A nucleotide segment encoding an activation-induced membrane-bound cytokine and use thereof

Abstract

This invention discloses a nucleotide fragment encoding an activation-inducible membrane-bound cytokine and its applications, belonging to the fields of genetic engineering and cell engineering. The nucleotide fragment encoding the activation-inducible membrane-bound cytokine includes a signal peptide nucleotide sequence, a CAR molecule sequence targeting CD19 or EGFRvIII, an activation-inducible promoter sequence containing 3-6 NFAT motifs, a membrane-bound IL-4 or IL-10 nucleotide sequence, and a Degron sequence. Delivering the membrane-bound IL-4 or IL-10 DNA fragment with adjustable expression levels provided by this invention to T cells using appropriate methods allows T cells to secrete membrane-bound IL-4 or IL-10 upon activation, enhancing T cell viability in the activated state while avoiding the immunosuppressive effects of IL-4 or IL-10 on resting T cells.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and cell engineering, and in particular to a nucleotide fragment encoding an activation-induced membrane-bound cytokine and its applications. Background Technology

[0002] T-cell therapy is an immunotherapy approach for cancer and other diseases. It utilizes the patient's own T lymphocytes, enhancing their ability to kill cancer cells or other pathogenic cells by altering their gene expression or introducing new genes. Depending on the source and modification method of the T cells, T-cell therapy can be categorized into CAR-T, TCR-T, and TIL cell therapies. CAR-T cell therapy is a genetically engineered T-cell therapy that introduces the chimeric antigen receptor (CAR) gene into the patient's T cells, enabling them to recognize and attack cancer cells. CAR-T cell therapy has shown significant efficacy in treating acute lymphoblastic leukemia, lymphoma, and other malignant tumors. TCR-T cell therapy is another genetically engineered T-cell therapy that introduces the T-cell receptor (TCR) gene into the patient's T cells, enabling them to recognize and attack cancer cells. TCR-T cell therapy has made some progress in treating melanoma, lung cancer, and other malignant tumors. TIL cell therapy is a T-cell therapy that modifies tumor-infiltrating lymphocytes (TILs) extracted from the patient. TIL cell therapy has shown some efficacy in treating melanoma, breast cancer, cervical cancer, and other malignant tumors.

[0003] However, T-cell therapy still faces challenges in practical application. T cells in the tumor microenvironment exhibit a loss of effector function and proliferative capacity, defined as T-cell "exhaustion," which may be caused by continuous antigen stimulation and other metabolic stresses in the tumor microenvironment (PMID: 27521269, 30923193). Due to the presence of T-cell exhaustion, some cancer patients who have received T-cell therapy may experience relapse or metastasis. Therefore, delaying T-cell exhaustion and improving its persistence are key to enhancing the therapeutic efficacy of T-cell therapy.

[0004] Interleukin-4 (IL-4) and interleukin-10 (IL-10) are both type II cytokines with complex biological functions and a significantly dual mechanism of action. On the one hand, IL-4 and IL-10 can inhibit the production of inflammatory cytokines by Th1 cells and suppress T cell-specific proliferation by reducing the expression of major histocompatibility complex II (MHC-II), thus playing a negative regulatory role in the inflammatory response. On the other hand, recent studies have reported that IL-4 and IL-10 play an active role in the tumor microenvironment. Specifically, IL-4 and IL-10 can promote the number and function of tumor-infiltrating lymphocytes (TILs), especially in terminally exhausted T cells, where IL-10 can restore their vitality through metabolic reprogramming, thereby enhancing the response to cancer immunotherapy (PMID: 34031618, 39322664, 39322665).

[0005] Given the dual role of IL-4 and IL-10 in immunomodulation, precise regulation of their secretion is necessary to fully realize their promoting effect on T-cell immune function. However, ensuring that IL-4 or IL-10 is released only when T cells are highly activated, and that the IL-4 or IL-10 secreted by T cells acts only on themselves, avoiding its effect on resting T cells, thereby reducing immunosuppression of T cells and further enhancing the long-term efficacy of T-cell therapy, remains a pressing problem in the current field of T-cell therapy research. Summary of the Invention

[0006] The purpose of this invention is to provide a nucleotide fragment encoding an activation-induced membrane-bound cytokine and its application, thereby addressing the problems existing in the prior art. By delivering the membrane-bound IL-4 or IL-10 nucleotide fragment with adjustable expression levels provided by this invention to T cells through an appropriate method, the T cells can secrete membrane-bound IL-4 or IL-10 upon activation, thereby enhancing the activity of T cells in the activated state while avoiding the immunosuppressive effects of IL-4 or IL-10 on T cells in the resting state.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a nucleotide fragment encoding an activation-induced membrane-bound cytokine, characterized in that the nucleotide fragment targets CD19 or EGFRvIII; and the cytokine is IL-4 or IL-10.

[0009] The nucleotide fragments include signal peptide nucleotide sequences, CAR molecule sequences targeting CD19 or EGFRvIII, activation-inducible promoter sequences containing 3-6 NFAT motifs, membrane-bound IL-4 or IL-10 nucleotide sequences, and Degron sequences.

[0010] The signal peptide nucleotide sequence is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.34-39;

[0011] The nucleotide sequence of the CD19-targeting CAR molecule is shown in SEQ ID NO.2;

[0012] The nucleotide sequence of the CAR molecule targeting EGFRvIII is shown in SEQ ID NO.3;

[0013] The nucleotide sequence of the activation-inducible promoter sequence containing 3-6 NFAT motifs is any one of the sequences shown in SEQ ID NO.4-7;

[0014] The structure of the membrane-bound IL-4 includes a signal peptide, an IL-4 core sequence, and a transmembrane domain;

[0015] The structure of the membrane-bound IL-10 includes a signal peptide, an IL-10 core sequence, and a transmembrane domain.

[0016] The nucleotide sequence of the signal peptide is a nucleotide sequence capable of encoding the amino acid sequence shown in SEQ ID NO.35-40;

[0017] The nucleotide sequence of the IL-4 core sequence is a nucleotide sequence that can encode the amino acid sequence shown in SEQ ID NO.25;

[0018] The nucleotide sequence of the IL-10 core sequence is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.26-34;

[0019] The transmembrane domain includes any one or a combination of at least two of the following: the α chain of the T cell receptor, the β chain of the T cell receptor, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR transmembrane domain.

[0020] The nucleotide sequence of the Degron sequence is shown in SEQ ID NO.6.

[0021] Degron motifs are amino acid sequences that can be recognized and degraded by proteases, typically located at the N- or C-terminus of proteins. When degrons are recognized and degraded by proteases, it leads to the degradation or inactivation of the target protein. Degrons play a role in regulating protein stability and function within cells, such as regulating cell cycle, apoptosis, and signal transduction. To avoid excessive T cell proliferation caused by overproduction of membrane-bound IL-4 or IL-10, it is necessary to fuse degron motifs with membrane-bound IL-4 or IL-10, allowing the expression levels of these IL-4 or IL-10 to be regulated by drug molecules. This strategy effectively controls the expression levels of membrane-bound IL-4 or IL-10, ensuring they exert their effects at appropriate times and dosages, thereby improving the safety and efficacy of T-cell therapy.

[0022] The present invention also provides a vector comprising the nucleotide fragments described above, wherein the vector comprises a retroviral vector, an adeno-associated virus vector, a lentiviral vector, an adenovirus vector, or a liposome.

[0023] The present invention also provides a recombinant immune cell capable of stably expressing the nucleotide fragment, obtained by delivery of the immune cell to the vector.

[0024] The present invention also provides a membrane-bound cytokine produced by the recombinant immune cells, including membrane-bound IL-4 or IL-10.

[0025] Activation-induced membrane-bound IL-4 or IL-10 prevents T cells from secreting IL-4 or IL-10 when they are in a resting state, thus avoiding the inhibitory effect of IL-4 or IL-10 on their proliferation. Simultaneously, because membrane-bound IL-4 or IL-10 can fix IL-4 or IL-10 on the cell surface, compared to secreted cytokines, membrane-bound cytokines ensure that the IL-4 or IL-10 expressed by T cells acts only on themselves, without affecting other unactivated T cells, further reducing their immunosuppressive effect.

[0026] The present invention also provides a method for regulating the secretion of IL-4 or IL-10 by immune cells, comprising delivering the nucleotide fragment to the immune cells so that they produce membrane-bound IL-4 or IL-10 only when highly activated.

[0027] Furthermore, the immune cells are of autologous, allogeneic, or heterologous origin.

[0028] More preferably, the immune cells include any one or a combination of at least two of the following: T cells, CAR-T cells, NK cells, CAR-NK cells, Treg cells, CAR-Treg cells, or iPS-derived CAR-NK cells.

[0029] The present invention also provides a method for improving the viability of T cells in an activated state, comprising introducing the nucleotide fragment into the T cells to fix the generated IL-4 or IL-10 on the surface of the T cells.

[0030] Furthermore, the T cells include CAR-T cells and tumor-infiltrating T cells.

[0031] The present invention also provides the use of the nucleotide fragment, the vector, or the recombinant T cell in the preparation of a drug for treating cancer.

[0032] Furthermore, the cancers include acute lymphoblastic leukemia, lymphoma, and glioma.

[0033] The present invention discloses the following technical effects:

[0034] The present invention provides a nucleotide fragment of membrane-bound IL-4 or IL-10 with adjustable expression levels. When delivered to T cells by an appropriate method, the nucleotide fragment enables the T cells to secrete membrane-bound IL-4 or IL-10 upon activation.

[0035] This invention constructs an activation-induced CAR-T cell that targets CD19 or EGFRvIII and produces membrane-bound IL-4 or IL-10. The membrane-bound IL-4 or IL-10 is only secreted after the CAR-T cells are specifically activated by tumor cells, thereby exerting its role in metabolic reprogramming of CAR-T cells, improving the vitality of CAR-T cells in the activated state, and avoiding the immunosuppressive effect of IL-4 or IL-10 on CAR-T cells in the resting state.

[0036] This invention provides a membrane-bound IL-4 or IL-10 that can be degraded by drugs. When membrane-bound IL-4 or IL-10 is secreted in excess, it can be degraded by small molecule drugs, thereby improving the safety of membrane-bound IL-4 or IL-10.

[0037] Compared to existing CAR-T cells, the CAR-T cells expressing membrane-bound IL-4 or IL-10 provided by this invention have better long-term anti-tumor effects. Compared to CAR-T cells expressing continuously secreted IL-4 or IL-10, the CAR-T cells expressing membrane-bound IL-4 or IL-10 provided by this invention have better safety. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 A schematic diagram for constructing a CAR gene that targets CD19 or EGFRvIII and expresses activation-inducible IL-4 or IL-10;

[0040] Figure 2 Results of flow cytometry detection of CAR molecules expressed in different T cells;

[0041] Figure 3 The results of flow cytometry analysis of the expression levels of membrane-bound cytokines under different conditions;

[0042] Figure 4 This is a statistical analysis of the proliferation of different T cells during in vitro culture.

[0043] Figure 5 The results of flow cytometry analysis of T cells after treatment with different reagents;

[0044] Figure 6 The results of in vitro killing assays of different T cells;

[0045] Figure 7 The results of tumor volume detection in mice after the first (A) and second (B) tumor inoculations with different T cells. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0049] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0050] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0051] The term "nucleotide" refers to polymers of deoxyribonucleotides (such as DNA, cDNA) or nucleotides (such as RNA, mRNA), or combinations of deoxyribonucleotides and nucleotides (e.g., DNA / RNA), including linear or cyclic structures, and single-stranded or double-stranded forms. This term should not be construed as a limitation on polymer length and can include known natural nucleotide analogs, as well as nucleotides modified at the base, sugar, and / or phosphate moieties (e.g., thio groups). Generally, analogs of a particular nucleotide have the same base pair specificity, such as the A and T base pairs.

[0052] The term "vector" refers to an invention comprising a viral vector or nucleotide (DNA or RNA) molecule such as a plasmid or other tool containing one or more different nucleotide sequences for the purpose of transformation and / or amplification between different host cells. The terms "expression vector," "gene delivery vector," and "gene therapy vector" refer to any vector that efficiently fuses and expresses one or more nucleotides of the invention in cells, preferably under promoter regulation.

[0053] Any vector known in this technology can be used in this invention. Vector refers to a viral vector, which can be a retroviral vector (such as MSGV, MMLV, etc.), an adeno-associated virus vector (AAV), a lentiviral vector (such as pGAR), or an adenovirus vector (AD).

[0054] The term “treatment” or “therapy” means the use of any composition, pharmaceutical composition, therapeutic agent, compound, etc. disclosed to a party for the purpose of: (1) suppressing disease, i.e. preventing the development of clinical symptoms; and / or (2) alleviating disease, even if clinical symptoms subside.

[0055] The term "prevention" refers to the disclosure to a party of any composition, pharmaceutical composition, therapeutic agent, compound, etc., even if the clinical symptoms of a disease do not develop.

[0056] The immune cells of the present invention can be used alone or as a pharmaceutical composition. The pharmaceutical compositions of the present invention can be combined with one or more drugs or physiologically acceptable carriers or diluents, including the immune cells described herein, such as CAR-T cells. The composition may include buffers, such as neutral buffered saline, phosphate buffer, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0057] The pharmaceutical compositions of the present invention may further include at least one additional therapeutic agent or therapy. A variety of other additional therapeutic agents may be used in combination with the compositions described herein. Preferably, the at least one additional therapeutic agent or therapy is an anticancer agent or anticancer therapy useful for treating cancer, preferably hematologic malignancies. Preferably, one or more anticancer therapies will be selected from combinations of radiotherapy, chemotherapy, immune checkpoint inhibitors, immunotherapy, and hormone therapy, or a combination thereof.

[0058] Methods for treating and / or preventing cancer in patients or research subjects include: (1) removing and isolating immune cells, such as mononuclear cells, from the patients or research subjects; and (2) genetically engineering the immune cells with a recombinant structure encoding activation-induced membrane-bound IL-4 or IL-10 to construct CAR-T cells or tumor-infiltrating T cells (TILs) expressing activation-induced membrane-bound IL-4 or IL-10.

[0059] The amino acid sequence of the IL-4 or IL-10 core sequence is as follows:

[0060] The amino acid sequence of the IL-4 core sequence (SEQ ID NO.25):

[0061] HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS.

[0062] The amino acid sequence of the IL-10 core sequence (SEQ ID NO.26):

[0063] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0064] Furthermore, the amino acid sequence of the IL-10 core sequence may also be selected from any of the following variant sequences:

[0065] SEQ ID NO.27:

[0066] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0067] SEQ ID NO.28:

[0068] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRWRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0069] SEQ ID NO.29:

[0070] SPGQGTQSENSCTHFPGWLPMMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0071] SEQ ID NO.30:

[0072] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVMSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0073] SEQ ID NO.31:

[0074] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLMTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0075] SEQ ID NO.32:

[0076] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0077] SEQ ID NO.33:

[0078] SPGQGTQSENSCTFFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0079] SEQ ID NO.34:

[0080] SPGQGTQSENSCWHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVLSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0081] Preferably, the N-terminus of the IL-4 or IL-10 core sequence further includes a signal peptide;

[0082] More preferably, the amino acid sequence of the signal peptide is selected from any of the following sequences.

[0083] SEQ ID NO. 35: MHSSALLCCLVLLTGVRA.

[0084] SEQ ID NO. 36: MALPVTALLLPLALLLHAARP.

[0085] SEQ ID NO. 37: MYRMQLLSCIALSLALVTNS.

[0086] SEQ ID NO. 38: MYRMQLLSCIALSLALVVTNSISA.

[0087] SEQ ID NO. 39: MGAARSPSAVPGPLLGLLLLLLGVLAPGGAS.

[0088] SEQ ID NO. 40: MLCCMRRTKQVEKNDEDQKI.

[0089] Preferably, the transmembrane domain of the membrane-bound IL-4 or IL-10 is the CD8 transmembrane domain shown in SEQ ID NO 41.

[0090] SEQ ID NO 41:

[0091] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0092] For any techniques or conditions not specified in the embodiments of this invention, the procedures shall be performed in accordance with the technical conditions described in the literature in this field or according to the product instructions. All reagents or instruments without specified manufacturers are conventional products that can be purchased through legitimate channels.

[0093] Example 1: Construction of CAR plasmids expressing activation-inducible IL-4 or IL-10

[0094] The following sequences were obtained from the NCBI database: an activation-inducible promoter sequence containing 3-6 NFAT-binding motifs and an interleukin-2 core promoter sequence; the gene sequence of IL-4; the gene sequence of IL-10; the membrane-binding region sequence; and the CAR molecule sequence targeting CD19 or EGFRvIII. The CAR molecule targeting CD19 or EGFRvIII contains the following gene elements: scFv recognizing CD19 or EGFRvIII, hinge region, transmembrane region, co-stimulatory domain, and CD3ζ strand. The above DNA sequence information was submitted to Porton Biotech, which synthesized the following DNA fragments:

[0095] GMCSF signal peptide (SEQ ID NO.1):

[0096] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg.

[0097] CAR molecule targeting CD19 (SEQ ID NO.2):

[0098]

[0099] CAR molecules targeting EGFRvIII (SEQ ID NO.3):

[0100]

[0101] IL-2 core promoter sequences containing 3-6 NFAT motifs:

[0102] SEQ ID NO.4 (IL-2 core promoter sequence containing 3 NFAT motifs):

[0103] aattg ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaagg cgt caattg ggaggaaaaactgtttcatacagaag gcgt caattgcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgc (The underlined part is the NFAT motif).

[0104] SEQ ID NO.5 (IL-2 core promoter sequence containing 4 NFAT motifs):

[0105] aattg ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaagg cgt caattg ggaggaaaaactgtttcatacagaag gcgt caattg ggaggaaaaactgtttcatacagaaggcgt caattgcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttc cctatcactctctttaatcactactcacagtaacctcaactcctgc.

[0106] SEQ ID NO.6 (IL-2 core promoter sequence containing 5 NFAT motifs):

[0107] aattg ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaagg cgt caattg ggaggaaaaactgtttcatacagaag gcgt caattg ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaaggcgt caattgcattttgacacccccataatatttttcca gaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgc.

[0108] SEQ ID NO.7 (IL-2 core promoter sequence containing 6 NFAT motifs):

[0109] aatta ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaagg cgt caattg ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaaggcgt caattg ggaggaaaaactgtttcatacagaaggcgt caattgcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgc.

[0110] DNA sequence of membrane-bound IL-4 (SEQ ID NO.8):

[0111] atgctgctgcttgtgacaagtctcctcctttgtgaacttccgcaccctgcttttctcctgatcatgggtctcacctcccaactgcttccccctctgttcttcctgctagcatgtgccggcaactttgtccacggacacaagtgcgatatcaccttacaggagatcatcaaaactttgaacagcctcacagagcagaagactctgtgcaccgagttgaccgtaacagacatctttgctgcctccaagaacacaactgagaaggaaaccttctgcagggctgcgactgtgctccggcagttctacagccaccatgagaaggacactcgctgcctgggtgcgactgcacagcagttccacaggcacaagcagctgatccgattcctgaaacggctcgacaggaacctctggggcctggcgggcttgaatagctgtcctgtgaaggaagccaaccagagtacgttggaaaacttcttggaaaggctaaagacgatcatgagagagaaatattcaaagtgttcgagcaccactacaccagctccacgcccccctacacccgctcctacaatcgcttcccagcctttgagcttgaggcccgaggcatgtagaccagcagcaggaggcgcggttcatactagaggtcttgacttcgcatgcgacatatatatttgggcccccctggcgggaacgtgtggggtgctgcttctgagcctggtgattactctgtactgc。

[0112] DNA sequence of membrane-bound IL-10 (SEQ ID NO.9):

[0113] atgctgctgcttgtgacaagtctcctcctttgtgaacttccgcaccctgcttttctcctgatcggccagggaacccagtccgaaaattcctgcacccactttccaggaaatctgcctaatatgcttagagatcttcgcgatgcgttctctcgcgtgaaaacatttttccaaatgaaagaccagctggacaatctcctgttgaaagagtcactcctggaggatttcaagggctacctcggttgtcaagctctcagcgagatgatccagttttacctggaggaagtcatgccccaggcggagaaccaggacccagacatcaaggcccacgtgaacagcctcggcgagaaccttaaaacactgaggctccgcctgagacggtgccaccgcttccttccttgcgagaacaagagcaaagccgtggagcaggtgaagaacgccttcaataagctgcaggaaaaaggcatttacaaggcaatgtcagagtttgacatcttcatcaactacatcgaggcctatatgactatgaagatccgcaacaccactacaccagctccacgcccccctacacccgctcctacaatcgcttcccagcctttgagcttgaggcccgaggcatgtagaccagcagcaggaggcgcggttcatactagaggtcttgacttcgcatgcgacatatatatttgggcccccctggcgggaacgtgtggggtgctgcttctgagcctggtgattactctgtactgc。

[0114] Degron sequence (SEQ ID NO.10):

[0115] ctgcagtgtgaaatatgtggcttcacttgccgacagaaagcctccctgaactggcatatgaagaaacacttccagtgtaatcagtgtggtgctagttttactcaga aaggcaatctcttgcgacacatcaaattgcac。

[0116] The membrane-bound IL-4 or IL-10 DNA sequence containing an activation-inducible promoter of 3-6 NFAT motifs is constructed by splicing an IL-2 core promoter sequence (SEQ ID NO. 4-7) containing 3-6 NFAT motifs, a membrane-bound IL-4 or IL-10 DNA sequence (SEQ ID NO. 8 or SEQ ID NO. 9), and a DNA sequence encoding Degron. The DNA fragments from these regions are then integrated into the backbone vector pLVH-EF1A-BRD3R (Brand: Addgene, Catalog No.: #130696) as needed to construct CAR plasmids targeting CD19 or EGFRvIII and expressing activation-inducible IL-4 or IL-10, such as... Figure 1 As shown. The specific construction steps are as follows:

[0117] (1) Obtain the full-length fragment containing the desired gene.

[0118] Input the DNA sequence of the desired gene into the DNA synthesizer program, and then use the DNA synthesizer (Unique) to synthesize the DNA. 600) Synthesize the full-length fragment of the desired gene. After obtaining the full-length fragment of the target gene, perform PCR amplification on the target gene fragment. The target gene fragments are: antiCD19-IL-4-iCAR (SEQ ID NO. 11), antiCD19-IL-4-pCAR (SEQ ID NO. 12), antiCD19-IL-10-iCAR (SEQ ID NO. 13), antiCD19-IL-10-pCAR (SEQ ID NO. 14), antiEGFRvIII-IL-4-iCAR (SEQ ID NO. 15), and antiEGFRvIII-IL-4-pCAR (SEQ ID NO. 16). NO.16), antiEGFRvIII-IL-10-iCAR (SEQ ID NO.17), antiEGFRvIII-IL-10-pCAR (SEQ ID NO.18), antiCD19-nCAR (SEQ ID NO.19), and antiEGFRvIII-nCAR (SEQ ID NO.20) are DNA fragments that target EGFRvIII and stably express membrane-bound IL-10.

[0119] Using the full-length target gene fragment as a template, primers were designed to amplify the DNA fragments in the above-mentioned regions by PCR. The primers used are as follows:

[0120] Primers for amplifying antiCD19-IL-4-iCAR, antiCD19-IL-10-iCAR, antiEGFRvIII-IL-4-iCAR, and antiEGFRvIII-IL-10-iCAR:

[0121] Forward primer: gacccggtcgaattcatggccttaccagtgacc (SEQ ID NO.21);

[0122] Reverse primer: cttaaaggtaccaattaggaggaaaaactgtttc (SEQ ID NO.22);

[0123] Primers for amplifying antiCD19-IL-4-pCAR, antiCD19-IL-10-pCAR, antiEGFRvII-IL-4-pCAR, antiEGFRvII-IL-10-pCAR, antiCD19-nCAR, and antiEGFRvIII-nCAR:

[0124] Forward primer: gacccggtcgaattcatggccttaccagtgacc (SEQ ID NO.23);

[0125] Reverse primer: cattggtcttaaaggtaccaattatgaggtgtg (SEQ ID NO.24);

[0126] PCR reaction system: 100 ng of the full-length target gene fragment synthesized by a DNA synthesis instrument, KOD One TM PCRMasterMix 25μL, 10μM forward and reverse primers 2μL each, and ultrapure water to bring the total to 50μL.

[0127] PCR reaction conditions: 98℃ for 60s, 1 cycle; 98℃ for 10s, 60℃ for 5s, 68℃ for 10s, 35 cycles; 16℃ for 60s, 1 cycle.

[0128] PCR amplification was first performed using a CAR gene targeting CD19 and expressing activation-inducible membrane-bound IL-4 as a template. Primers SEQ ID NO.21 and SEQ ID NO.22 were selected, and the PCR amplification reaction was completed under the aforementioned conditions to construct a DNA fragment antiCD19-IL-4-iCAR containing the CAR gene targeting CD19 and expressing activation-inducible membrane-bound IL-4. After verification, the same method was used to obtain antiCD19-IL-4-pCAR, antiCD19-IL-10-iCAR, antiCD19-IL-10-pCAR, antiEGFRvIII-IL-4-iCAR, antiEGFRvIII-IL-4-pCAR, antiEGFRvIII-IL-10-iCAR, antiEGFRvIII-IL-10-pCAR, antiCD19-nCAR, and antiEGFRvIII-nCAR, respectively.

[0129] (2) Enzyme digestion

[0130] The pLVH-EF1A-BRD3R vector (Brand: Addgene, Catalog No.: #130696) was treated with Thermo's FastDigest BamHI (catalog number FD0054) and FastDigest KpnI (FD0524). The enzyme digestion system was as follows: 2 μg pLVH-EF1A-BRD3R vector, 1 μL FD BamHI, 1 μL FD KpnI, 2 μL 10×FD Buffer, and ddH2O to a final volume of 20 μL.

[0131] After 2 hours of enzyme digestion, 20 μL of the vector digestion product was subjected to agarose gel electrophoresis. A fragment of approximately 10,000 bp was extracted and recovered using Magen's HiPure Gel DNA Micro Kit (catalog number: D2110).

[0132] (3) Homologous recombination

[0133] Homologous recombination reaction was performed using the ClonExpress Homologous Recombination Kit (catalog number: C112-01 / 02) from Vazyme Biotech. The reaction system consisted of: 200 ng of linearized vector, 80 ng of antiCD19-IL-4-iCARDNA fragment obtained by PCR in step (1), 10 μL of 2×Exnase Buffer, 2 μL of Exnase, and ddH2O to bring the total to 20 μL.

[0134] After incubating at 37°C for 30 min, the cells were quickly placed on ice for 5 min, followed by the addition of 20 μL of Trans1-T1 competent cells. After standing for 30 min, the cells were heat-shocked at 42°C for 90 s and then plated.

[0135] After 16 hours, a single colony was picked from the plate and incubated in 25 mL of LB medium at 37°C in a shaker for 16 hours. The plasmid was then extracted using a plasmid extraction kit (supplier: Magen, catalog number: P1156), and DNA sequencing was used to verify successful plasmid construction. The plasmid with correct sequencing was selected as the plasmid expressing the antiCD19-IL-4-iCAR gene. Similarly, CAR plasmids expressing antiCD19-IL-4-pCAR, antiCD19-IL-10-iCAR, antiCD19-IL-10-pCAR, antiEGFRvIII-IL-4-iCAR, antiEGFRvIII-IL-4-pCAR, antiEGFRvIII-IL-10-iCAR, antiEGFRvIII-IL-10-pCAR, antiCD19-nCAR, and antiEGFRvIII-nCAR could be constructed.

[0136] Example 2: Preparation of CAR-T cells expressing activation-induced membrane-bound IL-4 or IL-10

[0137] 1. Preparation of lentiviruses

[0138] Prepare a 10cm culture dish and inoculate 5×10⁶ cells / year. 6 Two 293T cells were cultured overnight at 37°C in a 5% CO2 incubator in complete medium (DMEM containing 4500 mg / L glucose, 10% FBS, 1% ampicillin, and 1% streptomycin). 100 μM of PEI and plasmids were removed from the freezer, thawed at room temperature, and thoroughly mixed by pipetting. The plasmids were the CAR gene-containing plasmids prepared in Example 1, and two others: pMD2.G (Addgene, catalog number: #12259) and psPAX2 (Addgene, catalog number: #12260). 9 μg of the CAR plasmid from Example 1, 3 μg of pMD2.G, and 12 μg of psPAX2 were mixed thoroughly to form the plasmid mixture. The PBS solution was removed and warmed to room temperature. Add 2 mL of PBS solution to one well of a 6-well plate, add the plasmid mixture, and mix thoroughly by pipetting. Then add 18 μL of 100 μM PEI and immediately mix by pipetting. Let stand at room temperature for 10 min. Add the plasmid / PEI complex dropwise to a 10 cm culture dish, gently shaking to mix thoroughly. Incubate the dish at 37°C with 5% CO2 for 7 hours. After incubation, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. After 60 hours of continuous incubation, collect the supernatant containing the virus from the culture dish, filter it through a 0.45 μm filter membrane, transfer it to a centrifuge tube, balance it, and centrifuge at 20000g, 4°C for 2 hours. After centrifugation, carefully aspirate the liquid from the centrifuge tube in a biosafety cabinet, resuspend the precipitate with 500 μL of PBS buffer, aliquot the virus, and store at -80°C.

[0139] 2. Activation of primary T cells and lentiviral infection

[0140] After PBMC separation, wash the CD3 / CD28 beads: Vibrate to mix the CD3 / CD28 beads, then take 100 μL of CD3 / CD28 beads (4 × 10⁻⁶). 6 Add 100 μL of magnetic beads to each T cell in a prepared 1.5 mL centrifuge tube. Add 1 mL of separation buffer (PBS buffer containing 1% fetal bovine serum) and mix for 1 min. Place the centrifuge tube on a magnetic rack for 1 min and remove the supernatant. Add 4 mL of activation medium (X-VIVO15 medium containing 10% inactivated serum and 300 IU / mL IL-2), resuspend the cells and magnetic beads, and mix until the cell concentration is 1 × 10⁻⁶ cells / mL. 6Cells / mL; Centrifuge T cells stimulated with magnetic beads for 20 h to remove supernatant; After virus thawing, add virus solution (MOI 2), add 0.1% volume of protamine sulfate at a concentration of 10 mg / mL and 2% inactivated plasma according to the total volume of the suspension, add the cell suspension to the well plate, and incubate the well plate in an incubator for 5 h; Add 10 mL of amplification medium (X-VIVO15 medium containing 10% inactivated plasma, 1% ampicillin and streptomycin, 5 ng / mL IL-7 and 5 ng / mL IL-15), resuspend and mix the cells, add the cell suspension to a 6-well plate or T75 flask for culture, and change the medium every other day.

[0141] 3. Detection of CAR-T cell transduction.

[0142] Flow cytometry was used to detect CAR molecules expressed on T cells. Anti-G4S tags specifically recognize the GGGGSGGGGSGGGGS tag on the scFv of CAR molecules to detect the CAR-T cell positivity rate. The detection steps were as follows: Untransduced T cells (MOCK-T), conventional CAR-T cells targeting CD19 and EGFRvIII (antiCD19-nCAR-T, antiEGFRvIII-nCAR-T), CAR-T cells expressing membrane-bound IL-4 targeting CD19 and EGFRvIII (antiCD19-IL-4-iCAR-T, antiEGFRvIII-IL-4-iCAR-T), and CAR-T cells expressing membrane-bound IL-10 targeting CD19 and EGFRvIII (antiCD19-IL-10-iCAR-T, antiEGFRvIII-IL-10-iCAR-T) were counted, and a sample containing 5 × 10⁻⁶ cells was collected. 5 The cell suspension was transferred to 1.5 mL centrifuge tubes. After centrifugation at 500 g for 3 min, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS. The cells were then centrifuged again at 500 g for 3 min at 4°C, and the supernatant was discarded. 0.5 μL of anti-G4S-tagged antibody (ACROBiosystems, clone number 016) was added to 50 μL of PBS, and the tubes were incubated at 4°C for 30 min. 1 mL of PBS was added, and the cells were centrifuged at 500 g for 3 min at 4°C, and the supernatant was discarded. Each tube was resuspended in 500 μL of PBS, and the cells were analyzed by flow cytometry. Results are as follows: Figure 2 As shown, this indicates that CAR-T cells expressing activation-induced membrane-bound IL-4 or IL-10 were successfully prepared.

[0143] Example 3: Functional verification of CAR-T cells expressing activation-induced membrane-bound IL-4 or IL-10

[0144] According to the method described in Example 2, CAR-T cells that continuously express membrane-bound IL-4 and IL-10 (antiCD19-mIL-4-pCAR or antiCD19-mIL-10-pCAR) and CAR-T cells that do not contain membrane-bound IL-4 and IL-10 (antiCD19-nCAR-T) can be constructed as control groups for subsequent experiments.

[0145] 1. Flow cytometry detection of membrane-bound cytokine expression levels

[0146] Prepare a suspension of target cells (Raji cells), density: 1×10⁻⁶ 6 CAR-T cell suspension was added to each well at a ratio of 1:1 to target cells. CAR-T cell suspensions containing CD19-targeting expression activation-induced membrane-bound IL-4 or IL-10 were added at a 1:1 ratio. Each experimental group had three replicates, with 100 μL of CAR-T cell suspension added to each well. After incubation of CAR-T cells and target cells for 24 hours, cells were collected and the supernatant discarded. The samples were resuspended in 50 μL of PBS. 0.5 μL of flow cytometry antibody for detecting IL-4 (APC anti-human IL-4 Antibody, catalog number: 500812) or IL-10 (APC anti-human IL-10 Antibody, catalog number: 506807) was added to the samples. The mixture was incubated at 4°C for 30 min. 1 mL of PBS was added, and the mixture was centrifuged at 500 g for 3 min at 4°C, then the supernatant was discarded. Each tube was resuspended in 500 μL of PBS and analyzed by flow cytometry.

[0147] Experimental results are as follows Figure 3 As shown, after co-culturing with target cells, CAR-T cells expressing activation-induced membrane-bound IL-4 or IL-10 (antiCD19-mIL-4-iCAR-T or antiCD19-mIL-10-iCAR-T) and CAR-T cells continuously expressing membrane-bound IL-4 or IL-10 (antiCD19-mIL-4-pCAR-T or antiCD19-mIL-10-pCAR-T) produced large amounts of membrane-bound IL-4 or IL-10. However, CAR-T cells without membrane-bound IL-4 or IL-10 genes (antiCD19-nCAR-T) and antiCD19-mIL-4-iCAR-T or antiCD19-mIL-10-iCAR-T cells not co-cultured with target cells could not detect membrane-bound IL-4 or IL-10, indicating that activation-induced membrane-bound IL-4 or IL-10 CAR-T cells have the function of producing membrane-bound IL-4 or IL-10 after activation.

[0148] 2. Cell counting

[0149] Cryopreserved untransduced CAR gene T cells (MOCK-T), CAR-T cells without membrane-bound IL-4 or IL-10 genes (antiCD19-nCAR-T), CAR-T cells continuously expressing membrane-bound IL-4 or IL-10 (antiCD19-mIL-4-pCAR-T or antiCD19-mIL-10-pCAR-T), and activation-induced membrane-bound IL-4 or IL-10 (antiCD19-mIL-4-iCAR-T or antiCD19-mIL-10-iCAR-T) were revived and cultured in vitro with an initial cell number of 1×10⁻⁶ cells. 6 Cells were cultured continuously for 12 days. Cell counts were performed daily, and the cells were passaged according to their proliferation status.

[0150] The results are as follows Figure 4 As shown, Figure 4 The results showed that the number of antiCD19-mIL-4-iCAR-T or antiCD19-mIL-10-iCAR-T cells expanded in vitro was comparable to that of MOCK-T and nCAR-T cells and higher than that of antiCD19-mIL-4-pCAR-T or antiCD19-mIL-10-pCAR-T cells. This result demonstrates that activation-induced membrane-bound IL-4 or IL-10 is more conducive to the proliferation of CAR-T cells than continuously expressed membrane-bound IL-4 or IL-10.

[0151] 3. Degradation experiments of membrane-bound IL-4 or IL-10

[0152] Prepare a suspension of target cells (Raji cells), density: 1×10⁻⁶ 6 100 μL of target cell suspension was added to each well; 100 μL of CAR-T cell suspension expressing activation-induced membrane-bound IL-4 or IL-10 targeting CD19 was added at an effector-target ratio of 1:1. 100 nM and 1 μM lenalidomide (LE) were added to the experimental groups, respectively, while 100 μL of PBS was added to the control group. After incubation of CAR-T cells and target cells for 24 hours, cells were collected and the supernatant was discarded. The samples were resuspended in 50 μL of PBS. 0.5 μL of flow cytometry antibody for detecting IL-4 (APC anti-human IL-4 Antibody, catalog number: 500812) and IL-10 (APC anti-human IL-10 Antibody, catalog number: 506807) was added to the samples.

[0153] The results are as follows Figure 5As shown, CAR-T cells expressing membrane-bound IL-4 or IL-10 (antiCD19-mIL-4-iCAR-T or antiCD19-mIL-10-iCAR-T) treated with PBS produced large amounts of membrane-bound IL-4 or IL-10, while the membrane-bound IL-4 or IL-10 detected in antiCD19-mIL-4-iCAR-T or antiCD19-mIL-10-iCAR-T cells treated with lenalidomide was significantly reduced, indicating that activation-induced membrane-bound IL-4 or IL-10 can be degraded by lenalidomide in a concentration-dependent manner.

[0154] Example 4: In vitro long-term killing experiment of CAR-T cells expressing activation-induced membrane-binding IL-4 or IL-10

[0155] Prepare a suspension of target cells (U87 cells expressing GFP), density: 2 × 10⁻⁶ 5 Add 1 mL of target cell suspension to each well of a 6-well plate. Prepare CAR-T cell suspensions at an effector-to-target ratio of 4:1: untransduced T cells (MOCK-T), EGFRvIII-targeting CAR-T cells (antiEGFRvIII nCAR-T), and EGFRvIII-targeting expression activation-induced membrane-bound IL-4 or IL-10 (antiEGFRvIII-mIL-4-iCAR-T or antiEGFRvIII-mIL-10-iCAR-T). Increase the culture medium volume of the 6-well plate to 3 mL, with 3 replicates per group. Every 3 days, aspirate the supernatant from the 6-well plate, centrifuge at 300g for 5 min, discard the supernatant, and resuspend the cells in 1 mL of fresh culture medium. Add the resuspended cells to a new plate containing pre-seeded target cells (1 mL / well, 1 × 10⁶ cells / well). 5 T cells were placed in 6-well plates (cells / mL). U87 cells were adherent. After aspirating the supernatant containing suspended T cells, 0.5 mL of 0.25% trypsin was added to the existing 6-well plates to digest the U87 cells. After 2 min, medium containing 10% FBS was added to stop the digestion, and the U87 cells were resuspended. Then, 100 μL was taken for GFP-positive cell counting. The above steps were repeated 3 times.

[0156] The results are as follows Figure 6 As shown, CAR-T cells expressing activation-induced membrane-bound IL-4 or IL-10 can clear more target cells in multiple rounds of in vitro killing and have better long-term anti-tumor ability compared with CAR-T cells that do not express activation-induced membrane-bound IL-4 or IL-10.

[0157] Example 5: In vivo antitumor experiment of CAR-T cells expressing activation-induced membrane-bound IL-4 or IL-10.

[0158] Prepare a suspension of U87 target cells with a density of 5 × 10⁻⁶. 6 100 μL of U87 cell suspension was subcutaneously injected into the left groin of immunodeficient NSG mice, and tumor area was measured periodically. Five mice were in each group. On day 6 of tumor bearing, 200 μL of cell suspensions of untransduced T cells (MOCK-T), EGFRvIII-targeting CAR-T cells (antiEGFRvIII-nCAR-T), and EGFRvIII-targeting CAR-T cells expressing activation-inducible membrane-bound IL-10 (antiEGFRvIII-IL-10iCAR-T) were injected into the tumor-bearing mice via tail vein. The CAR-T cell density was 5 × 10⁶ cells / mL. 6 On day 28 after the initial tumor inoculation, except for the MOCK-T group, the tumors in the other groups had been cleared, so a second tumor cell inoculation was performed. The MOCK-T group was euthanized due to excessively large tumor volume exceeding ethical limits, and no second tumor cell transplantation was performed. The procedure for secondary tumor transplantation was as follows: 100 μL of LU87 cell suspension (cell density: 5 × 10⁻⁶) was used. 6 Tumors (number per mL) were injected subcutaneously into the right groin of immunodeficient mice, and the tumor area was measured periodically thereafter.

[0159] The results are as follows Figure 7 As shown, after secondary tumor inoculation, the tumor volume of tumor-bearing mice that were infused with CAR-T cells expressing activation-induced membrane-bound IL-10 was smaller than that of CAR-T cells that did not express IL-10. These results indicate that CAR-T cells expressing activation-induced membrane-bound IL-10 have better long-term anti-tumor ability in mice.

[0160] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

[0161] antiCD19-mIL-4-iCAR gene sequence (SEQ ID NO.11):

[0162] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgg

[0163] gagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatca

[0164] agattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaa

[0165] cagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggt

[0166] gaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcg

[0167] ccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactc

[0168] caagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactgggg

[0169] ccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagagg

[0170] cgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcct

[0171] gtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgta

[0172] gctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctat

[0173] aacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagg

[0174] aaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttac

[0175] cagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccggaggaagcggaactagttgagatccaccggatcta

[0176] ggcgtctggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatca

[0177] tgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgt

[0178] ttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgc

[0179] cttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctgga

[0180] ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgc

[0181] cctcagacgagtcggatctccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagcta

[0182] ccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcattagtgaagtttgatgtggcgcagcaggttccctttctgggt

[0183] aaaagaagctccacactgattgcactgaaaatgctttttcatgtgccaattcaaggacgccttctgtctgcaggtgaaaccacagatctcgcactgcaagcagtacaga

[0184] gtaatcaccaggctcagaagcagcaccccacacgttcccgccaggggggcccaaatatatatgtcgcatgcgaagtcaagacctctagtatgaaccgcgcctcctg

[0185] ctgctggtctacatgcctcgggcctcaagctcaaaggctgggaagcgattgtaggagcgggtgtaggggggcgtggagctggtgtagtggtgctcgaacactttgaa

[0186] tatttctctctcatgatcgtctttagcctttccaagaagttttccaacgtactctggttggcttccttcacaggacagctattcaagcccgccaggccccagaggttcctgtcg

[0187] agccgtttcaggaatcggatcagctgcttgtgcctgtggaactgctgtgcagtcgcacccaggcagcgagtgtccttctcatggtggctgtagaactgccggagcaca

[0188] gtcgcagccctgcagaaggtttccttctcagttgtgttcttggaggcagcaaagatgtctgttacggtcaactcggtgcacagagtcttctgctctgtgaggctgttcaaa

[0189] gttttgatgatctcctgtaaggtgatatcgcacttgtgtccgtggacaaagttgccggcacatgctagcaggaagaacagagggggaagcagttgggaggtgagacc

[0190] catgatcaggagaaaagcagggtgcggaagttcacaaaggaggagacttgtcacaagcagcagcatgcaggagttgaggttactgtgagtagtgattaaagagagt

[0191] gatagggaactcttgaacaagagatgcaatttatactgttaattctggaaaaatattatgggggtgtcaaaatgcaattgacgccttctgtatgaaacagtttttcctcccaa

[0192] ttgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcctaattggtacctttaag。

[0193] antiCD19-mIL-4-pCAR gene sequence (SEQ ID NO.12):

[0194] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgg

[0195] gagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatca

[0196] agattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaa

[0197] cagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggt

[0198] gaaactgcaggatcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcatcatacccgactatggtgtaagctggattcg

[0199] ccagcctccacgaaagggtctggagtggctgggagtaatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactc

[0200] caagagccaagttttcttaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactgggg

[0201] ccaaggaacctcagtcaccgtctcctcaaccaccgacgccagcgccggaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagagg

[0202] cgtgccggccagccggcggggggcgcagtgcacacgaggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcct

[0203] gtcactggttatcaccctttactgcaaacggggcaagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagaagaatggctgta

[0204] gctgccgatttccagaagaagaagagaggatgtgaactgagagtgaagttcagcaggagcgcagacgccccccgcgtaccagcagggccagaaccagctctat

[0205] aacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagg

[0206] aaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttac

[0207] cagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccggaggaagcggagagggcagaggaagtctgctaac

[0208] atgcggtgacgtcgaggagaatcctggacctgctagcatgctgctgcttgtgacaagtctcctcctttgtgaacttccgcaccctgcttttctcctgatcatgggtctcacc

[0209] tcccaactgcttccccctctgttcttcctgctagcatgtgccggcaactttgtccacggacacaagtgcgatatcaccttacaggagatcatcaaaactttgaacagcctc

[0210] acagagcagaagactctgtgcaccgagttgaccgtaacagacatctttgctgcctccaagaacacaactgagaaggaaaccttctgcagggctgcgactgtgctccg

[0211] gcagttctacagccaccatgagaaggacactcgctgcctgggtgcgactgcacagcagttccacaggcacaagcagctgatccgattcctgaaacggctcgacagg

[0212] aacctctggggcctggcgggcttgaatagctgtcctgtgaaggaagccaaccagagtacgttggaaaacttcttggaaaggctaaagacgatcatgagagagaaata

[0213] ttcaaagtgttcgagcaccactacaccagctccacgcccccctacacccgctcctacaatcgcttcccagcctttgagcttgaggcccgaggcatgtagaccagcagc

[0214] aggaggcgcggttcatactagaggtcttgacttcgcatgcgacatatatatttgggcccccctggcgggaacgtgtggggtgctgcttctgagcctggtgattactctgt

[0215] actgcttgcagtgcgagatctgtggtttcacctgcagacagaaggcgtccttgaattggcacatgaaaaagcattttcagtgcaatcagtgtggagcttcttttacccaga

[0216] aagggaacctgctgcgccacatcaaacttcactaagatccaccggatctaggcgtctggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattctta

[0217] actatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctcttta

[0218] tgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccggg

[0219] actttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtc

[0220] ggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt

[0221] cccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattaattctgcagt

[0222] cgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcataattggtacctttaagaccaatg。

[0223] antiCD19 - mIL - 10 - iCAR gene sequence (SEQ ID NO.13):

[0224] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgg

[0225] gagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatca

[0226] agattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaa

[0227] cagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggt

[0228] gaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcg

[0229] ccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactc

[0230] caagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactgggg

[0231] ccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagagg

[0232] cgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcct

[0233] gtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgta

[0234] gctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctat

[0235] aacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagg

[0236] aaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttac

[0237] cagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccggaggaagcggaactagttgagatccaccggatcta

[0238] ggcgtctggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatca

[0239] tgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgt

[0240] ttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgc

[0241] cttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctgga

[0242] ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgc

[0243] cctcagacgagtcggatctccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagcta

[0244] ccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcattagtgaagtttgatgtggcgcagcaggttccctttctgggt

[0245] aaaagaagctccacactgattgcactgaaaatgctttttcatgtgccaattcaaggacgccttctgtctgcaggtgaaaccacagatctcgcactgcaagcagtacaga

[0246] gtaatcaccaggctcagaagcagcaccccacacgttcccgccaggggggcccaaatatatatgtcgcatgcgaagtcaagacctctagtatgaaccgcgcctcctg

[0247] ctgctggtctacatgcctcgggcctcaagctcaaaggctgggaagcgattgtaggagcgggtgtaggggggcgtggagctggtgtagtggtgttgcggatcttcata

[0248] gtcatataggcctcgatgtagttgatgaagatgtcaaactctgacattgccttgtaaatgcctttttcctgcagcttattgaaggcgttcttcacctgctccacggctttgctct

[0249] tgttctcgcaaggaaggaagcggtggcaccgtctcaggcggagcctcagtgttttaaggttctcgccgaggctgttcacgtgggccttgatgtctgggtcctggttctc

[0250] cgcctggggcatgacttcctccaggtaaaactggatcatctcgctgagagcttgacaaccgaggtagcccttgaaatcctccaggagtgactctttcaacaggagattg

[0251] tccagctggtctttcatttggaaaaatgttttcacgcgagagaacgcatcgcgaagatctctaagcatattaggcagatttcctggaaagtgggtgcaggaattttcggac

[0252] tgggttccctggccgatcaggagaaaagcagggtgcggaagttcacaaaggaggagacttgtcacaagcagcagcatgcaggagttgaggttactgtgagtagtga

[0253] ttaaagagagtgatagggaactcttgaacaagagatgcaatttatactgttaattctggaaaaatattatgggggtgtcaaaatgcaattgacgccttctgtatgaaacagt

[0254] ttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcc。

[0255] antiCD19-mIL-10-pCAR gene sequence (SEQ ID NO.14):

[0256] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgg

[0257] gagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatca

[0258] agattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaa

[0259] cagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggt

[0260] gaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcg

[0261] ccagcctccacgaaagggtctggagtggctgggagtaatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactc

[0262] caagagccaagttttcttaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactgggg

[0263] ccaaggaacctcagtcaccgtctcctcaaccaccgacgccagcgccggaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagagg

[0264] cgtgccggccagccggcggggggcgcagtgcacacgaggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcct

[0265] gtcactggttatcaccctttactgcaaacggggcaagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagaagaatggctgta

[0266] gctgccgatttccagaagaagaagagaggatgtgaactgagagtgaagttcagcaggagcgcagacgccccccgcgtaccagcagggccagaaccagctctat

[0267] aacgagctcaatctaggacgaagagagtacgatgttttggacaagagacgtggccgggaccctgagatgggggggaaagccgagaagaagaaccctcagg

[0268] aaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttac

[0269] cagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccggaggaagcggagagggcagaggaagtctgctaac

[0270] atgcggtgacgtcgaggagaatcctggacctgctagcatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgagccca

[0271] ggccagggcacccagtctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaa

[0272] atgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggag

[0273] gtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctgtcatcga

[0274] tttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttca

[0275] tcaactacatagaagcctacatgacaatgaagatacgaaacaccactacaccagctccacgcccccctacacccgctcctacaatcgcttcccagcctttgagcttga

[0276] ggcccgaggcatgtagaccagcagcaggaggcgcggttcatactagaggtcttgacttcgcatgcgacatatatatttgggcccccctggcgggaacgtgtggggt

[0277] gctgcttctgagcctggtgattactctgtactgcttgcagtgcgagatctgtggtttcacctgcagacagaaggcgtccttgaattggcacatgaaaaagcattttcagtg

[0278] caatcagtgtggagcttcttttacccagaaagggaacctgctgcgccacatcaaacttcactaagatccaccggatctaggcgtctggaacaatcaacctctggattac

[0279] aaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctc

[0280] ctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggc

[0281] attgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggct

[0282] gttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccc

[0283] ttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggcc

[0284] gcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcataattggtacctttaagaccaatg。

[0285] antiEGFRvIII-mIL-4-iCAR gene sequence (SEQ ID NO.15):

[0286] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaatccagctggtgcagtcaggcgccgaggtcaagaagccg

[0287] ggagagtcgctgagaatctcgtgcaagggctcggggttcaacatcgaggactactacattcactgggtcaggcagatgccgggaaagggactggaatggatgggc

[0288] cggatcgacccagaaaatgacgaaaccaaatacgggccgatttttcaaggccacgtgactatcagcgcagacacgagcatcaacactgtctacctccagtggtcctc

[0289] gcttaaggccagcgataccgctatgtactactgcgcattcagaggcggggtgtactggggacaaggaaccactgtgaccgtgagcagcggaggtggcggctcggg

[0290] aggaggtgggagcggaggaggaggttccggcggtggaggatcagatgtcgtgatgacccagtccccggactccctcgctgtctcactgggcgagcgcgcgacca

[0291] tcaactgcaaatcgagccagtcgctgttggactccgatggaaagacttatctgaattggctgcaacagaaaccaggacaacctcccaagcggctcatctcgcttgtgtc

[0292] aaaactcgattcgggagtgccagaccgcttctcggggtccgggagcggaactgactttactttgaccatttcctcactgcaagcggaggatgtggccgtgtattactgtt

[0293] ggcagggcacgcatttccctggaaccttcggtggcggaactaaggtggaaatcaagggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccac

[0294] catcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatct

[0295] gggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga

[0296] gaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacg

[0297] cccccgcgtaccaacagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgaga

[0298] tggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcga

[0299] gcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccg

[0300] gaggaagcggaactagttgagatccaccggatctaggcgtctggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttt

[0301] tacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtgg

[0302] cccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccc

[0303] cctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctga

[0304] cgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgc

[0305] tgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaa

[0306] aacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcattag

[0307] tgaagtttgatgtggcgcagcaggttccctttctgggtaaaagaagctccacactgattgcactgaaaatgctttttcatgtgccaattcaaggacgccttctgtctgcagg

[0308] tgaaaccacagatctcgcactgcaagcagtacagagtaatcaccaggctcagaagcagcaccccacacgttcccgccaggggggcccaaatatatatgtcgcatgc

[0309] gaagtcaagacctctagtatgaaccgcgcctcctgctgctggtctacatgcctcgggcctcaagctcaaaggctgggaagcgattgtaggagcgggtgtagggggg

[0310] cgtggagctggtgtagtggtgctcgaacactttgaatatttctctctcatgatcgtctttagcctttccaagaagttttccaacgtactctggttggcttccttcacaggacag

[0311] ctattcaagcccgccaggccccagaggttcctgtcgagccgtttcaggaatcggatcagctgcttgtgcctgtggaactgctgtgcagtcgcacccaggcagcgagt

[0312] gtccttctcatggtggctgtagaactgccggagcacagtcgcagccctgcagaaggtttccttctcagttgtgttcttggaggcagcaaagatgtctgttacggtcaactc

[0313] ggtgcacagagtcttctgctctgtgaggctgttcaaagttttgatgatctcctgtaaggtgatatcgcacttgtgtccgtggacaaagttgccggcacatgctagcaggaa

[0314] gaacagagggggaagcagttgggaggtgagacccatgatcaggagaaaagcagggtgcggaagttcacaaaggaggagacttgtcacaagcagcagcatgca

[0315] ggagttgaggttactgtgagtagtgattaaagagagtgatagggaactcttgaacaagagatgcaatttatactgttaattctggaaaaatattatgggggtgtcaaaatg

[0316] caattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcctaattggtacctttaag. antiEGFRvIII-mIL-4-pCAR gene sequence (SEQ ID NO.16):

[0317] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaatccagctggtgcagtcaggcgccgaggtcaagaagccg

[0318] ggagagtcgctgagaatctcgtgcaagggctcggggttcaacatcgaggactactacattcactgggtcaggcagatgccgggaaagggactggaatggatgggc

[0319] cggatcgacccagaaaatgacgaaaccaaatacgggccgatttttcaaggccacgtgactatcagcgcagacacgagcatcaacactgtctacctccagtggtcctc

[0320] gcttaaggccagcgataccgctatgtactactgcgcattcagaggcggggtgtactggggacaaggaaccactgtgaccgtgagcagcggaggtggcggctcggg

[0321] aggaggtgggagcggaggaggaggttccggcggtggaggatcagatgtcgtgatgacccagtccccggactccctcgctgtctcactgggcgagcgcgcgacca

[0322] tcaactgcaaatcgagccagtcgctgttggactccgatggaaagacttatctgaattggctgcaacagaaaccaggacaacctcccaagcggctcatctcgcttgtgtc

[0323] aaaactcgattcgggagtgccagaccgcttctcggggtccgggagcggaactgactttactttgaccatttcctcactgcaagcggaggatgtggccgtgtattactgtt

[0324] ggcagggcacgcatttccctggaaccttcggtggcggaactaaggtggaaatcaagggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccac

[0325] catcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatct

[0326] gggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga

[0327] gaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacg

[0328] cccccgcgtaccaacagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgaga

[0329] tggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcga

[0330] gcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccg

[0331] gaggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctgctagcatgctgctgcttgtgacaagtctcctcctttgtgaa

[0332] cttccgcaccctgcttttctcctgatcatgggtctcacctcccaactgcttccccctctgttcttcctgctagcatgtgccggcaactttgtccacggacacaagtgcgatat

[0333] caccttacaggagatcatcaaaactttgaacagcctcacagagcagaagactctgtgcaccgagttgaccgtaacagacatctttgctgcctccaagaacacaactga

[0334] gaaggaaaccttctgcagggctgcgactgtgctccggcagttctacagccaccatgagaaggacactcgctgcctgggtgcgactgcacagcagttccacaggcac

[0335] aagcagctgatccgattcctgaaacggctcgacaggaacctctggggcctggcgggcttgaatagctgtcctgtgaaggaagccaaccagagtacgttggaaaactt

[0336] cttggaaaggctaaagacgatcatgagagagaaatattcaaagtgttcgagcaccactacaccagctccacgcccccctacacccgctcctacaatcgcttcccagcc

[0337] tttgagcttgaggcccgaggcatgtagaccagcagcaggaggcgcggttcatactagaggtcttgacttcgcatgcgacatatatatttgggcccccctggcgggaac

[0338] gtgtggggtgctgcttctgagcctggtgattactctgtactgcttgcagtgcgagatctgtggtttcacctgcagacagaaggcgtccttgaattggcacatgaaaaagc

[0339] attttcagtgcaatcagtgtggagcttcttttacccagaaagggaacctgctgcgccacatcaaacttcactaagatccaccggatctaggcgtctggaacaatcaacct

[0340] ctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctt

[0341] tcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactg

[0342] gttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacagg

[0343] ggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttct

[0344] gctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctc

[0345] cctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcataattggtacctttaagaccaatg。

[0346] antiEGFRvIII-mIL-10-iCAR gene sequence (SEQ ID NO.17):

[0347] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaatccagctggtgcagtcaggcgccgaggtcaagaagccg

[0348] ggagagtcgctgagaatctcgtgcaagggctcggggttcaacatcgaggactactacattcactgggtcaggcagatgccgggaaagggactggaatggatgggc

[0349] cggatcgacccagaaaatgacgaaaccaaatacgggccgatttttcaaggccacgtgactatcagcgcagacacgagcatcaacactgtctacctccagtggtcctc

[0350] gcttaaggccagcgataccgctatgtactactgcgcattcagaggcggggtgtactggggacaaggaaccactgtgaccgtgagcagcggaggtggcggctcggg

[0351] aggaggtgggagcggaggaggaggttccggcggtggaggatcagatgtcgtgatgacccagtccccggactccctcgctgtctcactgggcgagcgcgcgacca

[0352] tcaactgcaaatcgagccagtcgctgttggactccgatggaaagacttatctgaattggctgcaacagaaaccaggacaacctcccaagcggctcatctcgcttgtgtc

[0353] aaaactcgattcgggagtgccagaccgcttctcggggtccgggagcggaactgactttactttgaccatttcctcactgcaagcggaggatgtggccgtgtattactgtt

[0354] ggcagggcacgcatttccctggaaccttcggtggcggaactaaggtggaaatcaagggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccac

[0355] catcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatct

[0356] gggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga

[0357] gaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacg

[0358] cccccgcgtaccaacagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgaga

[0359] tggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcga

[0360] gcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccg

[0361] gaggaagcggaactagttgagatccaccggatctaggcgtctggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttt

[0362] tacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtgg

[0363] cccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccc

[0364] cctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctga

[0365] cgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgc

[0366] tgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaa

[0367] aacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcattag

[0368] tgaagtttgatgtggcgcagcaggttccctttctgggtaaaagaagctccacactgattgcactgaaaatgctttttcatgtgccaattcaaggacgccttctgtctgcagg

[0369] tgaaaccacagatctcgcactgcaagcagtacagagtaatcaccaggctcagaagcagcaccccacacgttcccgccaggggggcccaaatatatatgtcgcatgc

[0370] gaagtcaagacctctagtatgaaccgcgcctcctgctgctggtctacatgcctcgggcctcaagctcaaaggctgggaagcgattgtaggagcgggtgtagggggg

[0371] cgtggagctggtgtagtggtgttgcggatcttcatagtcatataggcctcgatgtagttgatgaagatgtcaaactctgacattgccttgtaaatgcctttttcctgcagctta

[0372] ttgaaggcgttcttcacctgctccacggctttgctcttgttctcgcaaggaaggaagcggtggcaccgtctcaggcggagcctcagtgttttaaggttctcgccgaggct

[0373] gttcacgtgggccttgatgtctgggtcctggttctccgcctggggcatgacttcctccaggtaaaactggatcatctcgctgagagcttgacaaccgaggtagcccttga

[0374] aatcctccaggagtgactctttcaacaggagattgtccagctggtctttcatttggaaaaatgttttcacgcgagagaacgcatcgcgaagatctctaagcatattaggca

[0375] gatttcctggaaagtgggtgcaggaattttcggactgggttccctggccgatcaggagaaaagcagggtgcggaagttcacaaaggaggagacttgtcacaagcag

[0376] cagcatgcaggagttgaggttactgtgagtagtgattaaagagagtgatagggaactcttgaacaagagatgcaatttatactgttaattctggaaaaatattatgggggt

[0377] gtcaaaatgcaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcccaattgacgccttctgtatgaaacagtttttcctcc。

[0378] antiEGFRvIII - mIL - 10 - pCAR gene sequence (SEQ ID NO.18):

[0379] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaatccagctggtgcagtcaggcgccgaggtcaagaagccg

[0380] ggagagtcgctgagaatctcgtgcaagggctcggggttcaacatcgaggactactacattcactgggtcaggcagatgccgggaaagggactggaatggatgggc

[0381] cggatcgacccagaaaatgacgaaaccaaatacgggccgatttttcaaggccacgtgactatcagcgcagacacgagcatcaacactgtctacctccagtggtcctc

[0382] gcttaaggccagcgataccgctatgtactactgcgcattcagaggcggggtgtactggggacaaggaaccactgtgaccgtgagcagcggaggtggcggctcggg

[0383] aggaggtgggagcggaggaggaggttccggcggtggaggatcagatgtcgtgatgacccagtccccggactccctcgctgtctcactgggcgagcgcgcgacca

[0384] tcaactgcaaatcgagccagtcgctgttggactccgatggaaagacttatctgaattggctgcaacagaaaccaggacaacctcccaagcggctcatctcgcttgtgtc

[0385] aaaactcgattcgggagtgccagaccgcttctcggggtccgggagcggaactgactttactttgaccatttcctcactgcaagcggaggatgtggccgtgtattactgtt

[0386] ggcagggcacgcatttccctggaaccttcggtggcggaactaaggtggaaatcaagggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccac

[0387] catcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatct

[0388] gggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga

[0389] gaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacg

[0390] cccccgcgtaccaacagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgaga

[0391] tggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcga

[0392] gcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccg

[0393] gaggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctgctagcatggccttaccagtgaccgccttgctcctgccgc

[0394] tggccttgctgctccacgccgccaggccgagcccaggccagggcacccagtctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatct

[0395] ccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgcca

[0396] agccttgtctgagatgatccagttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctg

[0397] aagaccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaa

[0398] aggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaacaccactacaccagctccacgcccccctac

[0399] acccgctcctacaatcgcttcccagcctttgagcttgaggcccgaggcatgtagaccagcagcaggaggcgcggttcatactagaggtcttgacttcgcatgcgacat

[0400] atatatttgggcccccctggcgggaacgtgtggggtgctgcttctgagcctggtgattactctgtactgcttgcagtgcgagatctgtggtttcacctgcagacagaagg

[0401] cgtccttgaattggcacatgaaaaagcattttcagtgcaatcagtgtggagcttcttttacccagaaagggaacctgctgcgccacatcaaacttcactaagatccaccg

[0402] gatctaggcgtctggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgccttt

[0403] gtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgca

[0404] ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccg

[0405] cctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccac

[0406] ctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgcc

[0407] ttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcataattggtacctttaagaccaatg。

[0408] antiCD19-nCAR gene sequence (SEQ ID NO.19):

[0409] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctgg

[0410] gagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatca

[0411] agattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaa

[0412] cagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggt

[0413] gaaactgcaggatcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcatcatacccgactatggtgtaagctggattcg

[0414] ccagcctccacgaaagggtctggagtggctgggagtaatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactc

[0415] caagagccaagttttcttaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactgggg

[0416] ccaaggaacctcagtcaccgtctcctcaaccaccgacgccagcgccggaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagagg

[0417] cgtgccggccagccggcggggggcgcagtgcacacgaggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcct

[0418] gtcactggttatcaccctttactgcaaacggggcaagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagaagaatggctgta

[0419] gctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctat

[0420] aacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagg

[0421] aaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttac

[0422] cagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccggaggaagcggataagatccaccggatctaggcgtc

[0423] tggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctatt

[0424] gcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctg

[0425] acgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcc

[0426] cgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgc

[0427] gcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcag

[0428] acgagtcggatctccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcataattggtacctttaagaccaatg。

[0429] antiEGFRvIII - nCAR gene sequence (SEQ ID NO.20):

[0430] Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaatccagctggtgcagtcaggcgccgaggtcaagaagccg

[0431] ggagagtcgctgagaatctcgtgcaagggctcggggttcaacatcgaggactactacattcactgggtcaggcagatgccgggaaagggactggaatggatgggc

[0432] cggatcgacccagaaaatgacgaaaccaaatacgggccgatttttcaaggccacgtgactatcagcgcagacacgagcatcaacactgtctacctccagtggtcctc

[0433] gcttaaggccagcgataccgctatgtactactgcgcattcagaggcggggtgtactggggacaaggaaccactgtgaccgtgagcagcggaggtggcggctcggg

[0434] aggaggtgggagcggaggaggaggttccggcggtggaggatcagatgtcgtgatgacccagtccccggactccctcgctgtctcactgggcgagcgcgcgacca

[0435] tcaactgcaaatcgagccagtcgctgttggactccgatggaaagacttatctgaattggctgcaacagaaaccaggacaacctcccaagcggctcatctcgcttgtgtc

[0436] aaaactcgattcgggagtgccagaccgcttctcggggtccgggagcggaactgactttactttgaccatttcctcactgcaagcggaggatgtggccgtgtattactgtt

[0437] ggcagggcacgcatttccctggaaccttcggtggcggaactaaggtggaaatcaagggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccac

[0438] catcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatct

[0439] gggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatga

[0440] gaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacg

[0441] cccccgcgtaccaacagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgaga

[0442] tggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcga

[0443] gcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctccg

[0444] gaggaagcggataagatccaccggatctaggcgtctggaacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatg。

Claims

1. A nucleotide fragment encoding an activation-induced membrane-bound cytokine, characterized in that, The nucleotide fragment targets CD19 or EGFRvIII; the cytokine is IL-4 or IL-10. The nucleotide fragments include signal peptide nucleotide sequences, CAR molecule sequences targeting CD19 or EGFRvIII, activation-inducible promoter sequences containing 3-6 NFAT motifs, membrane-bound IL-4 or IL-10 nucleotide sequences, and Degron sequences. The signal peptide nucleotide sequence is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.34-39; The nucleotide sequence of the CD19-targeting CAR molecule is shown in SEQ ID NO.2; The nucleotide sequence of the CAR molecule targeting EGFRvIII is shown in SEQ ID NO.3; The nucleotide sequence of the activation-inducible promoter sequence containing 3-6 NFAT motifs is any one of the sequences shown in SEQ ID NO.4-7; The structure of the membrane-bound IL-4 includes a signal peptide, an IL-4 core sequence, and a transmembrane domain; The structure of the membrane-bound IL-10 includes a signal peptide, an IL-10 core sequence, and a transmembrane domain. The nucleotide sequence of the signal peptide is a nucleotide sequence capable of encoding the amino acid sequence shown in SEQ ID NO.35-40; The nucleotide sequence of the IL-4 core sequence is a nucleotide sequence that can encode the amino acid sequence shown in SEQ ID NO.25; The nucleotide sequence of the IL-10 core sequence is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.26-34; The nucleotide sequence of the Degron sequence is shown in SEQ ID NO.

6.

2. A vector comprising the nucleotide fragment of claim 1, characterized in that, The vectors include retroviral vectors, adeno-associated virus vectors, lentiviral vectors, adenovirus vectors, or liposomes.

3. A recombinant immune cell capable of stable expression of the nucleotide fragment of claim 1, wherein, Obtained by delivery to immune cells containing the vector of claim 2.

4. A membrane-bound cytokine, characterized in that, Produced by the recombinant immune cells of claim 3, including membrane-bound IL-4 or IL-10.

5. A method of modulating IL-4 or IL-10 secretion by immune cells, comprising, This includes introducing the nucleotide fragment of claim 1 into the immune cells, causing them to produce membrane-bound IL-4 or IL-10 only when highly activated.

6. The method of claim 5, wherein, The immune cells include autologous, allogeneic, or heterologous immune cells.

7. A method of increasing the viability of T cells in an activated state, characterized in that, This includes delivering the nucleotide fragment of claim 1 into the T cells, and immobilizing the resulting IL-4 or IL-10 onto the T cell membrane.

8. The method of claim 7, wherein, The T cells include CAR-T cells and tumor-infiltrating T cells.

9. The use of the DNA fragment of claim 1, the vector of claim 2, or the recombinant T cell of claim 3 in the preparation of a medicament for treating cancer.

10. Use according to claim 9, characterized in that, The cancers mentioned include acute lymphoblastic leukemia, lymphoma, and glioma.

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