Chimeric antigen receptors targeting cd20, gene expression constructs and uses thereof

Abstract

This invention belongs to the field of biomedical technology and discloses a chimeric antigen receptor targeting CD20, its gene expression construct, and its applications. The chimeric antigen receptor provided by this invention specifically includes an amino acid fragment with the sequence shown in SEQ ID NO:29 or a variant fragment having at least 85% sequence identity with SEQ ID NO:29. Its functional fragments, including the signal peptide, antigen-binding region, hinge region, transmembrane region, intracellular co-stimulatory domain, and signal transduction domain, form an organic whole, working synergistically to endow the chimeric antigen receptor with good CD20-targeting binding, T-cell activation, and cell-killing effects, demonstrating excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a chimeric antigen receptor targeting CD20, its gene expression construct, and its applications. Background Technology

[0002] CD20 is a non-glycosylated transmembrane phosphoprotein belonging to the MS4A family encoded by the MS4A1 gene on chromosome 11. It is stably and highly expressed on the surface of most malignantly proliferating and abnormally activated CD20-positive B cells, but not expressed in normal hematopoietic stem cells, plasma cells, and other unrelated cells. This makes CD20 a core target for precision-targeted intervention in B-cell-related diseases. Currently, several monoclonal antibodies and CAR-T cell therapies targeting CD20 have been approved.

[0003] Compared to monoclonal antibodies, CAR-T cell therapies possess sustained targeted killing ability, in vivo expansion capacity, and are independent of the body's immune status. They exhibit stronger and more persistent targeting and elimination of CD20-positive abnormal B cells. On one hand, in B-cell malignancies, tumor cells such as B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, and follicular lymphoma consistently overexpress CD20 protein. CAR-T cells targeting CD20 can specifically recognize and lyse cancerous CD20-positive B tumor cells, precisely eliminating lesions and inhibiting tumor proliferation and invasion, demonstrating significant therapeutic advantages for relapsed / refractory B-cell malignancies. On the other hand, in autoimmune diseases such as systemic lupus erythematosus (SLE), CAR-T cell therapy also has significant therapeutic advantages. In diseases such as leukemia, systemic sclerosis, rheumatoid arthritis (RA), antisynthetic antibody syndrome, and myasthenia gravis, the body contains abnormally activated and pathogenic CD20-positive B lymphocytes. These cells can secrete autoantibodies, mediate immune disorders and tissue damage, and are key drivers of disease development. CAR-T therapy targeting CD20 can target and deplete pathogenic activated B cells, block the production of autoantibodies, and inhibit abnormal autoimmune responses, thereby regulating immune imbalance at its source and achieving targeted intervention and disease control for autoimmune diseases.

[0004] However, because CD20 is a four-transmembrane phosphoprotein with only two short extracellular loops in its extracellular region, there are very few available antigenic epitopes. Tumor cells can easily escape through epitope modification and conformational changes. The hinge and transmembrane regions of CAR molecules may interfere with the aggregation state of scFv and CAR molecules on the membrane surface. Furthermore, since it is mainly transduced into T cells through lentiviral or retroviral vectors, there are problems such as unsatisfactory CD20 targeting and binding, poor T cell activation, insufficient T cell expression, and unsatisfactory cell killing effects, which limit its clinical translation potential and have significant limitations. Summary of the Invention

[0005] The primary objective of this invention is to address the problems of existing chimeric antigen receptors, such as unsatisfactory CD20 targeting binding, poor T cell activation, insufficient T cell expression, and unsatisfactory cell killing effects, and to provide a chimeric antigen receptor that targets the CD20 protein.

[0006] A second objective of this invention is to provide a CAR gene expression construct.

[0007] A third objective of this invention is to provide a CAR-T cell.

[0008] The fourth objective of this invention is to provide a method for constructing the above-mentioned CAR-T cells.

[0009] The fifth object of the present invention is to provide a pharmaceutical composition.

[0010] The sixth objective of this invention is to provide the use of the above-mentioned chimeric antigen receptor, CAR gene expression construct, CAR-T cell or pharmaceutical composition in the preparation of a therapeutic medicament for CD20-positive B-cell-related diseases.

[0011] Specifically, the chimeric antigen receptor targeting the CD20 protein provided by the present invention includes an amino acid fragment with the sequence shown in SEQ ID NO:29 or a variant fragment having at least 85% sequence identity with SEQ ID NO:29.

[0012] The CAR gene expression construct provided by the present invention includes a CDS fragment encoding the chimeric antigen receptor of claim 1, wherein the CDS fragment includes one or more nucleotide fragments with sequences such as SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:12.

[0013] Furthermore, the CAR gene expression construct is a circRNA, and the CAR gene expression construct includes, along the 5' to 3' direction, an IRES fragment, a Kozak fragment, a CDS fragment, and optionally a spacer-1 fragment and a spacer-2 fragment.

[0014] Further, the CAR gene expression construct includes one or more of the following technical features: (1) the IRES fragment includes one or more nucleotide fragments shown in SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:18 and SEQ ID NO:21; (2) the Kozak fragment includes one or more nucleotide fragments shown in SEQ ID NO:2, SEQ ID NO:9 and SEQ ID NO:23; (3) the CDS fragment includes the nucleotide fragments shown in SEQ ID NO:7 and / or SEQ ID NO:12; (4) the spacer-1 fragment includes the nucleotide fragments shown in SEQ ID NO:4 and / or SEQ ID NO:5; (5) the spacer-2 fragment includes the nucleotide fragments shown in SEQ ID NO:4 and / or SEQ ID NO:5.

[0015] Further, the CAR gene expression construct includes one or more of the following technical features: (1) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:6; (2) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:8; (3) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:11; (4) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:13; (5) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:15; (6) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:16; (7) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:17; (8) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:19; (9) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:20; (10) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:22; (11) the nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:19. (12) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:25; (13) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:26; (14) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:27; (15) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:28.

[0016] The CAR-T cells provided by this invention express the above-mentioned chimeric antigen receptor.

[0017] The method for constructing CAR-T cells provided by the present invention includes: transfecting T cells with a CAR gene expression construct to obtain the CAR-T cells; wherein the nucleotide sequence of the CAR gene expression construct is as shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

[0018] Furthermore, the method for transfecting T cells with the CAR gene expression construct is selected from one or more of the following: viral vector transfection, electroporation transfection, and liposome transfection.

[0019] The pharmaceutical composition provided by the present invention includes the above-mentioned chimeric antigen receptor, CAR gene expression construct or CAR-T cell.

[0020] This invention provides the application of the above-mentioned chimeric antigen receptor, CAR gene expression construct, CAR-T cell or pharmaceutical composition in the preparation of a therapeutic drug for CD20-positive B-cell-related diseases.

[0021] Beneficial effects: The chimeric antigen receptor provided by this invention specifically includes an amino acid fragment with a sequence as shown in SEQ ID NO:28 or a variant fragment having at least 85% sequence identity with SEQ ID NO:28. The signal peptide, antigen-binding region, hinge region, transmembrane region, intracellular co-stimulatory domain, signal transduction domain, and other functional fragments contained therein form an organic whole, working synergistically to endow the chimeric antigen receptor with good CD20 targeting binding, T cell activation, and cell killing effects, showing excellent application potential in the preparation of therapeutic drugs for CD20-positive B cell-related diseases.

[0022] In some specific embodiments, the CAR gene expression construct preferably includes a CDS fragment with sequences such as SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12. In this case, the CDS fragment not only has good compatibility with the protein expression system contained in human cells, but the secondary structure it forms also endows the CAR gene expression construct with good translation initiation efficiency, structural stability, and low immunogenicity, enabling better efficient, stable, and sustained expression of CAR molecules in human T cells. The resulting CAR... + T cells exhibit better CD20 targeting and binding effects, T cell activation effects, and cell killing effects.

[0023] In some specific embodiments, the CAR gene expression construct is preferably circRNA, and the preferred nucleotide sequence is as shown in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:22, or SEQ ID NO:27. In this case, the fragments in the CAR gene expression construct form secondary structures with different properties, which, while endowing the CAR gene expression vector with excellent translation initiation efficiency, provide appropriate ribosome movement resistance to induce brief ribosome dwell during translation and improve the overall balance of elongation rate. The resulting CAR gene expression construct not only has excellent high-activity CAR precursor protein production but also translation efficiency adapted to the post-translational processing, transport, and anchoring processes of CAR precursor proteins, effectively improving the final CAR membrane expression efficiency of human T cells. + The CAR molecules on the surface of T cells are ideally distributed, exhibiting excellent and stable CD20 targeting binding, T cell activation, and cell killing effects. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the CAR gene expression construct provided in Example 2 of the present invention; Figure 2 One of the experimental results (OAS1) of the immunogenicity test of the CAR gene expression construct provided for the test example of the present invention. Figure 3 Figure 2 (MDA5) shows the experimental results of the immunogenicity test of the CAR gene expression construct provided for the test example of this invention. Figure 4 Figure 3 shows the experimental results of the immunogenicity test of the CAR gene expression construct provided for the test example of this invention (IL-6). Figure 5Figure 4 (RIG-1) shows the experimental results of the immunogenicity test of the CAR gene expression construct provided for the test example of this invention. Figure 6 Figure 5 shows the experimental results of the immunogenicity test of the CAR gene expression construct provided for the test example of this invention (TNF-α). Figure 7 Figure 6 (INF-b) shows the experimental results of the immunogenicity test of the CAR gene expression construct provided for the test example of this invention. Figure 8 One of the experimental results (24h) of the CAR gene expression construct transfected into Jurkat cells as a test example of the present invention. Figure 9 Figure 2 (48h) shows the experimental results of CAR gene expression level testing after transfection of Jurkat cells with the CAR gene expression construct provided in the test example of this invention. Figure 10 Figure 3 (96h) shows the experimental results of CAR molecule expression level test after the CAR gene expression construct provided for the test example of this invention was transfected into Jurkat cells. Figure 11 The CAR gene expression construct provided in the test examples of this invention was transfected into Jurkat cells and then CAR was generated. + One of the experimental results of the cell generation test (24h); Figure 12 The CAR gene expression construct provided in the test examples of this invention was transfected into Jurkat cells and then CAR was generated. + Figure 2 shows the experimental results of the cell generation test (48h). Figure 13 The CAR gene expression construct provided in the test examples of this invention was transfected into Jurkat cells and then CAR was generated. + Figure 3 shows the experimental results of the cell generation test (96h). Figure 14 One of the experimental results (24h) of the CAR gene expression construct transfected into human primary T cells as a test example of the present invention. Figure 15 Figure 2 (48h) shows the experimental results of CAR gene expression construct transfected into human primary T cells for the test example of this invention. Figure 16 Figure 3 (96h) shows the experimental results of CAR gene expression construct transfected into human primary T cells for the test example of this invention. Figure 17The CAR gene expression construct provided in the test examples of this invention was transfected into human primary T cells and then CAR was generated. + One of the experimental results of the cell generation test (24h); Figure 18 The CAR gene expression construct provided in the test examples of this invention was transfected into human primary T cells and then CAR was generated. + Figure 2 shows the experimental results of the cell generation test (48h). Figure 19 The CAR gene expression construct provided in the test examples of this invention was transfected into human primary T cells and then CAR was generated. + Figure 3 shows the experimental results of the cell generation test (96h). Detailed Implementation

[0025] This invention addresses the shortcomings of existing CAR molecules targeting the CD20 protein and their corresponding CAR-T cells in terms of CD20 targeting binding, cell activation, and cytotoxic activity. Through extensive and in-depth research and numerous experiments, a chimeric antigen receptor with a specific structural composition has been creatively designed. Compared to commercially available Anti-CD20 CAR-T cells, CARs expressing this chimeric antigen receptor exhibit superior performance. + T cells exhibit excellent CD20-targeting binding, T cell activation, and cell-killing activity, demonstrating great potential for application in the preparation of therapeutic drugs for CD20-positive B-cell-related diseases.

[0026] However, in further research, the inventors discovered that using a CDS fragment optimized with simple human codons to genetically modify human T cells resulted in CARs. + The expression level, stable expression duration, and immunogenicity of CAR molecules in T cells remain unclear. Therefore, considering the characteristics of human cell protein expression systems, the base composition, secondary structure, and codon bias of the CDS fragment, further research and design were conducted on the CDS fragment. Through extensive substitution, calculation, and theoretical and experimental verification, a CDS fragment with a specific nucleotide sequence was creatively obtained. This CDS fragment not only exhibits good compatibility with the protein expression system contained in human cells, but its secondary structure also endows the CAR gene expression construct with good translation initiation efficiency, structural stability, and low immunogenicity. This allows for better efficient, stable, and sustained expression of CAR molecules in human T cells, thus enhancing CAR expression. + T cells exhibited superior CD20 targeting binding, T cell activation, and cell killing activity.

[0027] Furthermore, based on the nucleotide sequence of the obtained CDS fragment and its three-dimensional spatial conformation with several dispersed weak stem-loop structures, the inventors of this invention creatively selected circRNA as the vector backbone and expression template for the CDS fragment. Taking into account the interactions between other fragments in the CAR gene expression construct and the CDS fragment, as well as the overall structure they form, the inventors investigated the effects of numerous substitutions, calculations, and theoretical and experimental verifications on the "peptide chain elongation, co-translational folding, post-translational processing, transport, and anchoring of CAR precursor protein peptide chains in human T cells" and "the structural changes of the CAR gene expression construct during its presence and expression on cell state." This established connections between variables with ambiguous causal relationships and dynamic changes, creatively obtaining a CAR gene expression construct with a specific nucleotide sequence. This CAR gene expression construct possesses excellent stability, CAR membrane expression efficiency, and low immunogenicity, enabling CAR... + T cells exhibit excellent and stable CD20 targeting binding, T cell activation, and cell-killing activity. Based on this, the technical solution of this invention is obtained.

[0028] The chimeric antigen receptor targeting the CD20 protein provided by this invention specifically includes an amino acid fragment with the sequence shown in SEQ ID NO:29 or a variant fragment having at least 85% sequence identity with SEQ ID NO:29. The variant fragment refers to an amino acid fragment with the sequence shown in SEQ ID NO:29 obtained by substitution, deletion, or addition of one or more amino acids, and the substitution, deletion, or addition does not affect the activity of the chimeric antigen receptor. The sequence identity can be 85%, 89%, 90%, 92.5%, 98%, 99%, 99.9999%, or any value between them.

[0029] The CAR gene expression construct provided by the present invention includes a CDS fragment encoding the above-mentioned chimeric antigen receptor, and the CDS fragment specifically includes one or more nucleotide fragments with sequences such as SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:12.

[0030] In this invention, the key to achieving highly efficient, sustained, and low-immunogenic protein expression of chimeric antigen receptors in the CAR gene expression construct lies in the loading of the CDS fragment, which can be any existing gene engineering vector, including but not limited to one or more of mRNA, saRNA, circRNA, recombinant adeno-associated virus vectors, and plasmids.

[0031] In some specific embodiments, when the CAR gene expression construct is mRNA, the CAR gene expression construct includes, along the 5' to 3' direction, a 5'-cap structure, a 5'-UTR fragment, a kozak fragment, a CDS fragment, a 3'-UTR fragment, and a poly(A) tail.

[0032] In some specific embodiments, when the CAR gene expression construct is saRNA, the CAR gene expression construct sequentially includes a 5'-cap structure, a 5'-UTR fragment, a coding fragment for the RdRp non-structural protein region, a subgenomic promoter, a CDS fragment, a 3'-UTR fragment, and a poly(A) tail along the 5' to 3' direction. The coding fragment for the RdRp non-structural protein region and the subgenomic promoter are limited to those capable of enabling self-replication of the saRNA and can be of various existing types; this invention does not impose any particular limitation.

[0033] In some specific embodiments, when the CAR gene expression construct is a plasmid, the CAR gene expression construct can be constructed by carrying the CDS fragment onto an existing plasmid vector through homologous recombination. The plasmid vector can be any of the existing options, and the present invention does not impose any particular limitation. Specific examples include, but are not limited to, one or more of the following: pcDNA vector, pEF1α vector, and pCAG vector.

[0034] In this invention, the CAR gene expression construct is preferably circRNA, which carries a CDS fragment and has a covalently closed circular structure, which includes an IRES fragment, a Kozak fragment, a CDS fragment, and optionally a spacer-1 fragment and a spacer-2 fragment along the 5' to 3' direction.

[0035] In some specific embodiments, the IRES fragment preferably includes one or more of the nucleotide fragments shown in SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:18, and SEQ ID NO:21. In this case, the IRES fragment contained in the CAR gene expression construct, together with the CDS initiation region, forms a secondary structure with suitable looseness. This ensures that the translation initiation efficiency of the CAR gene expression construct is well-suited to the peptide chain elongation, co-translational folding, post-translational processing, transport, and anchoring processes of the CAR precursor protein peptide chain, thereby achieving better CAR molecule expression and CAR... + T-cell generation effect.

[0036] In some specific embodiments, the Kozak fragment preferably includes one or more segments of the nucleotide fragments shown in SEQ ID NO:2, SEQ ID NO:9, and SEQ ID NO:23. In this case, the Kozak fragment, together with the IRES fragment and the CDS initiation region, forms a secondary structure with suitable looseness. This ensures that the translation initiation efficiency of the CAR gene expression construct is well-suited to the peptide chain elongation, co-translational folding, post-translational processing, transport, and anchoring processes of the CAR precursor protein peptide chain, thereby achieving better CAR molecule expression and CAR... + T-cell generation effect.

[0037] In some specific embodiments, the CDS fragment preferably includes the nucleotide fragments shown in SEQ ID NO:7 and / or SEQ ID NO:12. In this case, the CDS fragment may form a secondary structure containing several dispersed and relatively uniform short stem-loop structures, so that the CAR gene expression construct has a peptide chain elongation rate adapted to the post-translational processing, transport, anchoring and other processes of CAR precursor proteins, which is beneficial to achieving better CAR membrane expression results.

[0038] In some specific embodiments, the spacer-1 fragment preferably includes the nucleotide fragments shown in SEQ ID NO:4 and / or SEQ ID NO:5. In this case, the spacer-1 fragment exhibits good steric hindrance and facilitates the maintenance of the closed-loop conformation of circRNA during translation, thus promoting the sustained and stable expression of CAR precursor proteins in cells by the CAR gene expression construct.

[0039] In some specific embodiments, the spacer-2 fragment preferably includes the nucleotide fragments shown in SEQ ID NO:4 and / or SEQ ID NO:5. In this case, the spacer-2 fragment exhibits good steric hindrance and facilitates the maintenance of the closed-loop conformation of circRNA during translation, thus promoting the sustained and stable expression of CAR precursor proteins in cells by the CAR gene expression construct.

[0040] In this invention, when the CAR gene expression construct is preferably circRNA, the method for obtaining the CAR gene expression construct by cyclization can be any of the existing methods, and this invention does not particularly limit it. Specific examples include, but are not limited to, the following: type I intron self-splicing method, type II intron self-splicing method, enzymatic ligation method, and ribozyme cyclization method. The type I intron self-splicing method refers to a cyclization process based on the autocatalytic splicing mechanism of type I introns, achieved through two transesterification reactions. The type I introns can be any of the existing methods, and specific examples include, but are not limited to, the type I introns of T4 phage ribozymes and / or the type I introns of the Fusarium oxysporum CoB gene. The type II intron self-splicing method refers to a cyclization process based on the autocatalytic splicing mechanism of type II introns, achieved through two transesterification reactions. The type II introns can be any of the existing methods, and specific examples include, but are not limited to, the PIE system and / or the Group IIC intron system. The enzymatic ligation method refers to the process of cyclization by catalyzing the formation of a phosphodiester bond between the 5' and 3' ends of linear RNA using an RNA ligase. The RNA ligase can be any of the existing options, including, but not limited to, T4 RNA ligase. The ribozyme cyclization method refers to the process of cyclization using the catalytic activity of a ribozyme. The ribozyme can be any of the existing options, including, but not limited to, hammerhead ribozymes and / or hepatitis D virus ribozymes.

[0041] In some specific embodiments, the CAR gene expression construct is preferably obtained by circularizing the corresponding precursor through a type I intron of the Fusarium oxysporum CoB gene. The structural design of the precursor is based on construct 5 disclosed in Chinese Invention CN118813602A, and the preparation method of the CAR gene expression construct has also been disclosed in detail. This invention does not impose any particular limitations on the method used to obtain the CAR gene expression construct. In this case, the CAR gene expression construct contains only a 5-nt exogenous residual base (TGGGT). This exogenous residual base not only does not contribute to the immunogenicity of the CAR gene expression construct but also further optimizes its secondary structure.

[0042] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:6.

[0043] In some specific embodiments, the CAR gene expression construct preferably includes the nucleotide fragment shown in SEQ ID NO:8. In this case, the CAR gene expression construct is expressed as CAR at 96 hours after introduction into human primary T cells. + The average fluorescence intensity on the surface of T cells is greater than 23000, and CAR +With a cell proportion of no less than 50%, it has the ability to continuously and stably express CAR molecules, showing excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

[0044] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:11.

[0045] In some specific embodiments, the CAR gene expression construct preferably includes the nucleotide fragment shown in SEQ ID NO:13. In this case, the CAR gene expression construct is expressed as CAR at 96 hours after introduction into human primary T cells. + The average fluorescence intensity on the surface of T cells is greater than 35,000, and CAR + With a cell proportion of no less than 50%, it has the ability to continuously and stably express CAR molecules, showing excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

[0046] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:15.

[0047] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:16.

[0048] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:17.

[0049] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:19.

[0050] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:20.

[0051] In some specific embodiments, the CAR gene expression construct preferably includes the nucleotide fragment shown in SEQ ID NO:22. In this case, the CAR gene expression construct is expressed as CAR at 96 hours after introduction into human primary T cells. + The average fluorescence intensity on the surface of T cells is greater than 23000, and CAR + With a cell proportion of no less than 40%, it has the ability to continuously and stably express CAR molecules, showing excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

[0052] In some specific embodiments, the CAR gene expression construct preferably includes a nucleotide fragment as shown in SEQ ID NO:24.

[0053] In some specific embodiments, the CAR gene expression construct preferably includes the nucleotide fragment shown in SEQ ID NO:25. In this case, the CAR gene expression construct is expressed as CAR at 96 hours after introduction into human primary T cells. + The average fluorescence intensity on the surface of T cells is greater than 18000, and CAR + With a cell proportion of no less than 30%, it has the ability to continuously and stably express CAR molecules, showing excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

[0054] In some specific embodiments, the CAR gene expression construct preferably includes the nucleotide fragment shown in SEQ ID NO:26. In this case, the CAR gene expression construct is expressed as CAR at 96 hours after introduction into human primary T cells. + The average fluorescence intensity on the surface of T cells is greater than 15000, and CAR + With a cell proportion of no less than 35%, it has the ability to continuously and stably express CAR molecules, showing excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

[0055] In some specific embodiments, the CAR gene expression construct preferably includes the nucleotide fragment shown in SEQ ID NO:27. In this case, the CAR gene expression construct is expressed as CAR at 96 hours after introduction into human primary T cells. + The average fluorescence intensity on the surface of T cells is greater than 25,000, and CAR + With a cell proportion of no less than 40%, it has the ability to continuously and stably express CAR molecules, showing excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

[0056] In some specific embodiments, the CAR gene expression construct preferably includes the nucleotide fragment shown in SEQ ID NO:28. In this case, the CAR gene expression construct is expressed as CAR at 96 hours after introduction into human primary T cells. + The average fluorescence intensity on the surface of T cells is greater than 23000, and CAR + With a cell proportion of no less than 40%, it has the ability to continuously and stably express CAR molecules, showing excellent application potential in the preparation of drugs for the treatment of relapsed / refractory B-cell malignancies.

[0057] The CAR-T cells provided by this invention express the aforementioned chimeric antigen receptor. More specifically, the chassis cells of the CAR-T cells are a conventional technique used in existing CAR-T therapy and can be of various existing types. This invention does not particularly limit them. Specific examples include, but are not limited to, the patient's own T cells and / or universal CAR-T cells. The universal CAR-T cells refer to allogeneic cells obtained through gene editing to reduce immune rejection. The source of the allogeneic cells can be a healthy donor. The gene editing specifically includes, but is not limited to, knockout of the T cell receptor gene and / or knockout of the HLA gene.

[0058] The method for constructing CAR-T cells provided by the present invention specifically includes: transfecting T cells with a CAR gene expression construct to obtain the CAR-T cells. The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

[0059] In this invention, the method of transfecting T cells with the CAR gene expression construct is a conventional technique used in existing CAR-T therapy. Various options are available, and this invention does not impose any particular limitation on it. Specific examples include, but are not limited to, one or more of the following: viral vector transfection, electroporation transfection, and liposome transfection.

[0060] The pharmaceutical composition provided by the present invention specifically includes the above-mentioned chimeric antigen receptor, CAR gene expression construct or CAR-T cell.

[0061] In this invention, the pharmaceutical composition preferably further includes pharmaceutically acceptable excipients, which can be of various existing types and are not particularly limited thereto.

[0062] This invention provides the application of the above-mentioned chimeric antigen receptor, CAR gene expression construct, CAR-T cell or pharmaceutical composition in the preparation of a therapeutic drug for CD20-positive B-cell-related diseases.

[0063] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0064] Example 1 This embodiment illustrates a CAR gene expression construct (CAR20-1), referring to... Figure 1 The CAR gene expression construct is a circular RNA, which includes the IRES fragment, Kozak fragment, CDS fragment, spacer-1 fragment, and spacer-2 fragment sequentially along the 5' to 3' direction. The CAR gene expression construct is circularized by taking the corresponding linear RNA precursor and using the type I intron of the Fusarium oxysporum CoB gene. There is also a 5nt exogenous residual base between the spacer-1 and spacer-2 fragments. The specific sequence information of each fragment is shown in Table 1.

[0065] Table 1.

[0066]

[0067] Example 2 The CAR gene expression construct (CAR20-2) provided in this embodiment is basically the same as that in Example 1. The difference is that the CDS fragment contained in the CAR gene expression construct is CAR-2, as shown in Table 2. Other structures are kept the same, thus obtaining the CAR gene expression construct.

[0068] Table 2.

[0069] Example 3 The CAR gene expression construct (CAR20-3) provided in this embodiment is basically the same as that in Example 1. The difference is that the CDS fragment contained in the CAR gene expression construct is CAR-3 and the Kozak fragment is Kozak-2, as shown in Table 3. Other structures are kept the same, thus obtaining the CAR gene expression construct.

[0070] Table 3.

[0071] Example 4 The CAR gene expression construct (CAR20-4) provided in this embodiment is basically the same as that in Example 1. The difference is that the CDS fragment contained in the CAR gene expression construct is CAR-4, as shown in Table 4. Other structures are kept the same, thus obtaining the CAR gene expression construct.

[0072] Table 4.

[0073] Example 5 The CAR gene expression construct (CAR20-5) provided in this embodiment is basically the same as that in Example 1. The difference is that the IRES fragment in the CAR gene expression construct is the b69 IRES fragment, as shown in Table 5. Other structures are kept the same, thus obtaining the CAR gene expression construct.

[0074] Table 5.

[0075] Example 6 The CAR gene expression construct (CAR20-6) provided in this embodiment is basically the same as that in Example 2. The difference is that the Kozak fragment in the CAR gene expression construct is Kozak-2 and does not contain spacer-1 or spacer-2 fragments, as shown in Table 6. All other structures are kept the same, and the CAR gene expression construct is obtained.

[0076] Table 6.

[0077] Example 7 The CAR gene expression construct (CAR20-7) provided in this embodiment is basically the same as that in Example 7, except that the Kozak fragment in the CAR gene expression construct is Kozak-1, as shown in Table 7. All other structures remain the same, thus obtaining the CAR gene expression construct.

[0078] Table 7.

[0079] Example 8 The CAR gene expression construct (CAR20-8) provided in this embodiment is basically the same as that in Example 2. The difference is that the IRES fragment in the CAR gene expression construct is the EVA IRES fragment, as shown in Table 8. Other structures are kept the same, thus obtaining the CAR gene expression construct.

[0080] Table 8.

[0081] Example 9 The CAR gene expression construct (CAR20-9) provided in this embodiment is basically the same as that in Example 2. The difference is that the spacer-1 fragment and the spacer-2 fragment in the CAR gene expression construct are both spacer sequence-2, as shown in Table 9. Other structures are kept the same, thus obtaining the CAR gene expression construct.

[0082] Table 9.

[0083] Example 10 The CAR gene expression construct (CAR20-10) provided in this embodiment is basically the same as that in Example 2. The difference is that the IRES fragment in the CAR gene expression construct is the b81 IRES fragment and does not contain the spacer-2 fragment, as shown in Table 10. All other structures are kept the same, and the CAR gene expression construct is obtained.

[0084] Table 10.

[0085] Example 11 The CAR gene expression vector (CAR20-11) provided in this embodiment is basically the same as that in Example 2. The difference is that the Kozak fragment in the CAR gene expression vector is Kozak-3 and does not contain the spacer-2 fragment, as shown in Table 11. Other structures are kept the same, and the CAR gene expression vector is obtained.

[0086] Table 11.

[0087] Example 12 The CAR gene expression construct (CAR20-12) provided in this embodiment is basically the same as that in Example 3. The difference is that the IRES fragment in the CAR gene expression construct is the b69 IRES fragment, the Kozak fragment is the Kozak-1 fragment, and the spacer-1 fragment and the spacer-2 fragment are both spacer sequence-2, as shown in Table 12. Other structures are kept the same, and the CAR gene expression construct is obtained.

[0088] Table 12.

[0089] Example 13 The CAR gene expression construct (CAR20-13) provided in this embodiment is basically the same as that in Example 4. The difference is that the IRES fragment in the CAR gene expression construct is the b81 IRES fragment, and it does not contain the spacer-1 fragment and the spacer-2 fragment, as shown in Table 13. All other structures are kept the same, and the CAR gene expression construct is obtained.

[0090] Table 13.

[0091] Example 14 The CAR gene expression construct (CAR20-14) provided in this embodiment is basically the same as that in Example 4. The difference is that the IRES fragment in the CAR gene expression construct is the b81 IRES fragment, as shown in Table 14. Other structures are kept the same, and the CAR gene expression construct is obtained.

[0092] Table 14.

[0093] Example 15 The CAR gene expression construct (CAR20-15) provided in this embodiment is basically the same as that in Example 14(N), except that the two spacer fragments in the CAR gene expression construct are spacer sequence-2, as shown in Table 15. All other structures are kept the same, thus obtaining the CAR gene expression construct.

[0094] Table 15.

[0095] Comparative Example This comparative example illustrates a CAR gene expression vector constructed by loading a CAR-2 CDS fragment (SEQ ID NO:7) encoding a CAR molecule targeting CD20 onto a pcDNA3.1 vector.

[0096] Test case This test case illustrates the performance of the CAR gene expression construct provided in the above embodiments. The test specifically includes: 1. Immunogenicity of CAR gene expression constructs (1) Using Lipofectamine 3000 reagent (Thermo Fisher Scientific, catalog number L3000150, the same below), the CAR gene expression constructs provided in Examples 1 to 15 were transfected into A549 cells under the same operating conditions according to the instructions, and then cultured at 37°C and 5% CO2 for 24 h.

[0097] (2) The expression levels of OAS1, MDA5, IL-6, RIG-1, TNF-α, and IFN-β in A549 cells of each group were detected by qPCR. The relative mRNA expression levels of the above genes in each group were calculated based on the mRNA content of the endogenous gene β-actin. Lipofectamine 3000 was used as a blank control and m1Ψ-Fluc-mRNA (Genscript, catalog number RP-A00024-50, the same below) was used as a positive control. The results are as follows: Figures 2-7 As shown.

[0098] Depend on Figures 2-7 The results show that, compared with the m1Ψ-Fluc-mRNA group, the relative expression levels of immune-related genes such as OAS1, MDA5, and IL-6 were less increased after A549 cells were treated with the CAR gene expression constructs provided in Examples 1-4, 9-11, and 13-15, indicating low immunogenicity.

[0099] 2. CAR molecular expression capability of CAR gene expression constructs (1) Using Jurkat cells as chassis cells: i. Using NanoLNP™ Primary Immune CellsTransfection Reagent (Shanghai Shitu Ke, catalog number CT0020, the same below), the CAR gene expression constructs provided in Examples 1-15 and the comparative examples were transfected into Jurkat cells under the same operating conditions according to the instructions, and then cultured at 37°C and 5% CO2 for 96 h.

[0100] ii. At 24h, 48h, and 96h of culture, cells were treated with fluorescently labeled CD20 antigen (Beijing Baipusai Biotechnology Co., Ltd., catalog number CD0-HP2E4, hereinafter the same) and then detected by flow cytometry. The mean fluorescence intensity (MFI) and CATR of positive cells were calculated. + Cell percentage (in %), with NanoLNP™ Primary Immune Cells Transfection Reagent alone as a blank control and Anti-CD20CAR-T Cells (BPS Bioscience, catalog number #78611, hereinafter the same) as a positive control (POS.CTRL). The results are as follows. Figures 8-13 As shown.

[0101] Depend on Figures 8-13The results show that, compared to POS.CTRL and the comparative example, the CAR gene expression constructs provided in Examples 1-15 exhibit good transduction efficiency, CAR molecule expression efficiency, and CAR expression rate in Jurkat cells. + Cell generation efficiency. Jurkat cells were treated with CAR20-2, CAR20-4, CAR20-10, CAR20-12, CAR20-13, CAR20-14, and CAR20-15. The MFI (Mean Free Intake) was not lower than 16000 at 24 h post-transfection. + The cell percentage was all above 95%, and the MFI was still above 1000 at 96 hours post-transfection, and the CAR... + The cell proportions are all above 70%, exhibiting both excellent transduction efficiency and CAR. + Cell generation efficiency and sustained expression capacity.

[0102] (2) Using human primary T cells as chassis cells: i. Using electrotransfection, the CAR gene expression constructs provided in Examples 1-15 were transfected into human primary T cells under the same operating conditions, and then cultured at 37°C and 5% CO2 for 96 h.

[0103] ii. At 24h, 48h, and 96h of culture, cells were treated with fluorescently labeled CD20 antigen and then detected by flow cytometry. The average fluorescence signal intensity and CATR of positive cells were calculated. + Cell percentage was determined, with NanoLNP™ Primary Immune Cells Transfection Reagent alone serving as a blank control and Anti-CD20 CAR-T Cells serving as a positive control (POS.CTRL). Results are as follows: Figures 14-19 As shown.

[0104] Depend on Figures 14-19 The results show that, compared to POS.CTRL, the CAR gene expression constructs provided in Examples 1-15 have better T cell transduction efficiency, CAR molecule expression efficiency, and CAR expression efficiency for human primary T cells. + Cell generation efficiency. Human primary T cells were treated with CAR20-2, CAR20-4, CAR20-10, CAR20-12, CAR20-13, CAR20-14, and CAR20-15. The MFI (metastatic fluid index) was not lower than 18,000 at 24 hours post-transfection. + The cell proportion was not less than 80%, and the MFI was still not less than 15,000 at 96 hours post-transfection, and the CAR... +The cell proportion is no less than 38%, with excellent transduction efficiency and CAR + Cell generation efficiency and sustained expression capacity.

[0105] 3. CARs constructed from CAR gene expression constructs + Cell-specific binding ability (1) Using NanoLNP TM Immune Cells Transfection Reagent for RNA (Shanghai Shitu Technology, catalog number CT0013, hereinafter the same) Following the instructions, the CAR gene expression constructs provided in Examples 1-15 and the comparative examples were transfected into Jurkat cells under the same operating conditions. The cells were then cultured at 37°C and 5% CO2 for 48 hours to obtain CARs. + cell.

[0106] (2) Take CAR according to the addition amount of 1:1 cell number ratio. + Cells or Anti-CD20 CAR-T Cells (POS.CTRL) and CFSE-labeled Raji cells were mixed and co-incubated at 37°C and 5% CO2 for 24 hours. CFSE was then detected using flow cytometry. + CAR + The percentage of double-positive cells (binding rate, in %), and the percentage of NanoLNP alone. TM Immune Cells Transfection Reagent for RNA (NC-Jurkat) or Jurkat cells without any treatment (Mock-Jurkat) were used as controls. The results are shown in Table 16.

[0107] Table 16.

[0108] As shown in Table 16, the CD20-targeting CAR molecule (including the amino acid sequence SEQ ID NO:28) provided by this invention has good specific binding ability to Raji cells. After transfecting Jurkat cells with the CAR gene expression constructs provided in Examples 1-15 and the comparative examples, the obtained CARs... + The cell binding rate to Raji cells was no less than 35.1% ± 2.7%. Furthermore, compared to the control group, the CAR gene expression constructs of Examples 1-15 exhibited better CAR molecule expression in Jurkat cells, and the resulting CAR... +The cell surface exhibits a more ideal distribution of CAR molecules, thus demonstrating a superior ability to specifically bind to Raji cells.

[0109] 4. CARs constructed from CAR gene expression constructs + Cellular killing activity (1) Using NanoLNP TM Following the instructions, the CAR gene expression constructs provided in Examples 1-15 and the comparative examples were transfected into Jurkat cells under the same operating conditions. The cells were then cultured at 37°C and 5% CO2 for 48 hours to obtain CARs. + cell.

[0110] (2) Take CAR according to the cell ratio of 1:1, 5:1 or 10:1. + Cells or Anti-CD20 CAR-TCells (POS.CTRL) were mixed with CFSE-labeled Raji cells and co-incubated at 37°C and 5% CO2 for 24 hours. Cells were then treated with a 7-ADD staining kit (Shanghai Sangon Biotech, catalog number E607304) according to the manufacturer's instructions. Flow cytometry was used to detect the 7-ADD staining. + CFSE + CAR + The percentage of positive cells (kill rate, in %) was determined by NanoLNP alone. TM Immune Cells Transfection Reagent for RNA (NC-Jurkat) or Jurkat cells without any treatment (Mock-Jurkat) were used as controls. The results are shown in Table 17.

[0111] Table 17.

[0112] As shown in Table 17, the CAR gene expression vectors provided in Examples 1-15 and the comparative examples were used to transfect Jurkat cells, resulting in CARs... + The cells exhibited a killing rate of no less than 17.9% ± 2.0% against Raji cells, demonstrating good killing activity, and this killing activity increased with the growth of CAR cells. + The expression of CARs increased with the increase in cell proportion. Furthermore, compared to the control group, the CAR gene expression vectors of Examples 1-15 exhibited better CAR molecule expression in Jurkat cells, resulting in CARs... +The cell surface exhibits a more ideal distribution of CAR molecules, thus demonstrating superior killing activity against Raji cells.

[0113] 5. CARs constructed from CAR gene expression vectors + Cellular cytokine secretion levels (1) Using NanoLNP TM Following the instructions, the CAR gene expression vectors provided in Examples 1-15 and the comparative examples were transfected into Jurkat cells under the same operating conditions. The cells were then cultured at 37°C and 5% CO2 for 48 hours to obtain CARs. + cell.

[0114] (2) CAR+ cells or Anti-CD20 CAR-T Cells (POS.CTRL) and CFSE-labeled Raji cells were added at a cell ratio of 5:1 and incubated at 37°C and 5% CO2 for 24 h. The supernatant of the culture medium was tested according to the instructions of the IL-2 ELISA kit (Sinochem, catalog number KIT11848), IFN-γ ELISA kit (Sinochem, catalog number KIT11725A) and TNF-α ELISA kit (Sinochem, catalog number KIT10602) to obtain the concentration of each cytokine. Jurkat cells treated with NanoLNP™ Immune Cells Transfection Reagent for RNA (NC-Jurkat) alone or without any treatment (Mock-Jurkat) were used as controls. The results are shown in Table 18.

[0115] Table 18.

[0116] As shown in Table 18, the CAR gene expression vectors provided in Examples 1-15 and the comparative examples were used to transfect Jurkat cells, resulting in CARs... + After co-incubation with Raji cells, CAR + The levels of cytokines IL-2, IFN-γ, and TNF-α secreted by the cells were significantly higher than those in the NC-Jurkat and Mock-Jurkat groups, effectively inducing hypersecretion of cytokines such as IL-2, IFN-γ, and TNF-α.

[0117] 6. CARs constructed from CAR gene expression constructs + Intracellular signaling activation capacity of cells (1) Using electrotransfection, the CAR gene expression constructs provided in Examples 1-15 and the comparative examples, or co-transfected with the NFAT-luciferase reporter plasmid, were introduced into Jurkat cells under the same operating conditions. The cells were then cultured at 37°C and 5% CO2 for 48 h to obtain CARs. + cell.

[0118] (2) Take CAR according to the addition amount of 5:1 cell number ratio. + Cells or Anti-CD20 CAR-T Cells (POS.CTRL) and CFSE-labeled Raji cells were mixed and co-incubated at 37°C and 5% CO2 for 24 h. CAR-T cells were then detected by flow cytometry. + The fluorescence intensity of cells was normalized based on the fluorescence intensity of the NC-Jurkat group to obtain the relative fluorescence intensity and signal activation fold, and compared with the fluorescence intensity of NanoLNP alone. TM Jurkat cells (NC-Jurkat) used as a control in the Immune Cells Transfection Reagent for RNA are shown in Table 19.

[0119] Table 19.

[0120] As shown in Table 19, the CAR gene expression vectors provided in Examples 1-15 and the comparative examples were used to transfect Jurkat cells, resulting in CARs... + After co-incubation with Raji cells, the activation fold of the intracellular NFAT signaling pathway was higher than 8.5 ± 0.9, indicating a good T cell activation effect.

[0121] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0122] The amino acid sequence of the chimeric antigen receptor targeting the CD20 protein involved in this invention is shown in Table 20.

[0123] Table 20.

Claims

1. A chimeric antigen receptor targeting the CD20 protein, characterized in that, The chimeric antigen receptor comprises an amino acid fragment with a sequence as shown in SEQ ID NO:29 or a variant fragment having at least 85% sequence identity with SEQ ID NO:

29.

2. A CAR gene expression construct, characterized in that, The CAR gene expression construct includes a CDS fragment encoding the chimeric antigen receptor of claim 1, wherein the CDS fragment includes one or more nucleotide fragments with sequences as shown in SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:

12.

3. The CAR gene expression construct according to claim 2, characterized in that, The CAR gene expression construct is a circRNA, and the CAR gene expression construct includes, along the 5' to 3' direction, an IRES fragment, a Kozak fragment, a CDS fragment, and optionally a spacer-1 fragment and a spacer-2 fragment.

4. The CAR gene expression construct according to claim 3, characterized in that, The CAR gene expression construct includes one or more of the following technical features: (1) The IRES fragment includes one or more of the nucleotide fragments shown in SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:18 and SEQ ID NO:21; (2) The Kozak fragment includes one or more of the nucleotide fragments shown in SEQ ID NO:2, SEQ ID NO:9 and SEQ ID NO:23; (3) The CDS fragment includes the nucleotide fragments shown in SEQ ID NO:7 and / or SEQ ID NO:12; (4) The spacer-1 fragment comprises the nucleotide fragments shown in SEQ ID NO:4 and / or SEQ ID NO:5; (5) The spacer-2 fragment includes the nucleotide fragments shown in SEQ ID NO:4 and / or SEQ ID NO:

5.

5. The CAR gene expression construct according to claim 3, characterized in that, The CAR gene expression construct includes one or more of the following technical features: (1) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:6; (2) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:8; (3) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:11; (4) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:13; (5) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:15; (6) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:16; (7) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:17; (8) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:19; (9) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:20; (10) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:22; (11) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:24; (12) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:25; (13) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:26; (14) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:27; (15) The nucleotide sequence of the CAR gene expression construct is shown in SEQ ID NO:

28.

6. A CAR-T cell, characterized in that, The CAR-T cells express the chimeric antigen receptor as described in claim 1.

7. The method for constructing CAR-T cells according to claim 6, characterized in that, The construction method includes: transfecting T cells with the CAR gene expression construct to obtain the CAR-T cells; The nucleotide sequences of the CAR gene expression constructs are shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:

28.

8. The method for constructing CAR-T cells according to claim 7, characterized in that, The method for transfecting T cells with the CAR gene expression construct is selected from one or more of the following: viral vector transfection, electroporation transfection, and liposome transfection.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor of claim 1, the CAR gene expression construct of any one of claims 2 to 5, or the CAR-T cell of claim 6.

10. The use of the chimeric antigen receptor of claim 1, the CAR gene expression construct of any one of claims 2 to 5, the CAR-T cell of claim 6, or the pharmaceutical composition of claim 9 in the preparation of a therapeutic agent for CD20-positive B-cell-related diseases.

Images