Chimeric antigen receptors based on cleaved intracellular regions

Abstract

The present invention provides a chimeric antigen receptor based on the intracellular domain, comprising sequentially linked extracellular domains, transmembrane domains, and intracellular domains, wherein the extracellular domain comprises an antigen recognition region and a hinge region, and the intracellular domain comprises sequentially linked CD3ε intracellular domain cleavage, a co-stimulatory signaling region, and a CD3ζ intracellular domain. Compared to prior art, the provided chimeric antigen receptor exhibits superior therapeutic effects and fewer side effects, as reflected in improved membrane surface CAR molecule levels of CAR-T cells, better immune synapse formation, superior antigen sensitivity, fewer cytokines, better growth capacity, better sustained proliferation capacity, better repeated killing capacity, reduced NK cell activation and cell depletion, and an increase in stem cell-like and memory cell subsets of CAR-T cells. Furthermore, the chimeric antigen receptor can further enhance the therapeutic effect on tumors and, at the same time, reduce the production of inflammatory cytokines associated with the activation of macrophages and monocytes by downregulating its own cytokines, thereby enabling early prevention of cytokine storms.

Description

[Technical Field]

[0001] This invention relates to the field of chimeric antigen receptors, and more specifically, to chimeric antigen receptors based on cleaved intracellular regions. [Background technology]

[0002] Chimeric antigen receptors (CARs) are T cells that possess CARs that target specific tumor antigens, and these cells are called CAR-T cells. CAR-T therapy is widely used in tumor treatment, but many limitations still exist, particularly in its use in immunotherapy for solid tumors. These limitations include cytokine storms encountered in clinical treatment of leukemia and solid tumors, the conversion of CAR-T cells to NK cells (NKification), easy cell depletion, and the need for improved therapeutic efficacy.

[0003] Prior art CN112500492A modified the linkage order of the intracellular and transmembrane domains of a chimeric antigen receptor, discovering that when the intracellular domain consists of the CD3ε intracellular domain + the co-stimulatory signaling region intracellular domain + the CD3ζ intracellular domain, the resulting chimeric antigen receptor exhibits superior tumor therapeutic efficacy and fewer side effects. However, this chimeric antigen receptor has a reduced molecular membrane level, making it prone to NK cell conversion and cell depletion. Further improvements are needed in terms of growth capacity, sustained proliferation capacity, killing capacity, and antigen sensitivity.

[0004] Therefore, it is necessary to design chimeric antigen receptors that are more effective and have fewer side effects, thereby providing new insights into CAR-T therapy. [Overview of the project]

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a chimeric antigen receptor and its use.

[0006] To achieve the above and other related objectives, the first aspect of the present invention is: It includes sequentially linked extracellular domains, transmembrane domains, and intracellular domains, The extracellular domain includes an antigen recognition region and a hinge region. The intracellular domain provides a chimeric antigen receptor comprising sequentially linked CD3ε intracellular domain cleavages, a co-stimulatory signaling region, and a CD3ζ intracellular domain.

[0007] A second aspect of the present invention is, (1) A polynucleotide sequence encoding the chimeric antigen receptor described in the claim, and / or (2) Provide a polynucleotide sequence selected from the complementary sequence of the polynucleotide sequence described in (1).

[0008] A third aspect of the present invention provides a nucleic acid construct comprising the polynucleotide sequence.

[0009] A fourth aspect of the present invention provides a lentiviral vector system comprising the nucleic acid construct and the lentiviral vector auxiliary component.

[0010] A fifth aspect of the present invention provides genetically modified T cells comprising the polynucleotide sequence, or comprising the nucleic acid construct, or infected with the lentiviral vector system.

[0011] A sixth aspect of the present invention provides the use of the chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, or the lentiviral vector system for the production of one or more application products for any of the following: (1) production of CAR-T cells, (2) increasing the level of CAR molecules on the membrane surface of CAR-T cells, (3) improving CAR-T cell antigen sensitivity, (4) suppressing the secretion of cytokines IFN-γ, IL-2, and TNF from CAR-T cells, (5) promoting spontaneous growth of CAR-T cells during the quiescent phase, (6) enhancing the sustained proliferative capacity of CAR-T cells after target cell stimulation, (7) improving the repeated killing capacity of CAR-T cells, (8) reducing NK cell conversion and cell depletion of CAR-T cells, and (9) increasing the stem cell-like and memory cell subsets of CAR-T cells.

[0012] A seventh aspect of the present invention provides the use of the chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, the lentiviral vector system, or the genetically modified T cells for the manufacture of a tumor therapeutic product.

[0013] As described above, the chimeric antigen receptor of the present invention and its use have the following beneficial effects.

[0014] 1. Compared to prior art, the chimeric antigen receptor of the present invention reflects improved levels of CAR molecules on the membrane surface of CAR-T cells, better formation of immune synapses, superior antigen sensitivity, fewer cytokines, better growth capacity, better sustained proliferation capacity, better repeated killing capacity, reduced NK cell activation and cell depletion, and an increase in stem cell-like and memory cell subsets of CAR-T cells, resulting in superior therapeutic effects and fewer side effects.

[0015] 2. The chimeric antigen receptor of the present invention can further improve the therapeutic effect on tumors and, at the same time, by downregulating its own cytokines, reduces the production of inflammatory cytokines associated with the activation of macrophages and monocytes, thereby enabling early prevention of cytokine storms. [Brief explanation of the drawing]

[0016] [Figure 1a] These are schematic diagrams of the 28Z CAR, E28Z CAR, and B6I-28Z CAR, which target CD19. [Figure 1b] Flow cytometry plots of anti-CD19 CAR levels on the membrane surface of 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1c] This is a flow cytometry plot of the CD4+ and CD8+ cell ratios in 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1d] These are flow cytometry plots of memory cell clusters in the quiescent phase of 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1e] Statistical charts of immune synapse imaging and ZAP70 recruitment ability in anti-CD19 28Z, E28Z, and B6I-28Z CAR-T after CD19 antigen stimulation. [Figure 1f] Binding ability of anti-CD19 28Z, E28Z, and B6I-28Z CAR-T to target cells with different antigen densities. [Figure 1g] Killing ability of anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells against target cells with different antigen densities. [Figure 1h] Antigen levels on the membranes of remaining target cells after co-culturing anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells with target cells of different antigen densities for 24 hours. [Figure 1i] Levels of cytokines secreted by anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells after CD19 antigen stimulation. [Figure 1j] Growth curves of anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells without antigen stimulation. [Figure 1k] Sustained proliferation ability of anti-CD19 28Z, E28Z, and B6I-28Z CAR-T after the first round of Raji stimulation. [Figure 1l] Repeated killing ability of anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells against Raji. [Figure 1m] NK-like levels in the late stage of repeated killing by anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1n] Cell depletion status in the late stage of repeated killing by anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1o] Cell depletion status in the late stage of repeated killing by anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1p]This shows the cell depletion status in the later stages of repeated killing by anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1q] These are the NK cell activation levels after repeated killing in the fifth round with anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1r] This shows the differential gene expression status after repeated killing in the fifth round with anti-CD19 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 1s] This is a flowchart of the Raji subcutaneous xenograft tumor model treated with CAR-T cell therapy. [Figure 1t] This shows the growth status of Raji subcutaneous xenograft tumors after CAR-T cell therapy. [Figure 1u] This is a flowchart of the Raji subcutaneous xenograft tumor model and tumor recurrence model induced by CAR-T cell therapy. [Figure 1v] This shows the growth status of Raji subcutaneous xenograft tumors and recurrent tumors treated with CAR-T cell therapy. [Figure 2a] These are schematic diagrams of the 28Z CAR, E28Z CAR, and B6I-28Z CAR, which target GPC3. [Figure 2b] Flow cytometry plots of anti-GPC3 CAR levels on the membrane surface of 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 2c] This is a flow cytometry plot of the CD4+ and CD8+ cell ratios in 28Z, E28Z, and B6I-28Z CAR-T cells. [Figure 2d] These are growth curves of anti-GPC3 28Z, E28Z, and B6I-28Z CAR-T cells without antigen stimulation. [Figure 3a] These are schematic diagrams of the structures of 28Z CAR, E28Z CAR, B0I-28Z CAR, B3I-28Z CAR, B6I-28Z CAR, and B9I-28Z CAR, which target mesothelin. [Figure 3b]This is a flow cytometry plot of anti-mesothelin CAR levels on the membrane surface of 28Z, E28Z, B0I-28Z, B3I-28Z, B6I-28Z, and B9I-28Z CAR-T cells. [Figure 3c] This is a statistical diagram of anti-mesothelin CAR levels on the membrane surface of 28Z, E28Z, B0I-28Z, B3I-28Z, B6I-28Z, and B9I-28Z CAR-T cells. [Figure 3d] These are schematic diagrams of the structures of the 28Z CAR, E28Z CAR, B4I-28Z CAR, B5I-28Z CAR, B6I-28Z CAR, B7I-28Z CAR, and B8I-28Z CAR, which target BCMA. [Figure 3e] These are flow cytometry plots and statistical diagrams of anti-BCMA CAR levels on the membrane surface of B4I-28Z, B5I-28Z, B6I-28Z, B7I-28Z, and B8I-28Z CAR-T cells. [Figure 3f] This is a growth curve diagram of anti-mesothelin CAR-T under repeated tumor stimulation conditions. [Figure 3g] This chart shows the regulatory effect of anti-mesothelin CAR-T therapy and animal survival curves in a xenograft HCT116 tumor model. [Figure 4a] This is a schematic diagram of the anti-CD19 B6I-BBZ CAR-T structure. [Figure 4b] This is a flow cytometry plot of the phosphorylation of downstream signals ERK and S6 after CAR-T stimulation in target cells with low CD19 expression. [Figure 4c] This represents the short-term killing level of tumors by B6I BBZ CAR-T cells. [Figure 4d] This is a growth curve diagram of anti-CD19 BBZ CAR-T under repeated tumor-killing conditions. [Figure 4e] This shows the CD56 expression status of anti-CD19 BBZ CAR-T after repeated killing in the 8th round. [Figure 4f] This study describes the regulatory effect of anti-CD19 BBZ CAR-T therapy in a xenograft HCT116 tumor model. [Figure 4g]This is an animal survival curve diagram of anti-CD19 BBZ CAR-T therapy in a xenograft HCT116 tumor model. [Modes for carrying out the invention]

[0017] In one aspect of the present invention, It includes sequentially linked extracellular domains, transmembrane domains, and intracellular domains, The extracellular domain includes an antigen recognition region and a hinge region. The intracellular domain provides a chimeric antigen receptor comprising sequentially linked CD3ε intracellular domain cleavages, a co-stimulatory signaling domain, and a CD3ζ intracellular domain.

[0018] The chimeric antigen receptor provided by the present invention has a CD3ε intracellular domain (see patent CN112500492A), and the cleaved CD3ε intracellular domain retains only sequentially linked BRS sequences and ITAM sequences, with further linking sequences included between the BRS sequences and ITAM sequences.

[0019] In some embodiments, the linking sequence may be a linker sequence well known in the art and suitable for antibodies, for example, a linker sequence containing G and S. In some embodiments, the linking sequence may be a combination of polyGly, polySer, or Gly-Ser. In addition to glycine (G) and serine (S), the linking sequence may contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. In one specific embodiment, the number of amino acid residues in the linking sequence of the present invention is 0 to 9, specifically 0 to 3, 3 to 6, or 6 to 9, etc.

[0020] In one specific example, a cleaved CD3ε intracellular region is modified based on the CD3ε intracellular region sequence, retaining the BRS and ITAM sequences of the CD3ε intracellular region, using six Gs as a ligation sequence, and named B6I. Furthermore, the amino acid sequence of the cleaved CD3ε intracellular region is shown in Sequence ID No. 1, and is specifically as follows: [ka]

[0021] In the chimeric antigen receptor provided by the present invention, the co-stimulatory signaling region is selected from one or more intracellular regions of CD27, CD28, CD134, 4-1BB, or ICOS.

[0022] In one specific embodiment, the co-stimulatory signaling region is selected from CD28.

[0023] The amino acid sequence of CD28 is shown in Sequence ID No. 2, and is specifically as follows: [ka]

[0024] In another specific embodiment, the co-stimulatory signaling region is selected from 4-1BB.

[0025] The amino acid sequence of 4-1BB is shown in Sequence ID No. 3 and is specifically as follows: [ka]

[0026] In the chimeric antigen receptor provided by the present invention, the amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO: 4, and is specifically as follows: [ka]

[0027] In the chimeric antigen receptor provided by the present invention, the antigen recognition region is selected from single-chain antibodies that target tumor surface antigens.

[0028] In some embodiments, the single-chain antibody is selected from one or more of the following: CD19, Glypican-3 (GPC3), mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, Fibroblast activation protein (FAP), CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, and GD2.

[0029] In some embodiments, the single-chain antibody includes a light chain variable region and a heavy chain variable region.

[0030] In some embodiments, the light chain variable region and the heavy chain variable region are connected via a linker array.

[0031] In one specific embodiment, the single-chain antibody is selected from CD19, GPC3, mesothelin, and BCMA, where the monoclonal antibodies corresponding to CD19, GPC3, and mesothelin are commercially available as FMC63, GC33, SS1, and BCMA monoclonal antibodies, respectively, from companies such as Shenzhen Pregene, and the above sequences are known from the disclosure and exhibit excellent target specificity.

[0032] In one specific embodiment, the single-chain antibody comprises a light chain variable region and a heavy chain variable region of the monoclonal antibody FMC63, the light chain variable region and the heavy chain variable region being optionally linked via a linker sequence.

[0033] In one specific embodiment, the single-chain antibody comprises a light chain variable region and a heavy chain variable region of the monoclonal antibody GC33, the light chain variable region and the heavy chain variable region being optionally linked via a linker sequence.

[0034] In one specific embodiment, the single-chain antibody comprises a light chain variable region and a heavy chain variable region of a monoclonal antibody SS1, the light chain variable region and the heavy chain variable region being optionally linked via a linker sequence.

[0035] The scFv amino acid sequence of FMC63 is shown in SEQ ID NO: 10, and is specifically as follows: [ka]

[0036] The scFv amino acid sequence of GC33 is shown in SEQ ID NO: 11, and is specifically as follows: [ka]

[0037] The scFv amino acid sequence of SS1 is shown in SEQ ID NO: 20, and is specifically as follows: [ka]

[0038] The scFv amino acid sequence of BCMA is shown in reference to patent CN109134665B.

[0039] Furthermore, the extracellular structure may contain an additional signal peptide, forming a sequentially linked signal peptide-antigen recognition region-hinge region.

[0040] In the chimeric antigen receptor provided by the present invention, the transmembrane domain is selected from CD28™, CD4, CD8α, OX40, or H2-Kb. In one specific embodiment, the transmembrane domain is selected from CD28™. The amino acid sequence of CD28™ is shown in Sequence ID No. 5 and is specifically as follows:FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0041] Furthermore, the extracellular domain contains an additional signal peptide, forming a sequentially linked signal peptide-antigen recognition region-hinge region.

[0042] In some embodiments, the amino acid sequences of the chimeric antigen receptors are shown in SEQ ID NO: 6 (anti-CD19 B6I-28ZCAR), SEQ ID NO: 7 (anti-GPC3 B6I-28ZCAR), and SEQ ID NO: 21 (anti-mesothelin B6I-28ZCAR).

[0043] Sequence ID 6 (Anti-CD19 B6I-28Z CAR): [ka]

[0044] The nucleotide sequence corresponding to Sequence ID No. 6 (anti-CD19 B6I-28Z CAR) is shown in Sequence ID No. 8 (anti-CD19B6I-28Z CAR), which is as follows: [ka]

[0045] Sequence ID 7 (Anti-GPC3 B6I-28Z CAR): [ka]

[0046] The nucleotide sequence corresponding to Sequence ID No. 7 (anti-GPC3 B6I-28Z CAR) is shown in Sequence ID No. 9 (anti-GPC3 B6I-28Z CAR), which is as follows: [ka]

[0047] Sequence ID 21 (Anti-mesoterin B6I-28Z CAR): [ka]

[0048] The nucleotide sequence corresponding to Sequence ID No. 21 (anti-mesothelin B6I-28Z CAR) is shown in Sequence ID No. 19 (anti-mesothelin B6I-28Z CAR). [ka]

[0049] The parts constituting the chimeric antigen receptor of the present invention may be directly linked to each other or linked via a linker sequence. The linker sequence may be a linker sequence well known in the art and suitable for antibodies, such as a linker sequence containing G and S. Generally, the linker contains one or more repeating motifs before and after it. For example, the motifs may be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent within the linker sequence and no amino acid residues are inserted between the repeats. The linker sequence may consist of 1, 2, 3, 4, or 5 repeating motifs. The length of the linker may be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In one embodiment, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited and is usually 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q).

[0050] In gene cloning operations, the need to design appropriate restriction enzyme sites inevitably means introducing one or more irrelevant residues to the ends of the expressed amino acid sequence, but it should be understood that this does not affect the activity of the target sequence. To facilitate the construction of fusion proteins, the promotion of recombinant protein expression, the acquisition of autosecreted recombinant proteins outside host cells, or the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other appropriate region within the recombinant protein (including, but not limited to, appropriate linker peptides, signal peptides, leader peptides, end extensions, etc.). Therefore, the amino-terminus or carboxyl-terminus of the fusion protein (i.e., CAR) of the present invention may further contain one or more polypeptide fragments as protein tags. Any appropriate tag can be used herein. For example, the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0051] Another aspect of the present invention provides polynucleotide sequences selected from the following: (1) A polynucleotide sequence encoding the chimeric antigen receptor described in the claim, and / or (2) (1) Complementary sequence of polynucleotide sequence.

[0052] The polynucleotide sequences of the present invention may be in DNA form or RNA form. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The present invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequence but have a different nucleotide sequence.

[0053] The polynucleotide sequences described herein can typically be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be obtained by amplification using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. For long sequences, two or more PCR amplifications are often required, after which the fragments obtained from each amplification are joined together in the correct order.

[0054] Another aspect of the present invention provides nucleic acid constructs comprising the polynucleotide sequence.

[0055] The nucleic acid construct further includes one or more regulatory sequences functionally linked to the polynucleotide sequence. The coding sequence of the CAR of the present invention can be manipulated in various ways to ensure protein expression. Before inserting the nucleic acid construct into a vector, the nucleic acid construct can be manipulated according to differences or requirements of the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0056] The regulatory sequence may also be an appropriate promoter sequence. Promoter sequences are typically functionally linked to the coding sequence of the expressed protein. Promoter sequences are any nucleotide sequences that exhibit transcriptional activity in a selected host cell, and include mutagenic promoters, cleavage promoters, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to those host cells.

[0057] The regulatory sequence may further be a suitable transcriptional terminator sequence recognized by the host cell to terminate transcription. The terminator sequence is functionally ligated to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that functions in selected host cells can be used in the present invention.

[0058] The regulatory sequence may be a suitable leader sequence having an untranslated region of mRNA important for translation in the host cell. The leader sequence is operably ligated to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that functions in a selected host cell can be used in the present invention.

[0059] In a preferred embodiment of the present invention, the nucleic acid construct is a vector.

[0060] Typically, the expression of a polynucleotide sequence encoding a CAR is achieved by operably ligating the sequence to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and incorporation in eukaryotic cells. Common cloning vectors include transcription and translation terminators, start sequences, and promoters that can be used to regulate the expression of a desired nucleic acid sequence.

[0061] The polynucleotide sequence encoding the CAR of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phage particles, phage derivatives, animal viruses, and cosmids. Furthermore, the vector is an expression vector. The expression vector may be supplied to cells in the form of a viral vector. Viral vector technology is well known in the art. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selection markers.

[0062] In one specific embodiment of the present invention, the nucleic acid construct is a lentiviral vector comprising an origin of replication, a 3'LTR, a 5'LTR, and the polynucleotide sequence.

[0063] One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can express at high levels any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the promoters listed below: namely, the simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMμLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters including but not limited to actin promoters, myosin promoters, heme promoters, and creatine kinase promoters. Furthermore, the use of inductive promoters can also be considered. The use of inductive promoters provides a molecular switch, which allows the expression of a polynucleotide sequence operably linked to the inductive promoter to be turned on when expression is desired and turned off when expression is not desired. Examples of inductive promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0064] Expression vectors introduced into cells to evaluate the expression of CAR polypeptides or parts thereof may include one or two selection marker genes or reporter genes that facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. Alternatively, the selection marker may be housed in a separate DNA segment and used in co-transfection procedures. Both the selection marker and reporter genes can be flanked by appropriate regulatory sequences to enable expression within host cells. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0065] Reporter genes can be used to identify potentially transfected cells and assess the functionality of regulatory sequences. After DNA is introduced into receptor cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are publicly known and can be manufactured using well-known techniques or purchased commercially.

[0066] In this art, methods for introducing genes into cells and expressing those genes within the cells are well known. Vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in this art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0067] Physical methods used to introduce polynucleotides into host cells include calcium phosphate precipitation, lipofection, gene guns, microinjection, and electroporation. Biological methods for introducing the desired polynucleotide into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include, for example, colloidal dispersions of lipid-based systems including polymer complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0068] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for gene insertion into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. Many virus-based systems have been developed for gene delivery into mammalian cells. For example, lentiviruses have provided a convenient platform for gene delivery systems. Using techniques well known in the art, selected genes can be inserted into vectors and packaged into lentiviral particles. This recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are well known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are well known in the art. In one embodiment, a lentiviral vector is used.

[0069] Another aspect of the present invention provides a lentiviral vector system comprising the nucleic acid construct and lentiviral vector auxiliary components.

[0070] The lentiviral auxiliary component includes a lentiviral packaging plasmid and a cell line. The lentiviral vector system is formed by viral packaging of the nucleic acid construct with the assistance of the lentiviral packaging plasmid and cell line. The method for constructing the lentiviral vector system is a conventional method in the art.

[0071] Another aspect of the present invention provides a method for activating T cells in vitro, comprising the step of infecting the T cells with the lentivirus.

[0072] The CAR-T cells of the present invention undergo robust in vivo T cell expansion, resulting in sustained high levels in the blood and bone marrow, and forming specific memory T cells. After encountering and subsequently eliminating target cells expressing surrogate antigens, the CAR-T cells of the present invention can differentiate into a central memory-like state in the body.

[0073] The present invention further includes a type of cell therapy in which T cells are genetically modified to express the CARs of this specification, and the CAR-T cells are injected into recipients who require them. The injected cells can kill tumor cells in the recipient. Unlike antibody therapy, CAR-T cells can replicate in the body, producing long-term persistence that results in sustained tumor control.

[0074] The antitumor immune response induced by CAR-T cells may be an active or passive immune response. Furthermore, the CAR-mediated immune response may be part of an adoptive immunotherapy step in which CAR-T cells induce an immune response specific to the antigen-binding portion within the CAR.

[0075] The treatable cancers may be non-solid tumors, such as hematological malignancies like leukemia and lymphoma. In particular, diseases that can be treated using the CARs, their coding sequences, nucleic acid constructs, expression vectors, viruses, and CAR-T cells of the present invention may be used. The diseases may be CD19 or GPC3-mediated diseases, and in particular CD19 or GPC3-mediated tumors.

[0076] Specifically, in this specification, “diseases mediated by CD19” include, but are not limited to, leukemia and lymphoma, for example, B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, pilocytic cell leukemia, and acute myeloid leukemia.

[0077] Specifically, in this specification, phosphatidylinositol proteoglycan-3 (GPC3) is an extracellular matrix protein expressed in embryonic tissues, particularly the liver and kidney, and is involved in organogenesis. "Diseases mediated by GPC3" include, but are not limited to, hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma.

[0078] Another aspect of the present invention provides genetically modified T cells comprising the polynucleotide sequence, or comprising the nucleic acid construct, or infected with the lentiviral vector system.

[0079] The CAR-modified T cells of the present invention can be administered alone or as part of a pharmaceutical composition in combination with other components such as diluents and / or associated cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may contain the CAR-T cells described herein and be combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such a composition may contain buffers such as neutral buffered saline or sulfate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0080] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease being treated (or prevented). The dosage and frequency are determined by factors such as the patient's condition, the type and severity of the patient's disease.

[0081] When specifying an "immunologically effective dose," "antitumor effective dose," "tumor-suppressing effective dose," "therapeutic effective dose," or "therapeutic dose," the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease state. Generally, the pharmaceutical compositions containing T cells described herein are 10 4 ~10 9Cells / kg body weight, preferably 10 5 ~10 6 It is indicated that the T cell composition may be administered in doses of cells / kg body weight. Multiple doses of the T cell composition may be administered at these doses. The cells can be administered using infusion techniques well known in immunotherapy. The optimal dose and treatment method for a particular patient can be easily determined by a healthcare professional by monitoring the patient's disease symptoms and thereby adjusting the treatment accordingly.

[0082] The composition of interest may be administered by any simple method, including spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intralymphatically, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous administration. The T cell composition may be injected directly into a tumor, lymph node, or infected site.

[0083] In some embodiments of the present invention, the CAR-T cells or compositions thereof of the present invention can be combined with other therapies known in the art. Therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, they can be combined with various radiotherapy agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, FK506, fludarabine, rapamycin, and mycophenolate. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, concurrently with, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external radiation therapy (XRT), cyclophosphamide, or T-cell ablation therapy using antibodies such as OKT3 or CAMPATH.

[0084] In this specification, “antitumor effect” refers to a biological effect expressed by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an extension of expected lifespan, or an improvement in various physiological symptoms associated with cancer.

[0085] The terms "patient," "subject," "individual," and "subject" are used interchangeably herein and refer to organisms capable of eliciting an immune response, such as mammals or non-mammals. Mammals are preferably rodents, artiodactyls, odd-toed ungulates, lagomorphs, primates, etc. Non-mammals include, for example, non-mammalian vertebrates such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and their transgenic species. In one specific embodiment, the subject may be a human, for example, a patient suffering from immunodeficiency or cancer.

[0086] (1) Production of CAR-T cells, (2) Increase in CAR molecular levels on the membrane surface of CAR-T cells, (3) Improvement of CAR-T cell antigen sensitivity, (4) Suppression of cytokine secretion of CAR-T cells (IFN-γ, IL-2, TNF), (5) Promotion of spontaneous growth of CAR-T cells during the quiescent phase, (6) Enhancement of sustained proliferative capacity of CAR-T cells after target cell stimulation, (7) Improvement of repeated killing capacity of CAR-T cells, (8) Reduction of NK cell conversion and cell depletion of CAR-T cells, (9) Increase in stem cell-like and memory cell subsets of CAR-T cells. The use of the chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, and the lentiviral vector system for the manufacture of one or more of the following application products.

[0087] The use of the chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, the lentiviral vector system, or the genetically modified T cells for the manufacture of tumor therapeutic products.

[0088] In one specific embodiment of the present invention, the tumor is selected from one or more of leukemia or solid tumors.

[0089] In preferred embodiments of the present invention, the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, pilocytic cell leukemia, acute myeloid leukemia, hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, or lung squamous cell carcinoma.

[0090] The present invention also provides a method for treating a tumor, comprising administering a therapeutically effective amount of the gene-modified T cells to a target subject of interest.

[0091] "Treatment" or "therapy" of a condition includes prevention or reduction of a condition, reduction of the onset or progression of a condition, reduction of the risk of developing a condition, prevention or delay of the onset of symptoms associated with a condition, reduction or elimination of symptoms associated with a condition, induction of complete or partial recovery of a condition, cure of a condition, or a combination thereof. In cancer, "treatment" or "therapy" may mean inhibiting or delaying the growth, regeneration, or metastasis of a tumor or malignant cells, or a combination of some of these. With respect to tumors, "treatment" or "therapy" includes removing all or part of a tumor, inhibiting or delaying the growth and metastasis of a tumor, preventing or delaying the development of a tumor, or a combination of some of these.

[0092] When administering to subjects, the dosage will vary depending on the patient's age and weight, the characteristics and severity of the disease, and the route of administration. The results of animal experiments and various circumstances may be referenced, and the total dosage must not exceed the prescribed range.

[0093] The following specific examples illustrate embodiments of the present invention, and those skilled in the art will readily understand other advantages and effects of the present invention from the disclosure herein. The present invention may be carried out or applied by further different specific embodiments, and each detail herein may be modified or altered in various ways based on different viewpoints and uses, without departing from the spirit of the invention.

[0094] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific implementations described below, and that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments and do not limit the scope of protection of the present invention. In the specification and claims of the present invention, unless otherwise explicitly indicated in the text, the singular forms "one," "one," and "this" include the plural forms.

[0095] Where the examples indicate numerical ranges, it should be understood that, unless otherwise specified in the Invention, any numerical values ​​at both endpoints of each numerical range and any values ​​between those endpoints may be used. Unless otherwise defined, all technical and scientific terms used in the Invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in the examples, the Invention may be carried out using any prior art methods, apparatus, and materials similar or equivalent to those described in the Examples of the Invention, based on the prior art knowledge of those skilled in the art and the description of the Invention.

[0096] Unless otherwise stated, the experimental methods, detection methods, and manufacturing methods disclosed in this invention utilize conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0097] Example 1 Anti-CD19 B6I-28Z was modified based on the anti-CD19 E28Z sequence (see patent CN112500492A), retaining the BRS and ITAM sequences of the CD3ε intracellular region. A cleaved portion of the CD3ε intracellular region was constructed using six Gs as a ligation sequence, and the resulting chimeric antigen receptor was named B6I-28Z. The nucleotide sequence of anti-CD19 B6I-28Z CAR is shown in Sequence ID No. 8, and is specifically as follows: [ka] Anti-CD19 B6I-28Z CAR was subcloned into the lentiviral expression plasmid pHAGE vector. The CAR expression plasmid was then mixed with packaging plasmids pSPAX2 and pMD2G in a ratio of 10:7.5:3.5, and introduced into 293FT cells by calcium phosphate transfection to produce lentiviral particles. By ultracentrifugation, 28Z CAR, E28Z CAR (see patent CN112500492A), and B6I-28Z CAR lentiviruses were expressed, and small-volume, high-titer viruses (~10) were produced. 8 After concentration ( / ml), primary T cells (MOI=10) activated with αCD3 / αCD28Ab-beads for 1 day were transfected. Primary T cells were cultured in T cell complete medium (XIVO-15 + 1% PS + 10 ng / ml human IL-7 + 10 ng / ml human IL-15 + 5% human AB serum + 10 mM neutralizing NAC), removed 1 day after viral transfection, and removed 4 days after antibody stimulation. CAR-T cells (GFP-positive) were selected on 5 days after antibody stimulation. After amplification for a predetermined period, a series of indicators were evaluated for CAR-T cells, such as CAR membrane level, CD4 and CD8 subset ratio, extracorporeal cytokine secretion capacity, extracorporeal killing capacity, extracorporeal proliferation capacity, extracorporeal apoptosis level, extracorporeal survival capacity, and extracorporeal antitumor effect. The structures of the 28Z CAR, E28Z CAR, and B6I-28Z CAR, which target CD19, are shown in Figure 1a. Flow cytometry using anti-HA rabbit primary antibody and anti-rabbit AF647 secondary antibody measured CAR levels on the cell membrane surfaces of 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells. The flow cytometry results are shown in Figure 1b, demonstrating that B6I-28Z CAR restores the CAR molecular membrane level reduction caused by E28Z CAR. Flow cytometry using anti-human CD4-PB and anti-human CD8-PE antibodies detected 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells. The flow cytometry results are shown in Figure 1c, and the CD4 levels of the three cells were detected. +CAR-T cells and CD8 + The ratio of CAR-T cells was shown to be similar. Flow cytometry using anti-human CD62L-APC and anti-human CD45RA-PE-cy7 antibodies was used to detect the memory subsets of 28Z CAR-T, Ε28Z CAR-T, and B6I-28Z CAR-T cells, and the flow cytometry results are shown in Figure 1d. B6I-28Z CAR-T cells have the highest ratio of stem cell-like central memory cell subsets (CD62L + CD45RA + ), followed by E28Z CAR-T cells, and 28Z CAR-T cells have the lowest ratio. When cells were stimulated with 10 nM concentration of CD19 antigen and adhesion molecule ICAM-1, the immune synapses (Synapse) formed by 28Z CAR-T, Ε28Z CAR-T, and B6I-28Z CAR-T cells are shown in Figure 1e. The B6I-28Z CAR molecule showed higher fluorescence intensity and simultaneously recruited downstream Zap70 signaling molecules more effectively. Raji cells, low antigen-expressing K562 cells (K562L), and extremely low antigen-expressing K562 cells (K562VL) were co-cultured with CAR-T cells. The binding rate between target cells and CAR-T was detected at two time points of 10 minutes and 30 minutes, and the results are shown in Figure 1f. B6I-28Z CAR-T cells and Ε28Z CAR-T cells have stronger cell-to-cell contact ability compared to 28Z CAR-T cells, indicating that B6I CAR-T cells have better antigen sensitivity. K562 H, K562 L, and CAR-T cells were mixed at a ratio of 1:1:2 to simulate the antigen expression down-regulation scenario during tumor escape. After culturing for 30 hours, the number of K562 H and K562 L cells was detected, and the results are shown in Figure 1g. Compared with 28Z CAR-T and Ε28Z CAR-T cells, B6I-28Z CAR-T has fewer remaining K562 L cells after killing, indicating that it has better antigen sensitivity. Raji, K562 H, K562 L, and CAR-T cells were co-cultured in a 1:1 ratio. After 24 hours of culture, the mean CD19 fluorescence intensity on the surface of the remaining Raji, K562 H, and K562 L cells was detected using anti-CD19-PE antibody, and the results are shown in Figure 1h. Compared to 28Z CAR-T and E28Z CAR-T cells, B6I-28Z CAR-T cells showed lower fluorescence intensity of the remaining target cell surface antigen after killing, indicating superior antigen sensitivity. 100,000 CAR-T cells and 100,000 CD19+ lymphoma cell line Raji were seeded in a 1:1 ratio in a round-bottom 96-well plate, homogeneously mixed, and then centrifuged at room temperature (400g, 1 min) to promote cell-cell contact. After culturing in complete medium in a 37°C incubator for 1 day, the samples were collected. Six hours before sample collection, 1× BFA was added to inhibit cytokine efflux. After sample collection, the cells were washed once with PBS, then fixed with 4% PFA at room temperature for 5 minutes, and the wells were punched in with 0.1% TritonX-100 at room temperature for 5 minutes. Subsequently, IL-2, IFN-γ, and TNF-α were detected by intracellular staining according to standard procedures. The results are shown in Figure 1i, where E28Z CAR-T cells and B6I-28Z CAR-T cells were found to be CD19+. + The amounts of cytokines IL-2, IFN-γ, and TNF-α produced after specific cell stimulation were all significantly lower compared to 28Z CAR-T cells. CAR-T cells were cultured for a predetermined period without antigen stimulation and counted every 3-4 days. The results are shown in Figure 1j, demonstrating that the growth capacity of B6I-28Z CAR-T cells was superior to that of E28Z CAR-T cells and 28Z CAR-T cells. 100,000 CAR-T cells and CD19 + Lymphoma cell line Raji was seeded in a 1:1 ratio in a round-bottom 96-well plate, mixed uniformly, and then centrifuged at room temperature (400g, 1 minute) to promote cell-to-cell contact. The cells were then cultured in complete medium in a 37°C incubator for a predetermined period. Cell counts were performed every 3-4 days during the period, and the results are shown in Figure 1k. The sustained proliferative capacity of B6I-28Z CAR-T cells was shown to be superior to that of E28Z CAR-T cells and 28Z CAR-T cells. 100,000 CAR-T cells and CD19 + Lymphoma cell line Raji was seeded in a 1:1 ratio in a round-bottom 96-well plate, mixed uniformly, and counted after 3 days. After haploid elimination, the cells were seeded in a new round-bottom 96-well plate, and based on the counting results, the same number of CD19 cells as CAR-T cells were counted. + The lymphoma cell line Raji was added, homogeneously mixed, and counted after 3 days. This process was repeated for 6-7 rounds. The results are shown in Figure 1. The repeated killing ability of B6I-28Z CAR-T cells was shown to be superior to that of E28Z CAR-T cells and 28Z CAR-T cells. In the later stages of repeated cell killing, cells were washed once with PBS and then surface-stained with anti-CD56-PE, anti-TIM3-PE, anti-PD1-APC, and anti-TIGIT-BV421. The results are shown in Figure 1-mp. B6I-28Z CAR-T cells showed reduced NK conversion and cell depletion compared to E28Z CAR-T cells and 28Z CAR-T cells. 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells were collected after repeated killing in the fifth round, RNA was extracted and subjected to RNA-seq, and the sequencing results were analyzed using a NK cell activating database under GSEA. The results are shown in Figure 1q, indicating that B6I CAR-T cells had the lowest degree of NK cell activating, while 28Z CAR-T cells had the most severe NK cell activating. 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells were collected after repeated killing in the fifth round, RNA was extracted and subjected to RNA-seq, and differential gene expression analysis was performed. The results are shown in Figure 1r, and it was shown that B6I CAR-T cells highly expressed stem cell-related transcription factors such as KLF2. 3 × 10 by aseptic technique 7A Raji cell-PBS suspension was prepared at a concentration of / ml, and 3 million Raji cells in 100 μL were subcutaneously inoculated into the left dorsal region of 6-week-old B-NDG female mice, with this point designated as day 0. After 7 days, when the diameter of the Raji subcutaneous tumors reached 5-6 mm, the mice were randomly divided into groups, and 6 million CAR-T cells in 100 μL were intravenously injected into the tail vein of each mouse. The model is shown in Figure 1s. Subsequently, the tumor size was measured periodically using calipers, and the data was recorded. Tumor area = length × width. The results are shown in Figure 1t. The results showed that tumor growth was fastest in the vector control group, followed by the 28Z group. The B6I-28Z CAR-T group showed the best therapeutic effect, while the E28Z group showed an intermediate therapeutic effect between the 28Z group and the B6I-28Z group. 3 × 10 by aseptic technique 7 A Raji cell-PBS suspension was prepared at a concentration of / ml, and 3 million Raji cells in 100 μL were subcutaneously inoculated into the left dorsal region of 6-week-old B-NDG female mice. This point was designated as day 0. After 7 days, when the diameter of the Raji subcutaneous tumors reached 5-6 mm, the mice were randomly divided into groups. Subsequently, 6 million CAR-T cells in 100 μL were intravenously injected into the tail vein of each mouse. The tumor size was then measured periodically using calipers, and the data was recorded. Tumor area = length × width. The results are shown on the left side of Figure 1v, and the tumors completely disappeared after 1 month. Mice with completely disappeared tumors were selected and 1 × 10⁶ mice were removed using aseptic techniques. 7 A low-expression CD19 K562 cell-PBS suspension was prepared at a concentration of / ml, and 100 μL of CD19-low-expression K562 cells were inoculated into the right dorsal region of each mouse to simulate a tumor recurrence scenario, the model of which is shown in Figure 1u. Subsequently, the tumor size was measured periodically using calipers, and the data was recorded, with tumor area = length × width. The results are shown on the right side of Figure 1v. The results showed that the B6I-28Z CAR-T group exhibited the best therapeutic effect against re-transplanted low-antigen tumors, followed by the E28Z group, and the 28Z group exhibited the worst therapeutic effect.

[0098] Example 2 As shown in Figure 2a, by sequentially substituting the FMC63 scFv (anti-CD19) sequence with GC33 scFv (anti-GPC3) in the CD19-targeting 28Z CAR, E28Z CAR, and B6I-28Z CAR, we obtained 28Z CAR, E28Z CAR, and B6I-28Z CAR, respectively, that target GPC3. The nucleotide sequence of anti-GPC3 B6I-28Z CAR is shown in Sequence ID No. 9, and is specifically as follows: [ka] Flow cytometry using anti-HA rabbit primary antibody and anti-HA rabbit AF647 secondary antibody measured CAR expression levels on the cell membrane surfaces of 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells. The flow cytometry results are shown in Figure 2b, demonstrating that B6I-28Z CAR restores the decrease in CAR molecular membrane levels caused by E28Z CAR. Flow cytometry using anti-human CD4-PB and anti-human CD8-PE antibodies detected 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells. The flow cytometry results are shown in Figure 2c, and the CD4 levels of the three cells were detected. + CAR-T cells and CD8 + The ratio of CAR-T cells was shown to be similar. CAR-T cells were cultured for a predetermined period without antigen stimulation and counted every 3-4 days. The results are shown in Figure 2d, and B6I-28Z CAR-T and E28Z CAR-T cells showed superior growth ability compared to 28Z CAR-T cells.

[0099] Comparative Example 1 As shown in Figures 3a and 3d, the FMC63 scFv (anti-CD19) sequence in the CD19-targeting 28Z CAR, E28Z CAR, and B6I-28Z CAR was sequentially replaced with SS1 scFv (anti-mesothelin) and anti-BCMA, respectively, resulting in CARs that target mesothelin and BCMA. In CARs targeting mesoserine, if the ligation sequence of the CD3ε intracellular domain has 0, 3, 6, or 9 Gs, the resulting CARs are named anti-Meso B0I-28Z, anti-Meso B3I-28Z, anti-Meso B6I-28Z, and anti-Meso B09I-28Z, respectively. In CARs targeting BCMA, if the ligation sequence of the CD3ε intracellular domain has 4, 5, 6, 7, or 8 Gs, the resulting CARs are named anti-BCMA B4I-28Z, anti-BCMA B5I-28Z, anti-BCMA B6I-28Z, anti-BCMA B7I-28Z, and anti-BCMA B8I-28Z, respectively. The sequences of the chimeric antigen receptors are as follows: The nucleotide sequence of anti-Meso B0I-28Z is shown in Sequence ID No. 12, and is specifically as follows: [ka] The nucleotide sequence of anti-Meso B3I-28Z is shown in Sequence ID No. 13, and is specifically as follows: [ka] The amino acid sequence structure of anti-BCMA B4I-28Z is BCMA single-chain antibody (see patent CN109134665B) + CD28™ (SEQ ID NO: 5) + CD3ε intracellular region cleavage (KNRKAKAKGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 14) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4). The amino acid sequence structure of anti-BCMA B5I-28Z is BCMA single-chain antibody (see patent CN109134665B) + CD28™ (SEQ ID NO: 5) + CD3ε intracellular region cleavage (KNRKAKAKGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 15) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4). The amino acid sequence structure of anti-BCMA B6I-28Z is BCMA single-chain antibody (see patent CN109134665B) + CD28™ (SEQ ID NO: 5) + CD3ε intracellular region cleavage (SEQ ID NO: 1) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4). The nucleotide sequence of anti-Meso B6I-28Z is shown in Sequence ID No. 19, and is specifically as follows: [ka] The amino acid sequence structure of anti-BCMA B7I-28Z is BCMA single-chain antibody (see patent CN109134665B) + CD28™ (SEQ ID NO: 5) + CD3ε intracellular region cleavage (KNRKAKAKGGGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 16) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4). The amino acid sequence structure of anti-BCMA B8I-28Z is BCMA single-chain antibody (see patent CN109134665B) + CD28™ (SEQ ID NO: 5) + CD3ε intracellular region cleavage (KNRKAKAKGGGGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 17) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4). The nucleotide sequence of anti-Meso B9I-28Z is shown in Sequence ID No. 18, and is specifically as follows: [ka] The results are shown in Figure 3, and the structures of the mesothelin-targeting 28Z CAR, E28Z CAR, B0I-28Z CAR, B3I-28Z CAR, B6I-28Z CAR, and B9I-28Z CAR are shown in Figure 3a. Flow cytometry using anti-HA rabbit primary antibody and anti-rabbit AF647 secondary antibody was performed to measure CAR levels on the cell membrane surface of 28Z CAR-T, E28Z CAR-T, B0I-28Z CAR-T, B3I-28Z CAR-T, B6I-28Z CAR-T, and B9I-28Z CAR-T cells. Flow cytometry results are shown in Figure 3b, and statistical results are shown in Figure 3c. The results showed that B0I-28Z, B3I-28Z, B6I-28Z, and B9I-28Z CARs could partially restore membrane surface CAR molecular levels compared to E28Z, with B6I-28Z CAR showing the most effective restoration. The structures of the BCMA-targeting 28Z CAR, E28Z CAR, B4I-28Z CAR, B5I-28Z CAR, B6I-28Z CAR, B7I-28Z CAR, and B8I-28Z CAR are shown in Figure 3d. Flow cytometry using anti-HA rabbit primary antibody and anti-rabbit AF647 secondary antibody was performed to measure CAR levels on the cell membrane surface of B4I-28Z CAR-T, B5I-28Z CAR-T, B6I-28Z CAR-T, B7I-28Z CAR-T, and B8I-28Z CAR-T cells. The flow cytometry results are shown in Figure 3e, indicating that B6I-28Z CAR-T cells had the highest membrane surface CAR molecular level. Using solid tumor HCT116 expressing mesothelin antigen as the target cell, the aforementioned in vitro repeated killing experiment was performed using the target mesothelin B6I-28Z CAR, and changes in the number of CAR-T cells were monitored. As shown in Figure 3f, anti-mesothelin B6I-28Z CAR-T cells showed superior long-term proliferation compared to conventional 28Z CARs. HCT116 tumor cells were injected subcutaneously into mice to construct a subcutaneous tumor model, and CAR-T cells were reinjected after 5 days, as shown in Figure 3g. The results showed that reinjection of anti-mesothelin B6I-28Z CAR-T cells resulted in superior tumor inhibition compared to 28Z CAR-T cells, and extended mouse survival.

[0100] Comparative Example 2 As shown in Figure 4a, based on the aforementioned CD19.B6I 28Z CAR design, the CD28 costimulatory domain was replaced with the 4-1BB costimulatory domain to construct a BBZ-based B6I CAR, which was named the anti-CD19 B6I-BBZ CAR. The amino acid sequence structure of anti-CD19 B6I-BBZ is as follows: CD19 single-chain antibody (SEQ ID NO: 10) + CD8™ (SEQ ID NO: 5) + CD3ε intracellular domain cleavage (KNRKAKAKGGGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 16) + 4-1BB intracellular domain (SEQ ID NO: 4) + CD3ζ intracellular domain (SEQ ID NO: 4). The amino acid sequence is shown in SEQ ID NO: 22: [ka] The nucleotide sequence is shown in Sequence ID No. 23: [ka] When CAR-T cells were stimulated with CD19-low-expressing K562 cells, strong downstream signaling, including pERK and pS6, was observed in the anti-CD19 B6I-BBZ CAR, demonstrating its strong antigen sensitivity, as shown in Figure 4b. Using the above CAR-T cells, CD19-low-expressing or high-expressing K562 cells were killed, and the cell counts were counted at 24, 48, and 72 hours, respectively. This confirmed that the anti-CD19 B6I-BBZ CAR was more effective in eliminating tumor cells, as shown in Figure 4c. Using Raji cells, which naturally express CD19, as target cells, we performed continuous in vitro tumor killing using the cells designed above, and monitored the proliferation of CAR-T cells during the killing process. The CD19 B6I BBZ CAR showed stronger sustained proliferation ability, as shown in Figure 4d. CAR-T cells were collected after the 8th round of killing, and the NK cell marker CD56 was detected. The results showed that B6I BBZ exhibited weaker NK cell conversion and better functional persistence, as shown in Figure 4e. To simulate heterogeneity in tumor antigen expression within the body, equal volumes of K562 tumor cells expressing high and low levels of CD19 were mixed and injected subcutaneously into mice to establish a tumor model. When CAR-T cells were reinjected after 5 days, the B6I BBZ group showed better tumor control, as shown in Figure 4f, and also effectively extended the survival time of the mice, as shown in Figure 4g. The above description is merely a preferred embodiment of the present invention and does not limit the invention in any form or content. Those skilled in the art should note that various improvements and additions can be made without departing from the methods of the present invention, and these improvements and additions are also within the scope of protection of the present invention. Those skilled in the art can make any modifications, changes, or variations to the disclosed technical content without departing from the spirit and scope of the present invention, and all such modifications, changes, and variations are equivalent embodiments of the present invention. Furthermore, modifications, changes, or variations made to the above embodiments based on the essential art of the present invention are still within the scope of the technical solutions of the present invention.

Claims

1. A chimeric antigen receptor comprising sequentially linked extracellular domains, transmembrane domains, and intracellular domains, The extracellular domain includes an antigen recognition region and a hinge region. The intracellular domain is a chimeric antigen receptor comprising sequentially linked cleaved CD3ε intracellular regions, a co-stimulatory signaling region, and a CD3ζ intracellular region.

2. The chimeric antigen receptor according to claim 1, characterized in that, with respect to the intracellular region of CD3ε, the cleaved fragments of the intracellular region of CD3ε retain only sequentially linked BRS sequences and ITAM sequences, and further linking sequences are included between the BRS sequences and ITAM sequences.

3. The chimeric antigen receptor according to claim 2, characterized in that the linked sequence comprises a plurality of amino acid residues, the amino acid residues of the linked sequence include glycine and / or serine, the number of amino acid residues of the linked sequence is 0 to 9, and the amino acid sequence of the cleaved CD3ε intracellular region is shown in SEQ ID NO:

1.

4. The chimeric antigen receptor according to claim 1, characterized in that the co-stimulatory signaling region is selected from one or more intracellular regions of CD27, CD28, CD134, 4-1BB, and ICOS.

5. The chimeric antigen receptor according to claim 4, further comprising one or more of the following features. (1) The amino acid sequence of the intracellular region of CD28 has the characteristics shown in SEQ ID NO:

2. (2) The amino acid sequence of the 4-1BB intracellular region has the characteristics shown in SEQ ID NO:

3. (3) The amino acid sequence of the CD3ζ intracellular region is as shown in Sequence ID No.

4.

6. The chimeric antigen receptor according to claim 1, further comprising one or more of the following features. a. The antigen recognition region is selected from single-chain antibodies that target tumor surface antigens, and the single-chain antibody is characterized by being selected from one or more of the following: CD19, GPC3, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38, CD138, BCMA, fibroblast activation protein, CEA, EGFRvIII, PSMA, Her2, IL13Rα2, CD171, and GD2. b. The transmembrane domain is characterized by being selected from CD28™, CD4, CD8α, OX40, or H2-Kb.

7. The chimeric antigen receptor according to claim 6, further comprising one or more of the following features. c. The amino acid sequence of the single-chain antibody has the characteristics shown in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

20. d. The transmembrane domain is selected from CD28™, and the amino acid sequence of CD28™ is characterized as shown in Sequence ID No.

5.

8. The chimeric antigen receptor according to any one of claims 1 to 7, characterized in that the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 21, or SEQ ID NO:

22.

9. (1) A polynucleotide sequence encoding a chimeric antigen receptor according to any one of claims 1 to 8, and / or (2) Complementary sequence of the polynucleotide sequence described in (1) above A polynucleotide sequence selected from the following.

10. The polynucleotide sequence according to claim 9, characterized in that the polynucleotide sequence is shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, or SEQ ID NO:

23.

11. A nucleic acid construct comprising the polynucleotide sequence described in any one of claims 9 to 10, Preferably, the nucleic acid construct is a vector, More preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3'LTR, a 5'LTR, and a polynucleotide sequence according to any one of claims 9 to 10.

12. A lentiviral vector system comprising the nucleic acid construct and lentiviral vector auxiliary component according to claim 11.

13. Genetically modified T cells characterized by comprising a polynucleotide sequence according to any one of claims 9 to 10, or comprising a nucleic acid construct according to claim 11, or being infected with a lentiviral vector system according to claim 12.

14. Use of a chimeric antigen receptor according to any one of claims 1 to 8, or a polynucleotide sequence according to any one of claims 9 to 10, or a nucleic acid construct according to claim 11, or a lentiviral vector system according to claim 12, for the manufacture of one or more of the following application products. (1) Production of CAR-T cells, (2) Increase in CAR molecular levels on the membrane surface of CAR-T cells, (3) Improvement of CAR-T cell antigen sensitivity, (4) Suppression of cytokine secretion of IFN-γ, IL-2, and TNF by CAR-T cells, (5) Promotion of spontaneous growth of CAR-T cells during the quiescent phase, (6) Enhancement of the sustained proliferative capacity of CAR-T cells after target cell stimulation, (7) Improvement of the repeated killing capacity of CAR-T cells, (8) Reduction of NK cell activation and cell depletion in CAR-T cells, (9) Increase in stem cell-like and memory cell subsets of CAR-T cells.

15. Use of a chimeric antigen receptor according to any one of claims 1 to 8, or a polynucleotide sequence according to any one of claims 9 to 10, or a nucleic acid construct according to claim 11, or a lentiviral vector system according to claim 12, or genetically modified T cells according to claim 13, for the manufacture of a tumor treatment product.

16. A method for treating a tumor, characterized by comprising administering a therapeutically effective amount of gene-modified T cells according to claim 13 to the target in question.

17. The treatment method according to claim 16, wherein the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, pilocytic cell leukemia, acute myeloid leukemia, hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, or lung squamous cell carcinoma.

Images