Double-target chimeric antigen receptor and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a double-target chimeric antigen receptor and application thereof, an extracellular domain of the double-target chimeric antigen receptor sequentially comprises any one of the following components from an N end to a C end: CIgG scFv-MSLN scFv, MSLN scFv-CIgG scFv, CIgG scFv-CLDN18.2 scFv and CLDN18.2 scFv-CIgG scFv; wherein '-' is a connecting peptide. The four novel double-target chimeric antigen receptors can target multiple tumor antigens at the same time by combining different antigen recognition modules, so that more tumor cells are covered, the risk of antigen escape is reduced, and the killing efficiency of immune cells on the tumor cells is enhanced; the optimization of the double-target CAR structure also significantly increases the secretion of key effector cytokines, contributes to enhancing the immunocompetence in a tumor microenvironment, and further enhances the anti-tumor effect.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a dual-target chimeric antigen receptor and its application. Background Art

[0002] CAR-T cell therapy is a T-cell immunotherapy method modified by genetic engineering technology, which can specifically recognize and kill tumor cells. CAR-T therapy initially achieved significant results in hematological malignancies such as acute lymphoblastic leukemia (ALL) and large B-cell lymphoma. However, solid tumors often face great challenges in CAR-T cell therapy due to their complex tumor microenvironment (TME) and heterogeneity of tumor cells.

[0003] The tumor microenvironment in solid tumors usually has immunosuppressive characteristics, such as the functional inhibition of immune cells, the accumulation of immunosuppressive factors (such as TGF-β, IL-10, etc.), abnormal tumor blood vessels, and antigenic heterogeneity of tumor cells. These factors greatly limit the penetration, proliferation, and persistence of CAR-T cells in tumors, and changes in tumor antigens can easily lead to unstable anti-tumor effects of single-target CAR-T cells.

[0004] In order to overcome these difficulties in the treatment of solid tumors, researchers have begun to explore dual-target or multi-target CAR-T cell therapy strategies. This strategy aims to simultaneously target two or more tumor-associated antigens, enhance the recognition ability of CAR-T cells, reduce the risk of antigen escape, and improve the therapeutic effect. Dual-target CAR-T cells usually adopt a multi-specific design, such as fusing two different CAR structures into the same T cell, or enhancing the T cell's ability to attack tumor cells through multiple signaling mechanisms.

[0005] At present, some studies have shown that dual-target CAR-T cells can effectively overcome the problem of single-target antigen escape when treating solid tumors. For example, by simultaneously targeting different tumor-associated antigens such as HER2 and EGFR, researchers have found that dual-target CAR-T cells can kill tumor cells more effectively and delay the occurrence of drug resistance. In addition, the design of dual-target CAR-T can also avoid the immune escape of tumor cells, which helps to improve the sustainability and efficacy of treatment.

[0006] Although dual-target CAR-T cells can theoretically enhance the efficiency of tumor cell clearance, they still face a series of problems in clinical application. The design and manufacturing process of dual-target CAR-T cells is complex and may require optimization of the ratio and function of multiple CARs to ensure the balanced effect of the two targets. Summary of the invention

[0007] This application covers the following technical solutions:

[0008] A dual-target chimeric antigen receptor, wherein the extracellular domain includes any of the following from N-terminus to C-terminus: CIgG scFv-MSLN scFv, MSLN scFv- CIgG scFv, CIgG scFv-CLDN18.2 scFv, CLDN18.2 scFv -CIgGscFv; wherein "-" is a connecting peptide; wherein:

[0009] The amino acid sequence of the heavy chain complementary determining region of the CIgG scFv is shown in SEQ ID NOs: 1 to 3, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 4 to 6;

[0010] The amino acid sequence of the heavy chain complementary determining region of the MSLN scFv is shown in SEQ ID NOs: 7 to 9, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 10 to 12;

[0011] The amino acid sequence of the heavy chain complementary determining region of the CLDN18.2 scFv is shown in SEQ ID NOs: 13-15, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 16-18.

[0012] An isolated nucleic acid capable of expressing a dual-target chimeric antigen receptor as described above.

[0013] A vector comprising a nucleic acid as described above.

[0014] An immune cell expressing a dual-target chimeric antigen receptor as described above.

[0015] A pharmaceutical composition comprising the immune cell as described above.

[0016] Use of the immune cells as described above in the preparation of drugs for killing tumor cells that are CIgG-positive and MSLN or CLDN18.2-positive.

[0017] Beneficial effects:

[0018] This application designs and constructs four new dual-target chimeric antigen receptors, including CIgG-MSLN, CIgG-CLDN18.2, MSLN-CIgG and CLDN18.2-CIgG, which respectively target three key antigens highly expressed in pancreatic cancer - sialylated CIgG (SIA-CIgG), mesothelin (MSLN) and tight junction protein 18.2 (CLDN18.2). By combining different antigen recognition modules, these chimeric antigen receptors can simultaneously target multiple tumor antigens, thereby covering more tumor cells, reducing the risk of antigen escape, and thus enhancing the killing efficiency of immune cells against tumor cells.

[0019] The optimization of the dual-target CAR structure not only improves the activation level of T cells, but also significantly increases the secretion of key effector cytokines (such as IFN-γ, TNF-α and IL-6). These cytokines help enhance the immune activity in the tumor microenvironment and promote the synergistic effect of immune cells, thereby further enhancing the anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 A schematic diagram of the structure of a dual-target chimeric antigen receptor provided in one embodiment of the present application;

[0022] Figure 2 The test results of the lysis ability of different CAR-T cells on target cells provided in one embodiment of the present application;

[0023] Figure 3 The secretion of TFN-α by different CAR-T cells;

[0024] Figure 4 IFN-γ secretion of different CAR-T cells;

[0025] Figure 5 The GZMB secretion of different CAR-T cells;

[0026] Figure 6 The IL-6 secretion of different CAR-T cells;

[0027] Figure 7 The IL-10 secretion of different CAR-T cells. DETAILED DESCRIPTION

[0028] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature described or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0029] Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose this application are the same as those commonly understood by those of ordinary skill in the art to which this application belongs. By way of further guidance, the following definitions are used to better understand the teachings of this application. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0030] In this application, unless otherwise specified, the scientific and technical terms used in this application have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this application are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand this application, the definitions and explanations of the relevant terms are provided below.

[0031] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0032] As used in this application, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0033]

[0013] The use of numerical ranges as endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0034] References herein to "about" a value or parameter include (and describe) embodiments directed to the value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X".

[0035] The singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0036] In the present application, descriptions such as "plurality" and "multiple" refer to a quantity greater than or equal to 2 unless otherwise specified.

[0037] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0038] In this application, "preferred", "better", "more preferred", and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application. In this application, "optionally", "optionally", and "optional" refer to being optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "optional" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" item is independent of each other.

[0039] As used in this application, "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes referred to as a "chimeric receptor" or "chimeric immune receptor (CIR)". "Extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that can bind to a specific antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits signals to cause activation or inhibition of a biological process.

[0040] In this application, the term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed" US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the circumstances, the terms "CDR" and "CDRs" are used to refer to a region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope. In another specific embodiment, the CDR region or CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by Kabat.

[0041] As used in this application, the "region" or "domain" contained in the chimeric antigen receptor refers to a region in a polypeptide that can fold into a specific structure independently of other regions. These "regions" or "domains" can be sequences of mouse or other animal origin, preferably human sequences. In addition, when "regions" or "domains" are not specifically distinguished or emphasized, they should be understood as known sequences, which can be full-length or partially active segments.

[0042] In this application, the term "humanization" or "humanization treatment" refers to replacing an animal-derived (such as mouse-derived) antibody sequence with a human antibody sequence, thereby reducing or eliminating the human anti-mouse antibody (HAMA) response. This replacement can be a framework replacement, such as replacing the FR sequence in the variable region with a human one, and / or replacing the constant region of the antibody (if any) with a human one. This replacement can also be a chain replacement to convert a mouse monoclonal antibody into a fully human antibody (ie, DR is also replaced), and available means such as phage antibody library technology. It should be noted that during the humanization process, the replaced human sequence may contain partial amino acid substitutions or additions and subtractions, so that the replaced sequence may not be an exact copy of the expressed human immunoglobulin sequence or germline gene sequence. The antibody produced in this way can be called a human-mouse chimeric antibody, a humanized antibody, or a fully human antibody.

[0043] In the present application, the term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0044] In the present application, the term "connector peptide" may be a flexible or rigid peptide, for example, consisting of a repeated GGGGS amino acid sequence or a variant thereof, for example, a variant with 1 to 4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other connecting peptides that can be used in the present application are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0045] The term "CIgG" used in this application may also be referred to as SIA-CIgG, which refers to tumor-derived sialylated IgG. One of its functions is to inhibit the proliferation of effector T cells and significantly promote tumor growth.

[0046] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0047] The first aspect of the present application relates to a dual-target chimeric antigen receptor, the extracellular domain of which includes any one of the following from the N-terminus to the C-terminus: CIgG scFv-MSLN scFv, MSLN scFv- CIgG scFv, CIgG scFv-CLDN18.2 scFv, CLDN18.2 scFv -CIgG scFv; wherein "-" is a connecting peptide; wherein:

[0048] The amino acid sequence of the heavy chain complementary determining region of the CIgG scFv is shown in SEQ ID NOs: 1 to 3, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 4 to 6;

[0049] The amino acid sequence of the heavy chain complementary determining region of the MSLN scFv is shown in SEQ ID NOs: 7 to 9, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 10 to 12;

[0050] The amino acid sequence of the heavy chain complementary determining region of the CLDN18.2 scFv is shown in SEQ ID NOs: 13-15, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 16-18.

[0051] As known to those skilled in the art, the portion connecting the heavy chain variable region VH and the light chain variable region VL in scFv usually also has a linker peptide.

[0052] The number of amino acids in the connecting peptide can be 1 to 30; can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5 to 20.

[0053] In some embodiments, the amino acids of the connecting peptide are meaningless polypeptides that do not have additional functions other than connection (eg, protein localization, enzyme cleavage sites, etc.).

[0054] In some embodiments, the connecting peptide is a flexible connecting peptide;

[0055] In some embodiments, the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.

[0056] In some embodiments, the amino acid sequence of the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6. In some specific embodiments, the connecting peptide is selected from (GGGGS)3, (GGS)4GG, GGGS.

[0057] In some embodiments, the amino acid sequence of the CIgG scFv-MSLN scFv (hereinafter also abbreviated as CIgG-MSLN) is as shown in SEQ ID NO:19.

[0058] In some embodiments, the amino acid sequence of the MSLN scFv-CIgG scFv (hereinafter also abbreviated as MSLN-CIgG) is shown in SEQ ID NO: 20.

[0059] In some embodiments, the amino acid sequence of the CIgG scFv-CLDN18.2 scFv (hereinafter also referred to as CIgG-CLDN18.2) is as shown in SEQ ID NO: 21.

[0060] In some embodiments, the amino acid sequence of the CLDN18.2 scFv -CIgG scFv (hereinafter also referred to as CLDN18.2-CIgG) is as shown in SEQ ID NO: 22.

[0061] The modified forms of the above scFv are also within the scope of protection of the present application, such as those modified by covalent attachment of polyethylene glycol or other suitable polymers. Variants of scFv are also within the scope of the present application, wherein the heavy chain CDR1~CDR3 and light chain CDR1~CDR3 variants can respectively contain up to 3 amino acid mutations (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions or any combination thereof) compared to any of the complementary determining region combinations shown in SEQ ID NO: 1~6, 7~12, 13~18. scFv can be humanized. Obviously, the antibody framework region (FR) of scFv can also contain the above-mentioned types of modifications or mutations, and the amplitude of the changes can be greater than that of the CDR. As long as the resulting scFv still maintains the ability to bind to the target after modification or mutation, and the affinity is acceptable.

[0062] Preferably, the mutation is a conservative mutation. "Conservative substitution" refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein.

[0063] The substitutions generally considered as conservative substitutions are substitutions of each other in aliphatic amino acids Ala, Val, Leu and Ile, interchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr. It is known to those skilled in the art that, in general, single amino acid substitutions in non-essential regions of polypeptides do not substantially change biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, substitutions of amino acids with similar structures or functions are unlikely to destroy biological activity.

[0064] Variants of SEQ ID NO: 19-22 are also within the scope of protection of the present application, and the variants may have, for example, ≥ 85% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequences shown in SEQ ID NO: 19-22. The variants may also have modified forms or conservative substitutions as described above.

[0065] In some embodiments, the dual-target chimeric antigen receptor further comprises a hinge region, a transmembrane region, and an intracellular signaling region.

[0066] In some embodiments, the hinge region is selected from the hinge region of CD8, CD28, IgG1, IgG4, 4-1BB, ICOS, OX40, CD40, CD80, CD7, or CH3, CH2-CH3 constant region. In some specific embodiments, the hinge region is selected from the CD8 hinge region, and more preferably, the nucleotide sequence thereof is as shown in SEQ ID NO: 23.

[0067] The transmembrane region can be selected from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL 7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160( BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. In some embodiments, the transmembrane region is selected from the transmembrane region of CD8, CD28, CD4, ICOS, CD7, CD2, CD80, CD40, OX40, CD27, LFA-1, 4-1BB, ICOS, FcγR III, CD3ζ or CD3ε. In some specific embodiments, the hinge region is selected from the CD8 transmembrane region, and more preferably, its nucleotide sequence is as shown in SEQ ID NO: 24.

[0068] In some embodiments, the intracellular signaling region includes a CD3ζ signaling domain; in some specific embodiments, the nucleotide sequence of the CD3ζ signaling domain is as shown in SEQ ID NO: 25.

[0069] Further, the intracellular signal transduction region further comprises one or more selected from the following proteins or their intracellular signal transduction regions (or costimulatory regions): CD28, 4-1BB, OX40, ICOS, CD27, MYD88, HVEM, KIR2DS2, DAP10, DAP12, CD3ζ, TLRs, CD2, LFA-1, CD8α, CD40, CD80 and CD3ε. In some specific embodiments, the intracellular signal transduction region further comprises 4-1BB, and more preferably, its nucleotide sequence is shown in SEQ ID NO: 26.

[0070] According to yet another aspect of the present application, it also relates to an isolated nucleic acid that can express the dual-target chimeric antigen receptor as described above.

[0071] In the present application, nucleic acid comprises variants (such as replacement of degenerate codons) and complementary sequences thereof of conservative substitution, and also comprises variants optimized by codons to be expressed more efficiently in desired host cells. Nucleic acid is RNA or DNA normally, comprises gene, cDNA molecule, mRNA molecule and their fragments such as oligonucleotides. Nucleic acid molecule can be single-stranded or double-stranded, but preferably double-stranded DNA. When nucleic acid is placed in a functional relationship with another nucleic acid sequence, nucleic acid is "effectively connected". For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively connected to the coding sequence. It is preferred to adopt DNA nucleic acid when it is connected to a vector.

[0072] The present application also relates to a vector, which contains the nucleic acid as described above.

[0073] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; CRISPR / CAS plasmids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). In some embodiments, the vector described in the present application contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0074] The vector of the present application may also contain fragments such as nucleic acids for generating fluorescent proteins. Fluorescent proteins may be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein. Green fluorescent protein may use common GFP, or a modified GFP gene, such as enhanced GFP gene EGFP, etc.; blue fluorescent protein may be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein may be selected from EYFP, Ypct, PhiYFP, etc.; orange fluorescent protein may be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein may be selected from TagRFP, mRuby, mCherry, mKate, etc.

[0075] The present application also relates to immune cells expressing the dual-target chimeric antigen receptor as described above, such as one or more of T cells, B cells, NK cells, macrophages, dendritic cells, etc.

[0076] In some embodiments, the immune cells are T cells.

[0077] The T cells may be subtypes well known in the art, such as one or more of helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells, and γδ T cells.

[0078] According to yet another aspect of the present application, it also relates to a pharmaceutical composition comprising the immune cells as described above.

[0079] The pharmaceutical composition may also include a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" includes any material that allows the component to maintain biological activity when combined with an active ingredient and does not react with the subject's immune system. Examples include, but are not limited to, any of standard pharmaceutical carriers (such as phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions)) and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are prepared by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005).

[0080] According to another aspect of the present application, it also relates to the use of the immune cells as described above in the preparation of drugs for killing tumor cells that are CIgG-positive and MSLN- or CLDN18.2-positive.

[0081] In some embodiments, the tumor is pancreatic cancer.

[0082] According to yet another aspect of the present application, it also relates to a method for treating a tumor in a patient in need thereof, the method comprising administering a therapeutically effective amount of the immune cells or pharmaceutical composition as described above to the patient.

[0083] The tumor is preferably a solid tumor. In the present application, "solid tumor" includes: a tumor generated by lesions in any of the following: bones, bone connections, muscles, lungs, trachea, heart, spleen, arteries, veins, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testicle, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, thymus gland, gonads, sublingual gland and parotid gland.

[0084] In some embodiments, the tumor cells in the tumor are CIgG positive and are also MSLN or CLDN18.2 positive.

[0085] In some embodiments, the tumor is pancreatic cancer.

[0086] It should be understood that the contemplated treatment methods will also include the administration of other immunotherapeutic entities, with particular preference given to immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic Escherichia coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), and antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell transplants (e.g., allogeneic or autologous), and tumor-targeted cytokines (e.g., NHS-IL12, IL-12 conjugated to tumor-targeted antibodies or fragments thereof).

[0087] A "patient" is a mammal, including but not limited to humans, monkeys, pigs and other farm animals, sports animals, pets, primates, horses, dogs, cats, giant pandas, rodents (including mice, rats, guinea pigs), etc.

[0088] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0089] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0090] Example 1 Design of chimeric antigen receptor

[0091] The chimeric antigen receptor used in the example is composed of a VH1- (G4S1)3-VL1- G3S-VL2-(G2S)4GG-VH2 structure, and a total of four dual-target CAR-Ts were designed, namely CIgG-MSLN, CIgG-CLDN18.2, MSLN-CIgG, and CLDN18.2-CIgG, see Figure 1 The promoter is EF1α, the hinge region is CD8, the transmembrane region is CD8, the costimulatory domain is 4-1BB, CD3ζ, and the sequence is as follows:

[0092] Amino acid sequence of CIgG-MSLN: SEQ ID NO: 19;

[0093] Amino acid sequence of MSLN-CIgG: SEQ ID NO: 20;

[0094] Amino acid sequence of CIgG-CLDN18.2: SEQ ID NO: 21;

[0095] Amino acid sequence of CLDN18.2-CIgG: SEQ ID NO: 22;

[0096] CD8 hinge nucleic acid sequence: SEQ ID NO: 23;

[0097] CD8 TM nucleic acid sequence: SEQ ID NO: 24;

[0098] CD3ζ nucleic acid sequence: SEQ ID NO: 25;

[0099] 4-1BB nucleic acid sequence: SEQ ID NO: 26.

[0100] Example 2 Cell killing experiment

[0101] 1. Target Cell Preparation

[0102] Place the frozen target cells in a 37°C water bath to dissolve, then transfer to a 15 mL centrifuge tube containing 10 mL of culture medium, centrifuge (1200 rpm, 5 min) and remove the supernatant. Resuspend the cells with an appropriate amount of X-VIVO culture medium containing 10% FBS, and then count the cells. Cell counting: Take 20 μL of cell suspension, add 20 μL of 0.2% trypan blue, mix well, take 20 μL and add to the counting plate, and count the cells according to the cell counter operating requirements.

[0103] 2. Target cell calculation

[0104] Total number of target cells = number of target cells added per well × volume

[0105] Target cell volume = number of wells × 100 μL / well

[0106] 3. Target Cell Inoculation

[0107] Select the required number of wells in a 96-well cell culture plate and add 100 μL of target cells. Place the cell culture plate with the target cells in a 37°C, 5% carbon dioxide incubator for incubation.

[0108] 4. Access to effector cell killing

[0109] 5. CAR-T cell counting

[0110] Transfer the CART cells into a 15 mL centrifuge tube, centrifuge (500 g, 5 min), remove the supernatant, resuspend the cells in X-VIVO medium containing 10% FBS, and take a sample for counting.

[0111] Cell counting: Take 20 μL of cell suspension, add 20 μL of 0.2% trypan blue, mix well, take 20 μL and add it to the counting plate, and count the cells according to the operating requirements of the cell counter.

[0112] 6. Calculation of CAR-T Cell Amount

[0113] The required amount of CAR-T cells is calculated based on the effect-target ratio (E:T) and the positive rate. The calculation formula is as follows:

[0114] CAR-T cell amount (cells) = theoretical cell concentration C (cells / mL) × required cell volume V (mL)

[0115] Theoretical cell concentration C (cells / mL) = number of target cells per well × maximum effector-target ratio (E:T) / CAR-T cell addition volume (0.04 mL) / CAR-T cell positive rate.

[0116] Required cell volume V (mL) = number of target cells killed × 0.1.

[0117] 7. CAR-T cell gradient dilution

[0118] ① According to the calculation results of the CAR-T cell amount, aspirate the required amount of CAR-T cells, centrifuge (500g, 5 min), remove the supernatant, and add the required cell volume V of X-VIVO medium containing 10% FBS to resuspend the cells.

[0119] ② Gradient dilution: Perform gradient dilution on the CAR-T cells with adjusted cell density in ①, and dilute several tubes in a gradient manner according to the effector-target ratio set in the experiment.

[0120] 8. CAR-T Cell Vaccination

[0121] Take out 96 plates, take 40 μL of adjusted CAR-T and add it to the corresponding cell wells, and make the corresponding plate layout.

[0122] 9. Incubate and Read Plate

[0123] (1) Place the cell culture plate with the cells in a 37°C, 5% carbon dioxide incubator.

[0124] (2) Before the end of the incubation, take out the reagents in the ONE-lite Luciferase Assay System kit from the -20℃ refrigerator and place them at room temperature until the reagents melt. Dissolve the powder with the reagent according to the instructions. After it is completely dissolved, divide it into EP tubes and store it in a -20℃ refrigerator. You can take it out directly and restore it to room temperature for use in future experiments.

[0125] (3) Turn on the multifunctional microplate reader and software, select the Luminescence mode, and make the plate reading layout.

[0126] (4) Pipette 100 μL of the prepared reagent into a 96-well cell culture plate, pipette and mix well, and place at room temperature away from light for 4 min (the luminescence intensity is highest between 4 and 10 min, and the plate should be read 10 to 30 min after the reaction). Use a pipette to transfer 180 μL of the solution in the cell culture plate into a 96-well white flat-bottom plate, avoiding the formation of bubbles.

[0127] (5) Place the 96-well white flat-bottom plate on the microplate reader to read the data and export the data for storage.

[0128] 10. Experimental Results

[0129] The constructed dual-target CAR-T cells were used as effector cells, and the pancreatic cancer cell line BxPC-3 cells naturally expressing CIgG, MSLN and CLDN18.2 were used as target cells. A co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well was 50,000, and different numbers of CAR-T cells were added, respectively. The co-culture system was cultured in serum-free medium. After continuous culture for 4 hours, the well plate was taken out and centrifuged at 1200g for 10 minutes at room temperature to allow all suspended cells to precipitate to the bottom of the well plate, and then 30 microliters of supernatant was taken from each well to detect the release of LDH in the culture supernatant to reflect the lysis ability of CAR-T cells on target cells. The results are shown in FIG. Figure 2 As shown in the figure, when the effector-target ratio is 1:1, T cells can efficiently mediate the killing of tumor cells or recombinant cells; as the effector-target ratio increases, the killing effect of CAR-T cells on target cells also increases, reaching the highest level when the effector-target ratio is 4:1.

[0130] Example 3 Cytokine release assay

[0131] 1. Target Cell Preparation

[0132] (1) Adjust the target cells to the logarithmic growth phase and subculture them twice before the experiment to ensure the stability of the cells.

[0133] (2) Use trypsin to digest the adherent target cells, resuspend the cells in complete culture medium, and adjust the cell density.

[0134] (3) Take a new 96-well plate and add 100 μL of target cell suspension to each well. Note that 100 μL of sterile water should be added to each of the unused wells around the 96-well plate to reduce water evaporation in the middle experimental wells.

[0135] (4) Place the 96-well plate in a 5% CO2, 37°C incubator and culture overnight to allow the target cells to adhere.

[0136] 2. CAR-T Cell Preparation

[0137] The prepared CAR-T cells were collected by centrifugation and resuspended in serum-free 1640 medium.

[0138] 3. Add CAR-T cells

[0139] (1) Remove the 96-well plate from the incubator, aspirate the culture medium in the wells, and gently wash the cells once with sterile PBS, taking care to avoid excessive disturbance of the cells.

[0140] According to the predetermined effector-target ratio (E / T ratio), CAR-T cell suspension was added to each well, and the volume of each well was supplemented to 100 μL with serum-free medium.

[0141] (2) A control group was set up at the same time, in which only nonspecific T cells were added for comparative analysis.

[0142] The 96-well plate was placed back into the 5% CO2, 37°C incubator for 6 hours.

[0143] 4. Supernatant Collection and Analysis

[0144] (1) After the incubation period, remove the 96-well plate from the incubator and centrifuge at 1200 × g for 5 minutes at room temperature to sediment the cells.

[0145] (2) Gently remove the 96-well plate, being careful not to disturb the precipitate, and transfer 50 μL of culture supernatant from each well to a new collection tube.

[0146] (3) Use ELISA kit to detect the expression levels of IFN-γ and IL-2 in the supernatant according to the kit instructions.

[0147] (4) Use a microplate reader to read the OD value and analyze and plot the data.

[0148] 5. Experimental Results

[0149] CAR-T cells were used as effector cells, and pancreatic cancer cell line BxPC-3 cells naturally expressing CIgG, MSLN and CLDN18.2 were used as target cells. A co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well was 50,000, and different numbers of CAR-T cells were added, respectively. The co-culture system was cultured in serum-free medium. After continuous culture for 8 hours, the well plate was taken out and centrifuged at 1200g for 10 minutes at room temperature to allow all suspended cells to precipitate to the bottom of the well plate, and then 30 microliters of supernatant was taken from each well, and the expression levels of IFN-α, IFN-γ, GZMB, IL-10 and IL-6 secreted by CAR-T cells after being activated by tumor cells in the culture medium supernatant were detected by ELISA. The results are shown in FIG. Figure 2-Figure 6 As shown, after the BCMA-CD38-CART chimeric antigen receptor binds to the targeted tumor cells, it can effectively activate primary T cells and cause an increase in the expression of cytokine secretion; when the effector-target ratio is 1:1, after the CAR-T cells are activated by tumor cells, they can secrete a large amount of TFN-α, IFN-γ, GZMB, IL-6 and IL-10, which are significantly higher than the control T cells; the secretion amount reaches the highest value when the effector-target ratio is 4:1.

[0150] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A dual-target chimeric antigen receptor, characterized in that: Its extracellular domain includes any of the following from N-terminus to C-terminus: CIgG scFv-MSLN scFv, MSLN scFv- CIgG scFv, CIgG scFv-CLDN18.2 scFv, CLDN18.2scFv -CIgG scFv; wherein "-" is a connecting peptide; wherein: The amino acid sequence of the heavy chain complementary determining region of the CIgG scFv is shown in SEQ ID NOs: 1 to 3, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 4 to 6; The amino acid sequence of the heavy chain complementary determining region of the MSLN scFv is shown in SEQ ID NOs: 7 to 9, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 10 to 12; The amino acid sequence of the heavy chain complementary determining region of the CLDN18.2 scFv is shown in SEQ ID NOs: 13-15, and the amino acid sequence of the light chain complementary determining region is shown in SEQ ID NOs: 16-18.

2. The dual-target chimeric antigen receptor according to claim 1, characterized in that: It also contains the hinge region, transmembrane region, and intracellular signaling region; The hinge region is selected from the hinge region of CD8, CD28, IgG1, IgG4, 4-1BB, ICOS, OX40, CD40, CD80, CD7, or CH3, CH2-CH3 constant region; The transmembrane region is selected from the transmembrane region of CD8, CD28, CD4, ICOS, CD7, CD2, CD80, CD40, OX40, CD27, LFA-1, 4-1BB, ICOS, FcγR III, CD3ζ or CD3ε; The intracellular signaling region includes a CD3ζ signaling domain; and further comprises one or more selected from the following proteins or their intracellular signaling regions: CD28, 4-1BB, OX40, ICOS, CD27, MYD88, HVEM, KIR2DS2, DAP10, DAP12, CD3ζ, TLRs, CD2, LFA-1, CD8α, CD40, CD80 and CD3ε.

3. An isolated nucleic acid, characterized in that The dual-target chimeric antigen receptor according to claim 1 or 2 can be expressed.

4. A carrier, characterized in that Containing the nucleic acid as claimed in claim 3.

5. An immune cell, characterized in that Expressing the dual-target chimeric antigen receptor according to claim 1 or 2.

6. The immune cell according to claim 5, characterized in that The immune cells are selected from the group consisting of T cells, B cells, NK cells, macrophages and dendritic cells.

7. The immune cell according to claim 6, characterized in that The T cells are selected from helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells and γδ T cells.

8. A pharmaceutical composition, characterized in that Comprising the immune cell according to claim 6 or 7.

9. Use of the immune cell according to any one of claims 5 to 7 in the preparation of a drug for killing tumor cells that are CIgG positive and MSLN or CLDN18.2 positive.

10. The use according to claim 9, characterized in that: The tumor is pancreatic cancer.

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