Chimeric antigen receptors co-expressing fumarate hydratase and uses thereof

Abstract

The present application provides a kind of chimeric antigen receptor (CAR), it includes extracellular domain, transmembrane domain and intracellular domain, wherein the chimeric antigen receptor also expresses fumarase FH. By co-expression FH, the anti-tumor effect of the chimeric antigen receptor of the present application is enhanced. Taking CD19 antibody as an example, the anti-CD19 chimeric antigen receptor co-expressing FH is used to modify immune cells, and the modified immune cells can be more efficiently used for the treatment of surface CD19 positive tumor.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. More specifically, this invention relates to a chimeric antigen receptor (CAR) co-expressing fumarate hydratase (FH) and its application in the treatment of a broad spectrum of tumors. Background Technology

[0002] In early 2021, the International Agency for Research on Cancer (IARC) of the World Health Organization released data showing that in 2020, there were 19.29 million new cancer cases and 9.96 million cancer deaths globally. China had 4.57 million new cancer cases and 3 million cancer deaths, ranking first in the world in terms of new cancer cases. Scientists worldwide are dedicated to researching cancer therapies, with immunotherapy becoming the third revolution in cancer treatment after chemotherapy and targeted therapy. In 2013, the journal *Science* listed cancer immunotherapy as the top scientific breakthrough, and in 2015, it listed combined cancer immunotherapy as one of the four most noteworthy scientific advances.

[0003] Tumor immunotherapy refers to the application of immunological principles and methods to enhance the immunogenicity of tumors, utilizing the body's own immune system to attack tumor cells, thereby inhibiting or killing them. It is divided into two main categories based on its treatment strategy: immune checkpoint inhibitors (such as CTLA-4, PD-1, PD-L1, etc.) and cell immunotherapy (such as CAR T cells). CAR T cells, short for chimeric antigen receptor T cells, work by genetically engineering a single-chain variable region (Scfv) of an antibody that recognizes a specific tumor antigen to the intracellular region of the CD3-chain, creating a chimeric protein. This protein is then transfected into cultured patient T cells via gene transduction, causing them to express the chimeric antibody receptor (CAR). After being "reprogrammed," the patient's T cells generate a large number of cytotoxic CAR T cells that can specifically target tumor cells. Compared to traditional immunotherapy, CAR T cells offer significant advantages, including more precise treatment, more accurate targeting, broader tumor killing, and longer-lasting effects. As a novel and cutting-edge treatment, CAR-T therapy is primarily led by China and the United States, with China showing particularly strong performance. As of May 2019, there were 507 registered clinical trials for CAR-T therapy globally, mainly located in China and the United States, accounting for 44.2% and 36.7% of the total trials, respectively. The concept of CAR-T therapy was first proposed in 1989, and over the past thirty years, CAR-T therapy has undergone four generations of technological innovation. After years of research, CAR-T therapy has achieved great success in clinical practice, especially in the treatment of hematologic malignancies, with multiple products already approved for marketing. For example, in 2017, the FDA approved two CD19-targeted CAR-T drugs for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL) in children or adults, signifying that CAR-T therapy has become a new and legal treatment strategy for cancer.

[0004] Despite the continued success in CAR T cell therapy for cancer, several challenges remain. One major reason why CAR T cell infiltration and function within the tumor is that the therapy is only effective in a small percentage of patients, directly limiting its therapeutic potential. Therefore, promoting CAR T cell infiltration, activation, and function is a crucial step towards ensuring the effectiveness of CAR T cell therapy.

[0005] Fumarate hydratase (FH) encodes a mitochondrial protein in the tricarboxylic acid cycle. It is an L-malate hydratase that reversibly catalyzes the hydration of fumarate to L-malate. Summary of the Invention

[0006] Through in-depth research, the inventors have discovered that gene mutations or low expression of fumarate hydratase (FH) are prevalent in various tumors, particularly playing a significant role in the development of renal cell carcinoma. Both tumor patients and mouse models have shown that FH may promote CD8... + The function of T cells in killing tumors. Further research has confirmed that reducing fumarate levels by overexpressing FH can promote CAR T cell activation and improve the efficacy of tumor treatment.

[0007] Therefore, in one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the chimeric antigen receptor also expresses fumarate hydratase FH.

[0008] In some embodiments, in the chimeric antigen receptor of the present invention, the fumarate hydratase FH is expressed at the N-terminus or C-terminus of the extracellular domain and / or at the N-terminus or C-terminus of the intracellular domain.

[0009] In some embodiments, in the chimeric antigen receptor of the present invention, the extracellular domain comprises an antigen recognition domain (scFv) and a hinge region, and the intracellular domain comprises a co-stimulatory domain and / or a signal transduction domain. Preferably, the chimeric antigen receptor further comprises a signal peptide, a small molecule tag (preferably Strep II), a self-cleaving peptide, or a combination thereof. More preferably:

[0010] 1) The antigen recognition domain (scFv) specifically recognizes CD19, CD20, CD22, CD30, CD33, CD133, CD138, BCMA, CEA, EGFR, EGFRvIII, EphA2, EpCAM, GD2, GPC3, HER2, MSLN, MG7, MUC1, NY-ESO-1, LMP1, PSMA, Fra, NKG2D1, BCMA, IL13Rα2, LeY, CD70, B7-H3, ROR1, or PSCA;

[0011] 2) The hinge region is derived from the hinge of IgG or the extracellular region of CD8α / CD28;

[0012] 3) The transmembrane domains are derived from CD4, CD8α, CD28, or CD3ζ;

[0013] 4) The co-stimulatory domain is derived from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27); and / or

[0014] 5) The signal transduction domain is the T cell receptor TCR / CD3ζ chain or the immunoglobulin Fc receptor FcεRIγ chain.

[0015] In some embodiments, in the chimeric antigen receptor of the present invention, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide, a single-chain antibody ScFv (preferably targeting CD19), StrepII, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and a CD3ζ chain, a P2A peptide, and FH.

[0016] In another aspect, the present invention provides recombinant vectors and / or isolated polynucleotides encoding chimeric antigen receptors according to the present invention, preferably said vectors being transposon vectors, retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, or any combination thereof.

[0017] In another aspect, the present invention provides a chimeric antigen receptor immune cell that overexpresses fumarate hydratase FH.

[0018] In some embodiments, the chimeric antigen receptor immune cells according to the present invention express the chimeric antigen receptor according to the present invention or the recombinant vector and / or isolated polynucleotides according to the present invention; or wherein the chimeric antigen receptor (which itself does not express fumarate hydratase FH) and fumarate hydratase FH are expressed in different expression vectors respectively.

[0019] In some embodiments, the chimeric antigen receptor immune cells according to the present invention are autologous or allogeneic T cells, tumor-infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells or NK-T cells, preferably CAR T cells.

[0020] In another aspect, the present invention provides a pharmaceutical composition or kit comprising a chimeric antigen receptor according to the present invention, a recombinant vector and / or isolated polynucleotides according to the present invention, or chimeric antigen receptor immune cells according to the present invention.

[0021] On the other hand, the present invention provides a method for treating a disease or condition in a subject in need, the method comprising administering to the subject chimeric antigen receptor immune cells according to the present invention or a pharmaceutical composition according to the present invention, preferably the disease or condition being cancer, more preferably CD4+. + CD8 + and / or CD19 +The most preferred cancers are B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphomas, liver cancer, pancreatic cancer, brain cancer, breast cancer, ovarian cancer, colorectal cancer, acute myeloid leukemia (AML), multiple myeloma, lung cancer, gastric cancer, glioma, EGFR-positive solid tumor, glioblastoma, glioblastoma multiforme, nasopharyngeal carcinoma, esophageal cancer, prostate cancer, neuroblastoma, hepatocellular carcinoma, squamous cell lung cancer, MSLN-positive solid tumor, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumors, renal cell carcinoma, or central nervous system cancer.

[0022] In another aspect of the invention, the invention provides the use of an agent that reduces fumarate production in cells (e.g., fumarate hydratase FH) in the preparation of an antitumor drug, preferably wherein the tumor is a CD4+ tumor. + CD8 + and / or CD19 + The preferred cancers are B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphocytic malignancies, liver cancer, pancreatic cancer, brain cancer, breast cancer, ovarian cancer, colorectal cancer, acute myeloid leukemia (AML), multiple myeloma, lung cancer, gastric cancer, glioma, EGFR-positive solid tumors, glioblastoma, glioblastoma multiforme, nasopharyngeal carcinoma, esophageal cancer, prostate cancer, neuroblastoma, hepatocellular carcinoma, squamous cell lung cancer, MSLN-positive solid tumors, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumors, renal cell carcinoma, or central nervous system cancers. Preferably, the drug is used in CAR T-cell therapy, or in combination with another antitumor drug and / or CAR T-cell drug / therapy.

[0023] In another aspect, the present invention also provides a method for enhancing the therapeutic effect / tumor-killing effect of CAR T-cell drugs or therapies, the method comprising reducing the production, content, or expression level of fumaric acid in tumor cells. Preferably, the tumor is CD4. + CD8 + and / or CD19 +The most preferred cancers are B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphomas, liver cancer, pancreatic cancer, brain cancer, breast cancer, ovarian cancer, colorectal cancer, acute myeloid leukemia (AML), multiple myeloma, lung cancer, gastric cancer, glioma, EGFR-positive solid tumor, glioblastoma, glioblastoma multiforme, nasopharyngeal carcinoma, esophageal cancer, prostate cancer, neuroblastoma, hepatocellular carcinoma, squamous cell lung cancer, MSLN-positive solid tumor, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumors, renal cell carcinoma, or central nervous system cancer. Attached Figure Description

[0024] Figure 1 Low FH expression in renal cell carcinoma patients is associated with poorer survival.

[0025] Figure 2 Renal cell carcinoma patients with low FH expression also have lower functional CD8 levels. + T cell infiltration;

[0026] Figure 3 Knockdown of FH in mouse tumor cells significantly inhibited functional CD8. + T cell infiltration promotes tumor growth;

[0027] Figure 4 In the tumor microenvironment, the reduction of FH in tumor cells leads to the accumulation of large amounts of fumaric acid in the microenvironment;

[0028] Figure 5 Fumaric acid effectively inhibits CD8. + Activation of T cells;

[0029] Figure 6 Fumaric acid can succinate CD8. + ZAP70 protein in T cells;

[0030] Figure 7 Fumaric acid can inhibit the activity of ZAP70;

[0031] Figure 8 Fumaric acid can inhibit the activity of downstream signaling pathways of ZAP70;

[0032] Figure 9 Fumaric acid accumulated in the tumor microenvironment can inhibit ZAP70 and CD8. + T cell activation, blocking CD8 + The function of T cells in killing tumors;

[0033] Figure 10A schematic diagram of the DNA fragment containing anti-CD19 CAR in the examples;

[0034] Figure 11 A schematic diagram of the DNA fragment against-CD19 CAR-FH in the examples;

[0035] Figure 12 PTK-EF1α-anti-CD19 CAR plasmid map in the examples;

[0036] Figure 13 The PTK-EF1α-anti-CD19 CAR-FH plasmid map in the examples;

[0037] Figure 14 CAR expression efficiency in anti-CD19 CAR T cells and anti-CD19 CAR-FH T cells;

[0038] Figure 15 During the culture of anti-CD19 CAR-T cells, reducing the amount of FH added significantly blocked the killing ability of CAR-T cells against Raji cell lines;

[0039] Figure 16 Reducing FH levels decreases the release of cytokines IFN-γ, TNF-α, and Granzyme B from CAR-T cells;

[0040] Figure 17 Knockdown of FH in tumor cell lines significantly reduced the in vivo antitumor activity of CAR-T cells;

[0041] Figure 18 FHi significantly reduces the anti-tumor ability of CAR-T cells;

[0042] Figure 19 Schematic diagram of the in vitro killing effect of anti-CD19 CAR-FH T cells on Burkitt's lymphoma cell line Raji;

[0043] Figure 20 Schematic diagram showing the release of cytokines IFN-γ, TNF-α, and Granzyme B after in vitro co-incubation of anti-CD19 CAR-FH T cells with Burkitt's lymphoma cell line Raji;

[0044] Figure 21 Schematic diagram of the in vivo anti-tumor ability of anti-CD19 CAR-FH T cells.

[0045] Sequence List Description

[0046] The specific sequences used in the embodiments of this invention are as follows:

[0047] Amino acid sequence of anti-CD19-scfv of SEQ ID NO. 1

[0048] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTV

[0049] Nucleotide sequence of anti-CD19-scfv of SEQ ID NO. 2

[0050] GACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTG

[0051] SEQ ID NO. 3 Amino acid sequence of signal peptide (SP)

[0052] MDFQVQIFSFLLISASVIMSRMA

[0053] SEQ ID NO. 4 Nucleotide sequence of signal peptide (SP)

[0054] ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCTAGAATGGCC

[0055] Amino acid sequence of the CD8 hinge in SEQ ID NO.5

[0056] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0057] Nucleotide sequence of the CD8 hinge in SEQ ID NO.6

[0058] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0059] Amino acid sequence of the CD28 transmembrane region (CD28TM) of SEQ ID NO.7

[0060] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0061] Nucleotide sequence of the CD28 transmembrane region (CD28TM) of SEQ ID NO.8

[0062] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG

[0063] Amino acid sequence of the CD28 intracellular domain (CD28ICD) of SEQ ID NO.9

[0064] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0065] SEQ ID NO.10 Nucleotide sequence of the CD28 intracellular domain (CD28ICD)

[0066] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCACCACCACGCGACTTCGCAGCCTATCGCTCC

[0067] The amino acid sequence of the intracellular co-stimulatory domain 4-1BB of SEQ ID NO.11

[0068] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0069] Nucleotide sequence of intracellular co-stimulatory domain 4-1BB of SEQ ID NO.12

[0070] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0071] The amino acid sequence of SEQ ID NO.13 CD3ζ

[0072] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0073] SEQ ID NO.14 nucleotide sequence of CD3ζ nucleotide sequence of CD3ζ

[0074] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCTCTCGC

[0075] Amino acid sequence of SEQ ID NO.15 P2A peptide

[0076] ATNFSLLKQAGDVEENPGP

[0077] Nucleotide sequence of SEQ ID NO. 16 P2A peptide

[0078] GCAACAAACTTCTCACTACTCAAACAAGCAGGTGACGTGGAGGAGAATCCCGGACCC

[0079] Amino acid sequence of SEQ ID NO. 17 FH

[0080] MYRALRLLARSRPLVRAPAAALASAPGLGGAAVPSFWPPNAARMASQNSFRIEYDTFGELKVPNDKYYGAQTVRSTMNFKIGGVTERMPTPVIKAFGILKRAAAEVNQDYGLDPKIANAIMKAADEVAEGKLNDHFPLVVWQTGSGTQTNMNVNEVISNRAIEMLGGELGSKIPVHPNDHVNKSQSSNDTFPTAMHIAAAIEVHEVLLPGLQKLHDALDAKSKEFAQIIKIGRTHTQDAVPLTLGQEFSGYVQQVKYAMTRIKAAMPRIYELAAGGTAVGTGLNTRIGFAEKVAAKVAALTGLPFVTAPNKFEALAAHDALVELSGAMNTTACSLMKIANDIRFLGSGPRSGLGELILPENEPGSSIMPGKVNPTQCEAMTMVAAQVMGNHVAVTVGGSNGHFELNVFKPMMIKNVLHSARLLGDASVSFTENCVVGIQANTERINKLMNESLMLVTALNPHIGYDKAAKIAKTAHKNGSTLKETAIELGYLTAEQFDEWVKPKDMLGPK

[0081] Nucleotide sequence of SEQ ID NO. 18 FH

[0082]

[0083] SEQ ID NO.19 Nucleotide sequence of FH knockdown shRNA

[0084] CGCTGAAGTAAACCAGGATTACTCGAGTAATCCTGGTTTACTTCAGCGTTTTT Detailed Implementation

[0085] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this field, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0086] The expression of fumarate hydratase FH refers to its expression at two levels: one is expression at the DNA level; the other is expression at the RNA level.

[0087] The term "overexpression" refers to the situation where the tight control of gene / protein expression (transcription) is disrupted, and the gene may be inappropriately "turned off" or transcribed at a high rate. High-speed transcription leads to the production of large amounts of mRNA. For the overexpression of "fumarate hydratase FH" of the present invention, it means that its DNA, RNA, or protein expression level is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or 300% higher in chimeric antigen receptor immune cells (expressing the chimeric antigen receptor according to the present invention or the recombinant vector and / or isolated polynucleotides according to the present invention) than in the control (i.e., not expressing the corresponding chimeric antigen receptor according to the present invention or the recombinant vector and / or isolated polynucleotides according to the present invention), or even 4, 5, 6, 7, 8, 9, or 10 times or more the DNA, RNA, or protein expression level of fumarate hydratase FH in the control. The techniques and reagents used to detect gene / protein expression levels are well known to those skilled in the art.

[0088] As used herein, the term "chimeric antigen receptor (CAR)" is a protein that specifically recognizes a target antigen, such as a target antigen on a cancer cell. When bound to a target antigen, a CAR can activate immune cells to attack and destroy cells carrying that antigen (such as cancer cells). CARs can also incorporate costimulatory or signal transduction domains to increase their potency. Typically, chimeric antigen receptors contain extracellular, transmembrane, and intracellular domains.

[0089] The term "extracellular domain" used in chimeric antigen receptors (CARs) refers to a domain consisting of an antigen recognition domain (i.e., a single-chain variable fragment (scFv) of the monoclonal antibody responsible for recognizing and binding the antigen) and a hinge region that acts as a linker. The antigen recognition domain is fundamental to the specific binding of tumor antigens by CARs. Its main structure is the scFv, which is formed by the light chain (VL) and heavy chain (VH) of the monoclonal antibody linked by a polypeptide, retaining the antibody's specificity and affinity for the antigen. Most current CAR T studies target TAAs, such as CD19, CD20, CD22, CD30, CD33, CD133, CD138, BCMA, CEA, EGFR, EGFRvIII, EphA2, EpCAM, GD2, GPC3, HER2, MSLN, MG7, MUC1, NY-ESO-1, LMP1, PSMA, Fra, NKG2D1, BCMA, IL13Rα2, LeY, CD70, B7-H3, ROR1, or PSCA. The scFv used in this invention can be derived from mice or be humanized scFv.

[0090] The term "hinge region" used in chimeric antigen receptors refers to the region connecting the scFv and the transmembrane domain. Most CARs derive their hinge regions from the hinge of IgG or the extracellular region of CD8α / CD28. The length of the hinge region depends on the location and exposure of the target cell antigen epitope.

[0091] The term "transmembrane domain" used to describe chimeric antigen receptors connects the extracellular domain of the CAR to its intracellular signal transduction domain, anchoring the receptor to the T cell membrane. Commonly used transmembrane domains are derived from CD4, CD8α, CD28, and CD3ζ.

[0092] The term "co-stimulatory domain" used in chimeric antigen receptors typically comes from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). It can achieve dual activation of synergistic stimulatory molecules and intracellular signals, enabling T cells to proliferate continuously and release cytokines, thereby enhancing the anti-tumor ability of T cells.

[0093] The term "signal transduction domain" used to describe chimeric antigen receptors is typically the T-cell receptor TCR / CD3ζ chain or the immunoglobulin Fc receptor FcεRIγ chain, which contains immunoreceptor tyrosine-based activation motifs (ITAMs) and plays a role in T-cell signal transduction.

[0094] In some embodiments, the chimeric antigen receptor of the present invention includes a signal peptide at its N-terminus. In some embodiments, the signal peptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequence encoded by SEQ ID NO. 4. In some embodiments, the signal peptide is encoded by a nucleotide sequence comprising SEQ ID NO. 4.

[0095] In some embodiments, the chimeric antigen receptor of the present invention further includes a small molecule tag (preferably Strep II) for protein detection / labeling / purification at the C-terminus of the antigen recognition domain (scFv).

[0096] In some embodiments, the chimeric antigen receptor of the present invention further comprises fumarate hydratase FH. In some embodiments, the fumarate hydratase FH may be expressed at the N-terminus or C-terminus of the extracellular domain and / or at the N-terminus or C-terminus of the intracellular domain. Alternatively, the fumarate hydratase FH and the extracellular or intracellular domain may be expressed as a fusion protein, or a linker peptide (such as a self-cleaving peptide, preferably a P2A peptide) may be present between the fumarate hydratase FH and the extracellular or intracellular domain.

[0097] Fumarate hydratase (FH) encodes a mitochondrial protein in the tricarboxylic acid cycle. Mutations or low expression of its gene are prevalent in various tumors, particularly playing a crucial role in the development of renal cell carcinoma. Clinically, low FH expression has been observed in renal cell carcinoma patients (kidney chromophobe (KICH) and kidney renal papillary cell carcinoma (KIRP)) with associated poorer survival rates. Figure 1 Furthermore, FH low expression is associated with CD8 infiltration in tumors. + Inhibition of T cell functional gene (GZMA, GZMB, IFNG, MIKI67) expression ( Figure 2Knocking down FH in mouse B16-OVA (melanoma cells) tumor cells significantly inhibited functional CD8 in the tumor. + T cell infiltration promotes tumor growth. Figure 3 In vitro experiments revealed that tumor cell culture medium with low FH expression could effectively inhibit CD8. + T cell activation and function Figure 4 Further mechanistic studies revealed that the reduction of FH in tumor cells within the tumor microenvironment leads to the accumulation of large amounts of fumaric acid (FH) in the tumor microenvironment. Figure 4 Furthermore, the exogenous addition of FH substrates—fumaric acid derivatives (dimethyl fumarate, DMF)—or the use of FH inhibitors (FHi, Fumarate hydratase-IN-1, MCE, HY-100004) to accumulate endogenous fumarate effectively inhibited CD8. + T cell activation ( Figure 5 Fumaric acid can succinylate ZAP70 protein. Figure 6 ), inhibiting the activity of ZAP70 and its downstream signaling pathways ( Figure 7 , Figure 8 Furthermore, treatment with FH inhibitors or DMF-accumulated fumarate effectively inhibited CD8. + T cell activation, blocking CD8 + The function of T cells in killing tumors ( Figure 9 ).

[0098] Based on the above findings, the inventors reduced the level of fumaric acid by overexpressing FH, thereby promoting CAR T cell activation and improving the therapeutic effect on tumors.

[0099] Therefore, the present invention provides a chimeric antigen receptor (CAR) that co-expresses FH. CAR T cells carrying a ScFv sequence targeting CD19 can effectively kill any tumor cells that express CD19 on their surface, thereby improving the efficiency of tumor killing.

[0100] In one specific embodiment of the present invention, the chimeric antigen receptor CAR provided by the present invention is sequentially spliced ​​from the N-terminus to the C-terminus with a signal peptide, a single-chain antibody ScFv, strepII, a CD8 hinge, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB, and a CD3ζ chain; preferably, a P2A peptide and FH are further spliced ​​at the C-terminus of the CD3ζ chain; the nucleotide sequence of the single-chain antibody ScFv, which can recognize the CD19 antigen on the surface of tumor cells, is shown in SEQ ID NO.2. The nucleotide sequence of the signal peptide is shown in SEQ ID NO.4, the nucleotide sequence of the CD8 hinge is shown in SEQ ID NO.6, the nucleotide sequences of the CD28 transmembrane region and the CD28 intracellular domain are shown in SEQ ID NO.8 and SEQ ID NO.10, respectively, the nucleotide sequence of the intracellular co-stimulatory domain 4-1BB is shown in SEQ ID NO.12, and the nucleotide sequence of CD3ζ is shown in SEQ ID NO.14.

[0101] In some embodiments of the present invention, the signal peptide, single-chain antibody ScFv, strepII, CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, intracellular co-stimulatory domain 4-1BB, CD3ζ chain, P2A peptide, and FH are sequentially spliced ​​from the N-terminus to the C-terminus. Preferably, the amino acid sequence of the single-chain antibody ScFv is as shown in SEQ ID NO.1, and preferably, the nucleotide sequence of the single-chain antibody ScFv is as shown in SEQ ID NO.2; the amino acid sequence of the P2A peptide is as shown in SEQ ID NO.15, and preferably, the nucleotide sequence of the P2A peptide is as shown in SEQ ID NO.16; the amino acid sequence of the FH is as shown in SEQ ID NO.17.

[0102] A second objective of this invention is to provide a gene vector for a recombinant chimeric antigen receptor, using a viral or non-viral expression vector as a backbone, into which a lentivirus, adenovirus, adeno-associated virus, retrovirus, or transposon vector containing the aforementioned chimeric antigen receptor-encoding nucleotide sequence is inserted; preferably, the viral vector PTK-EF1α is used as a backbone, into which a lentiviral vector containing the aforementioned chimeric antigen receptor-encoding nucleotide sequence is inserted; the viral vector PTK-EF1α is a vector obtained by replacing the CMV promoter with the EF1α promoter using the PTK(Addgene,#36976) vector as a backbone.

[0103] A third objective of this invention is to provide chimeric antigen receptor immune cells, obtained by transfecting immune cells with the encoding nucleotide sequence of the chimeric antigen receptor or the recombinant chimeric antigen receptor gene vector described above. The immune cells are selected from T cells, NK cells, NKT cells, αβT cells, γδT cells, and CD4 cells derived from umbilical cord blood, peripheral blood, or iPSCs. + T cells, CD8 + The T cells, preferably peripheral blood-derived T cells, are CAR T cells that target CD19 for the treatment of broad-spectrum tumors. When the chimeric antigen receptor CAR single-chain antibody ScFv binds to CD19, it simultaneously overexpresses FH protein. The immune cells expressing the chimeric antigen receptor exhibit stronger anti-tumor activity. Preferably, the modification of immune cells is achieved by combining CRISPR, RNA interference, and other technologies with the expression of chimeric antigen receptor elements.

[0104] In some embodiments, when performing in vitro functional tests on immune cells expressing chimeric antigen receptors, cell lines expressing CD19 protein on their cell membrane surface are selected.

[0105] In some embodiments, the present invention provides a pharmaceutical composition or kit comprising the chimeric antigen receptor, recombinant vector and / or isolated polynucleotide, or chimeric antigen receptor immune cells of the present invention; and pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition or kit further comprises additional active agents. In some embodiments, said additional active agents are chemotherapeutic agents, PD-1 inhibitors, biological response modulators for preventing or reducing cytokine release syndrome (CRS) or neurotoxicity, cytokines, etc.

[0106] In some embodiments, the present invention provides a method for treating a disease or condition in a subject in need, the method comprising administering to the subject the chimeric antigen receptor immune cells of the present invention or the pharmaceutical composition of the present invention, preferably the disease or condition being cancer, more preferably CD4+. + CD8 + and / or CD19 +The preferred cancers are B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphomas, liver cancer, pancreatic cancer, brain cancer, breast cancer, ovarian cancer, colorectal cancer, acute myeloid leukemia (AML), multiple myeloma, lung cancer, gastric cancer, glioma, EGFR-positive solid tumors, glioblastoma, glioblastoma multiforme, nasopharyngeal carcinoma, esophageal cancer, prostate cancer, neuroblastoma, hepatocellular carcinoma, squamous cell lung cancer, MSLN-positive solid tumors, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumors, renal cell carcinoma, and central nervous system cancers. The preferred excipients are those with structurally and pharmaceutically acceptable domains.

[0107] The beneficial effects of this invention are as follows:

[0108] 1. This invention provides a chimeric antigen receptor (CAR) co-expressing FH. CAR T cells carrying a ScFv sequence targeting CD19 can effectively kill any tumor cells that express CD19 on their surface, thus improving the efficiency of tumor killing.

[0109] 2. This invention also provides a method for co-expressing FH chimeric antigen receptor (CAR), which involves activating isolated immune cells for 3 days before infecting them with lentiviruses expressing chimeric antigen receptors. Furthermore, when performing in vitro functional tests on immune cells expressing chimeric antigen receptors, the selected cell lines are those that highly or moderately express CD19 targets outside the cell membrane. This makes the evaluation of the tumor-killing effect of immune cells expressing chimeric antigen receptors more scientific.

[0110] Example

[0111] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments 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 embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0112] Example 1: Knockdown of FH in mouse tumor cells significantly inhibited the infiltration of functional CD8+ T cells and promoted tumor growth.

[0113] from Figure 1 As can be seen, in patients with clear cell renal cell carcinoma and patients with papillary renal cell carcinoma, based on the expression of FH in the GEPIA (http: / / gepia.cancer-pku.cn / detail.php) database, we divided them into two groups according to the level of FH expression. We found that patients with low FH expression had poor Kaplan-Meier survival curves.

[0114] Figure 2 The scatter plot shows that, in single-cell sequencing data (SCP1288) from eight renal cell carcinoma patients, the expression level of FH in tumor cells was correlated with the expression level of CD8+ in tumor-infiltrating cells. + The expression levels of functional genes (GZMA, GZMB, IFNG, and MIKI67) in T cells are positively correlated. The figure shows the Pearson correlation coefficient (r) and p-value.

[0115] Based on the above findings, we constructed a stable cell line in mouse melanoma B16-OVA cells by knocking down the expression of the FH gene using shRNA technology (the nucleotide sequence of the FH knockdown shRNA is shown in SEQ ID NO. 19). Subcutaneous tumorigenesis in mice was assessed after transplanting this knockdown stable cell line into C57BL / 6 mice. Figure 3 The results showed that B16-OVA cell xenografts with knocked-down FH had significantly higher growth rates and tumor sizes than wild-type B16-OVA cells. Figure 3 B). By analyzing the CD8 tumor-infiltrating CD8 in this transplanted mouse. + T cells and CD44 + CD8 + Flow cytometry analysis of T cells revealed that tumors with knocked-down FH cells contained CD8+. + T cells and CD44 + CD8 + T cells were significantly reduced ( Figure 3 (C). Furthermore, knockdown of FH in tumors resulted in CD8... + The expression of T cell cytokines INF-γ, TNFα, and GZMB was significantly reduced. Figure 3 D).

[0116] We further cultured CD8 cells in the culture medium after culturing B16-OVA cells for 24 hours. + T cells ( Figure 4 The results showed that B16-OVA cell culture medium with FH knockdown significantly inhibited CD8. + T cell activation ( Figure 4 B) and the expression of cytokines ( Figure 4 C). The content of fumarate in the extracellular space of FH-knocked tumor cells was detected using liquid chromatography-mass spectrometry (LC-MS / MS). The results showed that ( Figure 4 (D) The content of fumaric acid was significantly increased in tumors with knocked-down FH.

[0117] We also found that using fumarate analogs MMF (monomethyl fumarate) or DMF (dimethyl fumarate) ( Figure 5 , A) or FHi(Fumarate hydratase-IN-1,MCE,HY-100004)( Figure 5 B) Processing CD8 + One hour after T cells, CD8 + T cell activation was significantly inhibited, and this inhibition was concentration-dependent.

[0118] We further discovered that overexpression of Flag-ZAP70 protein in HEK 293T cells (Takara) followed by treatment with fumaric acid, MMF, DMF, or FHi enriched Flag-ZAP70 protein in lysed cells. Immunoblotting revealed that ZAP70 protein was succinylated (anti-2SC antibody, Discovery antibody (crb2005017)). Figure 6 A). The purified Flag-ZAP70 protein was incubated with fumarate, and mass spectrometry identified the succinylation modification sites as C96 and C102. Figure 6 B).

[0119] Furthermore, we found that the above-mentioned succinylation modification inhibited ZAP70 enzyme activity. Overexpression of Flag-ZAP70 protein in 293T cells, cell lysis to enrich Flag-ZAP70 protein, and incubation with DMF for 30 minutes followed by ZAP70 enzyme activity detection revealed that DMF gradually inhibited ZAP70 enzyme activity with increasing concentration gradient (see [link to relevant documentation]). Figure 7 ).

[0120] We will use mouse CD8 + T cells and different concentrations of FHi ( Figure 8 A) or DMF Figure 8 B) After incubation for 1 hour, cells were activated with anti-CD3 / CD28 antibody, and the activity of the T cell activation signaling pathway was detected by Western blotting. The results showed that phosphorylation at position Y394 of the ZAP70 protein and its downstream protein phosphorylation were blocked by FHi and DMF.

[0121] We also subcutaneously transplanted B16-OVA cells into C57BL / 6 mice and irradiated them to eliminate CD8+. + Following the T cell administration, OT-I CD8 was injected into the tail vein of mice. +T cells were activated by intraperitoneal injection of DMSO (Ctrl) or FHi (25 mg / kg mice) or oral administration of DMF (50 mg / kg mice) for four days, followed by intraperitoneal injection of OVA peptide to activate OT-I CD8. + T cells were used to assess subcutaneous tumor formation in mice. Figure 9 The results showed that the tumor growth rate and size treated with FHi and DMF were significantly higher than those in the control group (A). Figure 9 B and Figure 9 Furthermore, fumaric acid levels in the tumor microenvironment significantly increased after FHi and DMF treatment (C). Figure 9 D). In tumors treated with FHi and DMF, infiltrated CD44 + CD8 + T cells were significantly reduced ( Figure 9 (E). Furthermore, the processing of FHi and DMF leads to CD8... + The expression of T cell cytokines INF-γ, TNFα, and GZMB was significantly reduced. Figure 9 F).

[0122] In summary, we can conclude that gene mutations or low expression of fumarate hydratase (FH) are prevalent in various tumors, particularly playing a significant role in the development of renal cell carcinoma. Both tumor patient and mouse models indicate that FH may promote CD8 activation. + The function of T cells to kill tumors.

[0123] Example 2: Construction of PTK-EF1α-CD19, PTK-EF1α-CD19-FH plasmids

[0124] 1. Artificially synthesize fragments CD19 and FH, and artificially synthesize fragments SP and strepII-CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ.

[0125] 2. Overlap PCR was used to amplify the structures of CD99-CAR and CD99-CAR-FH fragments with EcoRI and BamHI restriction sites, respectively, using SP, CD99, and CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ or SP, CD99, and CD8 hinge-CD28TM+ICD-4-1BB-CD3ζ+P2A peptide+FH as templates. The schematic diagrams are shown below. Figure 10 , Figure 11 As shown.

[0126] The amino acid sequence of the signal peptide (SP) is shown in SEQ ID NO.3, the amino acid sequence of the CD8 hinge is shown in SEQ ID NO.5, the amino acid sequence of CD28TM is shown in SEQ ID NO.7, the amino acid sequence of CD28ICD is shown in SEQ ID NO.9, the amino acid sequence of 4-1BB is shown in SEQ ID NO.11, the amino acid sequence of CD3ζ is shown in SEQ ID NO.13, the amino acid sequence of the P2A peptide is shown in SEQ ID NO.15, and the amino acid sequence of FH is shown in SEQ ID NO.17. More preferably, the nucleotide sequence of the signal peptide (SP) is shown in SEQ ID NO.4, the nucleotide sequence of the CD8 hinge is shown in SEQ ID NO.6, the nucleotide sequence of CD28TM is shown in SEQ ID NO.8, the nucleotide sequence of CD28ICD is shown in SEQ ID NO.10, the nucleotide sequence of 4-1BB is shown in SEQ ID NO.12, the nucleotide sequence of CD3ζ is shown in SEQ ID NO.14, the nucleotide sequence of the F2A peptide is shown in SEQ ID NO.16, and the nucleotide sequence of FH is shown in SEQ ID NO.17. Shown in NO.18.

[0127] 3. The plasmid PTK881-EF1α-Kan was double-digested with EcoRI and BamHI restriction endonucleases. The product was subjected to 0.8% agarose gel electrophoresis, and the gel was excised and collected in Eppendorf tubes. The corresponding fragments were recovered using an Axygen agarose gel recovery kit, and the purity and concentration of the product were determined.

[0128] 4. Add the fragment to an Eppendorf tube at a 1:2 molar ratio, along with Exnase II ligase (Vazyme) and homologous recombinase 5×CE II buffer, and incubate at 37°C for 0.5 hours. Take 10 μL of the ligation solution and add it to 100 μL of DH5α competent cells. Incubate on ice for 30 min, then heat shock at 42°C for 90 s. After this, add 500 μL of SOC medium and incubate at 37°C and 220 rpm for 2 hours. After 2 hours, centrifuge the Eppendorf tube at 4000g for 1 min to remove 400 μL of excess liquid. Spread the remaining liquid onto LB agar plates and incubate at 37°C for 12 hours. Pick a single colony from the plate and inoculate it into 5 mL of LB liquid medium, incubating at 37°C and 220 rpm for 12 hours.

[0129] 5. Plasmids were extracted using the Axygen miniprep kit to obtain plasmids PTK881-EF1α-CD19 and PTK881-EF1α-CD19-FH. After verification by first-generation sequencing at Sangon Biotech (Shanghai) Co., Ltd., complete chromatograms containing plasmids PTK-EF1α-CD19 and PTK-EF1α-CD19-FH were obtained, as shown in the schematic diagrams below. Figure 12 , Figure 13 As shown. 20 μL (500 ng) of plasmid DNA was taken and sent for sequencing. Based on the original seed sequence, the target gene of the product produced from the plasmid was checked for any changes. Under stable process conditions, the target gene of the working seed will not change during fermentation and amplification, and can be used for the next stage of production and correct protein expression.

[0130] Example 3: Preparation and live droplet detection of PTK-EF1α-CD19 and PTK-EF1α-CD19-FH lentiviral vectors

[0131] 1. Preparation of lentiviral vectors

[0132] 293T cells (Takara) at a density of 130.0–140.0 × 10⁶ were inoculated into a multilayer cell culture flask (Hyperflask) in 560 mL of complete DMEM medium (50 mL fetal bovine serum, 5 mL Antibiotic-Antimycotic (100×)) and cultured at 37°C in a 5% CO₂ incubator for 24 hours. Then, 320 μg of DMEM medium containing plasmids (PTK-EF1α-CD19, PTK-EF1α-CD19-FH:pLP1 plasmid (Invitrogen):pLP2 plasmid (Invitrogen):Plp / VSV-G plasmid (Invitrogen) = 12:10:5:6) was added to a 960 μg PEI tube, vortexed, and equilibrated at room temperature for 10 min. Finally, 35 mL of the PEI and plasmid mixture was mixed with 525 mL of DMEM medium and transferred to the aforementioned multilayer cell culture flask. After incubating the multilayer cell culture flasks in a 37°C incubator with 5% CO2 for 3 days, the cell culture supernatant was collected.

[0133] Centrifuge the supernatant at 4000 rpm (or 3000 g) for 30 min, then add cryonase (Takara) to the supernatant and incubate at 4°C. After 6 hours, filter the lentivirus supernatant through a 0.22 μm filter membrane and centrifuge at 30000 g for 2.5 h at 4°C. Remove the supernatant and resuspend the pellet in 1 mL of T cell culture medium. After resuspending, reserve 20 μL for viral activity titer testing. Aliquot the remaining lentivirus concentrate, label it Lenti3-CD19-CAR and Lenti3-CD19-CAR-FH, and store at -80°C for later use.

[0134] 2. Detection of lentiviral vector activity titer

[0135] Principle: A commercially available antibody targeting FCM63 was used to validate CAR expression on the cell surface. The fluorescence signal detected by flow cytometry indirectly reflected the expression of CAR in 293T cells.

[0136] Method: Introduce 5.0 × 10⁻⁶ mm wire into a 6-well plate. 5 293T cells per well were added, with 0.1 μL, 0.5 μL, and 1 μL of lentiviral concentrate added to each well, and one negative control was included. Cells were incubated at 37°C in a 5% CO2 incubator. After three days, 293T cells were collected using Versene solution (Gibco) and analyzed by flow cytometry to determine the proportion of CAR-positive 293T cells. The activity titers of PTK-EF1α-CD19 and PTK-EF1α-CD19-FH lentiviral concentrate were then calculated.

[0137] The current lentivirus concentrate has an activity titer of 1×10⁻⁶. 8 ~10×10 8 The detection and analysis results within the range of (TU / mL) are shown in Table 1. This indicates that each lentiviral vector can achieve high activity titers and can be used for the subsequent preparation of chimeric antigen receptor immune cells.

[0138] Table 1. Results of Lentiviral Activity Titer Detection and Analysis

[0139] Lenti3-CD19-CAR <![CDATA[5.1×10 8 ]]> Lenti3-CD19-CAR-FH <![CDATA[5.8×10 8 ]]>

[0140] Example 4: Preparation of anti-CD19 CAR T cells and anti-CD19-FH CAR T cells

[0141] 1. Preparation of CAR T cell preparations:

[0142] 100 mL of peripheral blood was collected from healthy donors (donated by volunteers in our laboratory), and mononuclear cells were isolated using Ficoll lymphocyte separation medium. After counting, CD3-positive cells were sorted using an appropriate amount of CD3 MicroBeads, human (Medini), and counted at 1.0–2.0 × 10⁻⁶ cells / mL. 6 T cell density / mL in complete T cell culture medium (OpTmizer) TM CTS TM T-Cell Expansion BasalMedium, OpTmizer TM CTS T-Cell Expansion Supplement (Invitrogen), cultured in 500 IU / mL IL-2 (Shuanglu Pharmaceutical), while simultaneously administering 10 6 Add 25 μl of Dynabeads Human T-Activator CD3 / CD28 (Invitrogen) to each cell to activate T cells.

[0143] 48 hours later (Day 2), Lenti3-CD19-CAR and Lenti3-CD19-CAR-FH lentiviral vectors were added at an MOI of 3 for transduction. After mixing, the mixture was placed in a CO2 incubator for incubation. After 4 hours, an appropriate amount of complete T cell culture medium was added for further culture.

[0144] 24 hours after lentivirus transduction, the transduced cells were replaced with fresh complete T cell culture medium, and the viable cell density was adjusted to 1.0-2.0 × 10⁶ cells / year. 6 Cells / mL were cultured and expanded for 10–20 days, with daily observation and counting. Cells were replenished with additional medium to expand the culture, maintaining a cell culture density of 1.0–2.0 × 10⁶ cells / mL throughout. 6 per mL.

[0145] 2. Detection of transduction efficiency of anti-CD19 CAR T and anti-CD19-FH CAR T cells

[0146] Take 1.0 × 10 6 After transduction, T cells were incubated with 1 μg / mL FITC-Protein-L at room temperature for 30 minutes, washed twice with physiological saline, and then FITC fluorescence signal was detected by flow cytometry. The percentage of FITC-positive cells was measured, reflecting the percentage of CAR T cells in the total cell count. The transduction efficiency of anti-CD19 CAR T and anti-CD19-FH CAR T cells is shown in the figure below. Figure 14 As shown. Figure 14This indicates that CAR T cells were successfully prepared, and the CAR expression efficiencies of anti-CD19 CAR T cells and anti-CD19-FH CAR T cells were 16.3% and 16.9%, respectively.

[0147] Example 5: Effects of FH on the in vivo and in vitro function of antagonistic-CD19 CAR-T cells

[0148] 1. In vitro tumor-killing detection:

[0149] The in vitro tumor-killing function of T cells, anti-CD19 CAR-T cells, and anti-CD19 CAR-FH-T cells was detected by the calcein-AM fluorescence scanning assay (Tao Changli. Establishment of a method for determining cytotoxicity by Calcein-AM fluorescence scanning. doi:10.3969 / j.issn.1000-484X.2014.10014. Chinese Journal of Immunology). The target cells were CD19-positive, FH-knockdown shFH-Raji cells (constructed in the same way as in Example 1).

[0150] Take an appropriate amount of target cells, and adjust the culture density of target cells Raji (Raji B acute lymphoblastic tumor cell line (purchased from the National Biomedical Experimental Cell Resource Bank)) and CD19-positive, FH-knockdown shFH-Raji to 2×10⁻⁶ cells per cell line. 5 / mL, cultured for 24h, then centrifuged to obtain the supernatant of the target cells. Take an appropriate amount of anti-CD19 CAR-T and resuspend it in the supernatant of the two cell lines mentioned above, and incubate for 24h before use.

[0151] After 24 hours, an appropriate amount of target cells (shFH-Raji) were collected and processed at a concentration of 1×10⁻⁶ cells. 6 Add Calcein-AM to a final concentration of 25 μM in a 1 mL / mL cell suspension (PBS, 5% fetal bovine serum) and incubate for 30 min. After washing twice at room temperature, resuspend the cells at a concentration of 0.5 × 10⁶ cells / mL. 5 / mL, add 0.5×10 to each well of a 96-well plate. 5 At a target cell ratio of 25:1, anti-CD19 CAR-T (Raji) (after incubation in Raji cell culture medium for 24 h) and anti-CD19 CAR-T (shFH Raji) (after incubation in tumor cell supernatant for 24 h) were added separately, and incubated at 37°C for 2–3 h. After incubation, the supernatant was collected, and the fluorescence intensity of calcein was measured. The percentage of target cell lysis was calculated based on the spontaneous release control and the maximum release control.

[0152] The in vitro killing and lysis results of T cells, anti-CD19 CAR-T (Raji) cells, and anti-CD19 CAR-T (shFH Raji) cells on the CD19-overexpressing tumor cell line shFH-Raji are as follows: Figure 15 As shown in the results, the in vitro tumor-killing effects of anti-CD19 CAR-T (Raji) and anti-CD19 CAR-T (shFH Raji) are significant. Therefore, the killing ability of both anti-CD19 CAR-T (Raji) and anti-CD19 CAR-T (shFH Raji) cells is significantly enhanced compared to T cells, indicating that they both possess in vitro killing capabilities against CD19-positive tumor cell lines. More importantly, the killing ability of anti-CD19 CAR-T (Raji) cells is significantly higher than that of anti-CD19 CAR-T (shFH Raji) cells, indicating that FH in the CAR-T cell culture medium has a significant impact on the anti-tumor function of CAR-T cells.

[0153] 2. In vitro cytokine detection:

[0154] Take an appropriate amount of target cells and adjust the Raji and shFH-Raji cell culture density to 2×10⁶. 5 / mL, cultured for 24h, then centrifuged to obtain the supernatant of the target cells. Take an appropriate amount of anti-CD19 CAR-T and resuspend it in the supernatant of the two cell lines mentioned above, and incubate for 24h before use.

[0155] Take an appropriate amount of target cells and mix them at a concentration of 1×10⁻⁶. 6 Resuspend the cells in a 0.5 × 10⁶ mL cell suspension (PBS, 5% fetal bovine serum) at room temperature, wash twice, and then resuspend the cells to a concentration of 0.5 × 10⁶. 5 / mL, add 0.05×10 to each well of a 96-well plate. 5 At a target cell ratio of 25:1, anti-CD19 CAR-T (Raji) (after incubation in T cell culture medium for 24 h) and anti-CD19 CAR-T (shFH Raji) (after incubation in tumor cell supernatant for 24 h) were added. The mixture was centrifuged at 200g for 30 seconds and incubated at 37℃ for 18 h. After incubation, the supernatant was collected, and the concentrations of IFN-γ, TNF-α, and Granzyme B in the supernatant were measured.

[0156] The secretion of IFN-γ, TNF-α, and Granzyme B after in vitro incubation of T cells, anti-CD19 CAR-T (Raji) cells, anti-CD19 CAR-T (shFH Raji) cells, and the CD19-overexpressing cell line shFH-Raji were as follows: Figure 16As shown, consistent with the tumor-killing results, the levels of IFN-γ, TNF-α, and Granzyme B secreted by anti-CD19 CAR-T (Raji) and anti-CD19 CAR-T (shFH Raji) cells were significantly higher than those secreted by T cells, indicating their in vitro tumor-killing function against CD19-positive tumor cell lines. More importantly, the release levels of IFN-γ, TNF-α, and Granzyme B from anti-CD19 CAR-T (Raji) cells were significantly higher than those from anti-CD19 CAR-T (shFH Raji) cells, indicating that a reduction in FH in the CAR-T cell culture medium can significantly decrease the in vitro tumor-killing ability of CAR-T cells.

[0157] 3. Effects of FH knockdown in tumor cell lines on the in vivo anti-tumor function of CAR-T cells:

[0158] To verify the anti-tumor function of FH knockdown in tumor cell lines on CAR-T cells in vivo, we constructed mouse models using normal FH-positive cell lines and FH knockdown tumor cell lines, respectively, and observed and compared the anti-tumor ability of anti-CD19 CAR-T cells in different mouse models.

[0159] Raji-luci cell model: Twelve healthy female NPG mice were injected with Raji-luci cells via the tail vein, with each mouse receiving 1.5 × 10⁻⁶ cells. 6 Imaging to observe tumor growth begins 5-9 days later, and the fluorescence intensity (ROI value) is monitored until it reaches 10. 6 -10 7 A time interval of photons per second indicates successful model establishment. After successful model establishment, mice were randomly divided into two groups of six each. The first group received a tail vein injection of T cells as a control group, while the second group received a tail vein injection of 2.0 × 10⁻⁶ cells. 6 Anti-CD19 CAR-T cells.

[0160] shFH-Raji-luci cell model: Twelve healthy female NPG mice were injected with shFH-Raji-luci cells via the tail vein, with each mouse receiving 1.5 × 10⁻⁶ cells. 6 Imaging to observe tumor growth begins 5-9 days later, and the fluorescence intensity (ROI value) is monitored until it reaches 10. 6 -10 7 A time interval of photons per second indicates successful model establishment. After successful model establishment, mice were randomly divided into two groups of six each. The first group received a tail vein injection of T cells as a control group, while the second group received a tail vein injection of 2.0 × 10⁻⁶ cells. 6 Anti-CD19 CAR-T cells. Fluorescence imaging data were collected from mice every 4-6 days.

[0161] Fluorescence values ​​directly reflect the in vivo expansion of the Raji-luci tumor cell line. The fluorescence imaging data of Raji-luci in each mouse model group are as follows: Figure 17 As shown, in both the Raji-luci and shFH-Raji-luci cell models, anti-CD19 CAR-T cells significantly inhibited the proliferation of tumor cell lines in vivo compared to the control T cell group. More importantly, compared to the Raji-luci cell model, the anti-CD19 CAR-T cells in the shFH-Raji-luci cell model exhibited weaker anti-tumor activity, only temporarily inhibiting the expansion of shFH-Raji-luci cells, ultimately failing to prevent tumor recurrence. This indicates that knocking down FH in tumor cell lines significantly reduces the in vivo anti-tumor function of CAR-T cells.

[0162] 4. Effects of FH inhibitors on the in vivo anti-tumor function of CAR-T cells:

[0163] To verify the effect of intracellular ferrous hydroxyl (FH) on the anti-tumor function of CAR-T cells in vivo, we used the FH inhibitor FHi to inhibit FH function and observed the anti-tumor ability of CAR-T cells. Simultaneously, we added a zap70 inhibitor, MDF, as a positive control for FHi.

[0164] Raji-luci cell model: Sixteen healthy female NPG mice were injected with Raji-luci cells via the tail vein, with each mouse receiving 1.5 × 10⁻⁶ cells. 6 Imaging to observe tumor growth begins 5-9 days later, and the fluorescence intensity (ROI value) is monitored until it reaches 10. 6 -10 7 A time interval of photons per second indicates successful model establishment. After successful model establishment, mice were randomly divided into four groups of four mice each. Group one served as the control group, receiving a tail vein injection of T cells. Group two received a tail vein injection of 2.0 × 10⁻⁶ T cells. 6 Anti-CD19 CAR-T cells served as the control group for groups three and four. Group three received a tail vein injection of 2.0 × 10⁻⁶ cells. 6 In addition to anti-CD19 CAR-T cells, for the first four days after CAR-T injection, 12 mg / kg FHi was injected intraperitoneally daily, and in the fourth group, 2.0 × 10⁻⁶ mg / kg FHi was injected via tail vein. 6 In addition to anti-CD19 CAR-T cells, mice were administered 25 mg / kg DMF via gavage daily for the first four days after CAR-T injection. Fluorescence imaging data were collected in mice on days 5-6.

[0165] Fluorescence values ​​directly reflect the in vivo expansion of the Raji-luci tumor cell line. The fluorescence imaging data of Raji-luci in each mouse model group are as follows: Figure 18As shown, compared with the control T cell group, anti-CD19 CAR-T, anti-CD19 CAR-T+FHi, and anti-CD19 CAR-T+DMF all significantly inhibited the proliferation of tumor cell lines in vivo. More importantly, although both the anti-CD19 CAR-T+FHi and anti-CD19 CAR-T+DMF groups could inhibit tumor proliferation, their anti-tumor capabilities were significantly lower than those of the anti-CD19 CAR-T group alone. This indicates that the FH inhibitor FHi can significantly reduce the in vivo tumor-killing ability of CAR-T cells.

[0166] Example 6: In vitro functional detection of anti-CD19 CAR T and anti-CD19-FH CAR T cells

[0167] The results above show that FH inhibitors can significantly reduce the anti-tumor function of CAR-T cells. Therefore, we propose to overexpress FH protein in CAR-T cells to further improve their function.

[0168] 1. In vitro tumor-killing detection:

[0169] The in vitro tumor-killing function of T, anti-CD19 CAR-T, and anti-CD19 CAR-FH-T cells was detected using the calcein-AM fluorescence scanning assay (Tao Changli. Establishment of a method for determining cytotoxicity using Calcein-AM fluorescence scanning. doi:10.3969 / j.issn.1000-484X.2014.10014. Chinese Journal of Immunology). The target cells were the CD19-positive B acute lymphoblastic tumor cell line Raji (purchased from the National Biomedical Experimental Cell Resource Bank). Appropriate amounts of target cells were collected and analyzed at 1×10⁻⁶ cells / mL. 6 Add Calcein-AM to a final concentration of 25 μM in a 1 mL / mL cell suspension (PBS, 5% fetal bovine serum) and incubate for 30 min. After washing twice at room temperature, resuspend the cells at a concentration of 0.5 × 10⁶ cells / mL. 5 / mL, add 0.5×10 to each well of a 96-well plate. 5 At a target cell ratio of 25:1, T, anti-CD19 CAR-T, and anti-CD19 CAR-FH-T were added, and the cells were incubated at 37°C for 2–3 hours. After incubation, the supernatant was collected, and the fluorescence intensity of calcein was measured. The percentage of target cell lysis was calculated based on the spontaneous release control and the maximum release control.

[0170] The in vitro killing and lysis results of T, anti-CD19 CAR-T, and anti-CD19 CAR-FH-T cells against the CD19-overexpressing tumor cell line Raji are as follows: Figure 19As shown in the above in vitro tumor-killing results, among T, anti-CD19 CAR-T, and anti-CD19 CAR-FH-T, anti-CD19 CAR-FH-T has a significantly higher killing effect than ordinary anti-CD19 CAR-T. Therefore, anti-CD19 CAR-FH-T is preferred for tumor treatment.

[0171] 2. In vitro cytokine detection:

[0172] Take an appropriate amount of target cells and mix them at a concentration of 1×10⁻⁶. 6 Resuspend the cells in a 0.5 × 10⁶ mL cell suspension (PBS, 5% fetal bovine serum) at room temperature, wash twice, and then resuspend the cells to a concentration of 0.5 × 10⁶. 5 / mL, add 0.05×10 to each well of a 96-well plate. 5 T cells, anti-CD19 CAR-T cells, and anti-CD19 CAR-FH-T cells were added at an effector-to-target ratio of 25:1 per mL of target cells. The mixture was centrifuged at 200g for 30 seconds and incubated at 37℃ for 18 hours. After incubation, the supernatant was collected, and the concentrations of IFN-γ, TNF-α, and Granzyme B in the supernatant were measured.

[0173] The secretion of IFN-γ, TNF-α, and Granzyme B after in vitro incubation of T cells, anti-CD19 CAR-T cells, anti-CD19 CAR-FH-T cells, and the CD19-overexpressing cell line Raji were as follows: Figure 20 As shown, consistent with the tumor-killing results, the levels of IFN-γ, TNF-α, and Granzyme B secreted by anti-CD19 CAR-T and anti-CD19 CAR-FH-T cells were significantly increased compared to T cells, indicating their in vitro killing function against CD19-positive tumor cell lines. More importantly, the release levels of IFN-γ, TNF-α, and Granzyme B from anti-CD19 CAR-FH-T cells were significantly higher than those from anti-CD19 CAR-T cells; indicating that FH expression in CAR-T cells can significantly enhance the tumor-killing ability of CAR-T cells.

[0174] 3. In vivo anti-tumor function of anti-CD19 CAR-T cells and anti-CD19-FH CAR-T cells

[0175] To verify the anti-tumor function of FH on CAR-T cells in vivo, the anti-tumor ability of anti-CD19 CAR-T cells and anti-CD19-FH CAR-T cells was observed and compared.

[0176] Raji-luci cell model: Fifteen healthy female NPG mice were injected with Raji-luci cells via the tail vein, with each mouse receiving 1.5 × 10⁻⁶ cells. 6Imaging to observe tumor growth begins 5-9 days later, and the fluorescence intensity (ROI value) is monitored until it reaches 10. 6 -10 7 A time interval of photons per second indicates successful model establishment. After successful model establishment, mice were randomly divided into three groups of five mice each. Group one served as the control group, receiving a tail vein injection of T cells. Group two received a tail vein injection of 2.0 × 10⁻⁶ T cells. 6 Anti-CD19 CAR-T cells, group 3, 2.0 × 10⁻⁶ cells were injected via tail vein. 6 Anti-CD19-FH CAR-T cells. Fluorescence imaging data were collected from mice every 4-6 days.

[0177] Fluorescence values ​​directly reflect the in vivo expansion of the Raji-luci tumor cell line. The fluorescence imaging data of Raji-luci in each mouse model group are as follows: Figure 21 As shown, compared with the control T cell group, both anti-CD19 CAR-T cells and anti-CD19-FH CAR-T cells significantly inhibited the proliferation of tumor cell lines in vivo. More importantly, although both anti-CD19 CAR-T cells and anti-CD19-FH CAR-T cells could inhibit tumor proliferation, the anti-tumor ability of anti-CD19-FH CAR-T cells was significantly higher than that of anti-CD19 CAR-T cells, indicating that overexpression of FH in CAR-T cells can significantly enhance the tumor-killing ability of CAR-T cells in vivo.

[0178] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

Claims

1. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the chimeric antigen receptor further expresses fumarate hydratase FH, wherein the extracellular domain comprises an antigen recognition domain scFv that specifically recognizes CD19 and a hinge region, the intracellular domain comprises a co-stimulatory domain and a signal transduction domain, and wherein the fumarate hydratase FH is expressed at the C-terminus of the intracellular domain.

2. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor further comprises a signal peptide, a small molecule tag, a self-cleaving peptide, or a combination thereof.

3. The chimeric antigen receptor according to claim 1 or 2, wherein... 1) The hinge region is derived from the hinge of IgG or the extracellular region of CD8α / CD28; 2) The transmembrane domains are derived from CD4, CD8α, CD28, or CD3ζ; 3) The co-stimulatory domain is derived from the CD28 receptor family or the tumor necrosis factor receptor family; and / or 4) The signal transduction domain is the T cell receptor TCR / CD3ζ chain or the immunoglobulin Fc receptor FcεRIγ chain.

4. The chimeric antigen receptor according to claim 1 or 2, wherein the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide, a single-chain antibody ScFv that specifically recognizes CD19, StrepII, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and a CD3ζ chain, a P2A peptide and FH.

5. A recombinant vector and / or isolated polynucleotide encoding a chimeric antigen receptor according to any one of claims 1-4, wherein the vector is a DNA vector, an RNA vector, or any combination thereof.

6. The recombinant vector and / or isolated polynucleotide according to claim 5, wherein the DNA vector is selected from transposon vectors, plasmids, adenovirus vectors, and adeno-associated virus vectors.

7. The recombinant vector and / or isolated polynucleotide according to claim 5, wherein the RNA vector is selected from retroviral vectors and lentiviral vectors.

8. A chimeric antigen receptor immune cell expressing a chimeric antigen receptor according to any one of claims 1-4 or a recombinant vector and / or isolated polynucleotides according to any one of claims 5-7, wherein the immune cell is a CAR T cell.

9. A pharmaceutical composition or kit comprising a chimeric antigen receptor according to any one of claims 1-4, a recombinant vector and / or isolated polynucleotides according to any one of claims 5-7, or a chimeric antigen receptor immune cell according to claim 8.

10. The use of the chimeric antigen receptor immune cell of claim 8 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating cancer, wherein the cancer is CD19. + The cancers mentioned herein are selected from B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and B-cell lymphoma.

11. The application according to claim 10, wherein the drug is used in CAR T-cell therapy or in combination with another antitumor drug and / or CAR T-cell drug / therapy.

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