An immune cell that secretes type 2 cytokines under hypoxic conditions and its application

Abstract

This invention discloses an immune cell that secretes type 2 cytokines under hypoxic conditions and its applications, belonging to the fields of genetic engineering and cell engineering. The invention first constructs a nucleotide fragment encoding hypoxia-induced type 2 cytokines, including a signal peptide gene sequence, a CAR molecule nucleotide sequence targeting CD19 and CD22, and an IL-4 or IL-10 nucleotide sequence containing multiple HRE motifs of a hypoxia-inducible promoter. This nucleotide fragment is delivered to immune cells for stable expression, yielding an immune cell that secretes type 2 cytokines under hypoxic conditions. This immune cell secretes IL-4 or IL-10 only under hypoxic conditions, thereby leveraging the metabolic reprogramming effect of IL-4 or IL-10 on immune cells to enhance their activity under hypoxic conditions, thus achieving better long-term anti-tumor effects.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and cell engineering, and in particular to an immune cell that secretes type 2 cytokines under hypoxic conditions and its applications. Background Technology

[0002] CAR-T cell therapy is a revolutionary immunotherapy method that uses genetic engineering to modify normal T cells into "living drugs" capable of specifically recognizing and killing cancer cells. In the field of cancer treatment, particularly for relapsed or refractory acute B-cell lymphoma in children and adults, CAR-T cell therapy has demonstrated significant efficacy, with a complete remission rate as high as approximately 90%. However, CAR-T cell therapy still faces challenges such as tumor recurrence and insufficient long-term anti-tumor activity. Taking CD19-targeted CAR-T cell therapy for B-cell acute lymphoblastic leukemia (B-ALL) as an example, there are two main patterns of relapse after treatment: one is CD19-positive leukemia relapse that can still be detected by flow cytometry; the other is CD19-negative leukemia relapse that cannot be detected by flow cytometry. These relapses may be related to CAR-T cell depletion in some patients, and immune escape caused by decreased or lost expression of the tumor antigen CD19. Clinical studies have also found that leukemia often relapses when the number of CAR-T cells in a patient's peripheral blood decreases or disappears, and the effect of injecting additional CD19-targeted CAR-T cells is not ideal.

[0003] CAR-T cells targeting two B-cell specific antigens (such as CD19 and CD22) can effectively reduce tumor relapse caused by the loss of a single antigen. However, for CD19-positive leukemia relapse caused by CAR-T cell depletion, it is still necessary to enhance the long-term anti-tumor activity of CAR-T cells. In the tumor microenvironment, CAR-T cells exhibit a loss of effector function and proliferative capacity, which is called T cell "exhaustion," possibly due to the hypoxic tumor microenvironment, continuous antigen stimulation, and other metabolic stresses (PMID: 27521269; 30923193). During CAR-T cell therapy for lymphoma, the hypoxic microenvironment within the lymphoma can severely affect the long-term anti-tumor activity of CAR-T cells (PMID: 34440794, 37300573). Therefore, improving the tolerance of CAR-T cells to the hypoxic microenvironment and enhancing their long-term anti-tumor activity are key to improving the efficacy of CAR-T cell therapy.

[0004] Type II cytokines are a class of cytokines with complex biological functions. While playing a negative regulatory role in inflammatory responses, they also play an active role in the tumor microenvironment. Studies have shown that IL-4 and IL-10 can promote the number and function of tumor-infiltrating lymphocytes (TILs) (PMID: 34031618, 39322664, 39322665), 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). Given this dual role of IL-4 and IL-10 in immune regulation, precise regulation of their secretion is needed to fully realize their promoting effect on T cell immune function. However, ensuring that IL-4 or IL-10 is only secreted when CAR-T cells are in a hypoxic state, so as to exert the metabolic reprogramming effect of IL-4 or IL-10 on CAR-T cells and promote their vitality under hypoxia, while avoiding the immunosuppressive effect of IL-4 or IL-10 on CAR-T cells in the resting state, remains an urgent problem to be solved in the current field of CAR-T cell therapy research. Summary of the Invention

[0005] The purpose of this invention is to provide an immune cell that secretes type 2 cytokines under hypoxic conditions and its application, thereby addressing the problems existing in the prior art. By delivering the nucleotide fragments of IL-4 or IL-10 with adjustable expression levels provided by this invention to T cells through an appropriate method, the T cells can secrete IL-4 or IL-10 only under hypoxic conditions, enhancing the activity of T cells under hypoxic conditions and thus achieving better long-term anti-tumor effects.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a nucleotide fragment encoding a hypoxia-induced type 2 cytokine, wherein the nucleotide fragment can be translated into a CAR molecule that simultaneously targets CD19 and CD22; the type 2 cytokine is IL-4 or IL-10.

[0008] The nucleotide fragments include a signal peptide nucleotide sequence, a CAR molecule sequence targeting CD19 and CD22, a hypoxia-inducible promoter sequence containing 3-6 HRE motifs, and a nucleotide sequence of IL-4 or IL-10.

[0009] The signal peptide nucleotide sequence is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.26-31;

[0010] The hypoxia-inducible promoter sequence containing 3-6 HRE motifs is any one of the sequences shown in SEQ ID NO. 3-6;

[0011] The nucleotide sequence of the IL-4 is a nucleotide sequence capable of encoding the amino acid sequence shown in SEQ ID NO.16;

[0012] The nucleotide sequence of the IL-10 is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.17-25;

[0013] The CAR molecular sequences targeting CD19 and CD22 include the VH region of the scFv targeting CD19, the VL region of the scFv targeting CD22, the VH region of the scFv targeting CD22, the VL region of the scFv targeting CD19, the linker region, the transmembrane region, the co-stimulatory domain, and the intracellular signal transduction domain.

[0014] In the dual-target CAR molecule sequence targeting CD19 and CD22, the VH region of the scFv targeting CD19, the VL region of the scFv targeting CD22, the VH region of the scFv targeting CD22, the VL region of the scFv targeting CD19, and the linker region can be arranged and combined in different orders.

[0015] The amino acid sequence of the Linker region may be one or more combinations of the sequences shown in SEQ ID NO.32-35, but is not limited thereto;

[0016] The transmembrane region 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 the GITR transmembrane region.

[0017] The costimulatory domains include any one or a combination of at least two of 4-1BB, CD28, CD137, OX-40, or ICOS;

[0018] The intracellular signal transduction structures include any one or a combination of at least two of CD3ζ, BCR, NKp30, NKp44, NKp46, FcαR, FcRγ, CD16, or CD32.

[0019] Optionally, the nucleotide sequence of the CAR molecule targeting CD19 and CD22 is shown in SEQ ID NO.2.

[0020] 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.

[0021] The present invention also provides a recombinant immune cell capable of stably expressing the nucleotide fragment, obtained by delivering the carrier contained therein or in vitro to the immune cell.

[0022] The present invention also provides a hypoxia-inducible cytokine, produced by the recombinant immune cells, including IL-4 or IL-10.

[0023] 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 secrete IL-4 or IL-10 only under hypoxic conditions.

[0024] The present invention also provides a method for improving the activity of immune cells under hypoxic conditions, comprising delivering the nucleotide fragment to the immune cells so that they secrete IL-4 or IL-10 only under hypoxic conditions.

[0025] The present invention also provides the use of the nucleotide fragments, the vectors, or the recombinant immune cells in the preparation of medicaments for treating cancer.

[0026] In the above technical solution, optionally, the immune cells include autologous, allogeneic, or heterologous immune cells.

[0027] Further optionally, the immune cells include T cells, CAR-T cells, NK cells, CAR-NK cells, Treg cells, CAR-Treg cells, or iPS-derived CAR-NK cells.

[0028] Optionally, the cancer includes acute lymphoblastic leukemia and lymphoma.

[0029] The present invention discloses the following technical effects:

[0030] The present invention provides a nucleotide fragment for IL-4 or IL-10 with adjustable expression levels, which, when delivered to immune cells by an appropriate method, enables the immune cells to secrete IL-4 or IL-10 only under hypoxic conditions.

[0031] This invention constructs a hypoxia-induced CD19 / CD22 dual-target CAR-T cell that secretes IL-4 or IL-10. IL-4 or IL-10 is only secreted when the CAR-T cell is under hypoxia, thereby exerting the metabolic reprogramming effect of IL-4 or IL-10 on CAR-T cells, promoting the activity of CAR-T cells under hypoxia, and avoiding the immunosuppressive effect of IL-4 or IL-10 on CAR-T cells in the resting state.

[0032] Compared to existing CD19 / CD22 dual-target CAR-T cells, the dual-target CAR-T cells provided by this invention have better long-term anti-tumor effects. Compared to dual-target CAR-T cells expressing continuously secreted IL-4 or IL-10, the hypoxia-induced IL-4 or IL-10 dual-target CAR-T cells provided by this invention have better safety. Attached Figure Description

[0033] 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.

[0034] Figure 1 A schematic diagram for constructing CAR plasmids that target CD19 and CD22 and express hypoxia-inducible IL-4 or IL-10;

[0035] Figure 2 The results of flow cytometry detection of CAR molecules expressed on different T cells are shown below. A represents the results of detection on untransduced T cells; B represents the results of detection on conventional CAR-T cells targeting CD19 and CD22; C and D represent the results of detection on CAR-T cells expressing hypoxia-inducible IL-4 or IL-10, respectively; E and F represent the results of detection on CAR-T cells continuously expressing IL-4 or IL-10, respectively.

[0036] Figure 3 The results of flow cytometry detection of IL-4(A) and IL-10(B) expression levels under different conditions;

[0037] Figure 4 The cell counts of different T cells cultured in vitro at different time points;

[0038] Figure 5 The results of in vitro killing assays of different T cells;

[0039] Figure 6The results of tumor volume detection in mice after the first (A) and second (B) tumor inoculations with different T cells. Detailed Implementation

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The term "nucleotide" refers to polymers of deoxynucleotides (such as DNA, cDNA) or nucleotides (such as RNA, mRNA), or combinations of deoxynucleotides and nucleotides (such as 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 (such as thio groups). Generally, analogs of a particular nucleotide have the same base pair specificity, such as the A and T base pairs.

[0046] 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.

[0047] 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).

[0048] 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.

[0049] 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.

[0050] 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 T cells, CAR-T cells, NK cells, CAR-NK cells, Treg cells, or CAR-Treg cells or iPS-derived CAR-NK 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.

[0051] 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.

[0052] 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 patient or research subject; (2) genetically engineering the immune cells with a recombinant structure encoding hypoxia-inducible IL-4 or IL-10 to construct CAR-T cells expressing hypoxia-inducible IL-4 or IL-10; (3) an in vivo delivery system containing a CAR vector of hypoxia-inducible IL-4 or IL-10; and (4) reinfusing the in vivo delivery system containing a CAR vector of hypoxia-inducible IL-4 or IL-10 into the patient or research subject. Upon in vivo delivery of the CAR vector containing hypoxia-inducible IL-4 or IL-10 into the patient or research subject, T cells will be converted into CAR-T cells expressing hypoxia-inducible IL-4 or IL-10 and mediate a cellular immune response against the tumor.

[0053] The recombinant structure encoding hypoxia-inducible IL-4 or IL-10 includes a signal peptide nucleotide sequence, a CAR molecule nucleotide sequence targeting CD19 and CD22, a hypoxia-inducible promoter sequence containing 3-6 HRE motifs, and a nucleotide sequence of IL-4 or IL-10.

[0054] The signal peptide nucleotide sequence is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.26-31;

[0055] The CAR molecular sequences targeting CD19 and CD22 include the VH region of the scFv targeting CD19, the VL region of the scFv targeting CD22, the VH region of the scFv targeting CD22, the VL region of the scFv targeting CD19, the linker region, the transmembrane region, the co-stimulatory domain, and the intracellular signal transduction domain.

[0056] The hypoxia-inducible promoter sequence containing 3-6 HRE motifs is any one of the sequences shown in SEQ ID NO. 3-6;

[0057] In the dual-target CAR molecule sequence targeting CD19 and CD22, the VH region of the scFv targeting CD19, the VL region of the scFv targeting CD22, the VH region of the scFv targeting CD22, the VL region of the scFv targeting CD19, and the Linker region can be arranged and combined in different orders. The Linker region can be one or more combinations of the sequences shown in SEQ ID NO. 32-35, but is not limited to them.

[0058] SEQ ID NO.32:

[0059] GGGGSGGGGSGGGGS.

[0060] SEQ ID NO.33:

[0061] HHHHHH.

[0062] SEQ ID NO.34

[0063] EQKLISEEDL.

[0064] SEQ ID NO.35

[0065] EDLEEDLEEDLEEDL.

[0066] The amino acid sequences of the hypoxia-inducible IL-4 or IL-10 are as follows:

[0067] IL-4 amino acid sequence (SEQ ID NO.16):

[0068] HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS.

[0069] IL-10 amino acid sequence (SEQ ID NO.17):

[0070] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0071] Furthermore, the amino acid sequence of the IL-10 may also be selected from any of the following variant sequences:

[0072] SEQ ID NO.18:

[0073] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0074] SEQ ID NO.19:

[0075] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRWRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0076] SEQ ID NO.20:

[0077] SPGQGTQSENSCTHFPGWLPMMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0078] SEQ ID NO.21:

[0079] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVMSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0080] SEQ ID NO.22:

[0081] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLMTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0082] SEQ ID NO.23:

[0083] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0084] SEQ ID NO.24:

[0085] SPGQGTQSENSCTFFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0086] SEQ ID NO.25:

[0087] SPGQGTQSENSCWHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVLSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0088] Preferably, the N-terminus of the IL-4 or IL-10 further includes a signal peptide;

[0089] More preferably, the amino acid sequence of the signal peptide is selected from any of the following sequences.

[0090] SEQ ID NO. 26: MHSSALLCCLVLLTGVRA.

[0091] SEQ ID NO. 27: MALPVTALLLPLALLLHAARP.

[0092] SEQ ID NO. 28: MYRMQLLSCIALSLALVTNS.

[0093] SEQ ID NO. 29: MYRMQLLSCIALSLALVTNISA.

[0094] SEQ ID NO. 30: MGAARSPSAVPGPLLGLLLLLLGVLAPGGAS.

[0095] SEQ ID NO. 31: MLCCMRRTKQVEKNDEDQKI.

[0096] 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.

[0097] Example 1: Construction of CAR plasmids expressing hypoxia-inducible IL-4 or IL-10

[0098] The following sequences were obtained from the NCBI database: signal peptide sequence, hypoxia-inducible promoter sequence containing 3-6 HRE-binding motifs and an interleukin-2 core promoter sequence, IL-4 nucleotide sequence, IL-10 nucleotide sequence, and dual-target CAR molecule sequence targeting CD19 and CD22. The dual-target CAR molecule targeting CD19 and CD22 contains the following gene elements: VH region of scFv targeting CD19, VL region of scFv targeting CD22, VH region of scFv targeting CD22, VL region of scFv targeting CD19, linker region, transmembrane region, co-stimulatory domain, and intracellular signal transduction domain. The DNA sequence information is as follows:

[0099] Signal peptide (SEQ ID NO.1):

[0100] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg.

[0101] In the dual-target CAR molecule sequence targeting CD19 and CD22, the VH region of the scFv targeting CD19, the VL region of the scFv targeting CD22, the VH region of the scFv targeting CD22, the VL region of the scFv targeting CD19, and the linker region can be arranged and combined in different orders. This embodiment only presents one combination of fixed orders, and the specific sequence is as follows (SEQ ID NO.2):

[0102]

[0103] Hypoxia-inducible promoter sequence containing 3 HRE motifs (SEQ ID NO.3):

[0104] ggtaccactagtccacagtgcatacgtgggctccaacaggtcctcttggtcgaccccacagtgcatacgtgggctccaacaggtcctcttcggatccgccacagtgcatacgtgggctccaacaggtcct cttccggaattccggcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgc.

[0105] Hypoxia-inducible promoter sequence containing 4 HRE motifs (SEQ ID NO.4):

[0106] ggtaccactagtccacagtgcatacgtgggctccaacaggtcctcttgcggccgcccacagtgcatacgtgggctccaacaggtcctcttccatggccacagtgcatacgtgggctccaacaggtcctcttcggatccgccacagtgcat acgtgggctccaacaggtcctcttccggaattccggcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgc.

[0107] Hypoxia-inducible promoter sequence containing 5 HRE motifs (SEQ ID NO.5):

[0108] ggtaccactagtccacagtgcatacgtgggctccaacaggtcctcttggtcgaccccacagtgcatacgtgggctccaacaggtcctcttgcggccgcccacagtgcatacgtgggctccaacaggtcctcttccatggccacagtgcatacgtgggctccaacaggtcctcttcggatccgccacagtgcatacgtgggctccaacaggtcctcttccggaattccggcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgc。

[0109] Hypoxia-inducible promoter sequence containing 6 HRE motifs (SEQ ID NO.6):

[0110] ggtaccactagtccacagtgcatacgtgggctccaacaggtcctcttggtcgaccccacagtgcatacgtgggctccaacaggtcctcttggtcgaccccacagtgcatacgtgggctccaacaggtcctcttgcggccgcccacagtgcatacgtgggctccaacaggtcctcttccatggccacagtgcatacgtgggctccaacaggtcctcttcggatccgccacagtgcatacgtgggctccaacaggtcctcttccggaattccggcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgc。

[0111] IL-4 nucleotide sequence (SEQ ID NO.7):

[0112] cacaagtgcgatatcaccttacaggagatcatcaaaactttgaacagcctcacagagcagaagactctgtgcaccgagttgaccgtaacagacatctttgctgcctccaagaacacaactgagaaggaaaccttctgcagggctgcgactgtgctccggcagttctacagccaccatgagaaggacactcgctgcctgggtgcgactgcacagcagttccacaggcacaagcagctgatccgattcctgaaacggctcgacaggaacctctggggcctggcgggcttgaatagctgtcctgtgaaggaagccaaccagagtacgttggaaaacttcttggaaaggctaaagacgatcatgagagagaaatattcaaagtgttcgagc。

[0113] IL-10 nucleotide sequence (SEQ ID NO.8):

[0114] ggccagggcacccagtctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaacta。

[0115] The IL-4 or IL-10 DNA sequence containing 3-6 HRE motifs of an oxygen-inducible promoter is constructed by splicing an IL-4 or IL-10 DNA sequence containing 3-6 HRE motifs of an oxygen-inducible promoter (SEQ ID NO. 3-6) and an IL-4 or IL-10 DNA sequence (SEQ ID NO. 7 or SEQ ID NO. 8).

[0116] The DNA fragments from the above regions were integrated into pLVH-EF1A-BRD3R (brand: Addgene, catalog number: #130696) as needed to construct the target plasmid, 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: IL-4-iCAR (SEQ ID NO.9), a DNA fragment targeting CD19 and CD22 and expressing hypoxia-inducible IL-4; IL-10-iCAR (SEQ ID NO.10), a DNA fragment targeting CD19 and CD22 and expressing hypoxia-inducible IL-10; IL-4-pCAR (SEQ ID NO.11), a DNA fragment targeting CD19 and CD22 and continuously expressing IL-4; IL-10-pCAR (SEQ ID NO.12), a DNA fragment targeting CD19 and CD22 and continuously expressing IL-10; and nCAR (SEQ ID NO.13), a DNA fragment targeting CD19 and CD22.

[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] Forward primer: gacccggtcgaattcatggccttaccagtgacc (SEQ ID NO.14);

[0121] Reverse primer: cattggtcttaaaggtaccaattatgaggtgtg (SEQ ID NO.15).

[0122] PCR reaction system: 100 ng of the full-length target gene fragment synthesized by a DNA synthesis instrument, KOD One TMPCR MasterMix 25 μL, 10 μM forward and reverse primers 2 μL each, and ultrapure water to bring the total to 50 μL.

[0123] PCR reaction conditions: 98℃ for 60s, 1 cycle; 98℃ for 10s, 60℃ for 5s, 68℃ for 10s, 35 cycles; 16℃ for 60s, 1 cycle.

[0124] PCR amplification was first performed using IL-4-iCAR as a template and amplification primers SEQ ID NO.14 and SEQ ID NO.15 under the conditions described above to obtain the IL-4-iCAR gene fragment. After verification, the same method was used to obtain the gene fragments of IL-4-pCAR, IL-10-iCAR, IL-10-pCAR and nCAR, respectively.

[0125] (2) Enzyme digestion

[0126] 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.

[0127] 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).

[0128] (3) Homologous recombination

[0129] 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 IL-4-iCAR DNA 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.

[0130] 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.

[0131] 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 IL-4-iCAR gene. Similarly, plasmids expressing IL-4-pCAR, IL-10-iCAR, IL-10-pCAR, and nCAR can be constructed.

[0132] Example 2: Preparation of CAR-T cells expressing hypoxia-induced IL-4 or IL-10

[0133] 1. Preparation of lentiviruses

[0134] Lentivirals expressing the IL-4-iCAR, IL-4-pCAR, IL-10-iCAR, IL-10-pCAR and nCAR genes were prepared respectively.

[0135] Prepare a 10cm culture dish and inoculate 5×10⁶ cells / year. 6 293T cells were added to complete culture medium (DMEM medium containing 4500 mg / L glucose, 10% FBS, 1% ampicillin and 1% streptomycin) and placed in an incubator at 37°C and 5% CO2 for overnight culture. 100 μM of PEI and plasmid mixture was taken out of the freezer, thawed at room temperature, and then thoroughly mixed by pipetting.

[0136] The above plasmid mixture consists of three different plasmids: one is the plasmid containing the CAR gene prepared in Example 1, and the other two are pMD2.G (Addgene, catalog number: #12259) and psPAX2 (Addgene, catalog number: #12260). Take 9 μg of the CAR plasmid from Example 1, 3 μg of pMD2.G, and 12 μg of psPAX2, mix them evenly, and this mixture is called the plasmid mixture.

[0137] Remove the PBS solution and warm it to room temperature. Add 2 mL of PBS solution to one well of a 6-well plate, add three plasmids, and mix thoroughly by pipetting. Add 18 μL of 100 μM PEI and immediately mix by pipetting. Let stand at room temperature for 10 min. Add the DNA / 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. Remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. After 60 hours of continuous incubation, collect the virus-containing supernatant from the culture dish, filter through a 0.45 μm filter membrane, transfer to a centrifuge tube, balance, and centrifuge at 20000 g, 4°C for 2 hours. After centrifugation, carefully aspirate the liquid from the centrifuge tube in a biosafety cabinet, resuspend the precipitate in 500 μL of PBS buffer, aliquot the virus, and store at -80°C.

[0138] 2. Activation of primary T cells and lentiviral infection

[0139] CAR-T cells targeting CD19 and CD22 (nCAR-T), CAR-T cells expressing hypoxia-inducible IL-4 (IL-4-iCAR-T), CAR-T cells expressing hypoxia-inducible IL-10 (IL-10-iCAR-T), dual-target CAR-T cells continuously expressing IL-4 (IL-4-pCAR-T), and dual-target CAR-T cells continuously expressing IL-10 (IL-10-pCAR-T) were prepared respectively.

[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 15 mL centrifuge tube. Take an appropriate amount of separation buffer (PBS buffer containing 1% fetal bovine serum) and mix well for 1 min. Place the centrifuge tube on a magnetic block 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; Melt the lentivirus prepared in step 1, add viral 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 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 by T cells. Anti-G4S antibodies 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), dual-target CAR-T cells targeting CD19 and CD22 (nCAR-T), CAR-T cells expressing hypoxia-inducible IL-4 (IL-4-iCAR-T), CAR-T cells expressing hypoxia-inducible IL-10 (IL-10-iCAR-T), dual-target CAR-T cells continuously expressing IL-4 (IL-4-pCAR-T), and dual-target CAR-T cells continuously expressing IL-10 (IL-10-pCAR-T) were counted, and cells containing 5 × 10⁻⁶ cells were collected. 5 The cell suspension was transferred to 1.5 mL EP tubes. After centrifugation at 500 g for 3 min, the supernatant was discarded, and the cells were resuspended in 1 mL PBS. The cells were then centrifuged at 500 g for 3 min at 4 °C, and the supernatant was discarded. The samples were resuspended in 50 μL PBS, and 0.5 μL of anti-G4S-tagged antibody (ACRO Biosystems, clone number 016) was added. The mixture was incubated at 4 °C for 30 min. 1 mL 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 PBS, and flow cytometry analysis was performed. Results are as follows: Figure 2 As shown, this indicates that CAR-T cells expressing hypoxia-induced IL-4 or IL-10 were successfully prepared.

[0143] Example 3: Detection of the IL-4 or IL-10 secretion capacity of dual-target CAR-T cells expressing hypoxia-inducible IL-4 or IL-10 under hypoxic conditions.

[0144] Prepared with a density of 1×10 6400 μL of T cell suspension (untransduced T cells MOCK-T, nCAR-T, IL-4-iCAR-T, IL-10-iCAR-T) per well was added to each well of a 48-well plate, with three replicates per group. Two aliquots of the 48-well plates were prepared: one aliquot was incubated at 37°C in a normoxic incubator, and the other aliquot was incubated at 37°C in a hypoxic incubator. After 48 hours, the culture supernatant was collected, and cytokine levels were detected using an ELISA kit for detecting IL-4 (Daiyou, catalog number: 1110402) or IL-10 (Daiyou, catalog number: 1111002).

[0145] The results are as follows Figure 3 As shown, IL-4-iCAR-T and IL-10-iCAR-T cells can secrete large amounts of IL-4 or IL-10 after hypoxia culture, while IL-4-iCAR-T and IL-10-iCAR-T cells do not secrete IL-4 or IL-10 under normoxic conditions. This indicates that the constructed hypoxia-inducible IL-4 or IL-10 dual-target CAR-T cells have the function of responding to hypoxia signals and producing cytokines.

[0146] Example 4: Detection of the in vitro expansion of dual-target CAR-T cells expressing hypoxia-inducible IL-4 or IL-10.

[0147] Frozen untransduced T cells (MOCK-T), nCAR-T, IL-4-iCAR-T, IL-10-iCAR-T, IL-4-pCAR-T, and IL-10-pCAR-T cells were thawed and cultured in vitro, with an initial cell count of 1×10⁻⁶. 6 Cells were cultured continuously for 12 days. Cell counts were performed every 2 days.

[0148] The results are as follows Figure 4 As shown, the in vitro expansion trend of IL-4-iCAR-T or IL-10-iCAR-T cells is comparable to that of MOCK-T and nCAR-T cells, and higher than that of IL-4-pCAR-T or IL-10-pCAR-T cells. This result demonstrates that hypoxia-induced IL-4 or IL-10 is more conducive to the proliferation of dual-target CAR-T cells compared to continuously expressed IL-4 or IL-10.

[0149] Example 5: Long-term in vitro killing experiment of dual-target CAR-T cells expressing hypoxia-inducible IL-4 or IL-10

[0150] Prepare a suspension of target cells (Raji cells expressing GFP), density: 2 × 10⁻⁶ 5Add 1 mL of target cell suspension to each well of a 6-well plate. Add MOCK-T, nCAR-T, IL-4-iCAR-T, and IL-10-iCAR-T suspensions at an effector-to-target ratio of 4:1, bringing the culture medium volume of the 6-well plate to 3 mL. Set up 3 replicates per group. Incubate the cells in an oxygen-deficient incubator. Every 3 days, resuspend the cells in the 6-well plate, take 100 μL for GFP-positive cell counting, and then centrifuge the remaining cells (500 g, 5 min). Discard the supernatant and add 2 mL of fresh culture medium to resuspend the cells. Add the resuspended cells to a new 6-well plate, along with 1 mL of fresh target cells (concentration 1×10⁻⁶). 5 (number / mL). Repeat the above steps 3 times.

[0151] The results are as follows Figure 5 As shown, dual-target CAR-T cells expressing hypoxia-inducible 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 dual-target CAR-T cells that do not express hypoxia-inducible IL-4 or IL-10.

[0152] Example 6: In vivo antitumor experiment of dual-target CAR-T cells expressing hypoxia-inducible IL-4 or IL-10

[0153] Prepare Raji target cell suspension, density: 5 × 10⁻⁶ 6 100 μL of Raji cell suspension was subcutaneously injected into the right groin of NSG immunodeficient mice. Tumor area was measured every 3 days, with 5 mice per group. Six days later, 200 μL of untransduced T cell (MOCK-T), CD19 and CD22 dual-target CAR-T cell (nCAR-T), hypoxia-inducible IL-4-expressing CAR-T cell (IL-4-iCAR-T), and hypoxia-inducible IL-10-expressing CAR-T cell (IL-10-iCAR-T) suspensions were injected into the tumor-bearing mice via tail vein. The CAR-T cell density was 5 × 10⁶ cells / mL. 6 On day 33 after T cell infusion, tumor cells were re-seeded using 100 μL of L Aji cell suspension (cell density: 5 × 10⁻⁶ cells / mL). 6 Tumors (number per mL) were injected subcutaneously into the left groin of immunodeficient mice, and the tumor area was measured every 3 days thereafter.

[0154] The results are as follows Figure 6As shown, after secondary tumor inoculation, the tumor volume of tumor-bearing mice that received dual-target CAR-T cells expressing hypoxia-inducible IL-4 or IL-10 was smaller than that of tumor-bearing mice that received conventional dual-target CAR-T cells. These results indicate that dual-target CAR-T cells expressing hypoxia-inducible IL-4 or IL-10 have better anti-tumor persistence in mice.

[0155] 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.

[0156] IL-4-iCAR DNA sequence (SEQ ID NO.9):

[0157]

[0158] IL-10-iCAR DNA sequence (SEQ ID NO.10):

[0159]

[0160] IL-4-pCAR DNA sequence (SEQ ID NO.11):

[0161]

[0162] IL-10-pCAR DNA sequence (SEQ ID NO.12):

[0163]

[0164] nCAR DNA sequence (SEQ ID NO.13):

[0165]

[0166] ccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctg

[0167] gctagaagcacaagaggaggaggaggtgggttttccagtcacacctcataattggtacctttaagaccaatg。

Claims

1. A nucleotide fragment encoding a hypoxia-induced type 2 cytokine, characterized in that, The nucleotide fragment can be translated into a CAR molecule that simultaneously targets CD19 and CD22; the type 2 cytokine is IL-4 or IL-10; The nucleotide fragments include a signal peptide nucleotide sequence, a CAR molecule nucleotide sequence targeting CD19 and CD22, a hypoxia-inducible promoter sequence containing 3-6 HRE motifs, and a nucleotide sequence of IL-4 or IL-10. The signal peptide nucleotide sequence is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.26-31; The CAR molecular sequences targeting CD19 and CD22 include the VH region of the scFv targeting CD19, the VL region of the scFv targeting CD22, the VH region of the scFv targeting CD22, the VL region of the scFv targeting CD19, the linker region, the transmembrane region, the co-stimulatory domain, and the intracellular signal transduction domain. The hypoxia-inducible promoter sequence containing 3-6 HRE motifs is any one of the sequences shown in SEQ ID NO. 3-6; The nucleotide sequence of the IL-4 is a nucleotide sequence capable of encoding the amino acid sequence shown in SEQ ID NO.16; The nucleotide sequence of the IL-10 is a nucleotide sequence capable of encoding any of the amino acid sequences shown in SEQ ID NO.17-25.

2. The nucleotide fragment according to claim 1, characterized in that, The CAR molecule sequence targeting CD19 and CD22 is shown in SEQ ID NO.

2.

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

4. A recombinant immune cell capable of stably expressing the nucleotide fragment of claim 1 or 2, characterized in that, Obtained by delivery of the carrier of claim 3 to immune cells in vivo or in vitro.

5. A hypoxia-inducible cytokine, characterized in that, Produced by the recombinant immune cells of claim 4, including IL-4 or IL-10.

6. A method for regulating the secretion of IL-4 or IL-10 by immune cells, characterized in that, This includes delivering the nucleotide fragment of claim 1 or 2 into the immune cells, causing them to secrete IL-4 or IL-10 only under hypoxic conditions.

7. The method according to claim 6, characterized in that, The immune cells include autologous, allogeneic, or heterologous immune cells.

8. A method for enhancing the viability of immune cells under hypoxic conditions, characterized in that, This includes delivering the nucleotide fragment of claim 1 or 2 into the immune cells, causing them to secrete IL-4 or IL-10 only under hypoxic conditions.

9. The use of the nucleotide fragment of claim 1 or 2, the vector of claim 3, or the recombinant immune cell of claim 4 in the preparation of a medicament for treating cancer.

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

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