Chimeric antigen receptors containing internalizing peptides

Abstract

The invention relates to a chimeric antigen receptor containing an internalizing peptide, and particularly provides a chimeric antigen receptor which comprises an extracellular ligand binding domain, a transmembrane domain and a cytoplasm domain, and the cytoplasm domain comprises the internalizing peptide. The CAR provided by the invention can significantly improve the killing effect of CAR-T cells on target cells under a low-efficiency target ratio condition, reduces the depletion of immune effector cells, can be stored for a long time in a tumor immunosuppression environment, kills tumor cells, and is low in depletion level.

Description

[0001] This application claims priority to Chinese application No. 202411115845.5, filed on August 14, 2024, entitled "Chimeric Antigen Receptor Containing Sorted Peptides", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to a novel chimeric antigen receptor containing an internalized peptide. Background Technology

[0003] Since the first report of tumor-infiltrating lymphocytes (TILs) in clinical oncology treatment in 1988, TILs have demonstrated an increasingly important role in the treatment of solid tumors. In recent years, it has been found that TILs are particularly effective for large, difficult-to-treat tumors, especially solid tumors, for adoptive metastasis therapy (ACT). Iovance BioTherapeutics in the United States currently has one approved TIL drug, and several other TIL drugs have entered Phase II or pivotal Phase III clinical trials, with indications including melanoma, cervical cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, colon cancer, pancreatic cancer, and sarcoma, among others.

[0004] The main component of tumor cells (TILs) is T cells, and their killing of tumor cells mainly depends on the binding between TCRs and MHC-antigen peptide complexes. Immune cells expressing chimeric antigen receptors (CARs), such as CAR-T cells, are being used more and more widely in cancer therapy, especially in the treatment of hematological malignancies. Under natural conditions, the affinity between TCRs and MHC-antigen peptides is usually weak, much lower than the interaction between CARs and the tumor cell surface antigens they recognize. Expressing CARs in TILs may enhance their tumor-killing effect, but it is also necessary to prevent CAR depletion from reducing tumor-killing efficacy, which in turn affects the tumor-killing effect of TILs. For conventional CAR-T cells, appropriately reducing the contact between CARs and tumor cell surface antigens can better reduce depletion, enhance the killing power of CAR-T cells against tumor cells, and prolong their survival time. Previous reports have shown that introducing a peptide containing a YVKM motif with an endocytic effect at the cytoplasmic terminal of CTLA-4 into the CAR structure can continuously enhance cytotoxicity, prolong cell survival time, and reduce the production and activation of pro-inflammatory cytokines under repeated stimulation by tumor cells.

[0005] Therefore, there is still a need for novel transmembrane proteins, including CARs, that can reduce immune exhaustion, further enhance the killing power of tumor cells, and prolong the survival time of immune cells. Summary of the Invention

[0006] The present invention provides a chimeric antigen receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain comprises an internalized peptide.

[0007] In one or more embodiments, the internalized peptide is located at the C-terminus of the chimeric antigen receptor.

[0008] In one or more embodiments, the internalized peptide comprises one or more tyrosine internalization signals, preferably, the internalized peptide comprises (e.g., in tandem) 1, 2 or 3 tyrosine internalization signals.

[0009] In one or more embodiments, a linker may or may not be present between the one or more tyrosine internalization signals. In one or more embodiments, the linker is an amino acid sequence EP.

[0010] In one or more embodiments, the tyrosine internalization signal is based on Signals with motifs or NPXY motifs.

[0011] In one or more implementations, based on The motif's tyrosine internalization signal is a C-terminal sequence of a membrane protein containing YX1X2X3, or a sequence having at least 90% sequence identity with it, wherein X3 is M, L, I, F, or V. Preferably, X1 is V, E, or Q, and X2 is K, V, A, T, or Q. In one or more embodiments, the membrane protein is selected from: CTLA-4, CD63, CD68, LAMP-1, and LAMP-2a.

[0012] In one or more embodiments, the NPXY motif-based tyrosine internalization signal is a C-terminal sequence of a membrane protein containing NPXY or a sequence having at least 90% sequence identity with it, wherein X is V, E, D, T, L, M, I, or K. In one or more embodiments, the membrane protein is selected from: LDLR, LRP1, Megalin, Integrinβ, EGRR, Insulin receptor, APLP1, and APP.

[0013] In one or more embodiments, the carboxyl-terminal sequence is a sequence of at least 4, 4-20, 4-11, or 4-8 amino acids at the carboxyl terminus of a membrane protein.

[0014] In one or more embodiments, each of the one or more tyrosine internalization signals independently comprises any of the following sequences: YVKM, YEVM, YQAL, YQTI, YEQF, NPVY.

[0015] In one or more embodiments, each of the one or more tyrosine internalization signals independently has a sequence selected from:

[0016] The CTLA-4 carboxyl-terminal sequence containing YVKM

[0017] Contains the CD63 carboxyl-terminal sequence of YEVM.

[0018] Contains the CD68 carboxyl-terminal sequence of YQAL.

[0019] The LAMP-1 carboxyl-terminal sequence containing YQTI,

[0020] The LAMP-2a carboxyl-terminal sequence containing YEQF,

[0021] The LDLR carboxyl terminus sequence contains NPVY.

[0022] In one or more embodiments, the CTLA-4 carboxyl-terminal sequence containing YVKM is the fragment of SEQ ID NO.42 containing YVKM with a length of 4-9 (preferably 4 or 9) amino acids.

[0023] In one or more embodiments, the CD63 carboxyl-terminal sequence containing YEVM is the fragment of SEQ ID NO.44 containing YEVM and having a length of 4-6 (preferably 4 or 6) amino acids.

[0024] In one or more embodiments, the CD68 carboxyl-terminal sequence containing YQAL is the fragment of SEQ ID NO.46 containing YQAL with a length of 4-6 (preferably 4 or 6) amino acids.

[0025] In one or more embodiments, the LAMP-1 carboxyl-terminal sequence containing YQTI is the fragment of SEQ ID NO.49 containing YQTI and having a length of 4-7 (preferably 4 or 7) amino acids.

[0026] In one or more embodiments, the LAMP-2a carboxyl-terminal sequence containing YEQF is the fragment of SEQ ID NO. 50 containing YEQF and having a length of 4-7 (preferably 4 or 7) amino acids.

[0027] In one or more embodiments, the LDLR carboxyl-terminal sequence containing NPVY is the fragment of SEQ ID NO. 51 containing NPVY and having a length of 4-6 (preferably 4 or 6) amino acids.

[0028] In one or more embodiments, the internalized peptide comprises:

[0029] (1) One, two, three or more CTLA-4 carboxyl-terminal sequences containing YVKM, optionally also containing one, two, three or more sequences selected from: a CD63 carboxyl-terminal sequence containing YEVM, a CD68 carboxyl-terminal sequence containing YQAL, a LAMP-1 carboxyl-terminal sequence containing YQTI, a LAMP-2a carboxyl-terminal sequence containing YEQF, or an LDLR carboxyl-terminal sequence containing NPVY.

[0030] (2) One, two, three or more CD63 carboxyl-terminal sequences containing YEVM, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-1 carboxyl-terminal sequence containing YQTI, LAMP-2a carboxyl-terminal sequence containing YEQF, LDLR carboxyl-terminal sequence containing NPVY.

[0031] (3) One, two, three or more CD68 carboxyl-terminal sequences containing YQAL, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, LAMP-1 carboxyl-terminal sequence containing YQTI, LAMP-2a carboxyl-terminal sequence containing YEQF, LDLR carboxyl-terminal sequence containing NPVY,

[0032] (4) One, two, three or more LAMP-1 carboxyl-terminal sequences containing YQTI, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-2a carboxyl-terminal sequence containing YEQF, LDLR carboxyl-terminal sequence containing NPVY,

[0033] (5) One, two, three or more LAMP-2a carboxyl-terminal sequences containing YEQF, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-1 carboxyl-terminal sequence containing YQTI, LDLR carboxyl-terminal sequence containing NPVY.

[0034] (6) 1, 2, 3 or more LDLR carboxyl-terminal sequences containing NPVY, optionally also containing 1, 2, 3 or more sequences selected from the following: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-1 carboxyl-terminal sequence containing YQTI, and LAMP-2a carboxyl-terminal sequence containing YEQF.

[0035] In one or more embodiments, the internalized peptide comprises:

[0036] (1) One, two, three or more tandem CTLA-4 carboxyl-terminal sequences containing YVKM

[0037] (2) One, two, three or more CD63 carboxyl-terminal sequences containing YEVM in tandem.

[0038] (3) One, two, three or more CD68 carboxyl-terminal sequences containing YQAL in tandem.

[0039] (4) A tandem CD68 carboxyl-terminal sequence containing YQAL and a CD63 carboxyl-terminal sequence containing YEVM.

[0040] (5) One, two, three or more LAMP-1 carboxyl-terminal sequences containing YQTI in tandem.

[0041] (6) One, two, three or more LAMP-2a carboxyl-terminal sequences containing YEQF in tandem.

[0042] (7) One, two, three or more LDLR carboxyl-terminal sequences containing NPVY in tandem.

[0043] In one or more embodiments, the internalized peptide comprises:

[0044] (1) One or two tandem CTLA-4 carboxyl-terminal sequences containing YVKM,

[0045] (2) One or two CD63 carboxyl-terminal sequences containing YEVM,

[0046] (3) One or two CD68 carboxyl-terminal sequences containing YQAL,

[0047] (4) A tandem CD68 carboxyl-terminal sequence containing YQAL and a CD63 carboxyl-terminal sequence containing YEVM.

[0048] (5) One or two LAMP-1 carboxyl-terminal sequences containing YQTI,

[0049] (6) One or two LAMP-2a carboxyl-terminal sequences containing YEQF,

[0050] (7) One or two LDLR carboxyl-terminal sequences containing NPVY.

[0051] In one or more embodiments, the amino acid sequence of the internalized peptide is shown in SEQ ID NO:42-52.

[0052] In one or more embodiments, the cytoplasmic domain comprises a signal transduction domain and / or a co-stimulatory domain. In one or more embodiments, the co-stimulatory domain is an intracellular domain of a co-stimulatory signaling molecule or a functional fragment or mutant thereof that retains the biological function of the co-stimulatory signaling molecule in transmitting co-stimulatory signals and activating immune cells.

[0053] In one or more embodiments, the signal transduction domain is selected from one or more of the following:

[0054] The CD3ζ intracellular signal transduction region preferably contains the amino acid sequence encoded by SEQ ID NO:37 or a sequence having at least 80% sequence identity with it.

[0055] The CD3δ intracellular signal transduction region preferably contains the amino acid sequence encoded by SEQ ID NO:38 or a sequence having at least 80% sequence identity with it.

[0056] The intracellular signal transduction region of CD3γ preferably contains an amino acid sequence encoded by SEQ ID NO:39 or a sequence having at least 80% sequence identity with it.

[0057] In one or more embodiments, the co-stimulatory domain is an intracellular co-stimulatory region of CD28, which preferably contains an amino acid sequence encoded by SEQ ID NO:53 or a sequence having at least 80% sequence identity with it.

[0058] In one or more embodiments, the transmembrane domain is a CD28 transmembrane domain, which preferably contains an amino acid sequence encoded by SEQ ID NO:54 or a sequence having at least 80% sequence identity with it.

[0059] In one or more embodiments, the chimeric antigen receptor further comprises a hinge region, preferably located between the extracellular ligand-binding domain and the transmembrane domain. In one or more embodiments, the hinge region is a CD8 hinge region. Preferably, the CD8 hinge region comprises an amino acid sequence encoded by SEQ ID NO:55 or a sequence having at least 80% sequence identity with it.

[0060] In one or more embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably located at the N-terminus of the chimeric antigen receptor. Preferably, the signal peptide is the CD8 signal peptide.

[0061] In one or more embodiments, the extracellular ligand-binding domain is an antibody or antigen-binding fragment targeting a tumor antigen. In one or more embodiments, the tumor antigen is selected from one or more of the following: HER2, CD19, EpCAM.

[0062] In one or more embodiments, the extracellular ligand binding domain comprises a single-chain antibody as shown in any of the following sequences: SEQ ID NO:40, SEQ ID NO:41.

[0063] In one or more embodiments, the chimeric antigen receptor, preferably from the N-terminus to the C-terminus, comprises the following components:

[0064] Extracellular ligand-binding domain, transmembrane domain, and cytoplasmic domain containing internalized peptides.

[0065] Extracellular ligand-binding domain, hinge region, transmembrane domain, and cytoplasmic domain containing internalized peptides.

[0066] Signal peptide, extracellular ligand-binding domain, transmembrane domain, cytoplasmic domain containing internalized peptide, or

[0067] Signal peptide, extracellular ligand binding domain, hinge region, transmembrane domain, and cytoplasmic domain containing internalized peptides.

[0068] In one or more embodiments, the chimeric antigen receptor, preferably from the N-terminus to the C-terminus, comprises any one of the following groups of components:

[0069] Single-chain antibodies targeting HER2, CD8 extracellular hinge region, CD28 transmembrane region, CD28 intracellular co-stimulatory region, CD3ζ intracellular signal transduction region, and internalization peptides.

[0070] Single-chain antibodies targeting HER2, CD8 extracellular hinge region, CD28 transmembrane region, CD28 intracellular co-stimulatory region, CD3δ intracellular signal transduction region, CD3γ intracellular signal transduction region, and internalization peptides.

[0071] Single-chain antibodies targeting EpCAM, the CD8 extracellular hinge region, the CD28 transmembrane region, the CD28 intracellular co-stimulatory region, the CD3ζ intracellular signal transduction region, and the internalized peptide.

[0072] Single-chain antibodies targeting EpCAM, the CD8 extracellular hinge region, the CD28 transmembrane region, the CD28 intracellular co-stimulatory region, the CD3δ intracellular signal transduction region, the CD3γ intracellular signal transduction region, and the internalization peptide.

[0073] Signal peptides, single-chain antibodies targeting HER2, CD8 hinge region, CD28 transmembrane region, CD28 intracellular co-stimulatory region, CD3ζ intracellular signal transduction region, and internalization peptides.

[0074] Signal peptides, single-chain antibodies targeting HER2, CD8 extracellular hinge region, CD28 transmembrane region, CD28 intracellular co-stimulatory region, CD3δ intracellular signal transduction region, CD3γ intracellular signal transduction region, and internalization peptides.

[0075] Signal peptides, single-chain antibodies targeting EpCAM, CD8 extracellular hinge region, CD28 transmembrane region, CD28 intracellular co-stimulatory region, CD3ζ intracellular signal transduction region, and internalization peptides.

[0076] Signal peptides, single-chain antibodies targeting EpCAM, CD8 extracellular hinge region, CD28 transmembrane region, CD28 intracellular co-stimulatory region, CD3δ intracellular signal transduction region, CD3γ intracellular signal transduction region, and internalization peptides.

[0077] Preferably, the internalized peptide is any one of SEQ ID NO:42-52.

[0078] This invention also provides a polynucleotide having:

[0079] (1) The nucleic acid sequence encoding the chimeric antigen receptor described herein, and / or

[0080] (2)(1) complementary sequences.

[0081] The present invention also provides nucleic acid constructs comprising the polynucleotides described herein.

[0082] In one or more embodiments, the nucleic acid construct further comprises at least one regulatory element for expressing the chimeric antigen receptor operatively linked to the polynucleotide.

[0083] In one or more embodiments, the nucleic acid construct is an expression vector or a cloning vector.

[0084] In one or more embodiments, the nucleic acid construct is an expression vector; preferably a viral vector or a non-viral vector.

[0085] In one or more embodiments, the non-viral vector is a non-viral integrated vector based on a transposon subsystem.

[0086] The present invention also provides a host cell, wherein:

[0087] (1) Contains, expresses, or secretes the chimeric antigen receptor described in any embodiment of this document, and / or

[0088] (2) Contains a polynucleotide encoding the chimeric antigen receptor described in any embodiment of the present invention or a nucleic acid construct containing said polynucleotide.

[0089] In one or more embodiments, the host cell is an immune effector cell.

[0090] In one or more embodiments, the immune effector cells are T cells, NK cells, CIK cells, or tumor-infiltrating lymphocytes (TILs).

[0091] The present invention also provides a method for enhancing the activity of immune effector cells in an individual, comprising introducing immune effector cells into the individual, the immune effector cells comprising the chimeric antigen receptor described in any embodiment herein.

[0092] In one or more embodiments, the immune effector cells are T cells, NK cells, CIK cells, or tumor-infiltrating lymphocytes (TILs).

[0093] In one or more embodiments, the individual suffers from tumor antigen-associated cancer, wherein the tumor antigen is a tumor antigen targeted by the extracellular ligand-binding domain of the chimeric antigen receptor.

[0094] In one or more embodiments, the individual has HER2-related cancers, including but not limited to: melanoma, breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, and lung cancer.

[0095] In one or more embodiments, the individual suffers from EpCAM-related cancers, such as EpCAM-related adenocarcinoma, including but not limited to: esophageal cancer, gastric cancer, colon cancer, prostate cancer, lung cancer, ovarian cancer, breast cancer, kidney cancer, hepatocellular carcinoma, squamous cell carcinoma of the skin, and sarcoma.

[0096] In one or more embodiments, the individual has CD19-related cancer. In one or more embodiments, the cancer is a B-cell-derived cancer, such as B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, or B-cell non-Hodgkin lymphoma.

[0097] In one or more embodiments, the cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin lymphoma, and childhood acute lymphoblastic leukemia.

[0098] The present invention also provides a method for treating an individual suffering from cancer, comprising introducing immune effector cells into the individual, the immune effector cells comprising the chimeric antigen receptor as described in any embodiment herein.

[0099] In one or more embodiments, the immune effector cells are T cells, NK cells, CIK cells, or tumor-infiltrating lymphocytes (TILs).

[0100] In one or more embodiments, the individual suffers from tumor antigen-associated cancer, wherein the tumor antigen is a tumor antigen targeted by the extracellular ligand-binding domain of the chimeric antigen receptor.

[0101] In one or more embodiments, the individual has HER2-related cancers, including but not limited to: melanoma, breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, and lung cancer.

[0102] In one or more embodiments, the individual suffers from EpCAM-related cancers, such as EpCAM-related adenocarcinoma, including but not limited to: esophageal cancer, gastric cancer, colon cancer, prostate cancer, lung cancer, ovarian cancer, breast cancer, kidney cancer, hepatocellular carcinoma, squamous cell carcinoma of the skin, and sarcoma.

[0103] In one or more embodiments, the individual has CD19-related cancer. In one or more embodiments, the cancer is a B-cell-derived cancer, such as B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, or B-cell non-Hodgkin lymphoma.

[0104] In one or more embodiments, the cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin lymphoma, and childhood acute lymphoblastic leukemia.

[0105] The present invention also provides pharmaceutical compositions comprising one or more of the chimeric antigen receptor, polynucleotide, nucleic acid construct and host cell described in any embodiment of the present invention, wherein the pharmaceutical compositions further comprise pharmaceutically acceptable excipients.

[0106] The present invention also provides the use of any one or more of the chimeric antigen receptor, polynucleotide, nucleic acid construct and host cell described in any embodiment of the present invention in the preparation of a medicament for treating or preventing cancer.

[0107] In one or more embodiments, the cancer is a tumor antigen-associated cancer, and the tumor antigen is a tumor antigen targeted by the extracellular ligand-binding domain of the chimeric antigen receptor.

[0108] In one or more embodiments, the cancer is a HER2-related cancer, including but not limited to: melanoma, breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, and lung cancer.

[0109] In one or more embodiments, the cancer is an EpCAM-related cancer, such as EpCAM-related adenocarcinoma, including but not limited to: esophageal cancer, gastric cancer, colon cancer, prostate cancer, lung cancer, ovarian cancer, breast cancer, kidney cancer, hepatocellular carcinoma, squamous cell carcinoma of the skin, and sarcoma.

[0110] In one or more embodiments, the cancer is a CD19-related cancer. In one or more embodiments, the cancer is a B-cell-derived cancer, such as B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, or B-cell non-Hodgkin lymphoma.

[0111] In one or more embodiments, the cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin lymphoma, and childhood acute lymphoblastic leukemia. Detailed Implementation

[0112] This invention provides a chimeric antigen receptor (CAR) with a cytoplasmic domain containing an internalization signal. In one non-limiting example, the CAR includes an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain containing an internalized peptide, wherein the extracellular ligand-binding domain specifically recognizes tumor antigens. The CAR of this invention can significantly improve the killing of target cells by CAR-T cells under low target-to-cell ratio conditions, reduce the exhaustion of immune effector cells, survive for a long time in a tumor immunosuppressive environment, kill tumor cells, and have low exhaustion levels.

[0113] Terminology Definition

[0114] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following references provide general definitions for many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise stated, the following terms as used herein have the meanings given below.

[0115] As used herein, the term "about" or "approximately" refers to an acceptable margin of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to convention in the art, "about" may refer to within three or more standard deviations. Alternatively, "about" may indicate a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may indicate within an order of magnitude of the numerical value, preferably within five times, more preferably within two times.

[0116] The term "internalization signal" as used in this article refers to a protein-guided endocytosis signal that directs a protein to endosomes, lysosomes, or related organelles. "Tyrosine-based internalization signal" refers to an internalization signal based on a short tyrosine-containing 4-position peptide motif. Such a signal contains... Motifs and NPXY motifs. The motif can be recognized by adaptor protein (AP) complexes and participates in various functions such as internalization, lysosomal targeting, and basolateral targeting. Y represents tyrosine, and X represents any amino acid. This represents an amino acid residue with a large hydrophobic side chain. Including but not limited to M, L, I, F, or V. In one or more embodiments, The motif is YX1X2X3, where X1 is V, E, or Q, X2 is K, V, A, T, or Q, and X3 is M, L, I, F, or V. The NPXY motif mediates the rapid internalization of type I integrated membrane protein subsets, where X represents any amino acid, preferably V, E, D, T, L, M, I, or K. (Annu. Rev. Biochem. 2003. 72: 395–447).

[0117] Known tyrosine-based internalization signals originate from proteins, and in addition to the corresponding motif, these internalization signals may also contain amino acids at the corresponding positions of the protein from which they originate, at both ends of the motif. Tyrosine internalization signals based on motifs can originate from the C-terminal sequences of CTLA-4, CD63, CD68, LAMP-1, and LAMP-2a, while tyrosine internalization signals based on NPXY motifs can originate from the C-terminal sequences of LDLR, LRP1, Megalin, Integrinβ, EGRR, insulin receptor, APLP1, and APP. In this paper, the C-terminal sequence refers to the sequence containing the C-terminal region of membrane proteins. At least four amino acid sequences of motifs or NPXY motifs, such as those containing the C-terminus of membrane proteins. The amino acid sequence of the 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th amino acids of the motif or NPXY motif.

[0118] In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule containing amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope," as described above. As mentioned above, the antigen-binding domain typically includes a variable region (VL) and a variable region (VH) of the antibody light chain; however, it is not necessary to include both. Fd fragments, for example, have two VH regions and typically retain some of the antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab')2 fragments, bivalent fragments having two Fab fragments connected by disulfide bridges of hinge regions; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains of antibody single arms; (5) dAb fragments (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,” Nature 341:544-546 (1989), which are incorporated herein by reference in their entirety), having a VH domain; (6) separate complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv), for example derived from scFV libraries. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be conjugated using a recombination method via synthetic linkers, which allow it to be prepared as a single protein chain (called a single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, for example, Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichiacoli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). These antibody fragments are obtained using conventional techniques known to those skilled in the art and their function is evaluated in the same manner as that of intact antibodies. In this application, the term “antigen-binding fragment” also includes a ligand that specifically binds to an antigen, particularly a cell surface receptor molecule antigen. The ligand is typically a polypeptide or compound that can bind to a receptor protein (e.g., the said antigen) in a high-affinity and specific manner to elicit a functional response.

[0119] In this application, the term "tumor-infiltrating lymphocytes (TILs)" generally refers to infiltrating lymphocytes isolated from tumor tissue. Immunohistochemical staining shows that tumor-infiltrating lymphocytes may include CD3+ T cells, CD20+, CD79α+ B cells, plasma cells, and CD56+ NK cells; they may also include T cells, a small number of B cells, NK cells, macrophages, and dendritic cells. These tumor-infiltrating lymphocytes can produce a highly efficient and specific anti-tumor killing effect through direct cytotoxic T lymphocytes and secreted cytokines. In this application, the tumor-infiltrating lymphocytes may include monocytes that leave the bloodstream and migrate to a tumor. The tumor-infiltrating lymphocytes may be selected from a group consisting of T cells, B cells, natural killer cells, and natural killer T cells. This invention is suitable for TILs isolated from any tumor tissue, including tumor tissue obtained through open or minimally invasive surgery. Exemplary tumor tissues include tumor tissues of cancers such as gastric cancer, thyroid tumors, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, etc.

[0120] The term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0121] The term "effective dose" refers to a dose that can achieve therapeutic, preventive, alleviating and / or relieving disease or condition as described in this invention in a subject.

[0122] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0123] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0124] The term "extracellular" refers to the segment of membrane proteins (such as chimeric antigen receptors) located outside the cell.

[0125] The term "domain" refers to a region in a protein that has a specific structure and independent function. Common domains have between 100 and 400 amino acid residues, with the smallest domains having only 40 to 50 amino acid residues and the largest domains having more than 400 amino acid residues. Invention Details

[0127] This invention provides a chimeric antigen receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain comprises an internalizing peptide, and the internalizing peptide comprises (e.g., in tandem) one, two, or more tyrosine internalization signals. The one or more tyrosine internalization signals may or may not contain a linker.

[0128] In this document, the term "connector" or "hinge" refers to a polypeptide fragment that connects different proteins or polypeptides, with the purpose of maintaining the spatial conformation of the connected proteins or polypeptides to preserve their function or activity. Connectors can be readily determined by those skilled in the art based on practical needs. Exemplary connectors include those containing G, S, E, or P.

[0129] In this article, the extracellular ligand-binding domain includes an antibody targeting a tumor antigen or its antigen-binding fragment. The tumor antigen can be one or more selected from the following: CD19, CD20, CEA, GD2 (also known as B4GALNT1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL (pre-melanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE ​​(growth hormone-releasing factor) family proteins, AFP, MUC1 (also known as mucin1), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR. 1. VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR), and PROGRP (GRP gastrin-releasing peptide).

[0130] In one or more embodiments, the extracellular ligand-binding domain of the CAR comprises an antibody or an antigen-binding fragment thereof, wherein the LCDR1, LCDR2, and LCDR3 of the antibody are represented by the amino acid sequences encoded by SEQ ID NO:57-59, and / or, the HCDR1, HCDR2, and HCDR3 of the antibody are represented by the amino acid sequences encoded by SEQ ID NO:60-62, respectively. In one or more embodiments, the VL of the antibody is represented by the amino acid sequence encoded by SEQ ID NO:63 or a sequence having at least 80% sequence identity therewith, and the VH of the antibody is represented by the amino acid sequence encoded by SEQ ID NO:64 or a sequence having at least 80% sequence identity therewith.

[0131] In one or more embodiments, the extracellular ligand-binding domain of the CAR comprises an antibody or an antigen-binding fragment thereof, wherein the LCDR1, LCDR2, and LCDR3 of the antibody are represented by the amino acid sequences encoded by SEQ ID NO:65-67, and / or, the HCDR1, HCDR2, and HCDR3 of the antibody are represented by the amino acid sequences encoded by SEQ ID NO:68-70, respectively. In one or more embodiments, the VL of the antibody is represented by the amino acid sequence encoded by SEQ ID NO:71 or a sequence having at least 80% sequence identity therewith, and the VH of the antibody is represented by the amino acid sequence encoded by SEQ ID NO:72 or a sequence having at least 80% sequence identity therewith.

[0132] Typically, cytoplasmic domains contain signal transduction domains and / or co-stimulatory domains. In one or more embodiments, the co-stimulatory domain is a cytoplasmic domain of a co-stimulatory signaling molecule or a functional fragment or mutant of the co-stimulatory signaling molecule that retains the biological function of transmitting co-stimulatory signals and activating immune cells.

[0133] In this invention, the co-stimulatory signaling molecules include CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, CD80, and CD27. The chimeric antigen receptor of the present invention can be constructed using one or more of these co-stimulatory signaling molecules' cytoplasmic domains (intracellular regions) or functional fragments thereof, or mutants that retain the biological functions of the co-stimulatory signaling molecules in transmitting co-stimulatory signals and activating immune cells. The cytoplasmic domain of the co-stimulatory signaling molecule can also be the cytoplasmic domain of the co-stimulatory signaling molecule described in WO2021244486, which is incorporated herein by reference in its entirety.

[0134] In this invention, the signal transduction domains can be selected by those skilled in the art as needed, such as the CD3ζ intracellular signal transduction region, the CD3δ intracellular signal transduction region, and / or the CD3γ intracellular signal transduction region.

[0135] In this document, the transmembrane region includes, but is not limited to, regions selected from CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD4. The transmembrane regions of CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, CD80, and CD27, or any one or more of the mutants that retain transmembrane function.

[0136] In this invention, the extracellular domain (or extracellular region) can be connected to the transmembrane region via the hinge region. The hinge region includes, but is not limited to, the proximal membrane fragments of the natural extracellular domains selected from CD28, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, MyD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD27. Preferably, the hinge region is the extracellular hinge region of CD28 and / or the extracellular hinge region of IL7Rα.

[0137] It should be understood that the term "functional fragment" as used herein refers to a fragment that retains the desired biological function. For example, the functional fragment of the cytoplasmic domain as used herein refers to a fragment that retains the biological function of the co-stimulatory signaling molecule in transmitting co-stimulatory signals and activating immune cells. The functional fragments of the extracellular domains and the functional fragments of the cytoplasmic domains applicable to this invention can be readily determined by those skilled in the art in conjunction with existing technical means.

[0138] The term "mutant" as used in this article includes mutants of each domain, as long as the mutant retains the corresponding biological functions of the extracellular domain, transmembrane region, cytoplasmic domain, and internalized peptide of the chimeric antigen receptor. For example, mutants of the internalizing peptides suitable for use in this invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the internalizing peptide as a comparison; mutants of the extracellular domains suitable for use in this invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the extracellular domain as a comparison; mutants of the transmembrane regions suitable for use in this invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the transmembrane region as a comparison; mutants of the cytoplasmic domains suitable for use in this invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the cytoplasmic domain as a comparison. Alternatively, compared to the comparison sequence, the mutants described in this invention have one or more (e.g., up to 20, 15, 10, 8, 5, or 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted, or deleted. For example, in the art, conservative substitution with amino acids of similar or comparable properties typically does not alter the function of the protein or polypeptide. "Amino acids with similar or comparable properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0139] This invention also includes mutants of the chimeric antigen receptor described above, such as mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the chimeric antigen receptor. More specifically, this invention includes mutants that have one or more (e.g., up to 20, 15, 10, 8, 5, or 3, such as 1-20, 1-10, etc.) amino acid residues inserted, substituted, or deleted compared to the chimeric antigen receptor described above. Such mutants retain the biological functions of the chimeric antigen receptor described in this invention, including but not limited to the function of recognizing tumor antigens and activating immune effector cells into an activated and proliferating state. Mutations may occur in any one, any two, or all three of the extracellular domains, transmembrane domains, and cytoplasmic domains described herein.

[0140] The peptides described herein can be modified peptides. Modifications (typically without altering the primary structure) include chemical derivatizations of peptides, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylating enzymes, such as mammalian glycosylation or deglycosylation enzymes. Modifications also include sequences containing phosphorylated amino acid residues, such as phosphotyrosine, phosphotyserine, and phosphotythreonine. Furthermore, peptides modified to enhance their resistance to proteolytic hydrolysis or optimize their solubility are also included.

[0141] The exemplary chimeric antigen receptor of this invention, from the N-terminus to the C-terminus, includes, but is not limited to, containing, or composed of, the extracellular domain, hinge region, transmembrane region, and intracellular domain shown in each row of Table 1 below, or composed of, the extracellular domain, hinge region, transmembrane region, and cytoplasmic domain shown in each row of Table 1 below. Wherein, antigen-binding protein refers to the antibody or its antigen-binding fragment described herein.

[0142] Table 1, Chimeric antigen receptors

[0143]

[0144]

[0145] In some embodiments, the chimeric antigen receptor described herein further comprises a signal peptide. Preferably, the signal peptide is located at the N-terminus of the chimeric antigen receptor. The signal peptide can be any signal peptide conventional in the art capable of guiding a polypeptide out of the nucleus, including but not limited to CD8, CD4, CD28, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII, and antibody light chain signal peptides.

[0146] It should be understood that, as needed, the extracellular domains and transmembrane regions, and / or the transmembrane regions and intracellular domains described herein, can be connected by adapter sequences. Adapter sequences known in the art, such as those containing G and S, such as (GSSS)n or (GSSSS)n, where n is an integer from 1 to 8, can be used. The adapters can also be rigid or flexible.

[0147] In an exemplary embodiment, the amino acid sequence of the internalized peptide is shown in SEQ ID NO:42-52, and it is located at the C-terminus of the cytoplasmic structural domain.

[0148] This invention provides a polynucleotide molecule encoding the chimeric antigen receptor described herein. The invention also provides a complementary sequence to the coding sequence of the chimeric antigen receptor. The polynucleotide molecule may be a recombinant nucleic acid molecule or a synthetic one; it may comprise DNA, RNA, and PNA (peptide nucleic acid) and may be a hybrid thereof. Exemplarily, the polynucleotide molecule of this invention has a sequence in any of SEQ ID NO:3-36 that does not contain the easily detectable terminal 2A and EGFP portions.

[0149] This invention also provides an expression cassette for a chimeric antigen receptor, which is a nucleic acid construct containing a promoter, a chimeric antigen receptor coding sequence, and a PolyA tailing signal sequence. The nucleic acid construct may also contain other elements required for expression, including but not limited to enhancers.

[0150] This invention also provides a vector containing the polynucleotide molecule, expression cassette, or nucleic acid construct described herein. The vector can be a plasmid, granule, virus, or bacteriophage. The vector can be a viral vector or a non-viral vector. The vector can be a cloning vector, an integration vector, or an expression vector. The expression vector can be a transposon vector. In some embodiments, the expression vector is one or more transposon vectors selected from: piggybac, sleeping beauty, frogprince, Tn5, and Ty. In addition to the polynucleotide molecule described herein, the expression vector typically contains other elements commonly found in vectors, such as multiple cloning sites, resistance genes, replication initiation sites, etc. In some embodiments, the recombinant expression vector uses pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vectors, pUC57, pMAX, or pDC315 series vectors as a backbone. In other embodiments, the recombinant expression vector uses pCDNA3 series vectors, pCDNA4 series vectors, pCDNA5 series vectors, pCDNA6 series vectors, pRL series vectors, pUC57 vectors, pMAX vectors, or pDC315 series vectors as the backbone. In some embodiments, the present invention uses the pNB vector constructed by CN105154473A. In some embodiments, the present invention uses the pKB20 vector described in WO2022078310A1.

[0151] The CAR of this invention can also be expressed in the immune cells described in this invention using conventional vectors. The vector can be a conventional CAR expression vector, including but not limited to the various transposon vectors and recombinant expression vectors described above.

[0152] In some embodiments, the same vector simultaneously encodes the chimeric antigen receptor of the present invention and other proteins. This vector may be bicistronic. The coding sequences for other proteins may be positioned at the 5' or 3' end of the chimeric antigen receptor coding sequence. The expression of other proteins and the chimeric antigen receptor may be guided by the same or different regulatory sequences.

[0153] When the polynucleotide sequence is known, the polynucleotide molecules can be prepared using methods conventional in the art, and the corresponding vectors can be constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, the techniques described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory), Ausubel et al. (1989, Short Protocols in Molecular Biology, Wiley), or other standard textbooks. Alternatively, the nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the present invention can be transferred into host cells using well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al. (see above).

[0154] In this article, when expressing a heterologous nucleic acid sequence, "host cell" refers to a eukaryotic cell capable of replicating the vector and / or expressing the heterologous gene encoded by the vector. The host cell can serve as the recipient of the vector. The host cell can be "transfected" or "transformed," referring to the process of transfecting or transducing exogenous nucleic acids into the host cell. Transformed cells include primary target cells and their progeny. The terms "engineered" and "recombinant" cells or host cells used herein often refer to cells in which a exogenous nucleic acid sequence, such as a vector, has been introduced. Therefore, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.

[0155] The cells of this invention are preferably immune cells, which can be used for adoptive cell therapy for tumors. These cells are also referred to as chimeric antigen receptor-modified cells of this invention. More specifically, the cells of this invention are preferably immune effector cells, including T cells, such as cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or cytotoxic T cells), NK cells, NKT cells, CAR-T, CAR-NK, TCR-T, CIK, TIL, DN T cells; and other immune cells capable of inducing effector functions.

[0156] In this article, the cells can be autologous cells, homologous cells, allogeneic cells, and even xenotransplanted cells in some cases, in relation to the individuals that receive them.

[0157] The nucleic acid constructs / recombinant expression vectors of the present invention can be transferred into cells of interest. The transfer methods are conventional in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. In some embodiments, electroporation is used to transfer the nucleic acid constructs or recombinant expression vectors. When multiple expression cassettes are located in different nucleic acid constructs / recombinant expression vectors, these nucleic acid constructs / recombinant expression vectors can be transferred into cells simultaneously or sequentially.

[0158] In addition to carrying the chimeric antigen receptor and / or its coding sequence as described in this invention, the cells of this invention may also possess one or more other properties that can be used for cell immunotherapy (such as adoptive cell therapy for tumors). These other properties may be inherent to the cell or may be part of the cell after genetic manipulation in humans. For example, the cells of this invention may carry αβT cell receptors and / or antigen-specific receptors, such as tumor-specific receptors, or their coding sequences.

[0159] The immune cells of this invention can further express exogenous TCRs or contain coding sequences for genes expressing exogenous TCRs. The TCRs described in this invention can be various TCRs known in the art, such as HLA-matched TCRs with known sequences and structures, and whose binding antigen peptide sequences are also known. A single cell can express multiple exogenous TCRs, including exogenous TCRs targeting different tumor antigens.

[0160] The exogenous TCR described in this invention comprises an αβ double strand, which can form a complete TCR complex with the double-stranded structures of γε, δε, and ξξ endogenously expressed by immune effector cells such as T cells. The exogenous gene encoding the exogenous TCR described in this invention includes a gene encoding an αβ double strand. The coding sequences of the α and β strands are covalently linked by a DNA sequence encoding a cleavable adapter sequence, such as P2A, T2A, or F2A, or by a DNA fragment encoding an IRES sequence. In addition to the αβ double strand encoding the exogenous TCR, the gene encoding the exogenous TCR described in this invention may also include a tag protein gene fused with the αβ gene, such as EGFP, RFP, or YFP genes. The tag protein gene can be covalently linked to the gene encoding the αβ double strand by a cleavable adapter sequence, such as a 2A sequence, such as P2A, T2A, or F2A, or by a DNA sequence encoding an IRES sequence. The tag proteins, such as EGFP, RFP, and YFP genes, are co-expressed with the TCRαβ double strand and can serve as an identification indicator for detecting exogenous TCR expression.

[0161] The TCR-T of this invention can target one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL (pre-melanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer / testis antigen 1B), MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE ​​(growth hormone-releasing factor) family proteins, AFP, MUC1 (mucin 1) CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11R / , EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin) 16) CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR), microglobulin) and PROGRP (GRP gastrin-releasing peptide).

[0162] In this document, "pharmaceutical composition" refers to a composition intended for administration to an individual and encompassing a cell-based composition for immunotherapy. The pharmaceutical compositions of this invention may also contain pharmaceutically acceptable carriers. Examples of suitable drug carriers are those known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated using well-known conventional methods. These pharmaceutical compositions can be administered to the subject in appropriate doses.

[0163] Dosage regimens can be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any given patient depends on a variety of factors, including the patient's body size, body surface area, age, the specific compound to be administered, sex, timing and route of administration, overall health status, and any other medications administered concurrently.

[0164] The compositions of the present invention can be administered locally or systemically. In some embodiments, the compositions provided by the present invention (e.g., cells expressing the chimeric antigen receptor described herein) can be administered parenterally, for example, intravenously, intra-arterially, intrathecally, subdermally, or intramuscularly. In some other embodiments, DNA encoding the constructs provided by the present invention can be directly delivered to the target site, for example, via gene gun delivery to internal or external target sites or via catheter delivery to intra-arterial sites. In preferred embodiments, the pharmaceutical composition is administered subcutaneously, and in more preferred embodiments, intravenously. Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic solutions / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oil. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements (e.g., those based on ringer's dextran), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. Furthermore, the pharmaceutical compositions of the present invention may contain protein carriers, such as serum albumin or immunoglobulins, preferably human. In addition to protein-based chimeric cytokine receptor constructs or nucleic acid molecules or carriers encoding them, the pharmaceutical compositions of the present invention are envisioned to also contain bioactive agents, depending on the intended use of the pharmaceutical composition.

[0165] Compositions for parenteral (e.g., intravenous) administration of the cells described herein may also be stored in lyophilized form or in solution (e.g., cryopreservation formulations). Cryopreservation formulations may be stored in ready-to-use form or in a form that is further formulated prior to administration. Cryopreservation formulations can withstand long-distance transport without damaging the cells. In addition to the cells themselves, cryopreservation formulations typically include components such as cell cryopreservation solution and human serum albumin (HSA). Before administration (e.g., intravenous infusion), the cryopreserved pharmaceutical composition must be cryopreserved (e.g., placed in liquid nitrogen). After thawing, the cryopreservation formulation can be administered directly or formulated as an infusion composition to the patient. Those skilled in the art are familiar with the components and concentrations of conventional cryopreservation solutions. For example, cryopreservation solutions or infusion compositions may also contain dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, or magnesium chloride, the concentration of which can be determined by those skilled in the art (e.g., experienced physicians) based on the cells, disease, patient, and other conditions.

[0166] The chimeric antigen receptor, polynucleotide molecule, carrier, host cell, and pharmaceutical composition containing these substances described in this invention can be used to prevent, treat, or alleviate cancer, especially cancers in which cancer cells express corresponding tumor antigens on their surface, or to prepare drugs for the prevention, treatment, or alleviation of cancer.

[0167] As used herein, “treatment” or “treatment” includes any beneficial or necessary effect on the symptoms or lesions of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition (e.g., cancer). Treatment may optionally include a reduction or relief of symptoms of the disease or condition, or a delay in the progression of the disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of the disease or condition or its associated symptoms.

[0168] As used in this article, “prevention” refers to methods used to prevent, suppress, or reduce the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition, or delaying the appearance or recurrence of its symptoms. As used in this article, “prevention” also includes reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it occurs or recurs.

[0169] This invention includes the administration of cells, polynucleotide molecules, and vectors, alone or in any combination, using standard vectors and / or gene delivery systems, optionally in conjunction with pharmaceutically acceptable carriers or excipients. In some embodiments, after administration, the polynucleotide molecule or vector can be stably integrated into the genome of the subject.

[0170] In specific embodiments, viral vectors that specifically target certain cells or tissues and persist within said cells may be used. Suitable drug carriers and excipients are well known in the art. The compositions prepared according to the present invention can be used to prevent, treat, or delay the aforementioned identified diseases.

[0171] Furthermore, the present invention provides a method for preventing, treating, or alleviating cancer, comprising the steps of: administering an effective amount of cells to a subject in need, said cells carrying chimeric antigen receptors, polynucleotide molecules, and / or carriers as described in and / or generated by the methods described in the present invention.

[0172] The methods described herein can be used to prevent, treat, or alleviate various cancers, including various solid tumors and hematologic malignancies, including but not limited to lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, and glioma. More specifically, the cancers described herein include, but are not limited to, breast, prostate, lung, and colon cancers or epithelial cancers, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, and melanoma; genitourinary cancers, such as ovarian cancer, endometrial cancer, and cervical cancer; kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer. The compositions of this invention can be used for all stages and types of cancer, including for, for example, minimal residual disease, early-stage cancer, advanced cancer, and / or metastatic cancer and / or difficult-to-treat cancers.

[0173] By way of example, cancer patients, cancer-prone patients, or suspected cancer patients are treated as follows. The modified cells described herein can be administered to the individual and remain in the body for an extended period. The individual can receive the cells once or multiple times, with intervals between administrations ranging from days to weeks to months to years. In specific embodiments, multiple administrations can occur over weeks or months, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks or months. In some embodiments, the genetically modified cells are encapsulated to suppress immune recognition and are located at the tumor site. In cases where cells are provided to an individual after tumor recurrence following initial treatment with the cells of the present invention, these cells can be modified to recognize different target tumor antigens. For example, when the initial round includes cells carrying the chimeric antigen receptor of the present invention and another receptor specifically targeting a particular antigen, subsequent rounds (including after tumor recurrence) can use receptors targeting different specific antigens.

[0174] In some embodiments, an effective amount of therapeutic cells carrying or expressing the chimeric antigen receptor and optionally a CAR or exogenous transgenic TCR as described in any embodiment of the invention is provided to the individual in need. These cells may be delivered simultaneously or separately from one or more other cancer treatments. These cells and other cancer therapeutics may be delivered in the same or separate formulations. Cells and other cancer therapeutics may be delivered to the individual via separate delivery routes. Cells and / or other cancer therapeutics may be delivered, for example, by injection at the tumor site, intravenous administration, or oral administration. Conventional delivery routes for such compositions are known in the art.

[0175] The number of cells used will depend on a variety of factors, such as the purpose of introduction, cell lifespan, the protocol to be used, the number of administrations, cell proliferation capacity, and the stability of the recombinant construct.

[0176] Cells can be administered as needed. In some embodiments, multiple protocols can be used to adjust protocol parameters. In specific embodiments, the route or frequency or timing of administration, cell lifespan, and / or the number of cells present can vary. The frequency of administration may, for example, depend at least in part on the factors described above.

[0177] Any of the compositions described herein may be included in the kit. In a non-limiting embodiment, the kit may include cells expressing the chimeric antigen receptor described in any embodiment of the invention for cell therapy and / or reagents for generating one or more cells for cell therapy containing a recombinant expression vector. The kit components are provided in suitable containers.

[0178] In some embodiments, the kit is a medicine box containing the pharmaceutical composition described herein and instructions for use, the instructions for use specifying the indications for the pharmaceutical composition, such as melanoma. The instructions for use may also specify the method of using the pharmaceutical composition.

[0179] Some components of the kit may be packaged in an aqueous matrix or in lyophilized form. The container of the kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container in which the component can be placed, and preferably appropriately aliquoted. In kits containing more than one component, the kit typically also includes a second, third, or other container in which other components can be placed separately. However, various combinations of components can be contained in vials. The kit of the present invention typically also includes a device containing the component in a commercially available, closed-form constraint. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0180] When the kit components are provided in one or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred. In some cases, the container itself may be a syringe, pipette, and / or other such devices.

[0181] The kit components can also be provided in dry powder form. When reagents and / or components are provided as dry powder, the powder can be reconstituted by adding a suitable solvent. Therefore, the kit may also include a second container containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0182] The kit components can also be provided in the form of cryopreservation formulations (e.g., cryopreservation solutions). After thawing, these cryopreservation formulations can be administered directly to the patient or formulated into infusion compositions. Therefore, the kit may also include cell cryopreservation bags, cell cryovials, temperature maintenance devices (e.g., containers containing liquid nitrogen), thawing devices, etc.

[0183] In specific embodiments of the invention, cells intended for use in the cell therapies described herein are provided in the kit. In some embodiments, the cells are essentially the only component of the kit. The kit may contain reagents and materials for preparing the desired cells. In specific embodiments, the reagents and materials contain primers, nucleotides, suitable buffers or buffering agents, salts, etc., for amplifying the desired sequence, and in some cases, the reagents include DNA and / or vectors encoding the chimeric antigen receptor and / or its regulatory elements as described in any embodiment herein.

[0184] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press), relevant references, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0185] Example

[0186] Example 1: Design of transmembrane proteins and construction of their expression vectors

[0187] 1) Structure of CAR

[0188] 4D5-CAR-28Z: CD8 signal peptide-4D5 scFv-CD8 extracellular hinge region-CD28 transmembrane region-CD28 intracellular co-stimulatory region-CD3ζ intracellular signal transduction region;

[0189] 4D5-CAR-28DG: CD8 signal peptide-4D5 scFv-CD8 extracellular hinge region-CD28 transmembrane region-CD28 intracellular co-stimulatory region-CD3δ intracellular signal transduction region-CD3γ intracellular signal transduction region;

[0190] 17-1A-CAR-28Z: CD8 signal peptide-17-1A scFv-CD8 extracellular hinge region-CD28 transmembrane region-CD28 intracellular co-stimulatory region-CD3ζ intracellular signal transduction region

[0191] 2) motif signaling peptides

[0192] CCT: A CTLA-4 carboxyl-terminal polypeptide containing YVKM.

[0193] IN3: A CD63 C-terminal polypeptide containing YEVM

[0194] IN8: A CD68 carboxyl-terminal polypeptide containing YQAL.

[0195] INLP-1: A LAMP-1 carboxyl-terminal polypeptide containing YQTI.

[0196] INLP-2: A LAMP-2a carboxyl-terminal polypeptide containing YEQF.

[0197] 3) NPXY motif signaling peptide

[0198] INLDLR: A C-terminal polypeptide containing FDNPVY in LDLR.

[0199] 4D5 scFv is the extracellular single-chain antibody targeting the HER2 CAR, as described in CN106163547A. 17-1AscFv is a single-chain antibody targeting EpCAM.

[0200] The transmembrane proteins used in the following examples are all fused with EGFP via the P2A peptide. Their structures, sequences, and names are shown in Table 1 below:

[0201] Table 1

[0202]

[0203]

[0204]

[0205] 2) Construction of transmembrane protein expression vector

[0206] The pKB20 vector was constructed according to the method described in Example 1 on page 21 of PCT application WO2022078310A1. The pKB20-EGFP and the pKB20 vector containing the exogenous gene expression cassette were constructed according to the method described in that example.Specifically, the sequences shown in SEQ ID NO:2-21 and 23-36 were synthesized by a commissioned company. Adapters containing corresponding restriction sites were added to the two ends of these sequences using ligase, and then cloned into the prepared pKB20 vector according to the method described in Example 1 on page 21 of the WO2022078310A1 instruction manual. These were named pKB20-4D5-CAR-28Z, pKB20-4D5-CAR-28Z-CCT, pKB20-4D5-CAR-28Z-2CCT, pKB20-4D5-CAR-28Z-IN3, pKB20-4D5-CAR-28Z-2IN3, and pKB20-4D5-CAR-28Z-IN3, respectively. 8. pKB20-4D5-CAR-28Z-2IN8, pKB20-4D5-CAR-28Z-IN83, pKB20-4D5-CAR-28Z-INLP-1, pKB20-4D5-CAR-28Z-INLP-2, pKB20-4D5-CAR-28 DG, pKB20-4D5-CAR-28DG-CCT, pKB20-4D5-CAR-28DG-2CCT, pKB20-4D5-CAR-28DG-IN3, pKB20-4D5-CAR-28DG-2IN3, pKB20-4D5-CAR-28DG -IN8, pKB20-4D5-CAR-28DG-2IN8, pKB20-4D5-CAR-28DG-IN83, pKB20-4D5-CAR-28DG-INLP-1, pKB20-4D5-CAR-28DG-INLP-2, pKB20-17- 1A-CAR-28Z, pKB20-17-1A-CAR-28Z-CCT, pKB20-17-1A-CAR-28Z-2CCT, pKB20-17-1A-CAR-28Z-IN3, pKB20-17-1A-CAR-28Z-2IN3, pKB20 -17-1A-CAR-28Z-IN8, pKB20-17-1A-CAR-28Z-2IN8, pKB20-17-1A-CAR-28Z-IN83, pKB20-17-1A-CAR-28Z-INLP-1, pKB20-17-1A-CAR-28 Z-INLP-2, pKB20-4D5-CAR-28Z-INLDLR, pKB20-4D5-CAR-28Z-2INLDLR, pKB20-17-1A-CAR-28Z-INLDLR, and pKB20-17-1A-CAR-28Z-2INLDLR.

[0207] The recombinant plasmids obtained above were transformed into E. coli (DH5c). After correct sequencing, the plasmids were extracted and purified using a plasmid purification kit from Qiagen to obtain high-quality plasmids for each recombinant expression vector.

[0208] Example 2: Preparation and Detection of Cells Expressing Transmembrane Proteins

[0209] 1. Preparation of CAR-T cells

[0210] Following the steps outlined in Example 1, PBMCs were electroporated with the CAR-expressing vector and the control vector pKB20-EGFP to prepare CAR-T cells targeting HER2 and EpCAM, respectively. The PBMCs used were purchased from AllCells and derived from peripheral blood of healthy adults.

[0211] 1) Collect the suspended cells into a 50ml centrifuge tube and centrifuge at 1200rpm for 3min;

[0212] 2) Discard the supernatant, resuspend in physiological saline, centrifuge at 1200 rpm for 3 min, discard the physiological saline, and repeat this step to count the cells;

[0213] 3) Take 35 1.5ml centrifuge tubes, and add 5×10 to each tube. 6 1 cell, centrifuged at 1200 rpm for 3 min;

[0214] 4) Discard the supernatant, take the electroporation kit (purchased from Lonza), add 18 μL of solution I reagent and 82 μL of solution II reagent, and add 5 μg of each CAR expression plasmid constructed in Example 1 to each tube.

[0215] 5) Transfer the cell suspension containing plasmids from the centrifuge tube to the electroporation cuvette, place it in the electroporator, select program T020, and perform electroporation;

[0216] 6) Using the micropipettes provided in the kit, transfer the electroporated cell suspension to a 12-well plate containing X-VIVO 15 medium (X-VIVO 15 medium containing 5 v / v% human AB serum), mix well, and incubate at 37°C in a 5% CO2 incubator; at the same time, coat 35 wells of a 6-well plate with 1 mL of 5 μg / mL OKT-3 and 5 μg / mL CD28 antibody, and incubate the 6-well plate at 37°C.

[0217] 7) After 6 hours, the cells cultured at 37°C and 5% CO2 were transferred into six-well plates coated with OKT-3 and CD28 antibodies, and IL-2 was added to a final concentration of 100 IU / mL. X-VIVO containing 5 v / v% human AB serum was also added. Add 15 ml of culture medium to 3 mL and culture for 4–5 days. Observe the growth of T cells to obtain CAR-T cells targeting HER2 and EpCAM, respectively, and name them as follows: 4D5-28Z, 4D5-28Z-CCT, 4D5-28Z-2CCT, 4D5-28Z-IN3, 4D5-28Z-2IN3, 4D5-28Z-IN8, 4D5-28Z-2IN8, 4D5-28Z-IN83, 4D5-28Z-INLP-1, 4D5-28Z-INLP-2, 4D5-28DG, 4D5-28DG-CCT, 4D5-28DG-2CCT, 4D5-28DG-IN3, 4D5-28DG-2IN3, 4D5-28DG-IN8, 4D5- 28DG-2IN8, 4D5-28DG-IN83, 4D5-28DG-INLP-1, 4D5-28DG-INLP-2, 17-1A-28Z, 1 7-1A-28Z-CCT, 17-1A-28Z-2CCT, 17-1A-28Z-IN3, 17-1A-28Z-2IN3, 17-1A-28Z- IN8, 17-1A-28Z-2IN8, 17-1A-28Z-IN83, 17-1A-28Z-INLP-1, 17-1A-28Z-INLP-2, 4D5-28Z-INLDLR, 4D5-28Z-2INLDLR, 17-1A-28Z-INLDLR and 17-1A-28Z-2INLDLR. PBMCs electroporated with pKB20-EGFP are denoted as Mock-T.

[0218] 2. Preparation of CAR-TIL

[0219] A. Isolation and culture of TIL cells derived from melanoma tissue

[0220] Fresh melanoma tissue specimens showing HER2 expression positivity in immunohistochemical analysis were selected and immediately processed under aseptic conditions. The melanoma tissues of this embodiment were processed and cultured to obtain TILs according to the culture medium described in Example 1 of WO2022111571A1 and the tumor sample processing method and TIL culture method described in Example 2. WO2022111571A1 is incorporated herein by reference in its entirety.

[0221] Specifically as follows:

[0222] 1) Prepare physiological saline containing penicillin at a final concentration of 100 U / mL, streptomycin at a final concentration of 100 μg / mL, and gentamicin at a final concentration of 50 μg / mL for later use;

[0223] 2) In a sterile environment in a biosafety cabinet, the freshly isolated tumor tissue samples from the tumor patients were washed in a 10cm culture dish containing 30mL of physiological saline prepared in step 1), and then transferred to a new 10cm culture dish containing 30mL of physiological saline prepared in step 1). This washing process was repeated 3 times.

[0224] 3) Using a sterile scalpel blade, remove adipose and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm. 3 Forty-two randomly selected tumor tissue blocks were placed in two G-REX100 culture jars (purchased from Wilsonwolf). Seed cell culture medium was added to each jar, and the components of the seed culture medium included: 3000 IU / mL IL-2, 20 ng / mL IL-7, 20 ng / mL IL-15, 500 U / mL GM-CSF, 1000 IU / mL IFN-γ, 3 μg / mL anti-CD137 mAb, 3 μg / mL anti-CD28 mAb, 3 μg / mL anti-PD-1 mAb, 10 ng / mL TNF-α, 5% v / v human AB serum, 1×PS double antibody, and X-VIVO 15 basal medium to the final volume. Excess tumor tissue blocks were cryopreserved in liquid nitrogen using a programmed freezing system with CryoStor10 cryopreservation solution (purchased from BioLifeSolutions).

[0225] 4) After adding 1L of the above-mentioned seed cell culture medium to the G-REX100 culture vessel containing the tumor tissue block in step 3), the tumor tissue block was cultured at 37℃ with 5% CO2. Every 4 days, half the volume of the old seed cell culture medium was removed and half the volume of fresh seed cell culture medium was added. On the 12th day, the TIL seed cells were harvested by centrifugation, and the total number of cells and viability were counted. The harvested TIL seed cells were used for subsequent experiments.

[0226] B. Electroporation preparation of CAR-TIL

[0227] 1) Add %X-VIVO 15 medium containing 5v / v human AB serum to 12-well plates, for a total of 20 wells, 2mL per well, and then transfer to a cell culture incubator to preheat at 37°C with 5% CO2 for 1 hour.

[0228] 2) Prepare the electro-spreading fluid ratio for each well using the following table:

[0229] <![CDATA[100μL Nucleocuvette TM Strip(μL)]]> <![CDATA[Nucleofector TM Volume of solution 82 Electroporation replenishment solution 18

[0230] Prepare 35 sets of electroporation solutions for electroporation into TIL of the following plasmids constructed in Example 1: pKB20-4D5-CAR-28Z, pKB20-4D5-CAR-28Z-CCT, pKB20-4D5-CAR-28Z-2CCT, pKB20-4D5-CAR-28Z-IN3, pKB20-4D5-CAR-28Z-2IN3, and pKB20-4D5-CAR-28Z-IN8. , pKB20-4D5-CAR-28Z-2IN8, pKB20-4D5-CAR-28Z-IN83, pKB20-4D5-CAR-28Z-INLP-1, pKB20-4 D5-CAR-28Z-INLP-2, pKB20-4D5-CAR-28Z-INLDLR, pKB20-4D5-CAR-28Z-2INLDLR and pKB20-EGFP;

[0231] 3) Take the TIL obtained in A and put it into 13 EP tubes, adding 1×10 to each EP tube. 7 Centrifuge each cell at 800g for 5 minutes, discard the supernatant, then resuspend the cells in 500μL of physiological saline, and repeat the centrifugation steps to wash the cell pellet.

[0232] 4) Add 5 μg of each plasmid described in 2) to each of the electroporation solutions prepared in 2), and then let stand at room temperature for no more than 30 min;

[0233] 5) Resuspend all tubes in the plasmid-containing electroporation buffer prepared in step 4), 100 μL per tube. Carefully transfer the cell resuspended cell buffer into a LONZA 100 μL electroporation cuvette and place the cuvette into a LONZA Nucleofector. TM Inside the 2b electro-rotation tank, start the electro-rotation program and select program X001;

[0234] 6) After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to EP tubes. Add 200 μL of preheated X-VIVO 15 medium to each tube, then transfer the solution into the 12-well plate containing preheated X-VIVO 15 medium from step 1). TILs overexpressing different CARs were obtained and cultured in 15 wells of medium at 37°C and 5% CO2 for 5 days. They were named TIL-4D5-28Z, TIL-4D5-28Z-CCT, TIL-4D5-28Z-2CCT, TIL-4D5-28Z-IN3, TIL-4D5-28Z-2IN3, TIL-4D5-28Z-IN8, TIL-4D5-28Z-2IN8, TIL-4D5-28Z-IN83, TIL-4D5-28Z-INLP-1, TIL-4D5-28Z-INLP-2, TIL-4D5-28Z-INLP-1 and TIL-4D5-28Z-INLP-2. The TIL overexpressing pKB20-EGFP was named TIL-CTRL.

[0235] 3. Detection of CAR-T and CAR-TIL survival rates and transmembrane protein expression positivity rates

[0236] 1) Survival rate detection

[0237] The survival rates of CAR-T cells and HER2 CAR-TILs in each group were detected by trypan blue staining and cell counting. The results showed that the survival rates of CAR-T cells and CAR-TILs prepared in groups 1 and 2 were all above 95%.

[0238] 2) CAR expression positivity rate detection

[0239] Method 1: In all electroporated vectors, CAR was linked to EGFP via the P2A peptide. The percentage of cells expressing CAR gene positive could be obtained by detecting the percentage of cells expressing EGFP positive by flow cytometry.

[0240] Method 2: The proportion of CAR-expressing cells was determined by detecting the percentage of CAR-expressing cells on the cell surface, as detailed below:

[0241] (1) Collect CAR-T cells and CAR-TILs prepared in Example 2 respectively, with 1×10⁶ cells collected for each type of cell. 6 Centrifuge at 1000 rpm for 3 minutes;

[0242] (2) Discard the supernatant, add physiological saline to resuspend the cells, and centrifuge at 1000 rpm for 3 min;

[0243] (3) Discard the supernatant, add 100 μL of physiological saline to each tube to resuspend the cells, add 1 μL of biotin-labeled HER2 antigen (purchased from Kaika Biotechnology; catalog number: HER-HM402) to each tube, and incubate at 14℃ for 30 minutes;

[0244] (4) Add appropriate amount of physiological saline to each, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0245] (5) Add 100 μL of physiological saline to each tube to resuspend the cells, add 1 μL of PE-labeled streptavidin (purchased from ThermoFisher, catalog number: S20982) to each tube, mix well, and incubate at 4°C for 30 min.

[0246] (6) Add appropriate amount of physiological saline to each, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0247] (7) Resuspend in 400 μL of physiological saline and analyze by flow cytometer.

[0248] The proportions of positive CAR-T and CAR-TIL cells in each group are shown in Table 2:

[0249] Table 2

[0250]

[0251]

[0252] Table 2 shows that the proportion of CAR gene-positive cells detected by Method 1 in each group of CAR-T cells was approximately 30%. The proportion of cells with CAR expression on the cell surface detected by Method 2 varied among different cell structures. For CARs with an intracellular region structure of CD28 intracellular co-stimulatory region + CD3ζ intracellular signal transduction region (28Z), [the following is unclear and likely incomplete]... The positive rates of all groups of motif internalized peptides were significantly lower than those of wild-type peptides (4D5-28Z, 17-1A-28Z) that did not contain any sorting peptides.

[0253] For CARs with an intracellular region structure of CD28 intracellular co-stimulatory region + CD3δ intracellular region (28D) or CD3γ intracellular region (28G), the positive rate obtained by method 2 was significantly lower than that of method 1. When the above structure further includes... When motif internalization peptides are used, the positive rate of Method 2 is significantly lower than that of the former.

[0254] The results in CAR-TIL are similar to those in CAR-T, but do not include... When motif internalization peptides were detected, the CAR-TIL positivity rates obtained by Method 2 and Method 1 were essentially consistent. However, for peptides containing... The positive rates of all TILs containing motif internalized peptides were significantly lower than those of wild-type TILs that did not contain any sorting peptides.

[0255] For CAR-T and CAR-TIL containing NPXY, the positive rate results of Method 2 were basically consistent with those of Method 1, with no significant decrease.

[0256] The above results indicate that when the CAR structure contains When motifs are internalized into peptides, the positivity rate of CAR on the surface of CAR-T and CAR-TIL cells is significantly reduced.

[0257] Example 3: Repeated stimulation of HER2-targeting CAR-T cell tumor antigens

[0258] The HER2-positive cell line SKOV3 was selected as the tumor cell line to stimulate HER2 CAR-T therapy. Details are as follows:

[0259] 1) Take 1×10 6 SKOV3 cells were seeded into each well of a 6-well plate and incubated overnight at 37°C and 5% CO2 in DMEM 10 v / v% FBS serum. After adhesion, 1 × 10⁻⁶ cells were added to each well, resuspended in an equal volume of X-VIVO 15 5 v / v% human AB serum medium (previously electroporated but not stimulated by OKT-3 and anti-CD28 antibody) from step 6 of Example 2. 6 Her2 CAR-T cells and Mock-T cells in each group are now designated as D1.

[0260] 2) After co-incubating at 37℃ and 5% CO2 for 24 hours, the supernatant Her2 CAR-T suspension was collected, centrifuged at 800g for 5 minutes, and the cell pellet was resuspended in X-VIVO 15 5v / v% human AB serum medium and cultured at 37℃ and 5% CO2.

[0261] 3) The above operations were repeated on D4 and D7. After tumor cell stimulation on D7, Her2 CAR-T cells from each group were transfected into six-well plates coated with OKT-3 and CD28 antibodies, and IL-2 was added to a final concentration of 100 IU / mL. X-VIVO 15 medium containing 5 v / v% human AB serum was added to a final volume of 3 mL. The cells were incubated at 37°C with 5% CO2 for 24 h before further analysis. The positive rate and exhaustion phenotype of Her2 CAR-T cells from each group that underwent three repeated tumor cell stimulations were detected by flow cytometry, and IFN-γ secretion levels were detected using an HTRF assay kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the instructions. The positive rate was detected according to methods 1 and 2 of Example 2. The results are as follows:

[0262] Table 3. Positive rates of different HER2 CAR-T cells after repeated stimulation with tumor antigens.

[0263] Cell Name Method 1 Positive Rate (%) Method 2 Positive Rate (%) 4D5-CAR-28Z 51.9 48.7 4D5-CAR-28Z-CCT 54.7 38.6 4D5-CAR-28Z-2CCT 48.6 21.7 4D5-CAR-28Z-IN3 49.8 35.1 4D5-CAR-28Z-2IN3 47.2 21.4 4D5-CAR-28Z-IN8 51.1 31.1 4D5-CAR-28Z-2IN8 46.7 20.1 4D5-CAR-28Z-IN83 46.5 22.1 4D5-CAR-28Z-INLP-1 52.5 33.5 4D5-CAR-28Z-INLP-2 49.0 32.8 4D5-CAR-28DG 49.3 44.6 4D5-CAR-28DG-CCT 44.8 30.5 4D5-CAR-28DG-2CCT 54.7 23.4 4D5-CAR-28DG-IN3 44.6 37.7 4D5-CAR-28DG-2IN3 55.1 20.6 4D5-CAR-28DG-IN8 53.6 37.3 4D5-CAR-28DG-2IN8 54.0 23.3 4D5-CAR-28DG-IN83 51.9 22.6 4D5-CAR-28DG-INLP-1 45.6 36.7 4D5-CAR-28DG-INLP-2 51.1 34.3 4D5-28Z-INLDLR 51.7 49.5 4D5-28Z-2INLDLR 49.8 47.8 Mock-T N / A N / A

[0264] Table 4. Proportion of HER2 CAR-T cells positive for depletion markers after repeated tumor antigen stimulation

[0265] Cell Name PD-1 (%) TIGIT (%) LAG-3 (%) 4D5-CAR-28Z 41.3 35.4 32.5 4D5-CAR-28Z-CCT 29.9 25.5 23.4 4D5-CAR-28Z-2CCT 28.0 21.5 20.4 4D5-CAR-28Z-IN3 32.9 26.8 27.1 4D5-CAR-28Z-2IN3 30.6 21.8 22.3 4D5-CAR-28Z-IN8 33.5 31.2 28.1 4D5-CAR-28Z-2IN8 29.0 23.0 27.6 4D5-CAR-28Z-IN83 24.5 22.3 24.3 4D5-CAR-28Z-INLP-1 35.6 27.6 28.7 4D5-CAR-28Z-INLP-2 33.4 32.9 29.0 4D5-CAR-28DG 39.5 44.7 37.5 4D5-CAR-28DG-CCT 30.4 41.2 28.4 4D5-CAR-28DG-2CCT 20.7 25.9 26.0 4D5-CAR-28DG-IN3 32.7 36.7 34.9 4D5-CAR-28DG-2IN3 27.2 31.9 29.6 4D5-CAR-28DG-IN8 32.7 36.5 29.3 4D5-CAR-28DG-2IN8 20.6 25.8 21.5 4D5-CAR-28DG-IN83 22.6 26.2 24.5 4D5-CAR-28DG-INLP-1 30.9 37.7 32.8 4D5-CAR-28DG-INLP-2 32.9 36.4 29.5 4D5-28Z-INLDLR 40.8 35.1 30.6 4D5-28Z-2INLDLR 39.9 34.7 31.3 Mock-T 21.3 19.8 22.4

[0266] Table 5. IFN-γ secretion levels of each HER2 CAR-T cell after repeated stimulation with tumor antigens.

[0267]

[0268]

[0269] Table 3 shows that after repeated stimulation with HER2+ target cells, the proportion of CAR-positive T cells in each group of CAR-T cells significantly increased. The proportion of CAR-positive T cells detected by Method 1 in each group of CAR-T cells was significantly higher than that detected by Method 2. Table 4 shows that after repeated stimulation with tumor antigens, compared with CAR-T cells (4D5-28Z, 4D5-28DG) whose structures do not contain motif signaling peptides, those containing motif signaling peptides... The proportion of depletion marker-positive cells in all CAR-T groups containing the motif signaling peptide decreased to varying degrees. However, the proportion of depletion marker-positive cells in CAR-T groups containing the NPXY motif signaling peptide (4D5-28Z-INLDLR, 4D5-28Z-2INLDLR) did not decrease significantly compared to CAR-T groups without the motif signaling peptide. Table 5 shows that after repeated stimulation with tumor antigens, the proportion of depletion marker-positive cells in CAR-T groups containing the NPXY motif signaling peptide decreased significantly compared to CAR-T groups without the motif signaling peptide. The secretion levels of IFN-γ in CAR-T cells of all groups containing the motif signaling peptide were increased to varying degrees, while the secretion levels of IFN-γ in CAR-T cells of all groups containing the NPXY motif signaling peptide were not significantly increased.

[0270] Example 4: Killing of target cells by HER2 CAR-T cells under low target-to-cell ratio conditions

[0271] To better simulate the killing effect of HER2 CAR-T cells on tumor cells under repeated stimulation by tumor cells, the Real-Time Label-Free Cell Function Analyzer (RTCA) from Agilent Technologies was used to detect the killing activity of each group of HER2 CAR-T cells against HER2-expressing target cells SKOV3 under in vitro low target-to-cell ratio conditions. The specific steps are as follows:

[0272] (1) Zeroing: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and zero the instrument;

[0273] (2) Target cell plating: SKOV3 cells were plated at a density of 10 cells per well. 4 50 μL of cells were seeded in a plate containing the detection electrode and left to stand for several minutes until the cells stabilized. Then the cells were placed in the instrument to begin step 2, cell culture.

[0274] (3) Adding effector cells: After culturing target cells for 18-24 hours, observe the cell index. When the cell index is 0.8-1, add 50 μL of each group of HER2 CAR-T cells to each well, with an effector-to-target ratio of 1:16. Begin step 3. After co-culturing for more than 96 hours, observe the cell proliferation curve and killing level, and calculate the target cell killing rate. The formula for calculating the target cell killing rate is as follows:

[0275]

[0276] Where A represents the cell index of the group containing only target cells (i.e., tumor cells) without any effector cells, and B represents the cell index of each group with effector cells. The results are shown in Table 6:

[0277] Table 6. Results of HER2 CAR-T cell killing at low target-to-RTCA ratio in each group

[0278] Cell Name lethality (%) 4D5-CAR-28Z 18.2 4D5-CAR-28Z-CCT 21.9 4D5-CAR-28Z-2CCT 24.7 4D5-CAR-28Z-IN3 28.9 4D5-CAR-28Z-2IN3 30.9 4D5-CAR-28Z-IN8 27.3 4D5-CAR-28Z-2IN8 33.0 4D5-CAR-28Z-IN83 41.5 4D5-CAR-28Z-INLP-1 33.8 4D5-CAR-28Z-INLP-2 35.1 4D5-CAR-28DG 15.4 4D5-CAR-28DG-CCT 18.9 4D5-CAR-28DG-2CCT 18.3 4D5-CAR-28DG-IN3 19.7 4D5-CAR-28DG-2IN3 21.5 4D5-CAR-28DG-IN8 20.4 4D5-CAR-28DG-2IN8 23.9 4D5-CAR-28DG-IN83 25.2 4D5-CAR-28DG-INLP-1 19.7 4D5-CAR-28DG-INLP-2 21.1 4D5-28Z-INLDLR 18.4 4D5-28Z-2INLDLR 17.6 Mock-T 10.5

[0279] Table 6 shows the results, which are related to the structure that does not contain Compared to CAR-T therapy using motif signaling peptides, it contains different... CAR-T cells containing motif signaling peptides all showed a certain degree of increased killing rate against target cells. However, CAR-T cells containing NPXY motif signaling peptides (4D5-28Z-INLDLR, 4D5-28Z-2INLDLR) did not show a significant increase in target cell killing rate compared to their counterparts without NPXY motif signaling peptides.

[0280] Example 5: Repeated stimulation of EpCAM CAR-T cells with tumor antigens

[0281] The EpCAM-positive cell line MCF-7 (purchased from ATCC, Cat#HTB-22) was selected as the tumor cells to stimulate EpCAM CAR-T. Details are as follows:

[0282] 1) Take 1×10 6MCF-7 cells were seeded into each well of a 6-well plate and incubated overnight at 37°C and 5% CO2 in DMEM 10 v / v% FBS serum. After adhesion, 1×10⁻⁶ cells resuspended in an equal volume of X-VIVO 15 5 v / v% human AB serum medium (from step 6) of Example 2, which had been electroporated but not stimulated by OKT-3 and anti-CD28 antibody, were added to each well. 6 Each EpCAM CAR-T cell and Mock-T cell is designated as D1 at this point;

[0283] 2) After co-incubating at 37℃ and 5% CO2 for 24 hours, collect the supernatant EpCAM CAR-T suspension, centrifuge at 800g for 5 minutes, resuspend the cell pellet in X-VIVO 15 5v / v% human AB serum medium, and then culture at 37℃ and 5% CO2.

[0284] 3) The above operations were repeated on D4 and D7. After tumor cell stimulation on D7, EpCAM CAR-T cells from each group were transfected into six-well plates coated with OKT-3 and CD28 antibodies, and IL-2 was added to a final concentration of 100 IU / mL. X-VIVO 15 medium containing 5 v / v% human AB serum was added to a final volume of 3 mL. The cells were incubated at 37°C with 5% CO2 for 24 h before further analysis. The positivity rate and exhaustion phenotype of EpCAM CAR-T cells from each group after three repeated tumor cell stimulations were detected by flow cytometry, and IFN-γ secretion levels were detected using an HTRF assay kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the instructions. The positivity rate was detected according to methods 1 and 2 of Example 2. The results are as follows:

[0285] Table 7. Positive rates of each EpCAM CAR-T cell after repeated stimulation with tumor antigens.

[0286] Cell Name Method 1 Positive Rate (%) Method 2 Positive Rate (%) 17-1A-28Z 54.5 50.6 17-1A-28Z-CCT 51.6 38.4 17-1A-28Z-2CCT 53.9 26.9 17-1A-28Z-IN3 48.4 30.7 17-1A-28Z-2IN3 52.9 20.1 17-1A-28Z-IN8 53.6 31.3 17-1A-28Z-2IN8 50.7 21.5 17-1A-28Z-IN83 56.5 32.4 17-1A-28Z-INLP-1 49.2 23.2 17-1A-28Z-INLP-2 54.3 20.7 17-1A-28Z-INLDLR 47.9 43.2 17-1A-28Z-2INLDLR 51.2 46.2 Mock-T N / A N / A

[0287] Table 8. Proportion of cells positive for exhaustion markers of each EpCAM CAR-T cell type after repeated stimulation with tumor antigens.

[0288]

[0289]

[0290] Table 9. IFN-γ secretion levels of each EpCAM CAR-T cell after repeated stimulation with tumor antigens.

[0291] Cell Name IFN-γ (pg / mL) 17-1A-28Z 7696 17-1A-28Z-CCT 9635 17-1A-28Z-2CCT 11182 17-1A-28Z-IN3 10451 17-1A-28Z-2IN3 12987 17-1A-28Z-IN8 11649 17-1A-28Z-2IN8 13921 17-1A-28Z-IN83 14074 17-1A-28Z-INLP-1 12286 17-1A-28Z-INLP-2 13771 17-1A-28Z-INLDLR 8201 17-1A-28Z-2INLDLR 7993 Mock-T 9014

[0292] Table 7 shows that after repeated stimulation with EpCAM+ target cells, the proportion of CAR-positive T cells in each group of CAR-T cells significantly increased. The proportion of CAR-positive T cells detected by Method 1 in each group of CAR-T cells was significantly higher than that detected by Method 2. Table 8 shows that after repeated stimulation with tumor antigens, compared with CAR-T cells (17-1A-28Z) that do not contain motif signaling peptides in their structure, those containing motif signaling peptides in their structure... The proportion of depletion marker-positive cells in all CAR-T groups containing the motif signaling peptide decreased to varying degrees. However, the proportion of depletion marker-positive cells in CAR-T groups containing the NPXY motif signaling peptide (17-1A-28Z-INLDLR, 17-1A-28Z-2INLDLR) did not decrease significantly compared to CAR-T groups without the motif signaling peptide. Table 9 shows that after repeated stimulation with tumor antigens, the proportion of depletion marker-positive cells in CAR-T groups containing the NPXY motif signaling peptide decreased significantly compared to CAR-T groups without the motif signaling peptide. The secretion levels of IFN-γ in CAR-T cells of all groups containing the motif signaling peptide were increased to varying degrees, while the secretion levels of IFN-γ in CAR-T cells of all groups containing the NPXY motif signaling peptide were not significantly increased.

[0293] Example 6: Killing of target cells by EpCAM CAR-T cells under low target-to-cell ratio conditions

[0294] Following the procedure in Example 4, the killing effect of EpCAM CAR-T cells on target cells MCF-7 was tested in each group at a low target-to-cell ratio. The HER2 CAR-T cells in each group were replaced with the EpCAM CAR-T cells in each group, and the target cells were replaced with MCF-7 cells; all other conditions were the same as in Example 4. The results are shown in Table 10.

[0295] Table 10. Results of low-efficiency target-to-RTCA killing of EpCAM CAR-T cells in each group

[0296]

[0297]

[0298] Table 10 shows the results, which are consistent with structures that do not contain... Compared to CAR-T therapy using motif signaling peptides, it contains different... CAR-T cells containing motif signaling peptides all showed a certain degree of increased killing rate against target cells. However, CAR-T cells containing NPXY motif signaling peptides (17-1A-28Z-INLDLR, 17-1A-28Z-2INLDLR) did not show a significant increase in target cell killing rate compared to their counterparts without NPXY motif signaling peptides.

[0299] Example 7: Repeated stimulation of CAR-TIL cell tumor antigens

[0300] The HER2-positive cell line SKOV3 was selected as the tumor cells to stimulate CAR-TIL. Specific procedures are detailed in Example 3. The HER2 CAR-TIL cells in each group, after three repeated stimulations with tumor cells, were analyzed for positivity rate and exhaustion phenotype using flow cytometry. IFN-γ secretion levels were detected using an HTRF assay kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the manufacturer's instructions. The positivity rate was measured according to methods 1 and 2 of Example 2. The results are as follows:

[0301] Table 11. Positive rates of various CAR-TILs after repeated tumor antigen stimulation

[0302] Cell Name Method 1 Positive Rate (%) Method 2 Positive Rate (%) TIL-4D5-28Z 40.4 36.5 TIL-4D5-28Z-CCT 42.2 29.7 TIL-4D5-28Z-2CCT 39.8 20.3 TIL-4D5-28Z-IN3 33.1 21.2 TIL-4D5-28Z-2IN3 37.5 18.4 TIL-4D5-28Z-IN8 35.9 19.3 TIL-4D5-28Z-2IN8 45.6 24.1 TIL-4D5-28Z-IN83 40.9 23.6 TIL-4D5-28Z-INLP-1 42.7 26.2 TIL-4D5-28Z-INLP-2 38.4 22.0 TIL-4D5-28Z-INLDLR 47.7 42.8 TIL-4D5-28Z-2INLDLR 41.2 37.4 TIL-CTRL N / A N / A

[0303] Table 12. Proportion of cells positive for exhaustion markers of each CAR-TIL after repeated stimulation with tumor antigens.

[0304] Cell Name PD-1 (%) TIGIT (%) Lag-3 (%) TIL-4D5-28Z 15.8 39.1 35.3 TIL-4D5-28Z-CCT 9.5 30.1 29.2 TIL-4D5-28Z-2CCT 8.1 26.9 22.3 TIL-4D5-28Z-IN3 8.6 27.5 20.1 TIL-4D5-28Z-2IN3 7.3 20.5 17.9 TIL-4D5-28Z-IN8 9.2 28.6 24.6 TIL-4D5-28Z-2IN8 7.9 21.8 18.7 TIL-4D5-28Z-IN83 7.2 18.3 19.1 TIL-4D5-28Z-INLP-1 7.3 22.4 27.8 TIL-4D5-28Z-INLP-2 8.0 23.1 24.6 TIL-4D5-28Z-INLDLR 14.1 40.7 33.9 TIL-4D5-28Z-2INLDLR 16.3 38.8 34.6 ON-CTRL 15.2 33.3 38.7

[0305] Table 13. IFN-γ secretion levels of each CAR-TIL after repeated stimulation with tumor antigens.

[0306]

[0307] Table 11 shows that after repeated stimulation with HER2+ target cells, the proportion of CAR-positive TIL cells in each group of CAR-TIL cells significantly increased. The proportion of CAR-positive TIL cells detected by Method 1 in each group of CAR-TIL cells was significantly higher than that detected by Method 2. Table 12 shows that after repeated stimulation with tumor antigens, compared with CAR-TILs (TIL-4D5-28Z, TIL-4D5-28DG) whose structures do not contain motif signaling peptides, those with motif signaling peptides in their structures... The proportion of depletion marker-positive cells in all CAR-TIL groups containing the motif signaling peptide decreased to varying degrees. However, compared with CAR-T cells without the motif signaling peptide, the proportion of depletion marker-positive cells in CAR-TIL groups containing the NPXY motif signaling peptide (TIL-4D5-28Z-INLDLR, TIL-4D5-28Z-2INLDLR) did not show a significant decrease. Table 13 shows that after repeated stimulation with tumor antigens, the proportion of depletion marker-positive cells in CAR-TIL groups containing the NPXY motif signaling peptide decreased significantly compared with CAR-TILs without the motif signaling peptide. The secretion level of IFN-γ in CAR-TIL cells of all groups containing the motif signaling peptide was increased to varying degrees, while the secretion level of IFN-γ in CAR-TIL cells of all groups containing the NPXY motif signaling peptide was not significantly increased.

[0308] Example 10: Killing of target cells by HER2 CAR-TIL under low target-to-cell ratio conditions

[0309] The fresh melanoma tissue from Example 2 was cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and cultured to obtain primary melanoma cells according to the method described in the Materials and Methods section of Robert Suriano et al. Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines J Cancer 2013;4(5):371-382. Following the procedure in Example 4, the obtained primary melanoma cells were used as target cells to test the killing effect of each group of CAR-TIL cells on the target cells at a low target-to-cell ratio. Each group of CAR-T cells was replaced with its corresponding CAR-TIL, and the target cells were replaced with primary melanoma cells; other conditions remained the same as in Example 4. The results are shown in Table 14.

[0310] Table 14. CAR-TIL inefficient target ratio RTCA kill results for each group

[0311] cell name kill rate(%) TO-4D5-28Z 18.9 TIL-4D5-28Z-CCT 25.8 TO-4D5-28Z-2CCT 29.7 TO-4D5-28Z-IN3 27.1 TO-4D5-28Z-2IN3 31.3 TO-4D5-28Z-IN8 23.3 TO-4D5-28Z-2IN8 26.7 TO-4D5-28Z-IN83 27.1 TIL-4D5-28Z-INLP-1 28.5 TIL-4D5-28Z-INLP-2 30.5 TIL-4D5-28Z-INLDLR 20.2 TIL-4D5-28Z-2INLDLR 16.8 ON-CTRL 12.4

[0312] Table 14 shows the results, which are consistent with structures that do not contain... Compared to CAR-TILs containing motif signaling peptides, it contains different CAR-T cells containing motif signaling peptides all showed a certain degree of increased killing rate against target cells. However, CAR-TILs (TIL-4D5-28Z-INLDLR and TIL-4D5-28Z-2INLDLR) containing NPXY motif signaling peptides did not show a significant increase in target cell killing rate compared to their counterparts without NPXY motif signaling peptides.

[0313] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

[0314] Part of the sequence in this article

[0315]

[0316]

Claims

1. A chimeric antigen receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain comprises an internalizing peptide. Preferably, the internalizing peptide comprises one or more tyrosine-based internalization signals. More preferably, the tyrosine-based internalization signal is based on Signals with motifs or NPXY motifs.

2. The chimeric antigen receptor as described in claim 1, characterized in that, based on The motif signal is a C-terminal sequence of a membrane protein containing YX1X2X3, or a sequence with at least 90% sequence identity, wherein X3 is M, L, I, F, or V; preferably, X1 is V, E, or Q, and X2 is K, V, A, T, or Q. The NPXY motif-based signal is a C-terminal sequence of a membrane protein containing NPXY or a sequence that has at least 90% sequence identity with it, where X is V, E, D, T, L, M, I, or K. Preferably, the carboxyl-terminal sequence is a sequence of at least four amino acids at the carboxyl terminus of a membrane protein.

3. The chimeric antigen receptor as described in claim 1 or 2, characterized in that, Each of the one or more tyrosine-based internalization signals independently has a sequence selected from the following: The CTLA-4 carboxyl-terminal sequence containing YVKM Contains the CD63 carboxyl-terminal sequence of YEVM. Contains the CD68 carboxyl-terminal sequence of YQAL. The LAMP-1 carboxyl-terminal sequence containing YQTI, The LAMP-2a carboxyl-terminal sequence containing YEQF, The LDLR carboxyl-terminal sequence containing FDNPVY Preferably, The CTLA-4 carboxyl-terminal sequence containing YVKM is the 4-9 amino acid fragment containing YVKM in SEQ ID NO.

42. The CD63 C-terminal sequence containing YEVM is the 4-6 amino acid fragment containing YEVM as described in SEQ ID NO.

44. The CD68 C-terminal sequence containing YQAL is the 4-6 amino acid fragment containing YQAL in SEQ ID NO.

46. The LAMP-1 carboxyl-terminal sequence containing YQTI is the 4-7 amino acid fragment containing YQTI, as described in SEQ ID NO.

49. The LAMP-2a C-terminal sequence containing YEQF is the 4-7 amino acid fragment containing YEQF, as described in SEQ ID NO.

50. The LDLR carboxyl-terminal sequence containing NPVY is the 4-6 amino acid fragment containing NPVY in SEQ ID NO.

51.

4. The chimeric antigen receptor as described in claim 3, characterized in that, The internalized peptide comprises: (1) One, two, three or more CTLA-4 carboxyl-terminal sequences containing YVKM, optionally also containing one, two, three or more sequences selected from: a CD63 carboxyl-terminal sequence containing YEVM, a CD68 carboxyl-terminal sequence containing YQAL, a LAMP-1 carboxyl-terminal sequence containing YQTI, a LAMP-2a carboxyl-terminal sequence containing YEQF, or an LDLR carboxyl-terminal sequence containing NPVY. (2) One, two, three or more CD63 carboxyl-terminal sequences containing YEVM, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-1 carboxyl-terminal sequence containing YQTI, LAMP-2a carboxyl-terminal sequence containing YEQF, LDLR carboxyl-terminal sequence containing NPVY. (3) One, two, three or more CD68 carboxyl-terminal sequences containing YQAL, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, LAMP-1 carboxyl-terminal sequence containing YQTI, LAMP-2a carboxyl-terminal sequence containing YEQF, LDLR carboxyl-terminal sequence containing NPVY, (4) One, two, three or more LAMP-1 carboxyl-terminal sequences containing YQTI, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-2a carboxyl-terminal sequence containing YEQF, LDLR carboxyl-terminal sequence containing NPVY, (5) One, two, three or more LAMP-2a carboxyl-terminal sequences containing YEQF, optionally also containing one, two, three or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-1 carboxyl-terminal sequence containing YQTI, LDLR carboxyl-terminal sequence containing NPVY. (6) 1, 2, 3 or more LDLR carboxyl-terminal sequences containing NPVY, optionally also containing 1, 2, 3 or more sequences selected from: CTLA-4 carboxyl-terminal sequence containing YVKM, CD63 carboxyl-terminal sequence containing YEVM, CD68 carboxyl-terminal sequence containing YQAL, LAMP-1 carboxyl-terminal sequence containing YQTI, LAMP-2a carboxyl-terminal sequence containing YEQF, Preferably, the internalized peptide comprises: (1) One, two, three or more tandem CTLA-4 carboxyl-terminal sequences containing YVKM (2) One, two, three or more CD63 carboxyl-terminal sequences containing YEVM in tandem. (3) One, two, three or more CD68 carboxyl-terminal sequences containing YQAL in tandem. (4) A tandem CD68 carboxyl-terminal sequence containing YQAL and a CD63 carboxyl-terminal sequence containing YEVM. (5) One, two, three or more LAMP-1 carboxyl-terminal sequences containing YQTI in tandem. (6) One, two, three or more LAMP-2a carboxyl-terminal sequences containing YEQF in tandem. (7) One, two, three or more LDLR carboxyl-terminal sequences containing NPVY in tandem. More preferably, the internalized peptide comprises: (1) One or two tandem CTLA-4 carboxyl-terminal sequences containing YVKM, (2) One or two CD63 carboxyl-terminal sequences containing YEVM, (3) One or two CD68 carboxyl-terminal sequences containing YQAL, (4) A tandem CD68 carboxyl-terminal sequence containing YQAL and a CD63 carboxyl-terminal sequence containing YEVM. (5) One or two LAMP-1 carboxyl-terminal sequences containing YQTI, (6) One or two LAMP-2a carboxyl-terminal sequences containing YEQF, (7) One or two LDLR carboxyl-terminal sequences containing NPVY, More preferably, the amino acid sequence of the internalized peptide is shown in SEQ ID NO:42-52.

5. The chimeric antigen receptor as described in claim 1 or 2, characterized in that, The cytoplasmic domains include signal transduction domains and / or co-stimulatory domains. The chimeric antigen receptor further includes a hinge region, preferably located between the extracellular ligand-binding domain and the transmembrane domain. The chimeric antigen receptor further includes a signal peptide, preferably located at the N-terminus of the chimeric antigen receptor. The extracellular ligand-binding domain is an antibody or its antigen-binding fragment that targets tumor antigens.

6. A polynucleotide having: (1) The nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 1-5, and / or (2)(1) complementary sequences.

7. A nucleic acid construct comprising the polynucleotide of claim 6, Preferably, the nucleic acid construct is an expression vector or a cloning vector.

8. A host cell, wherein: (1) Containing, expressing, or secreting the chimeric antigen receptor of any one of claims 1-5, and / or (2) Containing a polynucleotide encoding the chimeric antigen receptor of any one of claims 1-5 or a nucleic acid construct containing said polynucleotide, Preferably, the host cell is an immune effector cell. More preferably, the immune effector cells are T cells, NK cells, CIK cells, or tumor-infiltrating lymphocytes.

9. A pharmaceutical composition comprising any one or more of the chimeric antigen receptor of any one of claims 1-5, the polynucleotide of claim 6, the nucleic acid construct of claim 7, and the host cell of claim 8, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

10. Use of any one or more of the chimeric antigen receptor of any one of claims 1-5, the polynucleotide of claim 6, the nucleic acid construct of claim 7, and the host cell of claim 8 in the preparation of a medicament for treating or preventing cancer. Preferably, the cancer is a tumor antigen-associated cancer, and the tumor antigen is a tumor antigen targeted by the extracellular ligand-binding domain of the chimeric antigen receptor. More preferably, the cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia.

11. A method for enhancing the activity of immune effector cells in an individual, comprising introducing immune effector cells into the individual, the immune effector cells comprising the chimeric antigen receptor of any one of claims 1-5. Preferably, The immune effector cells are T cells, NK cells, CIK cells, or tumor-infiltrating lymphocytes. The individual has tumor antigen-associated cancer, where the tumor antigen is a tumor antigen targeted by the extracellular ligand-binding domain of the chimeric antigen receptor, or The cancers mentioned are selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia.

12. A method of treating an individual with cancer, comprising introducing immune effector cells into the individual, the immune effector cells comprising the chimeric antigen receptor of any one of claims 1-5. Preferably, The immune effector cells are T cells, NK cells, CIK cells, or tumor-infiltrating lymphocytes. The individual has tumor antigen-associated cancer, where the tumor antigen is a tumor antigen targeted by the extracellular ligand-binding domain of the chimeric antigen receptor, or The cancers mentioned are selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma, acute lymphoblastic leukemia, small cell lung cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia.

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