Chimeric antigen receptors expressing cd47-cd28 costimulatory switching receptors and uses thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a chimeric antigen receptor expressing a CD47-CD28 co-stimulating conversion receptor and application thereof. The chimeric antigen receptor simultaneously and efficiently expresses a CAR targeting CD19 or MSLN and a CD47-CD28 chimera, so that a T cell simultaneously obtains tumor-targeting recognition capability, anti-macrophage phagocytosis capability and enhanced co-stimulating signals. The CD47-CD28 co-stimulating conversion receptor converts a CD47-mediated 'don't eat me' inhibiting signal into a CD28-mediated activating signal, significantly improves the persistence and killing efficiency of a CAR-T cell in a tumor microenvironment, and realizes good anti-tumor effects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a chimeric antigen receptor expressing a CD47-CD28 co-stimulatory switching receptor and its applications. Background Technology

[0002] In recent years, CAR-T cells have made groundbreaking progress in the treatment of relapsed / refractory B-cell-related hematological malignancies. However, in clinical cases of CD19-targeted CAR-T cell therapy, nearly half of the patients still experience disease relapse / drug resistance after treatment. The main reasons for this are insufficient long-term survival of adoptive cells due to macrophage-mediated T-cell phagocytosis and immune escape caused by decreased or absent tumor antigen expression, which are the main factors affecting clinical efficacy.

[0003] CD47, known as integrin-associated protein, is a five-transmembrane protein of the immunoglobulin superfamily that binds to integrins, thromboretin, and signal regulatory protein α (SIRPα). SIRPα is primarily expressed on macrophages and other myeloid cells. CD47 binding to SIRPα inhibits macrophage phagocytosis through phosphorylation of immune receptor tyrosine inhibitory motifs and recruitment of phosphatases SHP1 and SHP2. This protein is highly expressed in a variety of solid tumors, making it an attractive target for immunotherapy. However, CD47 is also expressed on T cells. Although systemic blockade of the CD47-SIRPα axis has mediated antitumor effects in several preclinical models, its clinical benefit as a single agent and in solid cancers remains lacking.

[0004] Recent studies have revealed that a key reason for insufficient persistence of CAR-T cells lies in reduced CD47 expression in CAR-T cells. This leads to rapid clearance of T cells expressing chimeric antigen receptors or engineered T cell receptors mediated by macrophages after infusion into clinical patients. Previous research has shown that CD47-knockout CAR-T cells cannot effectively proliferate and survive in vivo, and become sensitive to macrophage phagocytosis. In contrast, CD47-overexpressing CAR-T cells exhibit longer survival time and stronger proliferative capacity in mice compared to unmodified conventional CAR-T cells. This enhanced persistence directly translates into better tumor control and longer host survival. Therefore, CD47 expression is crucial for the survival of CAR-T cells in vivo.

[0005] CD47 provides a "don't eat me" signal to T cells, acting as a protective factor to ensure the persistence of T cells in vivo. When CD47 on the surface of CAR-T cells is blocked by antibodies or knocked out by genes, they are rapidly cleared by macrophages in the body, leading to treatment failure. Overexpression of CD47 on CAR-T cells helps resist phagocytosis by macrophages in the tumor microenvironment, thereby prolonging the duration of their anti-tumor effect. Therefore, increasing CD47 expression and engineering its expression can protect T cells and enhance anti-tumor immunity, potentially becoming an effective way to solve the problem of CAR-T cell persistence in vivo.

[0006] However, the anti-tumor effect of CD47 overexpression alone in T cells is limited. One common mechanism of relapse after CAR-T therapy is tumor cell immune escape caused by reduced or lost antigen density on tumor cells. This problem is prevalent in the clinical treatment of various malignant tumors (such as B-ALL, LBCL, etc.). For example, clinical data show that in 30-95% of patients with B-cell acute lymphoblastic leukemia who have progressed, reduced or absent expression of CD19 antigen on the surface of tumor cells can be detected. The expression density of tumor cell surface antigens is closely related to the efficacy of treatment. Decreased or absent antigen expression leads to tumor immune escape and relapse / drug resistance, which has become a key problem that must be solved in CAR-T cell therapy.

[0007] Given the persistent problems of long-term T-cell survival and tumor immune escape in CAR-T cell therapy, there is an urgent need to provide a more effective treatment product and method to address the relapse dilemma in immunotherapy for hematologic and solid tumors. Summary of the Invention

[0008] The purpose of this invention is to provide a chimeric antigen receptor expressing a CD47-CD28 co-stimulatory switching receptor and its application.

[0009] Studies have confirmed that constructing an inhibitory-to-stimulatory (ITS) co-stimulatory switching receptor in the molecular design of CARs can effectively address the problem of immune escape caused by decreased or absent tumor antigen expression. In this invention, the extracellular segment of the CAR uses an inhibitory receptor extracellular segment (CD47), and the intracellular segment uses a stimulatory receptor intracellular segment (CD28). During the effect, the extracellular molecule binds to the inhibitory ligand (SIRPa), activating the "don't eat me" signaling axis to prevent macrophage-mediated T cell phagocytosis in vivo. At the same time, it also transmits a continuous stimulatory signal (CD28) into the intracellular space, further enhancing the CAR's targeted recognition and killing function for tumor cells with low antigen density, thus solving the immune escape problem while maintaining the persistence of CAR-T cells.

[0010] In order to achieve the objectives of the present invention, in a first aspect, the present invention provides a chimeric antigen receptor expressing a CD47-CD28 co-stimulatory switching receptor, the chimeric antigen receptor comprising (a) a CAR; and (b) a CD47-CD28 co-stimulatory switching receptor;

[0011] The CD47-CD28 co-stimulatory switching receptor comprises an extracellular domain of the CD47 protein or a fragment thereof capable of binding SIRPα; and / or an intracellular signaling domain of the CD28 protein or a fragment thereof capable of transducing co-stimulatory signals.

[0012] Furthermore, the CAR and the CD47-CD28 co-stimulatory switching receptor also contain a self-cleaving peptide sequence.

[0013] Preferably, the self-cleaving peptide is a T2A peptide (structure Furin-linker-spacer-T2A), and its amino acid sequence is shown in SEQ ID NO:5.

[0014] Furthermore, the structure of the CD47-CD28 co-stimulatory switching receptor is selected from any of the following:

[0015] (i) A fusion protein containing the full-length CD47 protein and the CD28 intracellular domain (47OE28S CAR).

[0016] (ii) A fusion protein containing the CD47 extracellular domain, the CD47 transmembrane domain and the CD28 intracellular domain (47Δ28S CAR).

[0017] (iii) A fusion protein containing the CD47 extracellular domain, the CD28 transmembrane domain and the CD28 intracellular domain (47ΔΔ28L CAR).

[0018] The structure of the CD47-CD28 co-stimulatory switching receptor is preferably (i).

[0019] Optionally, the CD47-CD28 co-stimulatory switching receptor comprises the full-length CD47 (47OE CAR).

[0020] Preferably, the amino acid sequence of the CD47-CD28 co-stimulatory switching receptor is shown in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

[0021] Furthermore, the CAR is a CD19-targeting CAR or a mesothelin (MSLN)-targeting CAR, and their amino acid sequences are shown in SEQ ID NO:1 or 3, respectively.

[0022] In a second aspect, the present invention provides a nucleic acid construct encoding the chimeric antigen receptor or a biological material containing the nucleic acid construct.

[0023] The biological materials include, but are not limited to, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0024] Thirdly, the present invention provides a lentiviral vector comprising the nucleic acid construct, wherein the nucleic acid construct is operatively linked to a promoter (e.g., the SFFV2 promoter) capable of driving expression in T cells.

[0025] Fourthly, the present invention provides a genetically modified T cell comprising the nucleic acid construct or the lentiviral vector and expressing the CAR and the CD47-CD28 co-stimulatory switching receptor.

[0026] Fifthly, the present invention provides the use of the nucleic acid construct, the lentiviral vector, or the T cell in the preparation of antitumor drugs for enhancing the persistence and tumor-killing efficacy of T cells in the tumor microenvironment.

[0027] In a sixth aspect, the present invention provides the application of the nucleic acid construct, the lentiviral vector, or the T cell in CAR-T cell therapy.

[0028] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0029] (I) This invention provides a chimeric antigen receptor system expressing a CD47-CD28 co-stimulatory switching receptor and its use in CAR-T cell therapy. The chimeric antigen receptor simultaneously and efficiently expresses a CAR targeting CD19 or MSLN and a CD47-CD28 chimera, enabling T cells to simultaneously acquire tumor-targeting recognition ability, anti-macrophage phagocytic ability, and enhanced co-stimulatory signals. The CD47-CD28 co-stimulatory switching receptor converts the CD47-mediated "don't eat me" inhibitory signal into a CD28-mediated activation signal, significantly improving the persistence and killing efficacy of CAR-T cells in the tumor microenvironment.

[0030] (ii) The present invention also provides a lentiviral vector that simultaneously expresses a CAR targeting CD19 or MSLN and a CD47-CD28 co-stimulatory switching receptor, so that the T cells modified by this gene can not only target and recognize tumor cells expressing CD19 or MSLN, but also avoid being phagocytosed by macrophages in vivo, and further activate the immune response of T cells, enhance their persistence in vivo, and achieve good anti-tumor effects.

[0031] (III) Furthermore, this invention optimizes key promoter elements and CD47-CD28 co-stimulatory switching receptors of different lengths in the vector, enabling the target genes to be efficiently expressed in human T cells. By comparing the functions of CD47 and CD28 domain fusion proteins of different lengths in the vector, the tumor-killing efficiency of CD19 or MSLN CARs and CD47-CD28 chimeras in human T cells is optimized. Attached Figure Description

[0032] Figure 1 In Embodiment 3 of the present invention, the target is CD19 or MSLN. Figure 1 A schematic diagram of the CAR structure expressing the CD47-CD28 chimeric antibody.

[0033] Figure 2 This is a schematic diagram of the lentivirus vector structure in Embodiment 3 of the present invention, wherein T2A represents the combination of Furin-linker-spacer-T2A.

[0034] Figure 3 The percentage of CD19 or MSLN CAR expressed by peripheral blood T cells after infection with 10 lentiviral vectors in Example 3 of this invention.

[0035] Figure 4 The percentage of CD47 expression in peripheral blood T cells after infection with 10 lentiviral vectors in Example 3 of this invention.

[0036] Figure 5 This study analyzed the IFNγ production of CAR-T cells prepared by incubating peripheral blood T cells with target cells after infection with 10 lentiviral vectors, as described in Example 4 of this invention. The target cells for CD19-targeting CAR-T cell killing were human Burkitt's lymphoma Raji cell line, and the target cells for MSLN-targeting CAR-T cell killing were human non-small cell lung cancer A549 cell line.

[0037] Figure 6 This study analyzes the enhanced cell-killing effect of CAR-T cells targeting CD19 and simultaneously expressing CD47-CD28 co-stimulatory switching receptors on Raji cells in Example 4 of this invention.

[0038] Figure 7 This study analyzes the cell-killing enhancement effect of CAR-T cells targeting MSLN and simultaneously expressing CD47-CD28 co-stimulatory switching receptors on A549 cells in Example 4 of the present invention.

[0039] Figure 8 This study analyzes the in vivo antitumor enhancement effect of CAR-T cells targeting CD19 and simultaneously expressing CD47-CD28 co-stimulatory switching receptors on Raji cells in mice, as described in Example 5 of this invention.

[0040] Figure 9 This study analyzes the in vivo antitumor enhancement effect of CAR-T cells targeting MSLN and simultaneously expressing CD47-CD28 co-stimulatory switching receptors on A549 cells in mice, as described in Example 5 of this invention.

[0041] Figure 10 This study analyzes the enhanced cell activation and killing effects of CAR-T cells targeting CD19 and simultaneously expressing CD47-CD28 co-stimulatory switching receptors in tumors with low antigen expression, as described in Example 6 of this invention.

[0042] Figure 11 This study analyzes the enhanced cell activation and killing effects of CAR-T cells targeting MSLN and simultaneously expressing CD47-CD28 co-stimulatory switching receptors in tumors with low antigen expression, as described in Example 6 of this invention.

[0043] Figure 12 This study analyzes the antitumor enhancement effect of CAR-T cells that target CD19 or MSLN and simultaneously express CD47-CD28 co-stimulatory switching receptors under continuous antigen stimulation conditions, as described in Example 6 of this invention.

[0044] In the picture, express P <0.05, express P <0.01, express P <0.001, express P <0.0001, ns indicates no significant difference. Detailed Implementation

[0045] The present invention aims to provide a chimeric antigen receptor system expressing a CD47-CD28 co-stimulatory switching receptor and its use in CAR-T cell therapy.

[0046] The present invention adopts the following technical solution:

[0047] This invention provides a lentiviral vector that simultaneously expresses a CAR targeting CD19 or MSLN and a CD47-CD28 co-stimulatory switching receptor.

[0048] CD47 is an integrin-associated protein that can bind to SIRPa expressed by macrophages, forming a "don't eat me" signaling axis to prevent phagocytosis by macrophages in vivo. CD28 is a major co-stimulatory receptor for T cells and an essential element for T cell activation. Studies have found that chimeric proteins fusing the extracellular domain of CD28 and the intracellular domain of PD1 can exhibit activity similar to that of natural PD1 molecules in inhibiting T cell immune responses. In this invention, the extracellular domain of CD47 and the transmembrane and intramembrane domains of CD28 are fused. This chimeric protein can both bind to SIRPa, preventing macrophage-mediated T cell phagocytosis in vivo, and promote T cell activation, enhancing CAR-T cell cytokine release, promoting cell proliferation, and enhancing cell killing function. In in vivo experiments, reinfusion of T cells expressing the CD47-CD28 chimeric switching receptor into tumor-bearing mice completely eliminated the tumor. Further research revealed that the CD47-CD28 co-stimulatory switching receptor can convert the "don't eat me" signal transmitted by the CD47-SIRPα axis into a sustained stimulatory signal for T cell activation. Furthermore, CD47 is highly expressed in various hematologic and solid tumors. The CD47-CD28 co-stimulatory switching receptor, with the help of signals provided by CD47, not only avoids phagocytosis by macrophages in vivo but also further enhances the anti-tumor activity of T cells. In vivo and in vitro experiments have shown that cells expressing the switching receptor have stronger persistence and anti-tumor function compared to CAR-T cells expressing only CD47. The above experiments demonstrate that the different functional regions of the CD47-CD28 chimeric switching receptor protein can be effectively compatible, forming a novel artificial receptor that can regulate and enhance the anti-tumor immune response of T cells.

[0049] This invention constructs a CAR targeting CD19 or MSLN and a CD47-CD28 co-stimulatory switching receptor together in the same lentiviral vector and transfects it into patient T cells. The genetically modified T cells target and recognize tumor cells expressing CD19 or MSLN. At the same time, CD47 expression alone only provides a "don't eat me" signal, while CD28 alone provides a co-stimulatory signal. The fusion of the two achieves signal switching and synergistic enhancement, which can not only avoid being phagocytosed by macrophages in vivo, but also further activate the immune response of T cells, enhance their persistence in vivo, and achieve good anti-tumor effects.

[0050] Furthermore, when multiple target genes are expressed on the same vector, the activity and expression efficiency of the expressed proteins decrease. To promote efficient expression of both CARs targeting CD19 or MSLN and CD47-CD28 co-stimulatory switching receptors in T cells, this invention further optimizes the type of lentiviral vector promoter and the connection method of these two target genes, aiming to achieve optimal infection efficiency on T cells.

[0051] To this end, the present invention enables two target genes to share a single promoter SFFV2 in a lentiviral expression vector and to link them using Furin-linker-spacer-T2A, so that the lentivirus produced by the lentiviral vector can efficiently infect peripheral blood T cells, and the CAR targeting CD19 or MSLN and the CD47-CD28 chimera can be expressed on the surface of the T cells respectively.

[0052] More specifically, the lentiviral vector carries two target genes. First, lentivirus is generated by transfecting 293FT cells with a transfection reagent. Then, the lentivirus infects peripheral blood T cells, thereby integrating the two target genes into the T cell genome, thus achieving the expression of the two target genes in T cells.

[0053] This invention provides a lentiviral vector that simultaneously encodes and expresses a chimeric antigen receptor (CAR) and a CD47-CD28 co-stimulatory conversion receptor. The CD47-CD28 co-stimulatory conversion receptor comprises four chimeric construction methods: full-length CD47 (47OE CAR), CD47 extracellular domain + CD28 transmembrane and intracellular domains (47ΔΔ28L CAR), CD47 extracellular domain and transmembrane region + CD28 intracellular domain (47Δ28S CAR), and full-length CD47 + CD28 intracellular domain (47OE28S CAR). The amino acid sequences of the CD47-CD28 co-stimulatory conversion receptor are shown in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13. The CD47-CD28 co-stimulatory conversion receptor includes, but is not limited to, the structures shown in the above sequences, and may also include other functionally equivalent variants.

[0054] Furthermore, a CAR targeting CD19 or MSLN and a CD47-CD28 co-stimulatory switching receptor are simultaneously expressed. The amino acid sequence of the CD19 CAR is shown in SEQ ID NO:1, and the amino acid sequence of the MSLN-targeting CAR is shown in SEQ ID NO:3. The nucleotide sequences are ligated using Furin-linker-spacer-T2A to obtain a fusion gene, which is then constructed into a lentiviral vector. The amino acid sequence of Furin-linker-spacer-T2A is shown in SEQ ID NO:5.

[0055] The Furin-linker-spacer-T2A, referred to as T2A peptide in this invention, is... Figure 1 The Chinese character is represented as T2A.

[0056] Due to the cleavage of Furin and the T2A peptide, the C-terminus of the upstream protein (CD47 or CD47-CD28 co-stimulatory switching receptor) contains only 1 to 3 additional amino acid residues, while the N-terminus of the downstream protein (Flag) contains only an additional proline residue. Studies have shown that these amino acid residues do not affect the normal expression of these two proteins.

[0057] Furthermore, the nucleotide sequence of the CAR expressing CD19 is shown in SEQ ID NO:2; the nucleotide sequence of the CAR expressing MSLN is shown in SEQ ID NO:4; the nucleotide sequence of the Furin-linker-spacer-T2A is shown in SEQ ID NO:6; and the nucleotide sequences of the CD47-CD28 co-stimulatory switching receptor are shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14.

[0058] Furthermore, a promoter is inserted upstream of the fusion gene, which can be efficiently expressed in T cells.

[0059] Preferably, the lentiviral vector is selected from lentiviral vectors with the SFFV2 promoter. In this way, there is no need to replace the promoter of the lentiviral vector, and the SFFV2 promoter (SEQ ID NO:15) that comes with the lentiviral vector can be used directly.

[0060] More preferably, the promoter is located 308-519 bp upstream of the fusion gene.

[0061] The present invention also provides a method for constructing the aforementioned lentiviral vector, comprising the following steps:

[0062] (1) The nucleotide sequence of the CAR expressing CD19 or MSLN and the CD47-CD28 co-stimulatory switching receptor is linked together using Furin-linker-spacer-T2A to synthesize a fusion gene. When synthesizing the fusion gene, XbaI and SalI restriction sites are respectively included at both ends, and it is loaded onto a plasmid.

[0063] (2) The plasmid described in step (1) was digested with XbaI and SalI, and the target gene fragment was recovered by gel digestion;

[0064] (3) The original lentiviral vector was digested with XbaI and SalI, and the vector fragments were recovered by gel digestion.

[0065] (4) Use DNA ligase to ligate the target gene fragment recovered in step (2) with the vector fragment recovered in step (3) to obtain the final product.

[0066] To further improve the expression efficiency of the two target genes, this invention also compared several promoters that are highly efficient in T cells. The study found that the SFFV2 promoter has significant advantages over other commonly used strong promoters in the field (such as CMV, EF1α, etc.). Therefore, this invention preferably uses a lentiviral original vector that inherently carries the SFFV2 promoter.

[0067] The present invention further provides the application of the aforementioned lentiviral vector in antitumor therapy, and its application in the preparation of antitumor drugs.

[0068] The raw materials or reagents involved in this invention are all commercially available products, and the operations involved are all routine operations in the field unless otherwise specified.

[0069] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined to obtain specific implementation methods.

[0070] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0071] The amino acid sequence of the CAR targeting CD19 is shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2; the amino acid sequence of the CAR targeting MSLN is shown in SEQ ID NO:3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:4; the amino acid sequence of the T2A peptide is shown in SEQ ID NO:5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:6; the amino acid sequence of CD47 is shown in SEQ ID NO:7, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:8; the amino acid sequence of 47OE28S is shown in SEQ ID NO:9, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:10; the amino acid sequence of 47Δ28S is shown in SEQ ID NO:11, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:12; the amino acid sequence of the 47ΔΔ28L CAR is shown in SEQ ID NO:13, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:14.

[0072] Example 1: Construction of a T-cell lentiviral vector simultaneously expressing CD19 or MSLN CAR and CD47-CD28 co-stimulatory switching receptor

[0073] 1. Raw materials

[0074] The original vector pCDH-EF1-Luc2-T2A-tdTomato was purchased from Wuhan Miaoling Biotechnology Co., Ltd. The inventors replaced its promoter with SFFV2 to construct the vector pCDH-SFFV2-Luc2-T2A-tdTomato, and used this as a basis for subsequent constructions.

[0075] XbaI and SalI endonucleases were purchased from New England Biolabs (Beijing) LTD. X-VIVO15 medium was purchased from Lonza. PEI was purchased from Sigma. IL-2 and OKT3 were purchased from ACRO Biosystems. CD28 antibody was purchased from Tongli Haiyuan. 293FT cells and Raji cells were purchased from the American Type Culture Collection (ATCC). Flow cytometry antibodies for CD3, CD45RA, and CCR7 were purchased from BD; FLAG antibody was purchased from Sigma; CD19 protein was purchased from ACRO Biosystems. ELISA kits for IFN-γ and IL-7 were purchased from R&D Systems and Ecosai Biotechnology Co., Ltd., respectively.

[0076] 2. Construction of the carrier

[0077] (1) The nucleotide sequences encoding the CD47-CD28 co-stimulatory switching receptor and the CAR targeting CD19 or MSLN were linked using Furin-linker-spacer-T2A to synthesize a fusion gene sequence. During synthesis, XbaI and SalI restriction sites were introduced at both ends of the gene sequence, and it was cloned into the pUC57 vector. To facilitate subsequent detection of the fusion protein, a Flag tag sequence was added before the coding sequence of CD19 or MSLN.

[0078] (2) The pUC57 vector containing the target gene was double-digested with XbaI and SalI, and the target gene fragment was then separated and recovered by agarose gel electrophoresis.

[0079] (3) The original vector pCDH-SFFV2-Luc2-T2A-tdTomato was double-digested with XbaI and SalI. After separation by agarose gel electrophoresis, the vector backbone fragment with a size of about 6.5 kb was recovered.

[0080] (4) Use DNA ligase to ligate the target gene fragment recovered in step (2) with the vector fragment recovered in step (3) to obtain the desired recombinant vector.

[0081] Example 2: Preparation of Lentiviral Virus

[0082] Lentiviral virus was prepared by transfecting the recombinant lentiviral vector described in Example 1 into 293FT cells using a PEI-based transfection method and lentiviral packaging system. The specific steps are as follows:

[0083] The lentiviral vector and packaging plasmid mixture (pLP1:pLP2:VSVG in a mass ratio of 1:1:1) were mixed at a mass ratio of 1:3 and added to 500 μL of serum-free Opti-MEM medium. The mixture was then vortexed to ensure thorough mixing. Separately, 35 μg of PEI was added to 500 μL of serum-free Opti-MEM medium and vortexed to mix. Subsequently, 500 μL of the plasmid mixture was mixed with 500 μL of the PEI solution and added to 293FT cells with approximately 90% confluence. Forty-eight hours after transfection, the cell supernatant was collected, concentrated by ultracentrifugation, and resuspended in an appropriate amount of culture medium to obtain concentrated lentivirus.

[0084] Example 3: Preparation of CAR-T cells expressing CD47-CD28 co-stimulatory switching receptor

[0085] This embodiment constructs multiple co-stimulatory switching receptors based on CD47 and CD28 fragments of different lengths, verifies their function by preparing CAR-T cells, and then screens for the optimal co-stimulatory switching receptor structure.

[0086] This embodiment compares and analyzes four chimeric construction methods based on two target genes: full-length CD47 (47OE CAR), CD47 extracellular domain + CD28 transmembrane and intracellular domains (47ΔΔ28L CAR), CD47 extracellular domain and transmembrane region + CD28 intracellular domain (47Δ28S CAR), and full-length CD47 + CD28 intracellular domain (47OE28S CAR). Figure 1 and Figure 2 ).

[0087] The designed CAR genes targeting CD19 or MSLN are then commissioned to a company for gene synthesis. Figure 1 Ten target gene sequences were constructed into the pCDH-SFFV2-Luc2-T2A-tdTomato vector by double digestion with XbaI and SalI. The ten constructed lentiviral vectors were then packaged into lentiviruses and used to infect peripheral blood T cells. Staining with an antibody against Flag protein was performed, and the results were analyzed by flow cytometry. Figure 3 As shown in the figure. In addition, the expression level of CD47 protein in various CAR-T cells was also examined, and the results are as follows. Figure 4 As shown.

[0088] Example 4: CAR-T cells expressing the CD47-CD28 co-stimulatory switching receptor exhibit enhanced in vitro antitumor effects and cytotoxic functions.

[0089] Raji and A549 cells were used as target cells and co-incubated with CD19 or MSLN CAR-T cells expressing CD47-CD28 co-stimulatory switching receptors, respectively. The specific recognition and cell killing functions of CAR-T cells were then detected.

[0090] The results are as follows Figure 5 As shown, all CAR-T cell groups exhibited significant anti-tumor function compared to control Mock T cells. More importantly, compared to conventional CAR-T cells overexpressing CD47, 47ΔΔ28L, 47Δ28S, and 47OE28S CAR-T cells expressing the CD47-CD28 co-stimulatory switching receptor had higher levels of IFNγ production, demonstrating stronger specific target cell recognition and killing effects.

[0091] like Figure 6 and Figure 7 In the detection of cell-killing effects on target cells, compared with traditional CAR-T cells targeting CD19 or MSLN and traditional CAR-T cell groups overexpressing CD47, the 47ΔΔ28L, 47Δ28S, and 47OE28S CAR-T cells expressing the CD47-CD28 co-stimulatory switching receptor of this product all showed more significant cell-killing functions.

[0092] Example 5: CAR-T cells expressing CD47-CD28 co-stimulatory switching receptors exhibit enhanced in vivo anti-tumor function.

[0093] Based on the in vitro confirmation of the antitumor efficacy of CAR-T cells expressing CD47-CD28 co-stimulatory switching receptors, we further verified their in vivo antitumor effects and safety in the NXG tumor-bearing mouse model.

[0094] In vivo antitumor experiments of CAR-T cells targeting CD19 to express CD47-CD28 co-stimulatory switching receptors, such as... Figure 8 As shown. Among them, Figure 8 Figure A shows the experimental procedure; the experimental results showed that, compared with the Mock and conventional CD19-targeted CAR-T groups, all CAR-T cell groups expressing CD47-CD28 co-stimulatory switching receptors had more significant anti-tumor effects, especially the CAR-T cells in the full-length CD47 + CD28 intracellular domain (47OE28S CAR) group, which achieved a 40-day sustained and complete elimination of tumor cells in mice. Figure 8 Similarly, in in vivo experiments using the solid tumor cell line A594 as the target cell, conclusions were obtained consistent with those in the Raji mouse model of hematologic malignancies (B). Figure 9(A and B). Therefore, 47OE28S CAR can be selected as the optimal structural molecule for overexpressing the CD47-CD28 co-stimulatory switching receptor.

[0095] Example 6: CAR-T cells expressing CD47-CD28 co-stimulatory switching receptors showed significantly enhanced killing effect against tumors with low antigen expression.

[0096] To investigate the function of novel co-stimulatory transducer-expressing CAR-T cells under conditions of low tumor antigen expression, we transduced wild-type K562 and H1299 cells with lentivirus and then sorted cells expressing different antigen densities. This yielded K562-CD19 cell lines expressing different CD19 densities and H1299-MSLN cell lines expressing different MSLN densities. Figure 10 A, and Figure 11 A).

[0097] Compared to traditional CAR T cells, 47OE28s CAR T cells exhibited stronger cytotoxic and cytokine secretion activities, regardless of whether they were co-incubated with high-density or low-density target cells. Figure 10 B and C, and Figure 11 (B and C). Furthermore, we quantified CD107a (a cytotoxic degranulation marker used to characterize cytotoxicity levels). The 47OE28s group showed significantly elevated CD107a secretion levels, indicating superior cytotoxicity. Figure 10 D, and Figure 11 D).

[0098] To simulate continuous antigen exposure in vivo, we established a chronic antigen stimulation model ( Figure 12 (A). Under three rounds of stimulation, in both hematologic malignancy and solid tumor models, 47OE28s CAR T cells showed stronger cytotoxicity and higher levels of cytokine secretion (IFN-γ, IL-2, and TNF-α) compared to traditional second-generation CAR-T cells and CAR T cells overexpressing CD47. Figure 12 ,BD).

[0099] Overall, 47OE28S CAR-Ts exhibited strong in vitro antitumor effects against target cells with different antigen densities, especially against tumor cells with low antigen expression, providing a new approach to improve clinical efficacy.

[0100] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A chimeric antigen receptor expressing a CD47-CD28 co-stimulatory switching receptor, characterized in that, The chimeric antigen receptor comprises (a) a CAR; and (b) a CD47-CD28 co-stimulatory switching receptor; the CAR and the CD47-CD28 co-stimulatory switching receptor also contain a self-cleaving peptide sequence; The structure of the CD47-CD28 co-stimulatory switching receptor is: a fusion protein comprising the full-length CD47 protein and the intracellular domain of CD28; The amino acid sequence of the CD47-CD28 co-stimulatory switching receptor is shown in SEQ ID NO:

13.

2. The chimeric antigen receptor according to claim 1, characterized in that, The CAR is a CD19-targeting CAR, and its amino acid sequence is shown in SEQ ID NO:

1.

3. A nucleic acid construct encoding the chimeric antigen receptor of claim 1 or 2, or a biological material containing the nucleic acid construct.

4. A lentiviral vector, characterized in that, It comprises the nucleic acid construct of claim 3, wherein the nucleic acid construct is operatively linked to a promoter capable of driving expression in T cells.

5. A genetically modified T cell, characterized in that, It comprises the nucleic acid construct of claim 3 or the vector of claim 4, and expresses the CAR and the CD47-CD28 co-stimulatory switching receptor.

6. The use of the nucleic acid construct of claim 3, the vector of claim 4, or the T cell of claim 5 in the preparation of an antitumor drug for enhancing the persistence and tumor-killing efficacy of T cells in the tumor microenvironment.

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