Dual-target chimeric antigen receptor combined with PD-1 and IL-21 fusion protein

Abstract

This application relates to the field of biomedical technology, and specifically to a dual-target chimeric antigen receptor combined with a PD-1 and IL-21 fusion protein. The extracellular domain of this dual-target chimeric antigen receptor includes, from the N-terminus to the C-terminus, any one of the following: CIgG scFv-MSLN scFv, CIgG scFv-CLDN18.2 scFv; wherein "-" is a connecting peptide; it also includes a hinge region, a transmembrane region, and an intracellular signaling region; and PD-1 scFv and IL-21 are also fused to the N-terminus of the intracellular signaling region. This application constructs two dual-target chimeric antigen receptors combined with PD-1 and IL21 fusion proteins, and through a synergistic anti-tumor strategy, enhances CAR-T cell function, overcomes tumor microenvironment (TME) inhibition, and improves T cell persistence to achieve synergy.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a dual-target chimeric antigen receptor combined with PD-1 and IL-21 fusion protein. Background Art

[0002] CAR-T cell therapy is a genetically engineered T-cell immunotherapy method that specifically recognizes and kills tumor cells. CAR-T therapy initially achieved remarkable results in hematologic malignancies such as acute lymphoblastic leukemia (ALL) and large B-cell lymphoma. However, solid tumors often present significant challenges to CAR-T cell therapy due to their complex tumor microenvironment (TME) and heterogeneous tumor cells.

[0003] The tumor microenvironment in solid tumors often exhibits immunosuppressive properties, such as suppressed immune cell function, accumulation of immunosuppressive factors (such as TGF-β and IL-10), abnormal tumor vasculature, and antigenic heterogeneity in tumor cells. These factors significantly limit the penetration, proliferation, and persistence of CAR-T cells within tumors. Furthermore, variations in tumor antigens can easily lead to unstable anti-tumor effects of single-target CAR-T cells.

[0004] Pancreatic cancer is a highly aggressive cancer that is challenging to treat, particularly because it is often diagnosed in the late stages and is highly drug-resistant and metastatic. Currently, immunotherapy, such as CAR-T therapy, is a cutting-edge treatment strategy that has emerged as a potential hope for pancreatic cancer treatment. CAR-T therapies for pancreatic cancer currently focus on two targets: CLDN18.2 (Claudin 18.2) and MSLN (Mesothelin).

[0005] PD-1 / PD-L1 blockade therapy is a major breakthrough in the field of tumor immunotherapy, restoring anti-tumor immune responses by relieving tumor suppression of T cells. A successful anti-tumor immune response following PD-1 blockade requires the reactivation and proliferation of antigen-experienced CD8+ T cells present in the tumor microenvironment (TME). In this regard, γC family cytokines, particularly IL-2, IL-15, and IL-21, play an important role in regulating the magnitude and function of CD8+ T cell responses. IL-21, in particular, plays a key role in the development and maintenance of memory CD8+ T cells by inducing an early differentiation phenotype.

[0006] Cytokine-based therapies present numerous challenges, including pharmacokinetic barriers and side effects. Most cytokines, including IL-21, exert their effects on a variety of cell types, including immune and non-immune cells. Under physiological conditions, cytokines restrict their effects to specific target cells through paracrine and autocrine pathways and short half-lives. Systemic administration of cytokines often results in poor therapeutic efficacy and side effects, as cytokines can also activate counterregulatory pathways and cause toxicity by acting on diverse target cells, while also activating immune cells to enhance anti-tumor immune responses. Summary of the Invention

[0007] This application covers the following technical solutions:

[0008] A dual-target chimeric antigen receptor, whose extracellular domain includes any of the following from N-terminus to C-terminus: CIgG scFv-MSLN scFv, CIgG scFv-CLDN18.2 scFv; wherein "-" is a connecting peptide; it also includes a hinge region, a transmembrane region and an intracellular signaling region; and PD-1 scFv and IL-21 are also fused to the N-terminus of the intracellular signaling region; wherein:

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

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

[0011] The amino acid sequences of the heavy chain complementary determining regions of the CLDN18.2 scFv are shown in SEQ ID NOs: 13 to 15, and the amino acid sequences of the light chain complementary determining regions are shown in SEQ ID NOs: 16 to 18;

[0012] The amino acid sequences of the heavy chain complementary determining regions of the PD-1 scFv are shown in SEQ ID NOs: 19 to 21, and the amino acid sequences of the light chain complementary determining regions are shown in SEQ ID NOs: 22 to 24;

[0013] The amino acid sequence of IL-21 is shown in SEQ ID NO: 25.

[0014] An isolated nucleic acid capable of expressing the dual-target chimeric antigen receptor as described above.

[0015] A vector comprising a nucleic acid as described above.

[0016] An immune cell expressing a dual-target chimeric antigen receptor as described above.

[0017] A pharmaceutical composition comprising the immune cell as described above.

[0018] Use of the immune cells described above in the preparation of a drug for killing CIgG-positive and MSLN- or CLDN18.2-positive tumor cells.

[0019] Beneficial effects:

[0020] This application designs and constructs a dual-target CAR-T therapy combined with a PD-1 inhibitor (PD-1 scFv) and IL-21. This therapy combines multiple mechanisms of immunotherapy and offers significant technical advantages. This combination strategy primarily addresses the limitations of single-agent therapies by enhancing the anti-tumor effects of T cells, overcoming immune escape, and improving the persistence of CAR-T cells.

[0021] The specific technical advantages of this combination therapy include at least:

[0022] 1. Enhance the persistence and efficacy of CAR-T cells:

[0023] The role of IL-21: IL-21 is a T cell and natural killer (NK) cell proliferation factor that promotes sustained T cell proliferation, enhances effector function, and prolongs their survival. IL-21 can effectively inhibit T cell exhaustion, improve the tolerance of CAR-T cells, and prolong their activity in the tumor microenvironment. The synergistic effect of dual-target CAR-T: By combining with IL-21, dual-target CAR-T cells can remain active in the tumor microenvironment for a longer period of time, enhancing the killing effect of T cells and generating a more potent antigen-specific attack on tumor cells. This helps address the poor persistence of single-target CAR-T cells in a persistent immunosuppressive environment.

[0024] 2. Overcoming immune escape mechanisms

[0025] The role of PD-1 inhibitors: Tumor cells express PD-L1, which binds to the PD-1 receptor on the surface of T cells, inhibiting T cell activity and proliferation. This is a common immune escape mechanism. PD-1 inhibitors (such as nivolumab or pembrolizumab) block the PD-1 / PD-L1 pathway, relieving immune suppression on T cells and thereby enhancing the anti-tumor effects of CAR-T cells.

[0026] Synergistic effects of dual-target CAR-T and PD-1 inhibitors: Combining dual-target CAR-T with PD-1 inhibitors can effectively relieve the immune suppression of CAR-T cells in the tumor microenvironment, making CAR-T cells more active and efficient. This combination strategy can significantly improve therapeutic efficacy, especially against solid tumors with strong immune evasion capabilities (such as pancreatic cancer and non-small cell lung cancer).

[0027] 3. Improve the recognition and killing efficiency of tumor cells

[0028] Advantages of dual targets: Dual-target CAR-T cells can simultaneously target two different antigens, enabling them to more comprehensively identify tumor cells within the tumor microenvironment and reducing the possibility of tumor cells escaping immune surveillance by downregulating a single antigen. Dual-target CAR-T cells can cover more tumor cell subpopulations, especially in cases of high tumor heterogeneity.

[0029] Synergistic Effects of IL-21 and Dual-Target CAR-T Cells: IL-21 can enhance the function of dual-target CAR-T cells, making them more sustained and efficient in identifying and killing tumor cells. This combination not only enhances the cytotoxicity of CAR-T cells but also strengthens their adaptability to the tumor microenvironment, further improving efficacy.

[0030] 4. Reduce CAR-T cell exhaustion

[0031] The challenge of immune exhaustion: CAR-T cells are prone to immune exhaustion after prolonged exposure to the tumor microenvironment, manifesting as loss of cell function, decreased proliferation, and diminished cytotoxicity. However, the introduction of IL-21 can effectively inhibit T cell exhaustion and promote CAR-T cell activity and effector function. IL-21's effects are not limited to T cell proliferation; it also regulates CAR-T cell metabolism and anti-exhaustion, improving cell efficacy and survival.

[0032] Combining PD-1 inhibition with IL-21: The combined use of PD-1 inhibitors and IL-21 can enhance the immune response of CAR-T cells in two ways. PD-1 inhibitors relieve immune checkpoint inhibition, enabling CAR-T cells to better exert their anti-tumor effects; IL-21 directly enhances T cell tolerance and sustained efficacy, preventing rapid exhaustion. The combined effects of these two mechanisms can effectively improve the anti-tumor ability and therapeutic durability of CAR-T cells.

[0033] 5. Reduce side effects and enhance targeting

[0034] Reduce the side effects of single-target therapy: Because dual-target CAR-T cells recognize two antigens simultaneously, they can more precisely target tumor cells, avoid accidental damage to normal cells, and reduce the occurrence of side effects. In addition, IL-21 enhances the immune response of CAR-T cells, not only making them more effective in killing tumor cells, but also reducing the risk of tumor recurrence.

[0035] Managing side effects of PD-1 inhibition: Although PD-1 inhibitors may cause certain immune-related side effects, combination therapy can improve the therapeutic efficiency of CAR-T cells, reduce the required treatment dose, and lower the risk of immune-related side effects. Furthermore, the enhancing effect of IL-21 on CAR-T cells can also mitigate the effects of immunosuppression on T cells to a certain extent, improving the safety and tolerability of treatment.

[0036] 6. Potential for personalized treatment to improve efficacy

[0037] Enhanced efficacy and tumor heterogeneity: For patients with high tumor heterogeneity, dual-target CAR-T combined with immunomodulatory therapy can minimize the risk of antigen loss or immune escape, and improve the adaptability and efficacy of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. 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 any creative work.

[0039] Figure 1 This is a schematic diagram of the structure of a dual-target chimeric antigen receptor combined with PD-1 and IL-21 fusion protein provided in one embodiment of the present application;

[0040] Figure 2 This is a comparison of the BxPC-3 lytic and killing abilities of dual-target CAR-T cells secreting PD-1 and IL-21 against target cells;

[0041] Figure 3 This figure shows the comparison of the IFN-γ secretion ability of dual-target CAR-T cells that secrete PD1 and IL-21;

[0042] Figure 4 This figure shows the in vivo efficacy results of dual-target CAR-T cells secreting PD1 and IL-21 in a pancreatic cancer animal model. DETAILED DESCRIPTION

[0043] 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 to illustrate, not to limit, the present application. Indeed, 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, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0044] As used herein, "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)". An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that can bind to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits a signal to cause activation or inhibition of a biological process.

[0045] As used herein, 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, the terms "CDR" and "CDRs" are used to refer to a region comprising 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 CDRs refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by Kabat.

[0046] As used herein, a "region" or "domain" within a chimeric antigen receptor refers to a region within a polypeptide that folds into a specific structure independently of other regions. These "regions" or "domains" can be sequences of murine or other animal origin, preferably human. Furthermore, unless otherwise specified or emphasized, "region" or "domain" should be understood to refer to a known sequence, which can be a full-length or partial active segment.

[0047] As used herein, the term "humanization" or "humanization process" refers to the replacement of animal-derived (e.g., mouse-derived) antibody sequences with human-derived sequences, thereby reducing or eliminating the human anti-mouse antibody (HAMA) response. Such replacements can involve framework replacements, such as replacing human FR sequences in the variable region and / or replacing human constant regions (if present). Alternatively, such replacements can involve converting a mouse monoclonal antibody into a fully human antibody (i.e., DRs are also replaced) through chain replacement, using methods such as phage antibody library technology. It should be noted that during the humanization process, the replaced human sequence may include partial amino acid substitutions or additions and deletions, resulting in the replacement sequence not being an exact copy of the expressed human immunoglobulin sequence or germline gene sequence. The resulting antibody may be referred to as a human-mouse chimeric antibody, a humanized antibody, or a fully human antibody.

[0048] In this application, the term "scFv" refers to 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 can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0049] In the present application, the term "connector peptide" can 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.

[0050] The term "CIgG" as used in this application, also known as SIA-CIgG, refers to tumor-derived sialylated IgG, one of whose functions is to inhibit the proliferation of effector T cells and significantly promote tumor growth.

[0051] The first aspect of the present application relates to a dual-target chimeric antigen receptor, characterized in that its extracellular domain comprises any one of the following from the N-terminus to the C-terminus: CIgG scFv-MSLN scFv, CIgG scFv-CLDN18.2 scFv; wherein "-" is a connecting peptide; it further comprises a hinge region, a transmembrane region and an intracellular signaling region; and PD-1 scFv and IL-21 are further fused to the N-terminus of the intracellular signaling region; wherein:

[0052] The amino acid sequences of the heavy chain complementary determining regions of the CIgG scFv are shown in SEQ ID NOs: 1 to 3, and the amino acid sequences of the light chain complementary determining regions are shown in SEQ ID NOs: 4 to 6;

[0053] The amino acid sequences of the heavy chain complementary determining regions of the MSLN scFv are shown in SEQ ID NOs: 7 to 9, and the amino acid sequences of the light chain complementary determining regions are shown in SEQ ID NOs: 10 to 12;

[0054] The amino acid sequences of the heavy chain complementary determining regions of the CLDN18.2 scFv are shown in SEQ ID NOs: 13 to 15, and the amino acid sequences of the light chain complementary determining regions are shown in SEQ ID NOs: 16 to 18;

[0055] The amino acid sequences of the heavy chain complementary determining regions of the PD-1 scFv are shown in SEQ ID NOs: 19 to 21, and the amino acid sequences of the light chain complementary determining regions are shown in SEQ ID NOs: 22 to 24;

[0056] The amino acid sequence of IL-21 is shown in SEQ ID NO: 25.

[0057] In view of the problems existing in dual-target CAR-T, this application constructed two dual-target chimeric antigen receptors combined with PD-1 and IL21 fusion proteins. Through a synergistic anti-tumor strategy, it enhances the function of CAR-T cells, overcomes the inhibition of the tumor microenvironment (TME), and improves the persistence of T cells to achieve synergy.

[0058] As known to those skilled in the art, a linker peptide is usually present in the portion of an scFv that connects the heavy chain variable region VH and the light chain variable region VL.

[0059] The number of amino acids in the connecting peptide defined in the present application 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.

[0060] In some embodiments, the amino acids of the connecting peptide are nonsense polypeptides that do not have additional functions other than connection (eg, protein localization, enzyme cleavage sites, etc.).

[0061] In some embodiments, the connecting peptide is a flexible connecting peptide;

[0062] In some embodiments, the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.

[0063] 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, (G2S)4GG, GGGGS.

[0064] In some embodiments, the VH amino acid sequence of the CIgG scFv is shown in SEQ ID NO: 26; and the VL amino acid sequence is shown in SEQ ID NO: 27.

[0065] In some embodiments, the VH amino acid sequence of the MSLN scFv is shown in SEQ ID NO: 28; and the VL amino acid sequence is shown in SEQ ID NO: 29.

[0066] In some embodiments, the VH amino acid sequence of the CLDN18.2 scFv is shown in SEQ ID NO: 30; and the VL amino acid sequence is shown in SEQ ID NO: 31.

[0067] In some embodiments, the VH amino acid sequence of the PD-1 scFv is shown in SEQ ID NO: 32; and the VL amino acid sequence is shown in SEQ ID NO: 33.

[0068] Modified forms of the above-mentioned scFv are also within the scope of protection of this application, such as those modified by covalent attachment of polyethylene glycol or other suitable polymers. Variants of the scFv are also within the scope of this application, wherein the heavy chain CDR1-CDR3 and light chain CDR1-CDR3 variants may each comprise up to three amino acid mutations (e.g., substitutions, deletions, or additions of one, two, or three amino acids, or any combination thereof) compared to any of the complementarity determining region combinations set forth in SEQ ID NOs: 1-6, 7-12, 13-18, and 19-24. The scFv may be humanized. Obviously, the antibody framework regions (FRs) of the scFv may also comprise the aforementioned types of modifications or mutations, and the magnitude of the changes may be greater than those in the CDRs. As long as the resulting scFv retains target binding ability and acceptable affinity after modification or mutation, the resulting scFv remains humanized.

[0069] Preferably, the mutation is a conservative mutation. "Conservative substitution" refers to replacing 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), such that changes can be made frequently without altering the biological activity of the protein.

[0070] 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 the non-essential regions of a polypeptide do not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)). In addition, substitutions of amino acids with similar structure or function are unlikely to destroy the biological activity.

[0071] Variants of SEQ ID NOs: 25-33 are also within the scope of this application. Variants may have, for example, ≥85% identity, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth in SEQ ID NOs: 25-33. Variants may also have modifications or conservative substitutions as described above.

[0072] 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, more preferably, the nucleotide sequence of which is as shown in SEQ ID NO: 39.

[0073] 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, more preferably, its nucleotide sequence is as shown in SEQ ID NO: 35.

[0074] 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 shown in SEQ ID NO: 37.

[0075] Furthermore, the intracellular signaling region further comprises one or more proteins or their intracellular signaling regions (or costimulatory regions) selected from the group consisting of 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 signaling region further comprises 4-1BB, more preferably, the nucleotide sequence of which is as shown in SEQ ID NO: 36.

[0076] 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.

[0077] In the present application, nucleic acid comprises variants of its conservative substitution (such as substitution of degenerate codons) and complementary sequences, and also comprises variants optimized by codons to be expressed more efficiently in the desired host cell. Nucleic acid is typically RNA or DNA, comprising genes, cDNA molecules, mRNA molecules and fragments thereof such as oligonucleotides. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". 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. When it is connected to a vector, preferably a DNA nucleic acid is used.

[0078] The present application also relates to a vector comprising a nucleic acid as described above.

[0079] The term "vector" refers to a nucleic acid delivery vehicle 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 (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); 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, and 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.).

[0080] The vector of the present application may also contain fragments such as nucleic acids for generating fluorescent proteins. Fluorescent proteins can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein. Green fluorescent protein can use the common GFP, or a modified GFP gene, such as the enhanced GFP gene EGFP; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP, etc.; orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate, etc.

[0081] 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, and the like.

[0082] In some embodiments, the immune cells are T cells.

[0083] 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.

[0084] According to yet another aspect of the present application, it also relates to a pharmaceutical composition comprising the immune cells as described above.

[0085] 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 the 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 solution, 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).

[0086] According to another aspect of the present application, it also relates to the use of the immune cells described above in the preparation of drugs for killing tumor cells that are CIgG-positive and MSLN- or CLDN18.2-positive.

[0087] In some embodiments, the tumor is pancreatic cancer.

[0088] 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, comprising administering a therapeutically effective amount of the immune cells or pharmaceutical composition as described above to the patient.

[0089] The tumor is preferably a solid tumor. In this application, "solid tumor" includes: a tumor generated by lesions in any of the 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, gonads, sublingual gland and parotid gland.

[0090] In some embodiments, the tumor cells in the tumor are CIgG positive and also MSLN or CLDN18.2 positive.

[0091] In some embodiments, the tumor is pancreatic cancer.

[0092] 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., that bind 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 a tumor-targeted antibody or fragment thereof).

[0093] 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.

[0094] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only 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 manuals or conventional conditions of this area, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0095] In the following specific examples, 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 testing accuracy or operational accuracy are allowed.

[0096] Example 1 Design of Chimeric Antigen Receptor

[0097] In the dual-target CAR-T combined with PD-1 and IL-21 fusion protein structure, the dual-target part is composed of EF1α, CD8α leader, VH1- (G4S1)3-VL1- G3S-VL2-(G2S)4GG-VH2, CD8 hinge region, CD8 transmembrane region, 4-1BB, CD3ζ, GSG, P2A, PD1 scFv, IL-21, and GFP. A total of two dual-target CAR-Ts were designed, namely CIgG-MSLN and CIgG-CLDN18.2, in which the fusion protein region structure is CIgG scFv and PD1 scFv, both of which are VH-GGGGS-VL, see Figure 1 , the sequence is as follows:

[0098] CIgG scFv: VH is shown in SEQ ID NO: 26; VL is shown in SEQ ID NO: 27.

[0099] MSLN scFv: VH is shown in SEQ ID NO: 28; VL is shown in SEQ ID NO: 29.

[0100] CLDN18.2 scFv: VH is shown in SEQ ID NO: 30; VL is shown in SEQ ID NO: 31.

[0101] PD-1 scFv: VH is shown in SEQ ID NO: 32; VL is shown in SEQ ID NO: 33.

[0102] CD8α leader amino acid sequence: shown in SEQ ID NO: 34.

[0103] CD8 TM amino acid sequence: shown in SEQ ID NO: 35.

[0104] 4-1BB amino acid sequence: shown in SEQ ID NO: 36.

[0105] CD3ζ amino acid sequence: shown in SEQ ID NO: 37.

[0106] P2A amino acid sequence: shown in SEQ ID NO: 38.

[0107] CD8 hinge amino acid sequence: shown in SEQ ID NO: 39.

[0108] Example 2 Cell Killing Experiment

[0109] 1) Cell preparation

[0110] 1. Target cell culture: Take luciferase-labeled target cells (Fluc+) in the logarithmic growth phase, wash them once with PBS, and count them with trypan blue staining. Adjust the cell concentration to 10 5 cells / mL.

[0111] 2. CAR-T cell preparation: Collect CAR-T cells and adjust the concentration to 10 6 cells / mL (adjusted according to the set effector-target ratio).

[0112] 2) Cell co-culture

[0113] 1. Inoculation of target cells: Take a 96-well white plate and add 100 μL of target cell suspension (i.e. 10 5 cells / well) and incubate at 37°C for 2–4 hours to allow the cells to adhere (suspension cells can be used directly).

[0114] 2. Add CAR-T cells: Add CAR-T cells at different effector-target ratios (E:T = 10:1, 5:1, 2.5:1, 1.25:1) and bring the total volume up to 200 μL.

[0115] 3. Set up control groups: target cell autolysis control (target cells + culture medium, no CAR-T); background fluorescence control (culture medium + luciferase substrate, no cells); maximum lysis control (target cells + Triton X-100, final concentration 2%)

[0116] Incubate at 37°C, 5% CO2 for 4–24 hours (usually 16 hours) to allow the CAR-T cells to kill target cells.

[0117] 3) Luciferase Assay

[0118] 1. After incubation, mix the cell culture medium (gently pipetting).

[0119] 2. Add luciferase substrate (e.g., Bright-Glo™): Add 100 μL of luciferase detection reagent to each well and gently pipette to mix. Incubate in the dark for 10–15 minutes (at room temperature).

[0120] 3. Detect fluorescence signal: Read the luminescence value (RLU, relative luminescence units) using a microplate reader.

[0121] 4) Data analysis: calculation of target cell lysis rate

[0122] Lysis rate (%) = (RLU of control group - RLU of experimental group) / RLU of control group × 100%

[0123] Wherein: RLU of the control group = fluorescence intensity of the group without CAR-T cells; RLU of the experimental group = fluorescence intensity after CAR-T cells killing.

[0124] 5) Analysis of results: Plot the E:T ratio vs. cell lysis rate curve to assess the cytotoxicity of CAR-T cells. Compare the cytotoxicity of CAR-T cells with different CAR structures (e.g., different scFv designs, co-stimulatory molecules) or under different culture conditions.

[0125] The results are as follows Figure 2 As shown in the figure, compared with CIgG-MSLN CAR-T or CIgG-CLDN18.2 CAR-T cells, CAR-T cells coupled with PD-1sc-Fv and IL-21 have the strongest BxPC-3 lytic and killing ability for target cells.

[0126] Example 3 Cytokine release assay

[0127] CAR-T cell IFN-γ, PD-1, and IL-21 secretion levels were detected. The specific experimental steps are as follows:

[0128] 1) Cell co-culture

[0129] 1. Target cell plating: In a 96-well U-bottom cell culture plate, add 100 μL of target cell suspension (10 5 cells / well) at 37°C for 2–4 hours to allow the cells to adhere.

[0130] 2. Add CAR-T cells: Add 100 μL of CAR-T cells at different effector-target ratios (E:T = 10:1, 5:1, 2.5:1, 1.25:1) to a final volume of 200 μL. Establish control groups: target cell control (no CAR-T), CAR-T cell control (no target cells), and blank control (no cells, culture medium only).

[0131] 3. Incubate cells at 37°C, 5% CO2 for 16–24 hours. Collect the cell culture supernatant and centrifuge at 1000 × g for 5 minutes. Take 50–100 μL of the supernatant for ELISA analysis.

[0132] 2) ELISA test

[0133] 1. Coating Antibody: Add 100 μL of cytokine capture antibody (e.g., IFN-γ antibody) to each well of a 96-well ELISA plate at a concentration of 1–2 μg / mL in carbonate buffer (pH 9.6). Incubate overnight at 4°C (or 2 hours at 37°C).

[0134] 2. Wash: Wash each well three times with PBS + 0.05% Tween-20, 200 μL each time, to remove unbound antibodies.

[0135] 3. Blocking: Add 200 μL of 5% BSA / PBS to each well and incubate at room temperature for 1 hour to reduce nonspecific adsorption. Wash three times with 200 μL each time.

[0136] 3) Cytokine detection

[0137] 1. Add samples and standards: Add 100 μL of cell culture supernatant or standard (0–2000 pg / mL, 2-fold serial dilution) to each well. Incubate at room temperature for 2 hours or at 4°C overnight (for increased sensitivity). Wash five times.

[0138] 2. Add enzyme-labeled secondary antibody (HRP-labeled): Add 100 μL of HRP-conjugated secondary antibody (1:2000 dilution) to each well.

[0139] Incubate at room temperature for 1 hour and wash 5 times.

[0140] 3. TMB color development: Add 100 μL of TMB substrate to each well and incubate in the dark for 15–30 minutes. After the reaction is complete, add 50 μL of stop solution (2M H2SO4) to each well to terminate the reaction.

[0141] 4. Reading: Read the absorbance (OD value) at a wavelength of 450 nm.

[0142] 4) Data Analysis

[0143] 1. Draw a standard curve (standard concentration vs. OD value) and calculate cytokine concentrations using four-parameter regression (4PL).

[0144] 2. Calculate CAR-T cytokine secretion (pg / mL) and compare secretion levels under different effector-target ratios and CAR-T designs.

[0145] The results are as follows Figure 3 As shown in Figure 3, CAR-T cells coupled with PD-1sc-Fv and IL-21 released the highest level of IFN-γ compared with CIgG-MSLN CAR-T or CIgG-CLDN18.2 CAR-T cells.

[0146] Example 4 CAR-T in vivo efficacy experiment

[0147] 1) Establishment of mouse tumor model

[0148] 1. Cell preparation: Take tumor cells in logarithmic growth phase and resuspend them in PBS to a concentration of 5 × 10 6 cells / mL. Pre-cooled 1:1 Matrigel was added to mix the cell suspension to ensure tumor growth.

[0149] 2. Tumor inoculation: 100 μL of cell suspension (5 × 10 6 Observe for 5–10 days and start CAR-T cell therapy when the tumor grows to 100–150 mm³.

[0150] 2) CAR-T cell therapy

[0151] 1. CAR-T cell infusion: CAR-T cells were revived the day before freezing and activated for 24 hours under IL-2 (100 U / mL) cytokine stimulation. 100–200 μL of CAR-T cell suspension (5 × 10 6 cells / mouse).

[0152] 2. Experimental groups: non-transduced T cell control group (Mock T cells), PBS group (negative control), CIgG-MSLN CAR-T and CIgG-CLDN18.2 CAR-T cell groups, and CIgG-MSLN CAR-T and CIgG-CLDN18.2 CAR-T cell groups secreting PD-1Ab21 (PD-1 and IL21 fusion protein).

[0153] 3) In vivo anti-tumor evaluation

[0154] Tumor growth monitoring: Tumor volume was monitored every 3–4 days using a caliper. The formula was: Tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0155] 4) Data Analysis: Survival Curves (Kaplan-Meier Analysis)

[0156] The results are as follows Figure 4 As shown, CAR-T cells expressing PD-1Ab21 have significant efficacy in mouse tumor models and are superior to general CAR-T cells expressing dual-target chimeric antigen receptors.

[0157] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make several modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the appended claims, and the specification and drawings may be used to interpret the claims.

Claims

1. A dual-target chimeric antigen receptor, characterized in that: Its extracellular domain includes CIgG scFv-CLDN18.2 scFv from N-terminus to C-terminus, wherein "-" is a connecting peptide; it also contains a hinge region, a transmembrane region and an intracellular signaling region; and the C-terminus of the intracellular signaling region is connected to PD-1 scFv and IL-21 directly fused from N-terminus to C-terminus via GSG and P2A peptides; wherein: The heavy chain variable region of the CIgG scFv is shown in SEQ ID NO: 26, and the light chain variable region is shown in SEQ ID NO: 27; The amino acid sequences of the heavy chain complementary determining regions CDR1 to 3 of the CLDN18.2 scFv are shown in SEQ ID NOs: 13 to 15, and the amino acid sequences of the light chain complementary determining regions CDR1 to 3 are shown in SEQ ID NOs: 16 to 18. The amino acid sequences of the heavy chain complementary determining regions CDR1 to 3 of the PD-1 scFv are shown in SEQ ID NOs: 19 to 21, and the amino acid sequences of the light chain complementary determining regions CDR1 to 3 are shown in SEQ ID NOs: 22 to 24. The amino acid sequence of IL-21 is shown in SEQ ID NO:

25.

2. The dual-target chimeric antigen receptor according to claim 1, characterized in that 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 of the costimulatory regions selected from the following proteins: CD28, 4-1BB, OX40, ICOS, CD27, DAP10, DAP12, 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 according to 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 T cells, NK cells, macrophages and dendritic cells.

7. A pharmaceutical composition, characterized in that Comprising the immune cell according to claim 5 or 6.

8. Use of the immune cells according to claim 5 or 6 in the preparation of a medicament for killing CIgG-positive and CLDN18.2-positive tumor cells; the tumor is pancreatic cancer.

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