor cells sorted and labeled with biotin-PNE; Blank: Tumor cells without sorting label.

[0034] Figure 9A and Figure 9BShows the tumor size curves of mice that received intratumoral injection of PNEscFv-CAR-T cells and the sorting marker. The vertical arrows indicate the time points when the mice were injected with one dose of the respective sorting marker mixture. PNE-CAR-T + PNE motif: Mice received intratumoral injection of PNEscFv-CAR-T cells and the sorting marker; UN-T + PNE motif: Mice received intratumoral injection of primary T cells and the sorting marker; Figure 9A and Figure 9B are data collected from two independent replicates.

[0035] Figure 10 shows the proliferation ( Figure 10A ) and viability ( Figure 10B ) of PNEscFv-CAR-T cells at 0 days (D0), 2 days (D2), 5 days (D5), 7 days (D7), and 10 days (D10) after lentiviral transduction. PNEscFv-CAR-T: Primary T cells with PNEscFv CAR expression; UN-T: Primary T cells without PNEscFv-CAR expression.

[0036] Figure 11A Shows the transduction efficiency and PNEscFv-CAR expression on the surface of T cells at 2 days (D2), 5 days (D5), 7 days (D7), and 10 days (D10) after transduction. The transduction efficiency is indicated by tagBFP. The PNEscFv-CAR expression on the surface of T cells is indicated by anti-biotin-PE, where anti-biotin-PE is linked to the CAR through biotin-PNE-AALPETG*G (SEQ ID NO:15).

[0037] Figure 11B Shows the quantification of biotin-PNE-AALPETG*G (SEQ ID NO:15) on various solid tumor cell lines by the sorting marker. SORT: Tumor cells were sorted and marked with biotin-PNE-AALPETG*G (SEQ ID NO:15); Blank: Tumor cells without sorting and marking. The sorting marker efficiency is indicated by the intensity of anti-biotin-PE.

[0038] Figure 11C Shows the expression of CD25 and CD69 on the surface of T cells when co-cultured with various solid tumor cells. PNEscFv-CAR-T: Primary T cells expressing PNEscFv-CAR; UN-T: Primary T cells without PNEscFv-CAR expression; SORT: Tumor cells sorted and marked with PNE-AALPETG*G (SEQ ID NO:16); Blank: Tumor cells without sorting and marking.

[0039] Figure 12Cytokine release by T cells against tumor cells is shown. PNEscFv-CAR-T: primary T cells expressing PNEscFv-CAR; UN-T: primary T cells without PNEscFv-CAR expression; terms ending with "blank" in the x-axis label represent tumor cells that are not sorted; terms ending with "SORT" in the x-axis label represent tumor cells sorted with PNE-AALPETG*G (SEQ ID NO: 16).

[0040] Figure 13 The cytotoxicity of PNEscFv-CAR-T cells against HCT-116, Hela or HepG2 solid tumor cell lines is shown. Blank: tumor cells without sorting labeling; SORT: tumor cells sorted and labeled with PNE-AALPETG*G (SEQ ID NO: 16).

[0041] Figure 14 Tumor size curves for mice receiving PNEscFv-CAR-T cells and intratumoral injection of sorting markers are shown. The dosing limit for intratumoral injection of sorting markers is 2 times. The vertical arrow indicates the time point at which a dose of the sorting marker mixture is injected into the mouse. PNEscFv-CAR-T+Sortag: mice receive intratumoral injection of PNEscFv-CAR-T cells and sorting markers; UN-T+Sortag: mice receive intratumoral injection of primary T cells and sorting markers; Blank: mice receive PBS and intratumoral injection of no sorting markers. Data were collected from four independent replicates.

[0042] FIG. 15 shows the Bi-CAR vector design for PNE / CLDN18.2-CAR, which recognizes PNE via PNEscFv and Claudin 18.2 via CLDN18.2scFv. Figure 15A The Smart CAR module design is shown to include PNE scFv, CLDN18.2 scFv, hinge domain, transmembrane domain, co-stimulatory domain and CD247. Figure 15B , Figure 15C , Figure 15D and Figure 15E It is shown how the binding segments recognizing PNE and Claudin 18.2, respectively (e.g., PNEscFv light chain, PNEscFv heavy chain, CLDN18.2scFv heavy chain, and CLDN18.2sccFv light chain) can be arranged on a Bi-CAR design, wherein the binding segments are optionally connected by one or more linkers known in the art. Figure 15BShows the binding segments arranged in the following order: CLDN18.2scFv heavy chain, PNEscFv light chain, PNEscFv heavy chain, and CLDN 18.2scFv light chain. Figure 15C Shows the binding segments arranged in the following order: PNEscFv light chain, CLDN18.2scFv heavy chain, CLDN 18.2scFv light chain, and PNEscFv heavy chain. Figure 15D Shows the binding segments arranged in the following order: PNEscFv light chain, PNEscFv heavy chain, CLDN18.2scFv heavy chain, and CLDN18.2scFv light chain. Figure 15E Shows the binding segments arranged in the following order: CLDN18.2scFv heavy chain, CLDN18.2scFv light chain, PNEscFv light chain, and PNEscFv heavy chain.

[0043] Figure 16 Shows the tumor sizes of mice that received intratumoral injection of PNE / CLDN18.2-CAR-T (Bi-CAR-T) cells and sorting markers. In the tumors, only 10% of the tumor cells expressed the antigen CLDN18.2. The vertical arrows indicate the time points of injection of a single dose of the respective sorting marker mixture into the mice. PNE / CLDN18.2-CAR-T + sorting marker (Sortag): mice received intratumoral injection of PNE / CLDN18.2-CAR-T cells and sorting markers; UN-T + sorting marker (Sortag): mice received intratumoral injection of primary T cells and sorting markers. Data were collected from two independent experimental replicates.

[0044] Figure 17 Shows an example workflow of cytokine release assay and activation assay. Primary T cells were transduced with a lentiviral vector and engineered into Smart CAR-T cells (E: effector cells). Tumor cells were sorted with an exogenous antigen (T: tumor cells). Effector cells and tumor cells were co-cultured at a cell ratio of 2:1 for 16 hours. Subsequently, the supernatant was collected for cytokine release assay, and cells were collected for activation assay by examining the expression of CD25 and CD69 on CAR-T cells.

[0045] Figure 18 Shows an example workflow of cytotoxicity assay. Primary T cells were transduced with a lentiviral vector and engineered into Smart CAR-T cells (E: effector cells). Tumor cells with stable luciferase expression were sorted with an exogenous antigen (T: tumor cells). CAR-T cells and tumor cells were co-cultured at serial cell ratios for 16 hours. Subsequently, luciferin was added to detect cytotoxicity.

[0046] Figure 19Shows an exemplary workflow of in vivo experiments for testing the Smart CAR-T system described herein. Eight days before T cell injection (d-8), tumor cells were subcutaneously (s.c.) inoculated into mice. One day before T cell injection (d-1), the mice were irradiated. At d0 (day 0), the T cells were intravenously (i.v.) injected into the mice. In the following days, until dX (day X, such as day 3, day 4, or day 5), a mixture of exogenous antigen and sortase was injected intratumorally multiple times. The body weight loss of the mice was monitored every three days after T cell injection until the animals died or the tumor size reached the limit of the animal experiment ethics protocol.

[0047] Figure 20 shows the structures of certain modified peptides used herein. Figure 20A Shows the chemical structure of biotin-AALPETG*G (SEQ ID NO:14), where G* refers to 2-hydroxyacetic acid and is enclosed by a dashed rectangle. Figure 20B Shows the chemical structure of FITC-Ahx-GGGGK(biotin)-NH2 (SEQ ID NO:9), where FITC-Ahx refers to fluorescein isothiocyanate with an aminohexanoic acid linker and is enclosed by a dashed rectangle. Detailed Description of the Invention

[0048] All publications cited in this specification are incorporated herein by reference as if fully set forth. If some content of the references cited herein conflicts or is inconsistent with the present disclosure, the present disclosure shall prevail.

[0049] Any one of the embodiments of the present disclosure described herein, including those described only in a part of the specification that describes a particular aspect of the present disclosure, and those described only in the examples or the drawings, may be combined with any other one or more embodiments, unless explicitly denied or inappropriate. Definitions

[0050] It should be understood that the terms used herein are only for describing particular embodiments and are not intended to limit the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0051] Although any methods and materials similar or equivalent to those described herein may be used in the testing practice of the present disclosure, exemplary materials and methods are described herein.

[0052] In this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of one, two, or more cells, etc.

[0053] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Polynucleotides disclosed herein can be modified, for example, with a labeling group such as a fluorophore, with biotin, or with phosphorothioate.

[0054] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. These terms also include polypeptides having co-translational (e.g., signal peptide cleavage) and post-translational modifications of polypeptides (e.g., disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, etc.). Peptides disclosed herein can be modified, for example, with a labeling group such as a fluorophore, biotin, His-tag, or phosphorothioate.

[0055] Furthermore, as used herein, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions, and substitutions (such as are typically conservative as known to those of skill in the art), provided that the protein still retains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host that produces the protein, or due to errors in PCR amplification or other recombinant DNA methods.

[0056] The term "subject" refers to any animal, such as a mammal, such as a human.

[0057] The terms "being labeled", "labeled", or "labeling" mean that a detectable or recognizable group is attached to an entity via a covalent or non-covalent bond. For example, a protein, nucleic acid, or polysaccharide can be labeled with a group such as a fluorophore, biotin, His-tag, or phosphorothioate. For another example, a cell can be labeled by a peptide promoted by sortase (also referred to herein as "conjugation", "linkage", "anchoring", "joining", or "attachment").

[0058] The terms "sortagging", "sortagged", or "sortag" refer to the covalent or non-covalent labeling of cells mediated by sortase (e.g., SrtA). For example, any molecule containing a sortag motif, such as the LPXTG (SEQ ID NO: 2) ("X" refers to any amino acid) motif, can be covalently or non-covalently labeled on cells mediated by sortase.

[0059] As used herein, the term "sorting tag motif" refers to a stretch of amino acid sequence that can be recognized by a sortase. For example, the sorting tag motif can include LPXTG ("X" refers to any amino acid) (SEQ ID NO: 2), NP(Q / K)TN (SEQ ID NO: 3), (I / L)(P / A)XTG (e.g., LAXTG (SEQ ID NO: 7), IPXTG (SEQ ID NO: 17), IAXTG (SEQ ID NO: 18), LPXTG (SEQ ID NO: 2)), LPNTA (SEQ ID NO: 5), AALPETGXG (SEQ ID NO: 1), LAXTG (SEQ ID NO: 7), etc. The sorting tag motif can contain encoded and non-encoded amino acids, chemically or biochemically modified or derived amino acids, or amino acid mimetics. For example, the sorting tag motif AALPETG*G contains G*, which is glycolic acid.

[0060] As used herein, the term "tumor" refers to any abnormal and excessive growth of tissue in a subject. A tumor can be a soft tumor or a solid tumor. A tumor can be benign, potentially malignant, or malignant. Non-limiting examples of tumors include lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, colon cancer, kidney cancer, esophageal cancer, cervical cancer, and bladder cancer.

[0061] The term "antigen" is a molecule or molecular structure that can bind to another molecule or molecular structure (e.g., a specific antibody or T cell receptor, such as an engineered CAR). In other words, the "antigen" as used herein is not limited to substances that can stimulate antibody production. In the Smart-CAR system disclosed herein, the antigen can be a molecule or molecular structure that does not necessarily stimulate antibody production. For example, the antigen can be biotin, a peptide (e.g., PNE peptide, NYHLENEVARLKKL (SEQ ID NO: 10)), a chemical substance (e.g., FITC, CY5), etc.

[0062] As used herein, the term "exogenous antigen ligand" refers to any molecule that is not normally expressed on the tissues of a subject. They include, but are not limited to, peptides such as PNE peptide or leucine zippers, polysaccharides, and small molecules such as biotin, FITC, or TAMRA.

[0063] As used herein, the term "endogenous antigen" refers to any molecule or molecular structure that is normally expressed on the tissues of a subject. Such antigens include, but are not limited to, claudin8.2, Her2, Claudin6, GPC3, RGFR, MSLN, etc.

[0064] The term "exogenous antigen" refers to an exogenous antigen ligand fused to a sorting tag motif. The exogenous antigen can be directly fused to the sorting tag motif or fused via a spacer or linker. For example, by linking a sorting tag motif (such as the "AALPETGXG" (SEQ ID NO:1) motif) to an exogenous antigen ligand, such as to the C-terminus of an exogenous peptide antigen ligand, the exogenous antigen can be labeled onto tumor cells mediated by sortase (such as SrtA).

[0065] The term "binding domain" refers to any entity or component of an entity that is capable of binding to an exogenous antigen ligand. Depending on the identity of the exogenous antigen ligand, for example, the binding domain can be a protein that binds to the exogenous antigen ligand, or an antibody or a functional fragment thereof that recognizes the exogenous antigen ligand. As another example, the exogenous antigen ligand can be a small molecule (such as biotin), and the binding domain can be a binding partner (such as streptavidin) that recognizes the small molecule.

[0066] The term "chimeric antigen receptor" (CAR, also known as chimeric immune receptor, chimeric T cell receptor or artificial T cell receptor) refers to a cell surface receptor that comprises an extracellular antigen-binding domain, a transmembrane domain, and one or more cytoplasmic co-stimulatory signaling domains (which do not naturally occur together on a single receptor protein). The CAR can comprise an extracellular hinge region (such as a flexible spacer) between the extracellular antigen-binding domain and the transmembrane domain. The CAR can combine the antigen-binding domain, the hinge domain, the transmembrane domain, the co-stimulatory domain, and CD247 into a single receptor. As used herein, "SmartCAR" refers to a CAR that comprises an extracellular antigen-binding domain that recognizes an exogenous antigen ligand.

[0067] The term "immune cell" refers to cells of the immune system in an animal, including neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells). T cells include CD4+ cells, CD8+ cells, γδ T cells (γδ T cells), NK T cells, and / or regulatory T cells (Tregs).

[0068] The terms "CAR cell", "engineered immune cell" or "CAR-engineered immune cell" refer to an immune cell (such as a Bi-CAR-T cell) that has been genetically engineered to express a chimeric antigen receptor or multiple (such as 2, 3, 4, etc.) chimeric antigen receptors. As used herein, the term "Smart CAR cell" refers to an immune cell that has been genetically engineered to express a Smart-CAR.

[0069] The term "CAR-T cell" refers to a T cell that has been genetically engineered to express a chimeric antigen receptor or multiple chimeric antigen receptors (such as Bi-CAR-T cells). T cells engineered with CAR proteins can have the new ability to target specific antigens. The term "Smart Car-T cell" as used herein refers to a CAR-T cell that has been genetically engineered to express a Smart CAR.

[0070] As used herein, "affinity" refers to the total strength of non-covalent interactions between a molecule or molecular structure (e.g., a ligand) and its binding partner (e.g., a receptor). The affinity of a molecule for its partner can generally be expressed by the equilibrium dissociation constant (K D )(or its reciprocal equilibrium binding constant, K A ). Affinity can be measured by common methods known in the art, including the methods described herein. See, for example, Pope M.E., Soste M.V., Eyford B.A., Anderson N.L., Pearson T.W., (2009) J. Immunol. Methods. 341(1-2):86-96 and the methods described therein.

[0071] As used herein, the terms "specifically bind" or "bind" to a specific antigen or "recognize" a specific antigen refer to binding that has a measurable difference from non-specific interactions. For example, in some embodiments, a binding molecule (e.g., a single-domain antibody) specifically binds to a target molecule (e.g., an antigen) when the reaction or association of the binding molecule with the specific target molecule is more frequent, more rapid, longer-lasting, and / or of greater affinity than its reaction or association with alternative molecules. A binding molecule (e.g., a binding partner of an exogenous antigen) "specifically binds" to an exogenous antigen if it binds to the exogenous antigen with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other molecules. It will be understood that a binding molecule (e.g., a binding partner of an exogenous antigen) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specifically bind" does not necessarily require (although it can include) exclusive binding. In some embodiments, specific binding can be determined, for example, by comparing the binding of a specific antibody to the binding of an antibody that does not bind to the specific antigen. For example, when the K D of the binding partner to the antigen is at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11M, at least about 10 -12 M or greater, can show specific binding to a specific antigen, where K D refers to the dissociation rate of the binding partner / antigen interaction. In some embodiments, the K of the binding partner that specifically binds to the antigen is D The K of the binding partner (or binding partner) that does not bind to the same antigen will be D 20, 50, 100, 500, 1000, 5000, 10000 or more times higher. In some embodiments, binding between a binding partner and a specific antigen can be demonstrated by an EC50 value determined using a suitable method known in the art, including, for example, flow cytometry assays.

[0072] As used herein, "composition" refers to any mixture of two or more products, substances or compounds, including but not limited to proteins, antibodies, polynucleotides, vectors or cells. The composition can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0073] As used herein, "pharmaceutical composition" refers to an active pharmaceutical agent formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals alone or in combination with one or more other treatment modalities. It should also be understood that, if necessary, the compositions of the present disclosure can be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. Other ingredients that can also be included in the composition are almost unlimited, as long as these additional agents do not adversely affect the ability of the composition to provide the intended treatment. Some non-limiting examples of ingredients that can be included in the composition are carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. Pharmaceutical compositions help to administer the antibodies or cells described herein to subjects. There are a variety of administration techniques in the art, including but not limited to intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0074] The term "pharmaceutically acceptable" as used herein refers to a relatively non-toxic material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the therapeutic compound, i.e., the material can be administered to a subject without causing unexpected biological effects or interacting in a harmful manner with any component of the composition in which it is contained. Pharmaceutically acceptable ingredients include compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues within the scope of reasonable medical judgment without causing excessive toxicity, irritation, allergic reactions or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0075] As used herein, an "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical composition sufficient to significantly and positively modify a symptom and / or condition to be treated (e.g., provide a positive clinical response). The effective amount of the pharmaceutical composition will vary depending on factors such as the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent treatments, the specific composition employed, the specific pharmaceutically acceptable excipient and / or carrier used, etc., as well as the knowledge and professional skill of the attending physician.

[0076] As used herein, the term "treatment" refers to alleviating a disease or disorder, e.g., slowing or halting or reducing the development of the disease or disorder or reducing at least one of its clinical symptoms. For example, in some embodiments, alleviating a disease or disorder may include obtaining a beneficial or desired clinical outcome, including but not limited to any one or more of the following: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing disease progression, alleviating the disease state, inhibiting or eliminating the disease or the progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and alleviating (in part or in whole). Smart CAR System and Method of Use

[0077] The present disclosure provides a Smart CAR system, wherein an exogenous antigen is labeled on a tumor cell, and the tumor cell labeled with the exogenous antigen can be recognized by an engineered immune cell expressing a chimeric receptor, the chimeric receptor comprising a binding domain that binds to the exogenous antigen, and the tumor is treated, e.g., eliminated or at least partially eliminated.

[0078] The CAR-engineered immune cell can recognize the exogenous antigen labeled on the tumor cell and can induce cytotoxicity against the tumor cell independent of its native tumor antigen. Thus, as long as the tumor is labeled with an antigen recognized by the CAR, the CAR-engineered immune cell can be used as a universal anti-tumor treatment for the tumor.

[0079] Labeling of the tumor cell can be achieved by "sorting and labeling" the tumor cell with an exogenous antigen. Sorting and labeling the tumor cell with an exogenous antigen can convert a cold tumor into a hot tumor by neoantigen exposure caused by local cytotoxicity.

[0080] Any two molecules or molecular structures that have a specific binding affinity for each other can be used as an exogenous antigen ligand and the corresponding binding domain in the Smart CAR system, where one molecule (or molecular structure) is expressed on the engineered immune cell as the prey, and the other molecule (or molecular structure) is labeled on the target tumor cell as the bait. For example, any antigen (such as biotin, PNE peptide, leucine zipper, etc.) that can be bound by an antibody or a functional fragment (such as a single-chain variable fragment (scFv)) or can be bound by a non-antibody binding partner but is not present in the human proteome can be used as an exogenous antigen in the Smart-CAR system.

[0081] In some embodiments, the present disclosure provides a method of treating a tumor in a subject in need thereof, comprising: (a) providing a sortase and an exogenous antigen to the tumor, wherein the exogenous antigen comprises an exogenous antigen ligand and a sort tag motif, and wherein the cells in the tumor are sort-tagged by the exogenous antigen, and (b) administering to the subject a therapeutically effective amount of genetically engineered immune cells that express a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-binding domain capable of binding the exogenous antigen.

[0082] In some embodiments, the method of treating a tumor further comprises administering to the subject genetically engineered immune cells that express at least one other CAR, wherein the one other CAR comprises a binding domain that recognizes an endogenous antigen or an exogenous antigen in the tumor. In one embodiment, the method of treating a tumor further comprises administering to the subject genetically engineered immune cells that express one other CAR that recognizes the tumor, wherein the one other CAR recognizes an endogenous antigen in the tumor. Such CARs that recognize various endogenous antigens are known in the art. See, e.g., Nat Med. 2022 Jun;28(6):1189-1198. Clin Cancer Res;2020 Aug 1;26(15):3979-3989; Nat Med. 2021 Sep;27(9):1544-1552.

[0083] In some embodiments, the present disclosure provides a method for treating a tumor in a subject in need thereof, comprising: (a) providing a sortase and an exogenous antigen to the tumor, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein cells in the tumor are sorted and tagged by the exogenous antigen, and (b) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of genetically engineered immune cells that express a chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding the exogenous antigen. Steps (a) and (b) in the methods disclosed herein can be carried out approximately simultaneously or sequentially. Step (a) can be carried out before step (b), or step (b) can be carried out before step (a). Step (a) can be carried out multiple times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 times. Step (b) can be carried out one or more times, such as 2 or 3 times. For example, step (a) can be carried out first on day 0, then step (b) can be carried out on day 0, and then step (a) can be carried out 5 more times on days 3, 6, 9, 12, and 15, respectively. Step (b) can be carried out on day 3 or day 6.

[0084] In some embodiments, the present disclosure provides genetically engineered immune cells for treating a tumor, wherein cells in the tumor are sorted and tagged by an exogenous antigen mediated by a sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein the genetically engineered immune cells express a CAR comprising an antigen-binding domain capable of binding the exogenous antigen.

[0085] In some embodiments, the present disclosure provides a pharmaceutical composition for treating a tumor comprising genetically engineered immune cells, wherein cells in the tumor are sorted and tagged by an exogenous antigen mediated by a sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein the genetically engineered immune cells express a CAR comprising an antigen-binding domain capable of binding the exogenous antigen.

[0086] In some embodiments, the present disclosure provides the use of genetically engineered immune cells in the preparation of a medicament for treating a tumor, wherein cells in the tumor are sorted and tagged by an exogenous antigen mediated by a sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein the genetically engineered immune cells express a CAR comprising an antigen-binding domain capable of binding the exogenous antigen.

[0087] In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising genetically engineered immune cells in the preparation of a medicament for treating a tumor, wherein cells in the tumor are sorted and labeled with an exogenous antigen mediated by a sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting label motif, and wherein the genetically engineered immune cells express a CAR comprising an antigen-binding domain capable of binding to the exogenous antigen.

[0088] In some embodiments, the CAR comprising an antigen-binding domain capable of binding to an exogenous antigen ligand further comprises at least one other binding domain capable of binding to at least one other antigen that is endogenous or exogenous in the tumor. For example, the CAR comprising an antigen-binding domain capable of binding to an exogenous antigen ligand may further comprise one, two, three, or more additional binding domains capable of binding to one, two, three, or more additional exogenous or endogenous antigens in the tumor.

[0089] In some embodiments, the CAR comprising an antigen-binding domain capable of binding to an exogenous antigen ligand further comprises a second binding domain capable of binding to an endogenous antigen in the tumor. For example, Figure 17 A CAR comprising two binding domains is shown: a first binding domain such as PNEscFv (anti-PNE single-chain variable fragment) and a second binding domain such as CLDN18.2scFv (anti-CLDN18.2 single-chain variable fragment). The first and second binding domains may optionally be linked by a hinge known in the art. As Figures 15A - 15E shown, the binding segments of the binding domains may have alternative arrangements as long as the binding domains can achieve the purpose of recognizing both exogenous and endogenous antigens. PNEscFv can recognize tumor cells labeled with PNE, while CLDN18.2scFv can recognize the endogenous antigen claudin 8.2 on tumor cells. This dual CAR (or Bi-CAR) system can enhance or provide synergistic tumor killing. As in Example 3 and Figure 16 discussed, we designed a Bi-CAR system in which two CARs are expressed on each T cell. One CAR is PNE-CAR, which recognizes PNE-AALPETG*G labeled on tumor cells by a sortase; the other CAR is a clinically used CAR, which recognizes the antigen claudin 8.2 (CLDN18.2) of gastric cancer. We established a tumor model with low antigen expression, in which only 10% of the tumors express CLDN18.2. Generally, such tumors with low antigen expression cannot be effectively eliminated by CAR-T cells. However, when a sorting label mixture and Bi-CAR-T cells are used in an animal model, we found that tumors with low antigen expression can be significantly controlled.

[0090] Based on the knowledge in the art, it is easy to obtain binding domains (such as antibodies, such as scFv) that recognize endogenous antigens. See, for example, Biomark Res. 2022 May 31; 10(1):38; Front Oncol. 2022 Feb 16; 12:824208; Nat Rev Clin Oncol. 2020 Jan; 17(1):33-48; Semin Immunol. 2020 Feb; 47:101391.

[0091] In some embodiments, at least some of the tumor cells sorted and labeled with exogenous antigens are eliminated by Smart CAR cells administered to a subject. In some embodiments, the tumor cells near the tumor cells sorted and labeled with exogenous antigens are also at least partially eliminated by Smart CAR cells. In some embodiments, the treatment with Smart CAR cells alters the tumor microenvironment and enhances the endogenous immunity against the tumor to improve the long-term survival rate of the patient.

[0092] In some embodiments, the tumor is a solid tumor.

[0093] In some embodiments, the tumor is selected from lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, kidney cancer, esophageal cancer, cervical cancer, bladder cancer, endometrial cancer, and thyroid cancer.

[0094] In some embodiments, the sortase is selected from sortase A, sortase B, and their variants. In some embodiments, the sortase is SrtA. In some embodiments, the sortase is mgSrtA.

[0095] In some embodiments, the exogenous antigen ligand is a peptide such as a PNE peptide or a leucine zipper, or a small molecule such as a molecular marker (such as biotin, FITC, or TAMRA).

[0096] In some embodiments, the sorting tag motif is selected from LPXTG (SEQ ID NO:2), NP(Q / K)TN (SEQ ID NO:3), (I / L)(P / A)XTG (e.g., LAXTG (SEQ ID NO:7), IPXTG (SEQ ID NO:17), IAXTG (SEQ ID NO:18), LPXTG (SEQ ID NO:2)), LPNTA (SEQ ID NO:5), AALPETGXG (SEQ ID NO:1), and LAXTG (SEQ ID NO:7).

[0097] In some embodiments, sortase and an exogenous antigen are provided to the tumor sequentially, e.g., within minutes of each other. In some embodiments, sortase is provided to the tumor first and then the exogenous antigen is provided to the tumor. In some embodiments, the exogenous antigen is provided to the tumor first and then sortase is provided to the tumor. In some embodiments, a pharmaceutically acceptable composition comprising sortase is provided to the tumor first and then a pharmaceutically acceptable composition comprising the exogenous antigen is provided to the tumor. In some embodiments, a pharmaceutically acceptable composition comprising the exogenous antigen is provided to the tumor first and then a pharmaceutically acceptable composition comprising sortase is provided to the tumor.

[0098] In some embodiments, a mixture of sortase and an exogenous antigen is provided to the tumor.

[0099] In some embodiments, a pharmaceutically acceptable composition comprising a mixture of sortase and an exogenous antigen is provided to the tumor.

[0100] In some embodiments, in step (a) of the methods disclosed herein, sortase and an exogenous antigen are provided to a subject in the same composition (e.g., a mixture), and in some embodiments, in separate compositions. When in separate compositions, sortase and the exogenous antigen can be provided to the subject approximately simultaneously or sequentially.

[0101] In some embodiments, sortase and an exogenous antigen are provided to the tumor or near the tumor by injection.

[0102] In some embodiments, sortase and an exogenous antigen are provided to the tumor by intratumoral injection. For example, intratumoral administration using image-guided injection can be accomplished in most organs (8). Intratumoral immunotherapy in clinical applications can increase in situ bioavailability and the efficiency of immunotherapy (9, 10).

[0103] In some embodiments, sortase is provided to the tumor in an amount of 1 to 1000 μM, e.g., 20 to 800 μM, e.g., 400 to 500 μM.

[0104] In some embodiments, the exogenous antigen is provided to the tumor in an amount of 1 to 1000 μM, e.g., 20 to 800 μM, e.g., 400 to 500 μM.

[0105] In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is a human.

[0106] In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling regions, and a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the antigen-binding domain is capable of binding an exogenous antigen.

[0107] In some embodiments, the cytoplasmic domain comprises a CD247 cytoplasmic domain.

[0108] In some embodiments, one or more co-stimulatory signaling regions are selected from CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0109] In some embodiments, the chimeric antigen receptor further comprises one or more extracellular leader domains and / or one or more extracellular hinge domains.

[0110] In some embodiments, the antigen-binding domain is a scFv capable of binding an exogenous antigen.

[0111] In some embodiments, the Smart CAR cell is an engineered immune cell, such as a lymphocyte, such as a T cell. In some embodiments, the Smart CAR cell is an engineered immune cell, such as a T cell, NK cell, macrophage, dendritic cell, or B cell. In a preferred embodiment, the Smart CAR cell is a Smart CAR-T cell.

[0112] In some embodiments, immune cells are harvested from a subject in need thereof prior to genetic engineering with the CAR. In some embodiments, the immune cells are harvested from a subject other than a subject having a tumor. In a preferred embodiment, T cells are harvested from a subject in need thereof prior to genetic engineering with the CAR.

[0113] A therapeutically effective amount of the engineered immune cell (such as an engineered T cell) can be determined based on factors specific to the subject, including the subject's size, age, sex, weight, and condition. One of ordinary skill in the art can determine and adjust the dosage based on the present disclosure and knowledge in the art.

[0114] In some embodiments, the engineered immune cell (such as an engineered T cell) can be administered in any pharmaceutically acceptable vehicle. In some embodiments, about 1×10 5 to 1×10 12 、1×10 6 to 1×10 11 、1×10 6 to 1×10 10 、1×10 6 to 1×10 9, 1×10 7 to 1×10 11 , 1×10 7 to 1×10 10 , 1×10 7 to 1×10 9 or 1×10 8 to 1×10 9 a therapeutically effective amount of cells. In some embodiments, about 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 or 1×10 11 cells are administered to a subject with a tumor. In some embodiments, the engineered T cells can be administered in one or two doses.

[0115] In some embodiments, the purity of a population comprising engineered immune cells (e.g., engineered T cells) ranges from about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, preferably from about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. For example, the present disclosure provides a population of T cells, wherein 70% to 80% of the T cells are engineered T cells and 20% to 30% of the T cells are non-engineered T cells.

[0116] The engineered immune cells (e.g., engineered T cells) can be administered, for example, by injection or catheter, such as by intravenous injection or infusion. The engineered immune cells (e.g., engineered T cells) can also be administered by minimally invasive surgical techniques.

[0117] In some embodiments, the engineered immune cells (e.g., engineered T cells) are administered to a subject with a tumor within a predetermined time after providing the sortase and the foreign antigen to the tumor. For example, the range of the predetermined time can be 1 hour to 8 hours, 2 hours to 8 hours, 3 hours to 7 hours, 3 hours to 6 hours, 3 hours to 5 hours, etc.

[0118] In some embodiments, the present disclosure provides a Smart CAR-T system for treating tumors, as Figure 1 shown: The sortase (designated as "enzyme") and the foreign antigen (designated as "foreign Ag") are delivered to a solid tumor via injection, such as intratumoral injection; The foreign antigen is labeled on the tumor mediated by the enzyme within several hours; The Smart CAR that recognizes the foreign antigen is stably expressed on primary T cells (Smart CAR-T cells); Delivery of Smart CAR-T cells to a subject having a tumor, and Smart CAR-T cells can be activated by tumors labeled with exogenous antigens. The CAR-T system can effectively label tumors with exogenous antigens and enable effective elimination of tumors by CAR-T cells.

[0119] The Smart CAR system (e.g., Smart CAR-T system) as described herein can achieve one or more of the following advantages. The Smart CAR system (e.g., Smart CAR-T system) can induce cytotoxicity against tumor cells independent of the native tumor antigens of the tumor cells. The Smart CAR system (e.g., Smart CAR-T system) bypasses the challenges of screening for tumor antigens or scFvs that specifically bind tumor antigens. The mode of operation independent of tumor antigens prevents acquired drug resistance in Smart CAR cells (e.g., Smart CAR-T cells) due to heterogeneity and antigen loss in tumors. The tumor cell cytotoxicity induced by SmartCAR cells (e.g., Smart CAR-T cells) can contribute to the exposure of neoantigens to primary T cells. The introduction of exogenous antigens can weaken immune escape caused by tumor mutations.

[0120] The Smart CAR system and method can also be used sequentially or simultaneously in combination with one or more other therapeutics suitable for treating tumors. For example, the Smart CAR system and method can also be used in combination with, for example, chemotherapy, cytokines, growth factors, hormones, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. Tumor cell labeling

[0121] In some embodiments, the present disclosure provides a method of labeling tumor cells with an exogenous antigen, comprising providing a sortase and an exogenous antigen to the tumor cells, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein the tumor cells are sorted and labeled with the exogenous antigen.

[0122] In some embodiments, the sortase is selected from sortase A, sortase B, and variants thereof. In some embodiments, the sortase is mgSrtA.

[0123] In some embodiments, the exogenous antigen ligand is a peptide or a molecular marker.

[0124] In some embodiments, the sorting tag motif is selected from LPXTG (SEQ ID NO:2), NP(Q / K)TN (SEQ ID NO:3), (I / L)(P / A)XTG (e.g., LAXTG (SEQ ID NO:7), IPXTG (SEQ ID NO:17), IAXTG (SEQ ID NO:18), LPXTG (SEQ ID NO:2)), LPNTA (SEQ ID NO:5), AALPETGXG (SEQ ID NO:1), and LAXTG (SEQ ID NO:7).

[0125] In some embodiments, the tumor cells are selected from primary cells and immortalized cells.

[0126] In some embodiments, the sortase and the exogenous antigen are injected into or near the tumor cells.

[0127] In some embodiments, the labeling method occurs in a subject.

[0128] In some embodiments, the labeling method occurs in vitro.

[0129] In some embodiments, the sortase and the exogenous antigen are provided to the tumor cells sequentially.

[0130] In some embodiments, a mixture of the sortase and the exogenous antigen is provided to the tumor cells.

[0131] In some embodiments, the present disclosure provides a method of sorting and labeling tumor cells with an exogenous antigen by the method of labeling tumor cells described herein.

[0132] In some embodiments, the present disclosure provides tumor cells sorted and labeled with an exogenous antigen.

[0133] In some embodiments, the exogenous antigen is conjugated to the plasma membrane of the tumor cells.

[0134] In some embodiments, the present disclosure provides an exogenous antigen comprising a sorting tag motif and an exogenous antigen ligand.

[0135] In some embodiments, the exogenous antigen ligand is a peptide or a molecular marker. Smart CAR kit

[0136] In some embodiments, the present disclosure provides a Smart CAR kit, which includes an exogenous antigen and a sortase. In some embodiments, the kit includes a mixture of the exogenous antigen and the sortase.

[0137] In some embodiments, the sortase is selected from sortase A, sortase B, and variants thereof. In some embodiments, the sortase is mgSrtA.

[0138] In some embodiments, the Smart CAR kit further comprises a vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling regions, and a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the antigen-binding domain is capable of binding to an exogenous antigen described herein.

[0139] In some embodiments, a CAR comprising an antigen-binding domain capable of binding to an exogenous antigen ligand further comprises at least one other binding domain capable of binding to at least one other antigen that is endogenous or exogenous in the tumor.

[0140] In some embodiments, a CAR comprising an antigen-binding domain capable of binding to an exogenous antigen ligand further comprises a second binding domain capable of binding to an endogenous antigen in the tumor. Sortase

[0141] The sortase used herein can be any naturally occurring sortase or a functional variant thereof. There are at least six classes of sortases, including classes A, B, C, D, E, and F, as shown in the following table

[12] . Table 1. Sortase classes, substrates, and substrate recognition motifs with species specificity

[0142] As described above, a variety of sortase variants have been developed, including sortase variant (eSrtA, 5M)

[13] , Srt7M

[14] , the evolved variants of the Chen group based on the 5M variant

[15] , the " promiscuous " SrtA variant of the Chen group, mgSrtA

[16] , and the SrtA variant that recognizes LMVGG

[17] .

[0143] In some embodiments, a sortase selected from classes A, B, C, D, E, and F of sortases and any variants thereof is used to label tumor cells with an exogenous antigen. In some embodiments, a sortase selected from SrtA and any variants thereof is used to label tumor cells with an exogenous antigen. In some embodiments, a sortase selected from SrtA, mgSrtA, 5M, and Srt7M is used to label tumor cells with an exogenous antigen. CAR and engineered immune cells

[0144] In some embodiments, the present disclosure provides a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling regions, and a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the antigen-binding domain is capable of binding to an exogenous antigen described herein.

[0145] In some embodiments, a CAR comprising an antigen-binding domain capable of binding to an exogenous antigen ligand further comprises at least one other binding domain capable of binding to at least one other antigen that is endogenous or exogenous in a tumor.

[0146] In some embodiments, a CAR comprising an antigen-binding domain capable of binding to an exogenous antigen ligand further comprises a second binding domain capable of binding to an endogenous antigen in a tumor.

[0147] In some embodiments, the cytoplasmic domain comprises a CD247 cytoplasmic domain.

[0148] In some embodiments, one or more co-stimulatory signaling regions are selected from CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

[0149] In some embodiments, the chimeric antigen receptor further comprises one or more extracellular leader domains and / or one or more extracellular hinge domains. In some embodiments, one or more extracellular hinge domains include a CD28 extracellular hinge domain, a CD8a extracellular hinge domain, or an IgG4 extracellular hinge domain.

[0150] In some embodiments, the transmembrane domain includes a CD28, CD8a, CD64, CD32a, CD32c, CD16a, TLR1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLRS, or TLR9 transmembrane domain.

[0151] In some embodiments, the antigen-binding domain is a scFv capable of binding to an exogenous antigen.

[0152] In some embodiments, the present disclosure provides a vector comprising the nucleic acid described herein. Non-limiting examples of vectors include retroviral vectors, AAV vectors, and oncolytic viral vectors. In some embodiments, the vector is a lentiviral vector or comprises a lentiviral vector.

[0153] In some embodiments, the present disclosure provides an engineered immune cell expressing a CAR, the CAR comprising an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling regions, and a cytoplasmic domain comprising an ITAM, wherein the antigen-binding domain is capable of binding to an exogenous antigen described herein.

[0154] In some embodiments, the present disclosure provides engineered immune cells comprising a nucleic acid or vector encoding a CAR, the CAR comprising an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling regions, and a cytoplasmic domain comprising an ITAM, wherein the antigen-binding domain is capable of binding to an exogenous antigen described herein.

[0155] In some embodiments, the present disclosure provides a method of modifying an immune cell, comprising delivering to the immune cell a vector or nucleic acid described herein.

[0156] In some embodiments, the immune cells are selected from macrophages, monocytes, dendritic cells, T cells, B cells, and / or NK cells. In some embodiments, the immune cells are T cells, NK cells, macrophages, dendritic cells, or B cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are selected from CD4+ cells, CD8+ cells, γδ T cells (γδ T cells), NKT cells, and / or regulatory T cells (Tregs).

[0157] In some embodiments, the immune cells are immune cells harvested from a human subject having a tumor. In some embodiments, the immune cells are T cells harvested from a human subject having a tumor.

[0158] For example, white blood cells are harvested from a subject diagnosed with a tumor, T cells are isolated and cultured, and the T cells are transformed with a vector containing a nucleic acid encoding the CAR described herein. The T cells prepared for administration to a subject may include a purified cell population, such as CD4+ T cells. One of ordinary skill in the art can readily determine the percentage of genetically modified immune cells in the population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Pharmaceutical composition

[0159] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the immune cells described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0160] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the sortase described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0161] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the exogenous antigen described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0162] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a sortase and an exogenous antigen described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0163] In some embodiments, a pharmaceutical composition comprising engineered immune cells (e.g., CAR-T cells) is provided in a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. In some embodiments, the pharmaceutically acceptable carrier is selected from water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0164] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a nucleic acid or vector described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. Examples

[0165] The following examples are provided to describe the present disclosure in more detail. They are intended to illustrate rather than limit the present disclosure. The results reported in Examples 1 and 2 and the experimental procedures described in Examples 3-13 demonstrate that the Smart CAR-T system disclosed herein can effectively target and treat tumors in vitro and in vivo. Example 1: mSA-Smart CAR-T System

[0166] In the mSA-Smart CAR-T system, tumor cells are labeled with an exogenous antigen comprising biotin and a sortase motif, and CAR-T cells (referred to as "mSA-CAR-T cells") are engineered to express a CAR comprising an affinity-enhanced monomeric streptavidin (mSA) biotin-binding domain that is capable of recognizing and binding biotin. Figure 2 The vector design of the SmartCAR module is shown, where the affinity-enhanced monomeric streptavidin (mSA) biotin-binding domain incorporates components from a typical CAR, including a hinge domain, transmembrane domain, co-stimulatory domain, and CD247. A tagBFP (blue fluorescent protein) marker is included to identify cells transduced by the Smart CAR vector ( Figure 2 ). tagBFP is a basic (constitutively fluorescent) blue fluorescent protein derived from the sea anemone Entacmaea quadricolor.

[0167]

[0168] As Figure 2 shown and as set forth in the sequence of SEQ ID NO:11, nucleotides 1 to 1179 in SEQ ID NO:11 represent EF-1a, nucleotides 1197 to 1259 encode LP (leader peptide), nucleotides 1260 to 1625 encode mSA, nucleotides 1638 to 1673 encode the hinge, nucleotides 1674 to 1745 encode the transmembrane domain, nucleotides 1746 to 1871 encode the co-stimulatory domain, nucleotides 1872 to 2207 encode CD247, nucleotides 2223 to 2276 encode T2A, and nucleotides 2277 to 2975 encode tagBFP. Alternative nucleotide codons can be used to generate the Smart CAR system.

[0169] As Figure 2 shown, the Smart CAR vector also contains the first intron of human elongation factor 1α (EF-1α), which can highly enhance the expression of foreign genes in mammalian cells.

[0170] As Figure 2 shown, the Smart CAR vector contains a component encoding a leader peptide (LP), which typically has a sequence of 16 to 20 amino acids at the N-terminus of some eukaryotic proteins, and this sequence determines the final destination of these eukaryotic proteins. Proteins that are produced and function in the cytosol lack a leader peptide. Proteins destined for specific organelles require signal sequences suitable for each organelle. The leader peptide for proteins destined for entry into the endoplasmic reticulum usually contains hydrophobic amino acids that are embedded in the lipid bilayer membrane, and its function is to direct the nascent protein to a receptor protein that marks the position of the pore in the membrane. Once the protein enters the cysternal lumen through the pore, the leader peptide is cleaved from the protein. In plasmid design, the leader peptide is usually located between the promoter and the coding region.

[0171] As Figure 2 shown, the Smart CAR vector also contains a component encoding T2A, which is a self-cleaving 2A peptide. The average length of T2A is 18 - 22 amino acids. T2A can be used to express more than one gene in cells. There is a high cleavage efficiency between the upstream and downstream genes of the T2A peptide. Quality inspection of mSA-CAR-T cells

[0172] Monitoring CAR expression and cell proliferation on T cells. The expression of mSA-CAR on the surface of T cells was detected using FITC-conjugated peptides modified with biotin at the N-terminus or C-terminus (Figure 3). Two FITC- and biotin-labeled peptides, biotin-GGGGK(FITC)-NH2 (SEQ ID NO:8) and FITC-AhX-GGGGK(biotin)-NH2 (SEQ ID NO:9), were synthesized for quantifying mSA-CAR expression on the surface of T cells. The fluorescent biotin-labeled peptides could bind to mSA-CAR-T with high efficiency regardless of the position of biotin modification at the peptide terminus.

[0173] After transducing T cells with mSA-CAR, biotin-GGGGK(FITC)-NH2 (SEQ ID NO:8) or FITC-AhX-GGGGK(biotin)-NH2 (SEQ ID NO:9) was added to the cell suspension and the cells were incubated. After washing with PBS, the transduction efficiency and mSA-CAR expression were examined by flow cytometry.

[0174] As Figure 3A shown, the transduction efficiency of the mSA-CAR vector into T cells was represented by the signal of tagBFP. The expression of mSA-CAR on the surface of T cells was represented by the signal of FITC. As Figure 3A shown, the fluorescence signals remained stable on the 2nd, 8th, and 11th days after transduction, indicating stable and consistent mSA-CAR expression on the surface of T cells.

[0175] We also found that mSA-CAR expression did not affect the proliferation or viability of T cells ( Figure 3B and 3C ). Labeling efficiency of exogenous antigens on tumor cells via mgSrtA in vitro

[0176] The biotinylated exogenous antigen sorting label was first mixed with mgSrtA in PBS buffer, and then tumor cells were added to the PBS buffer containing sortase and exogenous antigen for sorting and labeling. After incubation with PE-streptavidin, the labeling efficiency in tumor cells was detected. Multiple tumor cell lines showed efficient labeling by exogenous antigens in vitro within 30 minutes, which was confirmed and quantified by flow cytometry ( Figure 4A ). Labeling efficiency of exogenous antigens on tumor cells via mgSrtA in vivo

[0177] We established a cell line-derived xenograft (CDX) model to detect in vivo labeling by exogenous antigens. For the sorting label of Hela tumor cells, a single dose (40 μl volume containing 100 μM mgSrtA and 2 mM biotin-AALPETG*G (SEQ ID NO:14)) was injected intratumorally; for the sorting label of MDA-MB-231 tumor cells, intratumoral injections were given every 2 days for a total of 3 times. At certain time points (such as Figure 4B shown), the sorted-labeled tumors were collected and cut into small pieces. After homogenization through a 100-μm filter, the tumor cell suspension was collected and PE-streptavidin was added for the detection of biotin-AALPETG*G (SEQ ID NO:14) via flow cytometry.

[0178] After a single intratumoral injection, biotin signals of successfully labeled antigens were detected on tumor cells 6 h later and gradually decreased within 48 h ( Figure 4B , left). After three intratumoral injections, the biotin signals on tumor cells persisted for more than one week ( Figure 4B , right).

[0179] In the "activation phenotype assay", the expression of two activation markers, CD25 and CD69, on the surface of T cells was detected by flow cytometry. After in vitro sorting enzyme-mediated labeling, 50,000 sorted-labeled tumor cells were co-cultured with mSA-CAR-T cells at an E:T ratio of 2:1 (E refers to effector cells, such as engineered T cells; T refers to target cells, such as tumor cells) in 200 μl of RPMI-1640 in each falcon tube. After co-culture for 16 - 20 h, the cell pellets were collected for the detection of the expression of surface CD25 and CD69, which was detected via flow cytometry using anti-CD25 and anti-CD69 antibodies (Biolegend). mSA-Smart-CAR T cells successfully recognized the exogenous antigen sorted and labeled onto tumor cells. The mSA-CAR-T cells were subsequently activated, which led to an increase in the surface expression of CD25 and CD69 ( Figure 5A ).

[0180] In the "cytokine release assay", the concentrations of cytotoxic factors, including perforin, granzyme A, granzyme B, and IFN-γ, were detected in the supernatant of the co-culture of tumor cells and mSA-CAR-T cells ( Figure 5B) After in vitro sortase-mediated labeling, 50,000 sorted-labeled tumor cells were co-cultured with mSA-CAR-T cells at an E:T ratio of 2:1 in 200 μl of RPMI-1640 in each Falcon tube. After 16 - 20 hours, the supernatant was collected and cytokine release assay was performed via flow cytometry (Legendplex human CD8 / NK panel, Biolegend).

[0181] For the "cytotoxicity assay", we engineered the target tumor cells to stably express luciferase. Luciferase was introduced to indicate the number of surviving tumor cells after co-culture with mSA-CAR-T cells. After in vitro sortase-mediated labeling, 2000 sorted-labeled tumor target cells were co-cultured with mSA-CAR-T cells at different E:T ratios of 8:1, 4:1, 2:1, 1:1, and 1:2 in 50 μl of PRMI-10% FBS in each well of a 384-well plate. After 16 - 20 hours, the medium was removed, 25 μl was left in each well, and then 25 μl of the prepared luciferase detection reagent (L00877C, Genescript) was added to each well. Measurement was performed using a Varioskan LUX microplate reader (Thermo).

[0182] The cytotoxicity of the target tumor cells was calculated as follows: Specific cytotoxicity = 100 × [(luminescence of remaining tumor cells after lysis with blank T cells - luminescence of remaining tumor cells after lysis with mSA-CAR-T cells) / (luminescence of remaining tumor cells after lysis with blank T cells)]; Non-specific cytotoxicity = 100 × [(total target cell luminescence - luminescence of remaining tumor cells after lysis with blank T cells) / (total target cell luminescence)]. The non-specific cytotoxicity should be controlled within ±20%. Luciferin (substrate of luciferase) was added after 16 hours of co-culture, and bioluminescence was measured and converted into a percentage of specific cytotoxicity.

[0183] Obviously, the luciferase signal was negatively correlated with the cytotoxic activity of mSA-CAR-T cells. All three tumor cell lines tested in this assay showed strong cytotoxicity after co-culture with mSA-CAR-T cells, indicating the effectiveness of the mSA-Smart CAR-T system ( Figure 5C )。 Example 2: PNEscFv-Smart CAR-T System

[0184] We demonstrated that tumor cells labeled with exogenous antigens linked to a sorting tag motif (e.g., -AALPETG*G (SEQ ID NO: 19)) were recognized and eliminated by Smart CAR-T cells expressing the corresponding scFv that binds to the exogenous antigen.

[0185] A 14-amino acid sequence from the yeast transcription factor GCN4 (peptide neoepitope, PNE) was used as the exogenous antigen in this Smart CAR-T system (PNE-CAR-T). PNE and the corresponding high-affinity scFv have been used in the past to establish switchable CAR-T cells (11). In the PNEscfv-Smart CAR-T system, as shown by the Smart CAR module in Figure 7 , the PNE scFv was incorporated with the hinge domain, transmembrane domain, co-stimulatory domain, and CD247. A tagBFP (blue fluorescent protein) marker was included to identify transduced T cells ( Figure 7 ). Similar to the demonstration of the mSA-CAR-T system, we performed multiple experiments to show the effectiveness of the PNEscFv-CAR-T system.

[0186]

[0187] As Figure 7 shown and as set forth in the sequence of SEQ ID NO:12, nucleotides 1 to 1179 in SEQ ID NO:12 represent EF-1a, nucleotides 1197 to 1259 encode LP (leader peptide), nucleotides 1260 to 1976 encode PNEscFv, nucleotides 1989 to 2024 encode the hinge, nucleotides 2025 to 2096 encode the transmembrane domain, nucleotides 2097 to 2222 encode the co-stimulatory domain, nucleotides 2223 to 2558 encode CD247, nucleotides 2574 to 2627 encode T2A, and nucleotides 2628 to 3326 encode tagBFP. Alternative nucleotide codons can be used to generate the PNEscfv-Smart CAR system.

[0188] We also found that PNE-CAR-T expression did not affect T cell proliferation ( Figure 10A ) or viability ( Figure 10B ).

[0189] We examined the expression of PNEscFv-CAR on the surface of T cells. Biotin-PNE-AALPETG*G was used followed by anti-biotin-PE to detect PNE scFv-CAR expression. After transducing T cells with PNEscFv-Smart CAR, biotin-PNE-AALPETG*G was added to the T cell suspension and the cell suspension was incubated, and then anti-biotin-PE was added after washing with PBS. Transduction efficiency and PNEscFv-CAR expression were detected by flow cytometry after washing with PBS. As Figure 8A and 11A shown, PNEscFv-CAR was stably expressed on the surface of T cells.

[0190] We successfully labeled PNE foreign antigens to tumor cells in vitro ( Figure 8B and 11B ). Tumor cells were added to a PBS buffer containing sortase and biotin-PNE-AALPETG*G (foreign antigen) for sorting and labeling. Then, the labeling efficiency in tumor cells was detected after incubation with anti-biotin-PE.

[0191] Next, we investigated the cytotoxicity induced by PNEscFv-Smart CAR-T cells against sorted marker-expressing tumor cells. After in vitro sortase-mediated labeling, 500,000 sorted marker-expressing tumor target cells were co-cultured with PNEscFv-CAR-T cells at an E:T ratio of 2:1 in 200 μl of RPMI-1640 in each falcon tube. After 16 - 20 h, cell pellets were collected and the expression of surface CD25 and CD69 was detected by flow cytometry (Biolegend). A similar assay as described in Example 1 was used to determine the cytotoxicity induced by PNEscFv-Smart CAR-T cells. For example, in the "activation phenotype assay", the expression of two activation markers, CD25 and CD69, on the T cell surface was detected by flow cytometry. After PNE-AALPETG*G (SEQ ID NO:16) labeling, PNE on the tumor cells specifically activated PNEscFv-CAR T cells through antigen recognition, which was indicated by an increase in CD25 and CD69 surface expression ( Figure 8C and 11C ).

[0192] In the cytokine release assay, the concentrations of cytotoxic factors, including perforin, IL-2, granzyme B, and IFN-γ, were detected in the supernatant of co-cultured tumor cells and PNEscFv-CAR-T cells ( Figure 12 ). After in vitro sortase-mediated labeling, 50,000 sorted marker-expressing tumor cells were co-cultured with PNEscFv-CAR-T cells at an E:T ratio of 2:1 in 200 μl of RPMI-1640 in each falcon tube. After 16 - 20 h, the supernatant was collected and the cytokine release assay was performed by flow cytometry (Legendplex human CD8 / NK panel, Biolegend).

[0193] For cytotoxicity assays, we engineered target tumor cells to stably express luciferase. The luciferase was introduced to indicate the number of viable tumor cells after co - culture with PNEscFv - CAR - T cells. After in vitro sortase - mediated labeling, 2000 sorted and labeled tumor target cells were co - cultured with PNEscFv - CAR - T cells at different E:T ratios of 8:1, 4:1, 2:1, 1:1, 1:2 in 50 μl of PRMI - 10% FBS per well in a 384 - well plate. After 16 - 20 h, the culture medium was removed, 25 μl was left in each well, and then 25 μl of the prepared luciferase detection reagent (L00877C, Genescript) was added to each well. Measurements were performed using a Varioskan LUX microplate reader (Thermo).

[0194] The cytotoxicity of the target tumor cells was calculated as follows: Specific cytotoxicity = 100×[(luminescence of remaining tumor cells after lysis with blank T cells - luminescence of remaining tumor cells after lysis with PNEscFv - CAR - T cells) / (luminescence of remaining tumor cells after lysis with blank T cells)]; Non - specific cytotoxicity = 100×[(total target cell luminescence - luminescence of remaining tumor cells after lysis with blank T cells) / (total target cell luminescence)]. The non - specific cytotoxicity should be controlled within ±20%. Luciferin (the substrate of luciferase) was added 16 h after co - culture, and the bioluminescence was measured and converted to a percentage of specific cytotoxicity. Apparently, the luciferase signal was negatively correlated with the cytotoxic activity of PNEscFv - CAR - T cells. All three tumor cell lines tested in this assay showed strong cytotoxicity after co - culture with PNEscFv - CAR - T cells, indicating the effectiveness of the PNEscFv - CAR - T system( Figure 13 ). In animal models, we found that compared with UN - T treatment, treatment with PNEscFv - CAR - T and intratumoral injection of the sorted - label mixture could significantly control tumor size( Figure 9A 、 9B and 14). Example 3: Bi - CAR - T system

[0195] We demonstrated that low - antigen - expressing tumor cells labeled with an exogenous label linked to a sort - tag motif (e.g., - AALPETG*G) were recognized and eliminated by Bi - CAR - T cells that express a PNEscFv that binds to the exogenous label and an additional scFv that recognizes a tumor - associated antigen (e.g., CLDN18.2 here).

[0196] In the Bi - CAR - T system, as Figure 15AAs shown in the Smart CAR module, the PNE scFv and the CLDN18.2 scFv are combined with a hinge domain, a transmembrane domain, a co-stimulatory domain, and CD247. A tagBFP marker is included to identify transduced T cells.

[0197]

[0198] As Figure 15B shown and as set forth in the sequence of SEQ ID NO:13, nucleotides 1 to 1179 in SEQ ID NO:13 represent EF-1a, nucleotides 1197 to 1259 encode LP (leader peptide), nucleotides 1260 to 1613 encode the heavy chain of CLDN18.2 scFv, nucleotides 1629 to 1955 encode the light chain of PNEscFv, nucleotides 2019 to 2345 encode the heavy chain of PNEscFv, nucleotides 2361 to 2699 encode the light chain of CLDN18.2 scFv, nucleotides 2712 to 2747 encode the hinge, nucleotides 2748 to 2819 encode the transmembrane domain, nucleotides 2820 to 2945 encode the co-stimulatory domain, nucleotides 2946 to 3281 encode CD247, nucleotides 3297 to 3350 encode T2A, and nucleotides 3351 to 4049 encode tagBFP. Alternative nucleotide codons can be used to generate the Bi-CAR system.

[0199] In an animal model carrying tumors with low antigen expression (10% of tumor cells express CLDN18.2), we found that compared with UN-T treatment, treatment with PNE / CLGN18.2-CAR-T (Bi-CAR-T) and intratumoral injection of the sorting marker mixture could significantly control tumor size ( Figure 16 ). Example 4: Cell Line Preparation

[0200] Generate a lentiviral vector (lenti-EF-1α-luciferase-T2A-HygR) carrying the intracellular luciferase gene. Then, transduce various tumor cell lines with the lentivirus carrying luciferase. Forty-eight hours after transduction, select the cells by adding hygromycin to the medium. Then, maintain the cell lines expressing luciferase in a medium containing 2 μg / ml hygromycin. Example 5: Primary T Cell Isolation and Culture

[0201] Human PBMCs from healthy donors were obtained from Shanghai Liquan Hospital and isolated by Milestone Biotechnologies, which has been approved for research purposes. Human pan T cells, including CD4+ T cells and CD8+ T cells, were isolated from human PBMCs via negative selection (130-096-535, Miltenyi). For long-term culture, T cells were cultured in RPMI-10% FBS containing only 100 U / ml recombinant human IL-2. T cells were cryopreserved in In CS10 (07930, Stemcell Technologies). Example 6: Packaging and transduction of lentivirus in T cells

[0202] Lentivirus was packaged via transfection plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G. Viral supernatants were collected at 48 h and 72 h after transfection, passed through a 0.45 μm filter, and concentrated by ultracentrifugation at 70,000 g for 2 h at 4°C. Subsequently, the concentrated supernatant was aliquoted and stored at -80°C. Human T cells were thawed 2 days before transduction and cultured in RPMI-10% FBS (Gibco) containing T cell activator (130-111-160, Miltenyi) and 100 U / ml recombinant human IL-2 (200-02, Peprotech). After 48 h, lentivirus transduction was performed during a 2 h spin infection at 2,000 g at RT in the presence of 4 mg / ml Lentiboost-P. After spin infection, the T cells were cultured overnight at 37°C and transferred to G-REX for long-term culture. At different time points after transduction, T cells were collected and transduction efficiency and CAR expression were detected via flow cytometry. Example 7: Detection of transduction rate and Smart-CAR expression

[0203] CAR expression on Smart-CAR T cells was monitored at different days after transduction. In the mSA-Smart CAR-T system, FITC-AhX-GGGGK (biotin)-NH2 (SEQ ID NO:9) was synthesized as a detector. 1 μM of the detector was added to the cell suspension, and the cell suspension was incubated at 4°C for 20 min. After washing with PBS, transduction rate and Smart-CAR expression on the T cell surface were detected via flow cytometry (CytoflexS, Beckman). In the PNE-Smart CAR-T system, a PNE peptide with biotin modification was synthesized as a detector. 1 μM of the detector was added to the cell suspension and incubated with the cells at 4°C for 20 min. After washing with PBS, anti-biotin-PE (Biolegend) was added to the cell suspension and incubated with the cells at 4°C for 20 min. Then, the cells were washed again with PBS. Transduction rate and Smart-CAR expression on the cell surface were detected via flow cytometry. Example 8: Generation of sortase

[0204] Express and purify sortase (mgSrtA) from transformed Escherichia coli BL21(DE3). Clone the DNA sequence encoding mgSrtA into the pET-28a(+) vector and express it in Escherichia coli BL21(DE3) cells. Induce the transformed cells with 1 mM IPTG until the OD600 reaches 0.6. Incubate the cells at 37 °C for an additional 3 - 4 hours, then harvest by centrifugation and resuspend in lysis buffer (20 mM Tris-HCl, pH 7.8, and 500 mM NaCl). Load the sonicated lysate onto a Ni-NTA column (Histrap 5 ml, GE Healthcare), wash it with 30 ml of wash buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl, and 40 mM imidazole), and then elute the mgSrtA protein with elution buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl, and 250 mM imidazole). Add glycerol to the purified mgSrtA protein to a final concentration of 10%, aliquot the mgSrtA sample, and store it at -80 °C. Example 9: Perform cell surface sorting labeling in vitro

[0205] Carry out the sorting labeling reaction in PBS with a total volume of 50 μl at 37 °C for 30 min. Unless otherwise specified, use a final concentration of 20 μM sortase and 200 μM sorting label substrate (exogenous antigen), with a cell density of 1X10 7 / ml. After performing the sorting labeling, wash the cells 3 times with PBS. For detection of labeling efficiency, incubate the cells with anti-biotin-PE (Biolegend) or PE-streptavidin (Biolegend) on ice for 15 min, wash again, and then analyze via flow cytometry. Example 10: Perform cell sorting labeling in vivo

[0206] In each injection dose, mix mgSrtA and biotinylated sorting label at 100 μM and 2 mM respectively in 40 μl PBS. For sorting labeling of Hela, give 1 dose of intratumoral injection; for sorting labeling of MDA-MB-231, give intratumoral injection every 2 days for a total of 3 times. At the given specified time points, collect the tumors and cut them into small pieces. After mashing through a 100 μm filter, collect the tumor cell suspension and wash it with PBS. Then, add PE-streptavidin or anti-biotin-PE to the cell suspension and incubate with the cells at 4 °C for 20 min. Then, wash the cells again with PBS and detect biotin on the cell surface via flow cytometry. Example 11: Cytokine Release Assay and Activation Assay of Smart-CAR T Cells

[0207] As Figure 17 shown in the workflow, after in vitro sortase-mediated labeling, 5×10 5 sorted and labeled tumor target cells were co-cultured with Smart-CAR T cells at an E:T ratio of 2:1 in 200 μl of RPMI-1640 in each falcon tube. After 16 - 20 h, the supernatant was collected for cytokine release assay (Legendplex human CD8 / NK panel, Biolegend), and the cell pellet was collected for detection of the expression of surface CD25 and CD69 by flow cytometry. Example 12: Cytotoxicity Assay of Smart-CAR T Cells

[0208] As Figure 18 shown in the workflow, after in vitro sortase-mediated labeling, 2×10 3 sorted and labeled tumor target cells were co-cultured with Smart-CAR T cells at an E:T ratio of 8:1 in 50 μl of PRMI-10% FBS in each well of a 384-well plate. After 16 - 20 h, the medium was removed, 25 μl was left in each well, and then 25 μl of the prepared luciferase reagent (L00877C, Genescript) was added to each well. Measurement was performed using a Varioskan LUX microplate reader (Thermo). The cytotoxicity of the target tumor cells was calculated as follows: Specific cytotoxicity = 100×[(luminescence of remaining cells after lysis with blank T cells - luminescence of remaining cells after lysis with Smart-CAR T cells) / (luminescence of remaining cells after lysis with blank T cells)]; Non-specific cytotoxicity = 100×[(total luminescence of target cells - luminescence of remaining cells after lysis with blank T cells) / (total luminescence of target cells)]. The non-specific cytotoxicity should be controlled within ±20%. Example 13: Xenograft Mouse Model

[0209] As Figure 19 shown in the workflow for animal experiments. NOD-Prkdc scid Il2rg null / Shjh (NPSG) mice were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd. and were housed under standard single-ventilated and pathogen-free conditions at the Experimental Animal Resource Center of Westlake University. Female NOD-Prkdc scid Il2rgnull Tumor transplantation was performed in Shjh (NPSG) mice. The use of all mice complied with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Westlake University. Tumor cells expressing luciferase were mixed with Matrigel (1:1 volume) and injected subcutaneously into the right side of NPSG mice at 0.5×10 6 or 1×10 6 cells per recipient. At 7 - 10 days later or when the tumor burden reached 50 mm 3 , 3×10 6 Smart-CAR T cells (including mSA-CAR-T cells, PNEscFv-CAR-T cells, or PNE / CLDN18.2-CAR-T cells) were adoptively transferred into each recipient via tail vein injection.

[0210] Twenty-four hours later, the mice were given intratumoral injections of the mgSrtA and biotin-AALPETG*G mixture (SEQ ID NO:14) (or the mgSrtA and PNE-AALPETG*G (SEQ ID NO:16) mixture) multiple times every 3 days. For each mouse, the mixture was prepared in 40 ul PBS for each dose, with a final concentration of 10 uM for mgSrtA and 200 μM for the peptide substrate. Body weight loss and tumor size were measured every 3 days after T cell injection.

[0211] Further embodiments are described below. 1. A method of treating a tumor in a subject in need thereof, comprising: providing a sortase and an exogenous antigen to the tumor, wherein the exogenous antigen comprises an exogenous antigen ligand and a sort tag motif, and wherein cells in the tumor are sort-tagged by the exogenous antigen, and administering a therapeutically effective amount of genetically engineered immune cells expressing a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-binding domain capable of binding the exogenous antigen. 2. The method of embodiment 1, wherein the CAR further comprises a binding domain capable of binding an endogenous antigen in the tumor. 3. The method of embodiment 2, wherein the endogenous antigen is claudin8.2. 4. The method of any one of embodiments 1 - 3, wherein the immune cells are T cells. 5. The method of any one of embodiments 1 - 4, wherein the immune cells are harvested from the subject before being genetically engineered with the CAR. 6. The method of any one of embodiments 1 - 5, wherein the sortase is selected from sortase A, sortase B, and variants thereof. 7. The method of any one of embodiments 1-6, wherein the sortase is mgSrtA. 8. The method of any one of embodiments 1-7, wherein the exogenous antigen ligand is a peptide or a molecular marker. 9. The method of any one of embodiments 1-8, wherein the sorting tag motif is selected from LPXTG (SEQ ID NO:2), NP(Q / K)TN (SEQ ID NO:3), (I / L)(P / A)XTG (e.g., LAXTG (SEQ ID NO:7)), IPXTG (SEQ ID NO:17), IAXTG (SEQ ID NO:18), LPXTG (SEQ ID NO:2)), LPNTA (SEQ ID NO:5), AALPETGXG (SEQ ID NO:1) and LAXTG (SEQ ID NO:7). 10. The method of any one of embodiments 1-9, wherein the sortase and the exogenous antigen are provided to the tumor or near the tumor by injection. 11. The method of any one of embodiments 1-9, wherein the sortase and the exogenous antigen are provided to the tumor by intratumoral injection. 12. The method of any one of embodiments 1-11, wherein the subject is a human. 13. The method of any one of embodiments 1-12, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling regions, and a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the antigen-binding domain is capable of binding the exogenous antigen. 14. The method of any one of embodiments 1-13, wherein the antigen-binding domain is a ScFv capable of binding the exogenous antigen. 15. The method of any one of embodiments 1-14, wherein the sortase and the exogenous antigen are provided to the tumor sequentially. 16. The method of any one of embodiments 1-14, wherein a mixture of the sortase and the exogenous antigen is provided to the tumor. 17. A method for labeling tumor cells with an exogenous antigen, comprising providing a sortase and an exogenous antigen to the tumor cells, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein the tumor cells are sorted and labeled with the exogenous antigen. 18. The method of embodiment 17, wherein the sortase is selected from sortase A, sortase B, and variants thereof. 19. The method of any one of embodiments 17-18, wherein the sortase is mgSrtA. The method according to any one of embodiments 17 - 19, wherein the exogenous antigen ligand is a peptide or a molecular marker. The method according to any one of embodiments 17 - 20, wherein the sorting tag motif is selected from LPXTG (SEQ ID NO:2), NP(Q / K)TN (SEQ ID NO:3), (I / L)(P / A)XTG (such as LAXTG (SEQ ID NO:7), IPXTG (SEQ ID NO:17), IAXTG (SEQ ID NO:18), LPXTG (SEQ ID NO:2)), LPNTA (SEQ ID NO:5), AALPETGXG (SEQ ID NO:1), and LAXTG (SEQ ID NO:7). The method according to any one of embodiments 17 - 21, wherein the tumor cells are selected from primary cells and immortalized cells. The method according to any one of embodiments 17 - 22, wherein the sortase and the exogenous antigen are injected into or near the tumor cells. The method according to any one of embodiments 17 - 23, wherein the labeling method occurs in a subject. The method according to any one of embodiments 17 - 23, wherein the labeling method occurs in vitro. The method according to any one of embodiments 17 - 25, wherein the sortase and the exogenous antigen are provided to the tumor cells sequentially. The method according to any one of embodiments 17 - 25, wherein a mixture of the sortase and the exogenous antigen is provided to the tumor cells. A tumor cell sorted and labeled with an exogenous antigen by the method according to any one of embodiments 17 - 27. A tumor cell sorted and labeled with an exogenous antigen. The tumor cell according to any one of embodiments 28 - 29, wherein the exogenous antigen is conjugated to the plasma membrane of the tumor cell. An exogenous antigen comprising a sorting tag motif and an exogenous antigen ligand. The exogenous antigen according to embodiment 31, wherein the exogenous antigen ligand is a peptide or a molecular marker. A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen - binding domain, a transmembrane domain, one or more co - stimulatory domains, and a cytoplasmic domain comprising an immunoreceptor tyrosine - based activation motif (ITAM), and wherein the antigen - binding domain is capable of binding to the exogenous antigen according to any one of embodiments 31 - 32. 34. The nucleic acid of embodiment 33, wherein the CAR further comprises a binding domain capable of binding an endogenous antigen in a tumor. 35. The nucleic acid of embodiment 34, wherein the endogenous antigen is claudin8.2. 36. The nucleic acid of any one of embodiments 33-35, wherein the cytoplasmic domain comprises a CD247 cytoplasmic domain. 37. The nucleic acid of any one of embodiments 34-36, wherein the one or more co-stimulatory signaling regions are selected from CD28, CD27, CD134 (OX40), and CD137 (4-1BB). 38. A vector comprising the nucleic acid of any one of embodiments 33-37. 39. A human immune cell comprising the nucleic acid of any one of embodiments 33-37 or the vector of embodiment 38. 40. The human immune cell of embodiment 39, wherein the human immune cell is a T cell from a human subject suffering from a tumor. 41. A pharmaceutical composition comprising a population of the human immune cells of embodiment 39 or 40. 42. A method of modifying an immune cell, comprising delivering the vector of embodiment 38 or the nucleic acid of any one of embodiments 33-37 to the immune cell. 43. A kit comprising sortase and an exogenous antigen of any one of embodiments 31-32, wherein the kit optionally further comprises the nucleic acid of embodiments 33-37 or the vector of embodiment 38. 44. A genetically engineered immune cell for treating a tumor, wherein cells in the tumor are sorted and labeled by an exogenous antigen mediated by sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting and labeling motif, and wherein the genetically engineered immune cell expresses a chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding the exogenous antigen. 45. The genetically engineered immune cell for treating a tumor of embodiment 44, wherein the CAR further comprises a binding domain capable of binding an endogenous antigen in the tumor. 46. Use of a genetically engineered immune cell in the preparation of a medicament for treating a tumor, wherein cells in the tumor are sorted and labeled by an exogenous antigen mediated by sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting and labeling motif, and wherein the genetically engineered immune cell expresses a chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding the exogenous antigen. Use of the genetically engineered immune cells of embodiment 46 for the preparation of a medicament for treating a tumor, wherein the CAR further comprises a binding domain capable of binding an endogenous antigen in the tumor.

[0212] Although the present disclosure has been specifically shown and described with reference to specific embodiments, those skilled in the art should understand that various changes may be made in its form and details without departing from the spirit and scope of the present disclosure as disclosed herein. References 1. Martinez M, Moon EK. CAR T Cells for Solid Tumors: New Strategies for Finding, Infiltrating, and Surviving in the Tumor Microenvironment. Front Immunol. 2019;10:128. 2. Marofi F, Motavalli R, Safonov VA, Thangavelu L, Yumashev AV, Alexander M, et al. CAR T cells in solid tumors: challenges and opportunities. Stem Cell Res Ther. 2021;12(1):81. 3. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell. 2017;168(4):707-23. 4. Philip M, Schietinger A. CD8(+) T cell differentiation and dysfunction in cancer. Nat Rev Immunol. 2021. 5. Pishesha N, Ingram JR, Ploegh HL. Sortase A: A Model for Transpeptidation and Its Biological Applications. Annu Rev Cell Dev Biol. 2018;34:163-88. 6. Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV. Crystal structures of Staphylococcus aureus sortase A and its substrate complex. J Biol Chem. 2004;279(30):31383-9. 7. Ge Y, Chen L, Liu S, Zhao J, Zhang H, Chen PR. Enzyme-Mediated Intercellular Proximity Labeling for Detecting Cell-Cell Interactions. J Am Chem Soc. 2019;141(5):1833-7. 8. Marabelle A, Tselikas L, de Baere T, Houot R. Intratumoral immunotherapy: using the tumor as the remedy. Ann Oncol. 2017;28(suppl_12):xii33-xii43. 9. Melero I, Castanon E, Alvarez M, Champiat S, Marabelle A. Intratumoural administration and tumour tissue targeting of cancer immunotherapies. Nat Rev Clin Oncol. 2021;18(9):558-76. 10. Champiat S, Tselikas L, Farhane S, Raoult T, Texier M, Lanoy E, et al. Intratumoral Immunotherapy: From Trial Design to Clinical Practice. Clin Cancer Res. 2021;27(3):665-79. 11. Rodgers DT, Mazagova M, Hampton EN, Cao Y, Ramadoss NS, Hardy IR, et al. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies. Proc Natl Acad Sci U S A. 2016;113(4):E459-68. 12. Bradshaw, W.J. et al. Molecular features of the sortase enzyme family. FEBS J 282, 2097-2114 (2015). 13. Chen, I., Dorr, B.M. & Liu, D.R. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci U S A 108, 11399-11404 (2011). 14. Glasgow, J.E., Salit, M.L. & Cochran, J.R. In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant. J Am Chem Soc 138, 7496-7499 (2016). 15. Chen, L. et al. Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency. Sci Rep 6, 31899 (2016). 16. Ge, Y. et al. Enzyme-Mediated Intercellular Proximity Labeling for Detecting Cell-Cell Interactions. J Am Chem Soc 141, 1833-1837 (2019). 17. Podracky, C.J. et al. Laboratory evolution of a sortase enzyme that modifies amyloid-beta protein. Nat Chem Biol 17, 317 - 325 (2021).

Claims

1. A method for treating a tumor in a subject in need thereof, comprising: providing a sortase and an exogenous antigen to the tumor, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein cells in the tumor are sorted and labeled with the exogenous antigen, and administering a therapeutically effective amount of genetically engineered immune cells expressing a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-binding domain capable of binding the exogenous antigen ligand.

2. The method of claim 1, wherein the CAR further comprises a binding domain capable of binding an endogenous antigen in the tumor.

3. The method of claim 2, wherein the endogenous antigen is claudin8.

2.

4. The method of any one of claims 1-3, wherein the immune cells are T cells, NK cells, macrophages, dendritic cells or B cells.

5. The method of any one of claims 1-4, wherein the immune cells are harvested from the subject prior to being genetically engineered with the CAR.

6. The method of any one of claims 1-5, wherein the sortase is selected from sortase A, sortase B and variants thereof.

7. The method of any one of claims 1-6, wherein the sortase is mgSrtA.

8. The method of any one of claims 1-7, wherein the exogenous antigen ligand is a peptide or a molecular marker.

9. The method of any one of claims 1-8, wherein the sorting tag motif is selected from LPXTG (SEQ ID NO:2), NP(Q / K)TN (SEQ ID NO:3), (I / L)(P / A)XTG, LPNTA (SEQ ID NO:5), AALPETGXG (SEQ ID NO:1) and LAXTG (SEQ ID NO:7).

10. The method of any one of claims 1-9, wherein the sortase and the exogenous antigen are provided to the tumor or near the tumor by injection.

11. The method of any one of claims 1-9, wherein the sortase and the exogenous antigen are provided to the tumor by intratumoral injection.

12. The method of any one of claims 1-11, wherein the subject is a human.

13. The method of any one of claims 1-12, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, one or more co-stimulatory signaling regions, and a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the antigen-binding domain is capable of binding the exogenous antigen.

14. The method of any one of claims 1-13, wherein the antigen-binding domain is a ScFv capable of binding the exogenous antigen.

15. The method of any one of claims 1-14, wherein the sortase and the exogenous antigen are provided to the tumor sequentially.

16. The method of any one of claims 1-14, wherein a mixture of the sortase and the exogenous antigen is provided to the tumor.

17. A method for labeling tumor cells with an exogenous antigen, comprising providing a sortase and an exogenous antigen to the tumor cells wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting tag motif, and wherein tumor cells are sorted and labeled by the exogenous antigen.

18. The method of claim 17, wherein the sortase is selected from sortase A, sortase B, and variants thereof.

19. The method of any one of claims 17-18, wherein the sortase is mgSrtA.

20. The method of any one of claims 17-19, wherein the exogenous antigen ligand is a peptide or a molecular marker.

21. The method of any one of claims 17-20, wherein the sorting tag motif is selected from LPXTG (SEQ ID NO:2), NP(Q / K)TN (SEQ ID NO:3), (I / L)(P / A)XTG, LPNTA (SEQ ID NO:5), AALPETGXG (SEQ ID NO:1), and LAXTG (SEQ ID NO:7).

22. The method of any one of claims 17-21, wherein the tumor cells are selected from primary cells and immortalized cells.

23. The method of any one of claims 17-22, wherein the sortase and the exogenous antigen are injected into or near the tumor cells.

24. The method of any one of claims 17-23, wherein the labeling method occurs in a subject.

25. The method of any one of claims 17-23, wherein the labeling method occurs in vitro.

26. The method of any one of claims 17-25, wherein the sortase and the exogenous antigen are provided to the tumor cells sequentially.

27. The method of any one of claims 17-25, wherein a mixture of the sortase and the exogenous antigen is provided to the tumor cells.

28. A tumor cell sorted and labeled with an exogenous antigen by the method of any one of claims 17-27.

29. A tumor cell sorted and labeled with an exogenous antigen.

30. The tumor cell of any one of claims 28-29, wherein the exogenous antigen is conjugated to the plasma membrane of the tumor cell.

31. An exogenous antigen comprising a sorting tag motif and an exogenous antigen ligand.

32. The exogenous antigen of claim 31, wherein the exogenous antigen ligand is a peptide or a molecular marker.

33. A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, one or more co-stimulatory domains, and a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the antigen-binding domain is capable of binding to the exogenous antigen of any one of claims 31-32.

34. The nucleic acid of claim 33, wherein the CAR further comprises a binding domain capable of binding to an endogenous antigen in a tumor.

35. The nucleic acid of claim 34, wherein the endogenous antigen is claudin8.

2.

36. The nucleic acid of any one of claims 33-35, wherein the cytoplasmic domain comprises a CD247 cytoplasmic domain.

37. The nucleic acid of any one of claims 33-36, wherein the one or more co-stimulatory signaling regions are selected from CD28, CD27, CD134 (OX40), and CD137 (4-1BB).

38. A vector comprising the nucleic acid of any one of claims 33-37.

39. A human immune cell comprising the nucleic acid of any one of claims 33-37 or the vector of claim 38.

40. The human immune cell of claim 35, wherein the human immune cell is a T cell, NK cell, macrophage, dendritic cell, or B cell from a human subject having a tumor.

41. A pharmaceutical composition comprising a population of the human immune cells of claim 39 or 40.

42. A method of modifying an immune cell, comprising delivering to the immune cell the vector of claim 38 or the nucleic acid of any one of claims 33-37.

43. A kit comprising sortase and an exogenous antigen of any one of claims 31-32, wherein the kit optionally further comprises the nucleic acid of any one of claims 33-37 or the vector of claim 38.

44. A genetically engineered immune cell for treating a tumor, wherein cells in the tumor are sorted and labeled by an exogenous antigen mediated by sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting label motif, and wherein the genetically engineered immune cell expresses a chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding the exogenous antigen ligand.

45. The genetically engineered immune cell for treating a tumor of claim 44, wherein the CAR further comprises a binding domain capable of binding an endogenous antigen in the tumor.

46. Use of a genetically engineered immune cell in the preparation of a medicament for treating a tumor, wherein cells in the tumor are sorted and labeled by an exogenous antigen mediated by sortase, wherein the exogenous antigen comprises an exogenous antigen ligand and a sorting label motif, and wherein the genetically engineered immune cell expresses a chimeric antigen receptor (CAR) comprising an antigen-binding domain capable of binding the exogenous antigen ligand.

47. Use of the genetically engineered immune cell of claim 46 in the preparation of a medicament for treating a tumor, wherein the CAR further comprises a binding domain capable of binding an endogenous antigen in the tumor.

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