Chimeric antigen receptor based on intracellular region truncation

Abstract

Provided is a chimeric antigen receptor based on an intracellular region truncation. The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain which are linked in sequence, wherein the extracellular domain comprises an antigen recognition region and a hinge region, and the intracellular domain comprises a CD3ε intracellular region truncation, a costimulatory signaling region and a CD3ζ intracellular region which are linked in sequence. Compared with the prior art, the chimeric antigen receptor provided has a better efficacy and fewer side effects, which are reflected in the increased level of CAR molecules on the membrane surface of CAR-T cells, the formation of better immunological synapses, a better antigen sensitivity, fewer cytokines, a better growth ability, a better sustained proliferation ability, a better repetitive killing ability, attenuated transformation into NK cells and attenuated cell depletion, and the improved stemness and memory cell subset of CAR-T. Also, the chimeric antigen receptor can further improve the therapeutic effects on tumors. In addition, the chimeric antigen receptor down-regulates the cytokines thereof to reduce inflammatory cytokines produced by macrophage and monocyte activation, thereby preventing cytokine storms in the early stage.

Description

A chimeric antigen receptor based on a truncated intracellular domain Technical Field

[0001] The present invention relates to the field of chimeric antigen receptors, and in particular to a chimeric antigen receptor based on an intracellular region truncation. Background Art

[0002] Chimeric antigen receptors (CARs) are abbreviated as CARs, and T cells carrying CARs that target specific tumor antigens are called CAR-T cells. CAR-T therapy is widely used in tumor treatment, but it still has many limitations, such as the cytokine storm encountered in the clinical treatment of leukemia and solid tumors, the transformation of CAR-T cells into NK cells (NK cells), the proneness to cell exhaustion, and the need for improved efficacy, especially in the immunotherapy of solid tumors.

[0003] The prior art CN112500492A modified the connection order of the intracellular domain and transmembrane domain of the chimeric antigen receptor, and found that when the intracellular domain is: CD3ε intracellular region + co-stimulatory signal transduction region intracellular region + CD3ζ intracellular domain, the resulting chimeric antigen receptor has good tumor treatment effect and few side effects, but the molecular membrane level of the chimeric antigen receptor is reduced, it is prone to NK cell depletion and cell exhaustion, and its growth ability, sustained proliferation ability, killing ability and antigen sensitivity need to be further improved.

[0004] Therefore, it is necessary to design a chimeric antigen receptor with better efficacy and fewer side effects, so as to provide new ideas for CAR-T therapy.

[0005] Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a chimeric antigen receptor and use thereof.

[0007] To achieve the above-mentioned and other related objectives, the present invention provides a chimeric antigen receptor in its first aspect.

[0008] The extracellular domain, transmembrane domain and intracellular domain are connected in sequence;

[0009] The extracellular domain includes an antigen recognition region and a hinge region;

[0010] The intracellular domain includes a truncated CD3ε intracellular region, a costimulatory signal transduction region and a CD3ζ intracellular region connected in sequence.

[0011] The second aspect of the present invention provides a polynucleotide sequence selected from:

[0012] (1) a polynucleotide sequence encoding the chimeric antigen receptor of claim 1; and / or

[0013] (2) a complementary sequence of the polynucleotide sequence of (1);

[0014] The third aspect of the present invention provides a nucleic acid construct comprising the aforementioned polynucleotide sequence.

[0015] A fourth aspect of the present invention provides a lentiviral vector system, which comprises the aforementioned nucleic acid construct and lentiviral vector auxiliary components.

[0016] A fifth aspect of the present invention provides a genetically modified T cell, wherein the T cell contains the aforementioned polynucleotide sequence, or contains the aforementioned nucleic acid construct, or is infected with the aforementioned lentiviral vector system.

[0017] The sixth aspect of the present invention provides the use of the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct or the aforementioned lentiviral vector system in the preparation of any one or more of the following use products: (1) preparing CAR-T cells; (2) increasing the level of CAR molecules on the membrane surface of CAR-T cells; (3) improving the antigen sensitivity of CAR-T cells; (4) inhibiting the secretion of cytokines IFN-γ, IL-2, and TNF by CAR-T cells; (5) promoting the natural growth of CAR-T cells in the resting state; (6) enhancing the sustained proliferation ability of CAR-T cells after stimulation by target cells; (7) improving the repeated killing ability of CAR-T cells; (8) reducing NK cell activation and cell exhaustion of CAR-T cells; and (9) improving the stemness and memory cell subsets of CAR-T.

[0018] A seventh aspect of the present invention provides the use of the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system or the aforementioned gene-modified T cell in the preparation of a tumor treatment product.

[0019] As described above, the chimeric antigen receptor and its use of the present invention have the following beneficial effects:

[0020] 1. Compared with the existing technology, the chimeric antigen receptor of the present invention has better efficacy and fewer side effects, which is reflected in the increased level of CAR molecules on the membrane surface of CAR-T cells; the formation of better immune synapses; better antigen sensitivity; fewer cytokines; better growth ability; better sustained proliferation ability; better repeated killing ability; reduced NK cell activation and cell exhaustion; and improved CAR-T stemness and memory cell subsets.

[0021] 2. The chimeric antigen receptor of the present invention can further improve the therapeutic effect of tumors. At the same time, it can reduce the activation of macrophages and monocytes to produce inflammatory cytokines by downregulating its own cytokines, thereby preventing cytokine storms at an early stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: a. Schematic diagram of the structures of targeting CD1928Z CAR, E28Z CAR and B6I-28Z CAR.

[0023] Figure 1: b. Flow cytometry of anti-CD19 CAR on the surface of 28Z, E28Z and B6I-28Z CAR-T cells.

[0024] Figure 1: CD4 in c.28Z, E28Z and B6I-28Z CAR-T + and CD8 + Flow cytometric plot of cell ratios.

[0025] Figure 1: d. Flow cytometry of memory cell populations of 28Z, E28Z and B6I-28Z CAR-T in the resting state.

[0026] Figure 1: e. Immune synapse imaging and ZAP70 recruitment ability statistics of anti-CD1928Z, E28Z and B6I-28Z CAR-T after CD19 antigen stimulation.

[0027] Figure 1: f. The ability of anti-CD1928Z, E28Z and B6I-28Z CAR-T to bind to target cells with different antigen densities.

[0028] Figure 1: g. Killing ability of anti-CD1928Z, E28Z and B6I-28Z CAR-T cells against target cells with different antigen densities.

[0029] Figure 1: h. Antigen levels on the membrane of remaining target cells after anti-CD1928Z, E28Z and B6I-28Z CAR-T cells were co-incubated with target cells at different antigen densities for 24 hours.

[0030] Figure 1: i. Levels of cytokines secreted by anti-CD1928Z, E28Z and B6I-28Z CAR-T cells after stimulation with CD19 antigen.

[0031] Figure 1: Growth curves of anti-CD1928Z, E28Z and B6I-28Z CAR-T cells without antigen stimulation.

[0032] Figure 1: k. Sustained proliferation capacity of anti-CD1928Z, E28Z and B6I-28Z CAR-T cells after one stimulation with Raji.

[0033] Figure 1: l. The ability of anti-CD1928Z, E28Z and B6I-28Z CAR-T cells to repeatedly kill Raji.

[0034] Figure 1: NK activation levels in the late stage of repeated killing by m.anti-CD1928Z, E28Z and B6I-28Z CAR-T cells.

[0035] Figure 1: np. Cell exhaustion in the late stage of repeated killing by anti-CD1928Z, E28Z and B6I-28Z CAR-T cells.

[0036] Figure 1: q. NK activation levels after 5 rounds of repeated killing by anti-CD1928Z, E28Z and B6I-28Z CAR-T cells.

[0037] Figure 1: Differentially expressed genes after 5 rounds of repeated killing by r.anti-CD1928Z, E28Z and B6I-28Z CAR-T cells.

[0038] Figure 1: Flowchart of CAR-T cell therapy for Raji subcutaneous xenograft tumor model.

[0039] Figure 1: Growth of Raji xenograft tumors after t.CAR-T cell therapy.

[0040] Figure 1: Flowchart of u.CAR-T cell therapy for Raji subcutaneous xenograft tumor model and tumor recurrence model.

[0041] Figure 1: Growth of Raji subcutaneous xenografts and recurrent tumors treated with v.CAR-T cells.

[0042] Figure 2: a. Schematic diagram of the structures of targeting GPC328Z CAR, E28Z CAR and B6I-28Z CAR.

[0043] Figure 2: b. Flow cytometry of anti-GPC3 CAR on the surface of 28Z, E28Z and B6I-28Z CAR-T cells.

[0044] Figure 2: CD4 in c.28Z, E28Z and B6I-28Z CAR-T + and CD8 + Flow cytometric plot of cell ratios.

[0045] Figure 2: d. Growth curves of anti-GPC328Z, E28Z and B6I-28Z CAR-T cells without antigen stimulation.

[0046] Figure 3: a. Schematic diagram of the structures of Mesothelin 28Z CAR, E28Z CAR, B0I-28Z CAR, B3I-28Z CAR, B6I-28Z CAR, and B9I-28Z CAR targeting.

[0047] Figure 3: b. Flow cytometry of anti-Mesothelin CAR on the surface of 28Z, E28Z, B0I-28Z, B3I-28Z, B6I-28Z, and B9I-28Z CAR-T cells.

[0048] Figure 3: c. Statistical graph of the surface levels of anti-Mesothelin CAR on 28Z, E28Z, B0I-28Z, B3I-28Z, B6I-28Z, and B9I-28Z CAR-T cells.

[0049] Figure 3: d. Schematic diagram of the structures of BCMA-targeting 28Z CAR, E28Z CAR, B4I-28Z CAR, B5I-28Z CAR, B6I-28Z CAR, B7I-28Z CAR, and B8I-28Z CAR.

[0050] Figure 3: Flow cytometry and statistical graphs of e.anti-BCMACAR on the surface of B4I-28Z, B5I-28Z, B6I-28Z, B7I-28Z, and B8I-28Z CAR-T cells.

[0051] Figure 3: f. Anti-Mesothelin CAR-T growth curve under repeated tumor stimulation conditions

[0052] Figure 3: g. Anti-Mesothelin CAR-T treatment control effect of xenograft HCT116 tumor model and animal survival curve

[0053] Figure 4: a. Schematic diagram of Anti-CD19 B6I-BBZ CAR-T construction

[0054] Figure 4: b. Flow cytometry of downstream signals ERK and S6 phosphorylation after CAR-T stimulation by weakly expressing CD19 target cells

[0055] Figure 4: Short-term tumor killing by c.B6I BBZ CAR-T cells

[0056] Figure 4: d. Growth curve of Anti-CD19 BBZ CAR-T under repeated tumor killing conditions

[0057] Figure 4: e. CD56 expression of Anti-CD19 BBZ CAR-T after the eighth round of repeated killing

[0058] Figure 4: f. Control effect of Anti-CD19 BBZ CAR-T in the xenograft HCT116 tumor model

[0059] Figure 4: g. Survival curve of animals in the HCT116 xenograft tumor model treated with Anti-CD19 BBZ CAR-T DETAILED DESCRIPTION

[0060] On the one hand, the present application provides a chimeric antigen receptor, comprising:

[0061] The extracellular domain, transmembrane domain and intracellular domain are connected in sequence;

[0062] The extracellular domain includes the antigen recognition region and the hinge region;

[0063] The intracellular domain includes a truncated CD3ε intracellular region, a costimulatory signaling region, and a CD3ζ intracellular region, which are sequentially connected.

[0064] In the chimeric antigen receptor provided in the present application, relative to the CD3ε intracellular region (see patent CN112500492A), the truncated CD3ε intracellular region only retains the BRS sequence and ITAM sequence connected in sequence, and also includes a linker sequence between the BRS sequence and the ITAM sequence.

[0065] In some embodiments, the linker sequence can be a linker sequence suitable for antibodies well known in the art, such as a linker sequence containing G and S. In some embodiments, the linker sequence can be poly-Gly, poly-Ser, or a Gly-Ser combination. In addition to glycine (G) and serine (S), the linker sequence can also 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 a specific embodiment, the number of amino acid residues in the linker sequence of the present invention is 0 to 9, specifically, it can be 0 to 3, 3 to 6, or 6 to 9, etc.

[0066] In one embodiment, a truncated form of the CD3ε intracellular region is modified based on the CD3ε intracellular region sequence, retaining the BRS and ITAM sequences of the CD3ε intracellular region and using six Gs as a linker sequence, and is named B6I. Furthermore, the amino acid sequence of the truncated form of the CD3ε intracellular region is shown in SEQ ID NO: 1; specifically:

[0067] In the chimeric antigen receptor provided by the present application, the co-stimulatory signaling region is selected from one or more intracellular regions of CD27, CD28, CD134, 4-1BB or ICOS.

[0068] In a specific embodiment, the costimulatory signaling domain is selected from CD28.

[0069] The amino acid sequence of CD28 is shown in SEQ ID NO: 2, specifically:

[0070] In another specific embodiment, the costimulatory signaling region is selected from 4-1BB.

[0071] The amino acid sequence of 4-1BB is shown in SEQ ID NO: 3, specifically:

[0072] In the chimeric antigen receptor provided in this application, the amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO: 4. Specifically:

[0073] In the chimeric antigen receptor provided by the present application, the antigen recognition region is selected from a single-chain antibody against a tumor surface antigen.

[0074] In some embodiments, the single-chain antibody is selected from one or more of 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.

[0075] In some embodiments, a single-chain antibody contains a light chain variable region and a heavy chain variable region.

[0076] In some embodiments, the light chain variable region and the heavy chain variable region are connected by a linker sequence.

[0077] In a specific embodiment, the single-chain antibody is selected from CD19, GPC3, mesothelin, and BCMA, wherein the corresponding monoclonal antibodies for CD19, GPC3, and mesothelin are FMC63, GC33, and SS1, respectively. The monoclonal antibody for BCMA is commercially available, for example, from companies such as Shenzhen Ruipujin. The above sequences are publicly known and have good targeting specificity.

[0078] In one embodiment, the single-chain antibody comprises the light chain variable region and the heavy chain variable region of the monoclonal antibody FMC63, and the light chain variable region and the heavy chain variable region are optionally connected by a linker sequence.

[0079] In one embodiment, the single-chain antibody comprises the light chain variable region and the heavy chain variable region of monoclonal antibody GC33, and the light chain variable region and the heavy chain variable region are optionally connected by a linker sequence.

[0080] In one embodiment, the single-chain antibody comprises the light chain variable region and the heavy chain variable region of monoclonal antibody SS1, and the light chain variable region and the heavy chain variable region are optionally connected by a linker sequence.

[0081] The amino acid sequence of the scFv of FMC63 is shown in SEQ ID NO: 10. Specifically:

[0082] The amino acid sequence of the scFv of GC33 is shown in SEQ ID NO: 11. Specifically:

[0083] The amino acid sequence of the scFv of SS1 is shown in SEQ ID NO: 20. Specifically:

[0084] For the amino acid sequence of BCMA scFv, please refer to patent CN109134665B.

[0085] Furthermore, the extracellular structure may also include a signal peptide, forming a signal peptide-antigen recognition region-hinge region connected in sequence.

[0086] In the chimeric antigen receptor provided by the present application, the transmembrane domain is selected from CD28TM, CD4, CD8α, OX40 or H2-Kb.

[0087] In a specific embodiment, the transmembrane domain is selected from CD28TM, the amino acid sequence of which is shown in SEQ ID NO: 5, specifically: FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0088] Furthermore, the extracellular domain also includes a signal peptide, forming a signal peptide-antigen recognition region-hinge region connected in sequence.

[0089] In some embodiments, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO: 6 (Anti-CD19 B6I-28Z CAR), SEQ ID NO: 7 (Anti-GPC3 B6I-28Z CAR), or SEQ ID NO: 21 (Anti-mesothelin B6I-28Z CAR).

[0090] SEQ ID NO: 6 (Anti-CD19 B6I-28Z CAR):

[0091] The nucleotide sequence corresponding to SEQ ID NO: 6 (Anti-CD19 B6I-28Z CAR) is SEQ ID NO: 8 (Anti-CD19 B6I-28Z CAR), and SEQ ID NO: 8 is as follows:

[0092] SEQ ID NO: 7 (Anti-GPC3 B6I-28Z CAR):

[0093] The nucleotide sequence corresponding to SEQ ID NO: 7 (Anti-GPC3 B6I-28Z CAR) is SEQ ID NO: 9 (Anti-GPC3 B6I-28Z CAR), and SEQ ID NO: 9 is as follows:

[0094] SEQ ID NO: 21 (Anti-mesothelin B6I-28Z CAR):

[0095] The nucleotide sequence corresponding to SEQ ID NO: 21 (Anti-mesothelin B6I-28Z CAR) is SEQ ID NO: 19 (Anti-mesothelin B6I-28Z CAR)

[0096] The various parts forming the chimeric antigen receptor of the present invention can be directly linked to each other or connected via a linker sequence. The linker sequence can be a linker sequence known in the art for use in antibodies, such as a linker sequence containing G and S. Typically, the linker contains one or more repeated motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the linker sequence, with no intervening amino acid residues between the repeats. The linker sequence can be composed of 1, 2, 3, 4, or 5 repeating motifs. The linker length can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine residues in the linker sequence is not particularly limited and is typically 2 to 20, such as 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), glutamine (Q), etc.

[0097] It should be understood that in gene cloning operations, it is often necessary to design suitable enzyme cleavage sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, and this does not affect the activity of the target sequence. In order to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate the purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions within the protein of the recombinant protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino terminus or carboxyl terminus of the fusion protein (i.e., CAR) of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tag can 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 to purify the protein.

[0098] On the other hand, the present application provides a polynucleotide sequence selected from:

[0099] (1) a polynucleotide sequence encoding the chimeric antigen receptor of the aforementioned claims; and / or

[0100] (2) The complementary sequence of a polynucleotide sequence.

[0101] The polynucleotide sequences of the present application may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The present invention also encompasses degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

[0102] The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification into the correct order.

[0103] On the other hand, the present application provides a nucleic acid construct comprising the aforementioned polynucleotide sequence.

[0104] The nucleic acid construct also includes one or more regulatory sequences operably connected to the aforementioned polynucleotide sequence. The coding sequence of the CAR of the present application can be manipulated in a variety of ways to ensure protein expression. Before the nucleic acid construct is inserted into the vector, the nucleic acid construct can be manipulated according to the different or required expression vectors. The technology of using recombinant DNA methods to change polynucleotide sequences is known in the art.

[0105] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is generally operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0106] The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0107] The regulatory sequence may also be a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention.

[0108] In a preferred embodiment of the present application, the nucleic acid construct is a vector.

[0109] Typically, the expression of the polynucleotide sequence encoding the CAR is achieved by operably connecting the polynucleotide sequence encoding the CAR to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0110] The polynucleotide sequence encoding the CAR of the present application can be cloned into many types of vectors. For example, it can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Further, the vector is an expression vector. The expression vector can be provided to the cell 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, herpes viruses, and lentiviruses. Typically, a suitable vector comprises a replication origin that works in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers.

[0111] In a specific embodiment of the present application, the nucleic acid construct is a lentiviral vector containing a replication origin, a 3'LTR, a 5'LTR and the aforementioned polynucleotide sequence.

[0112] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence high-level expression that can be operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMμLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, it is also possible to consider the use of inducible promoters. The use of inducible promoters provides a molecular switch that can open the expression of the polynucleotide sequence that can be operably connected to the inducible promoter when the deadline is expressed, and close expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0113] In order to evaluate the expression of the CAR polypeptide or its portion, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers can be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0114] The reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of the regulatory sequence. After DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at the appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are known and can utilize known technology to prepare or commercially obtain.

[0115] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0116] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0117] 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 inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, lentiviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into lentiviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0118] On the other hand, the present application provides a lentiviral vector system, which contains the aforementioned nucleic acid construct and lentiviral vector auxiliary components.

[0119] Lentiviral auxiliary components include lentiviral packaging plasmids and cell lines. Lentiviral vector systems are constructed by viral packaging of the aforementioned nucleic acid constructs with the assistance of lentiviral packaging plasmids and cell lines. Methods for constructing lentiviral vector systems are commonly used in the art.

[0120] On the other hand, the present application provides a method for activating T cells in vitro, comprising the step of infecting the T cells with the aforementioned lentivirus.

[0121] The CAR-T cells of the present application can undergo robust in vivo T cell expansion and persist at high levels in the blood and bone marrow for an extended period of time, and form specific memory T cells. After encountering and subsequently eliminating target cells expressing alternative antigens, the CAR-T cells of the present invention can differentiate into a central memory-like state in vivo.

[0122] The present application also includes a class of cell therapies in which T cells are genetically modified to express the CARs described herein, and the CAR-T cells are infused into a recipient in need thereof. The infused cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0123] The anti-tumor immune response elicited by CAR-T cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, in which the CAR-T cells induce an immune response specific for the antigen-binding portion of the CAR.

[0124] Treatable cancers can be non-solid tumors, such as blood tumors, for example, leukemia and lymphoma. In particular, diseases that can be treated using the CAR, its coding sequence, nucleic acid construct, expression vector, virus, and CAR-T cell of the present invention. The disease can specifically be a CD19 or GPC3-mediated disease, especially a CD19 or GPC3-mediated tumor.

[0125] Specifically, herein, "CD19-mediated diseases" include, but are not limited to, leukemias and lymphomas, such as B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0126] Specifically, herein, Glypican-3 (GPC3) is expressed in embryonic tissues, particularly the liver and kidney, and is an extracellular matrix protein associated with organ formation. "GP3-mediated diseases" include, but are not limited to, hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma.

[0127] On the other hand, the present application provides a genetically modified T cell, which contains the aforementioned polynucleotide sequence, or contains the aforementioned nucleic acid construct, or is infected with the aforementioned lentiviral vector system.

[0128] The CAR-modified T cells of the present application may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell groups. Simply put, the pharmaceutical composition of the present application may include CAR-T cells as herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0129] The pharmaceutical composition of the present application can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0130] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," "therapeutically effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6 The T cell composition can be administered at a dose of 10 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy. The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the medical field by monitoring the patient's disease signs and adjusting treatment accordingly.

[0131] Administration of the subject compositions can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[0132] In some embodiments of the present application, the CAR-T cells of the present invention or their compositions can be combined with other therapies known in the art. Therapies include but are not limited to chemotherapy, radiotherapy and immunosuppressants. For example, various radiotherapy preparations can be combined for treatment, including cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, fludarabine, rapamycin and mycophenolic acid. In a further embodiment, the cell composition of the present invention is combined with bone marrow transplantation, T cell ablation therapy using chemotherapeutics such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH (for example, before, simultaneously or after) and administered to the patient.

[0133] As used herein, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

[0134] "Patient," "subject," "individual," "subject," and the like are used interchangeably herein to refer to a living organism, such as a mammal or non-mammal, in which an immune response can be elicited. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, or the like. The non-mammal includes, 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 transgenic species thereof. In one embodiment, the subject can be a human, such as a patient who is immunocompromised or has cancer.

[0135] The use of the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, and the aforementioned lentiviral vector system in the preparation of any one or more of the following products: (1) preparing CAR-T cells; (2) increasing the level of CAR molecules on the membrane surface of CAR-T cells; (3) improving the antigen sensitivity of CAR-T cells; (4) inhibiting the secretion of cytokines IFN-γ, IL-2, and TNF by CAR-T cells; (5) promoting the natural growth of CAR-T cells in the resting state; (6) enhancing the sustained proliferation ability of CAR-T cells after stimulation by target cells; (7) improving the repeated killing ability of CAR-T cells; (8) reducing NK cell activation and cell exhaustion of CAR-T cells; and (9) improving the stemness and memory cell subsets of CAR-T.

[0136] Use of the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system or the aforementioned gene-modified T cell in the preparation of a tumor treatment product.

[0137] In a specific embodiment of the present application, the tumor is selected from one or more of leukemia or solid tumor.

[0138] In a preferred embodiment of the present application, the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute myeloid leukemia, hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, or lung squamous cell carcinoma.

[0139] The present invention also provides a method for treating tumors, comprising administering a therapeutic amount of the gene-modified T cells to a subject in need thereof.

[0140] "Treatment" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, causing complete or partial reversal of a condition, curing a condition, or some combination thereof. With respect to cancer, "treat" or "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. With respect to tumors, "treat" or "treatment" includes eliminating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination thereof.

[0141] When administered to a subject, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The results of animal experiments and various circumstances may be referenced, and the total dosage cannot exceed a certain range.

[0142] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0143] Before further describing the 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 specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0144] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0145] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0146] Example 1

[0147] 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 and using 6 Gs as the connecting sequence to construct a truncated CD3ε intracellular region. The resulting chimeric antigen receptor was named B6I-28Z.

[0148] The nucleotide sequence of Anti-CD19 B6I-28Z CAR is shown in SEQ ID NO: 8. Specifically:

[0149] The Anti-CD19 B6I-28Z CAR was subcloned into the lentiviral expression plasmid pHAGE vector. The CAR expression plasmid was mixed with the packaging plasmids pSPAX2 and pMD2G at a ratio of 10:7.5:3.5, and then transformed into 293FT cells using calcium phosphate transfection to produce lentiviral particles. The 28Z CAR was expressed by ultrahigh-speed centrifugation, and the E28Z CAR (see patent CN112500492A) and B6I-28Z CAR lentiviruses were concentrated into small-volume high-titer viruses (~10 8 / ml), and then transfected with primary T cells activated for one day with αCD3 / αCD28 Ab-beads (MOI = 10). Primary T cells were cultured in complete T cell medium (XIVO-15 + 1% PS + 10ng / ml human IL-7 + 10ng / ml human IL-15 + 5% human AB serum + 10mM neutralized NAC). The cells were removed one day after viral transfection and four days after antibody stimulation. CAR-T cells (GFP-positive) were sorted on day 5 after antibody stimulation. After a period of expansion, the CAR-T cells were evaluated for a series of indicators, including CAR epitope levels, CD4 and CD8 subset ratios, in vitro cytokine secretion, in vitro killing ability, in vitro proliferation ability, in vitro apoptosis level, in vitro persistent survival ability, and in vivo anti-tumor efficacy.

[0150] The structures of CD19-targeting 28Z CAR, E28Z CAR, and B6I-28Z CAR are shown in FIG1: a.

[0151] Anti-HA-rabbit primary antibody and anti-rabbit-AF647 secondary antibody were used to detect the CAR levels on the cell membrane surface of 28Z CAR-T, E28Z CAR-T and B6I-28Z CAR-T. The flow cytometric results are shown in Figure 1: b, indicating that B6I-28Z CAR compensated for the decrease in CAR molecular membrane levels caused by E28Z CAR.

[0152] 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells were detected by flow cytometry using anti-human CD4-PB and anti-human CD8-PE antibodies. The flow cytometric results are shown in Figure 1:c, indicating that the ratios of CD4+ CAR-T cells and CD8+ CAR-T cells in the three cells were similar.

[0153] Anti-human CD62L-APC and anti-human CD45RA-PE-cy7 antibodies were used to detect the memory subsets of 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells by flow cytometry. The flow cytometric results are shown in Figure 1: d, showing that the stem cell-like central memory cell subset of B6I-28Z CAR-T cells (CD62L + CD45RA + ) had the highest ratio, followed by E28Z CAR-T cells, and 28Z CAR-T cells had the lowest ratio.

[0154] When cells were stimulated with 10 nM CD19 antigen and the adhesion molecule ICAM-1, the immune synapses formed by 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells are shown in Figure 1e. The B6I-28Z CAR molecule exhibited higher fluorescence intensity and was better able to recruit the downstream Zap70 signaling molecule.

[0155] Raji cells, K562 cells with low antigen expression (K562 L), and K562 cells with very low antigen expression (K562 VL) were co-incubated with CAR-T cells. The percentage of target cell-CAR-T binding was measured at 10 and 30 minutes, as shown in Figure 1f. B6I-28Z CAR-T cells and E28Z CAR-T cells demonstrated stronger cell-cell contact than 28Z CAR-T cells, indicating that B6I CAR-T cells possess superior antigen sensitivity.

[0156] K562 H, K562 L, and CAR-T cells were mixed in a 1:1:2 ratio to simulate the scenario of antigen downregulation during tumor escape. After 30 hours of incubation, the number of K562 H and K562 L cells was measured, as shown in Figure 1(g). Compared to 28Z CAR-T and E28Z CAR-T cells, B6I-28Z CAR-T cells showed fewer K562 L cells remaining after killing, indicating better antigen sensitivity.

[0157] Raji, K562 H, K562 L, and CAR-T cells were co-cultured at a 1:1 ratio. After 24 hours of incubation, the mean fluorescence intensity of CD19 on the surface of remaining Raji, K562 H, and K562 L cells was detected using anti-CD19-PE antibody. The results are shown in Figure 1h. Compared with 28Z CAR-T and E28Z CAR-T cells, the fluorescence intensity of the target cell surface antigen remaining after killing by B6I-28Z CAR-T cells was lower, indicating its superior antigen sensitivity.

[0158] 100,000 CAR-T cells and 100,000 CD19+ lymphoma cell line Raji cells were seeded at a 1:1 ratio in a round-bottom 96-well plate. The cells were mixed and centrifuged at room temperature (400g, 1 minute) to promote cell-cell contact. The cells were cultured in complete medium at 37°C for one day before harvesting. Six hours before harvesting, 1x BFA was added to inhibit cytokine efflux. After harvesting, the samples were washed once with PBS and fixed with 4% PFA for 5 minutes at room temperature. The wells were then perforated with 0.1% TritonX-100 for 5 minutes at room temperature. Intracellular staining for IL-2, IFN-γ, and TNF-α was performed according to standard procedures. The results, shown in Figure 1i, show that E28Z CAR-T cells and B6I-28Z CAR-T cells produced significantly less IL-2, IFN-γ, and TNF-α than 28Z CAR-T cells after CD19+ cell-specific stimulation.

[0159] CAR-T cells were cultured for a period of time without antigen stimulation and counted every 3-4 days. The results are shown in Figure 1:j. The growth ability of B6I-28Z CAR-T cells is better than that of E28Z CAR-T cells and 28Z CAR-T cells.

[0160] 100,000 CAR-T cells and the CD19+ lymphoma cell line Raji were seeded at a 1:1 ratio in a round-bottom 96-well plate. The cells were mixed and centrifuged at room temperature (400g, 1 minute) to promote cell-cell contact. The cells were then cultured in complete medium in a 37°C incubator for a period of time. Cells were counted every 3-4 days. The results, shown in Figure 1k, demonstrate that B6I-28Z CAR-T cells exhibited superior sustained proliferation compared to E28Z and 28Z CAR-T cells.

[0161] 100,000 CAR-T cells and CD19+ lymphoma cell line Raji were seeded at a 1:1 ratio in a round-bottom 96-well plate, mixed, and counted after 3 days. Half of the killed cells were plated into a new round-bottom 96-well plate. Based on the counted cells, an equal number of CD19+ lymphoma cell line Raji cells were added, mixed, and counted after 3 days. This process was repeated 6-7 times. The results are shown in Figure 1:1. It can be seen that the B6I-28Z CAR-T cell has a better repetitive killing ability than E28Z CAR-T cells and 28Z CAR-T cells.

[0162] At the end of repeated killing, cells were washed with PBS and surface stained with anti-CD56-PE, anti-TIM3-PE, anti-PD1-APC, and anti-TIGIT-BV421. The results are shown in Figure 1-mp. It can be seen that B6I-28Z CAR-T cells reduced NK activation and cell exhaustion compared to E28Z CAR-T cells and 28Z CAR-T cells.

[0163] 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells were obtained after five rounds of repeated killing, and RNA was extracted and sent for RNA-seq. The sequencing results were analyzed by GSEA using the NK cell database. The results are shown in Figure 1:q. It can be seen that B6I CAR-T cells have the lowest degree of NK cell activation, while 28Z CAR-T cells have the most severe NK cell activation.

[0164] After five rounds of repeated killing, 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells were collected, and RNA was extracted and sent for RNA-seq. The results of differentially expressed genes were analyzed, as shown in Figure 1:r. It was found that B6I CAR-T cells highly expressed some stemness-related transcription factors, such as KLF2.

[0165] Aseptically prepare a 3*10^7 / ml Raji cell-PBS suspension and subcutaneously inoculate 3 million Raji cells (100 μL) on the left dorsal surface of each 6-week-old B-NDG female mouse. Day 0 is designated as day 0. Seven days later, when the Raji subcutaneous tumors reached a diameter of 5-6 mm, the mice were randomly divided into groups and each mouse was injected with 100 μL of 6 million CAR-T cells via the tail vein. The model is shown in Figure 1:s. Tumor size was then measured regularly with a vernier caliper and recorded. Tumor area = length * width. The results, shown in Figure 1:t, show that tumors in the vector control group grew fastest, followed by the 28Z group. The B6I-28Z CAR-T group showed the best therapeutic effect, with the E28Z group showing an intermediate therapeutic effect between the 28Z and B6I-28Z groups.

[0166] Aseptically prepare a 3*10^7 / ml suspension of Raji cells in PBS and subcutaneously inoculate 100μL of 3 million Raji cells on the left dorsal surface of each 6-week-old B-NDG female mouse. This time point is designated as day 0. Seven days later, when the Raji subcutaneous tumors reached 5-6 mm in diameter, the mice were randomly divided into groups and each mouse was injected intravenously with 100μL of 6 million CAR-T cells via the tail vein. Tumor size was measured regularly with a vernier caliper and recorded. Tumor area = length x width. The results are shown in Figure 1:v (left). One month later, the tumors were completely eliminated. Mice with completely eliminated tumors were selected and aseptically prepare a 1*10^7 / ml suspension of CD19-low expressing K562 cells in PBS. 100μL of these cells were inoculated on the right dorsal surface of each mouse to simulate tumor recurrence. The model is shown in Figure 1:u. Tumor size was measured regularly with a vernier caliper and recorded. Tumor area = length x width. The results are shown in Figure 1:v (right). The results showed that for re-implanted low-antigen tumors, the B6I-28Z CAR-T group had the best therapeutic effect, followed by the E28Z group, and the 28Z group had the worst therapeutic effect.

[0167] Example 2

[0168] As shown in Figure 2: a, the FMC63 scFv (anti-CD19) sequence in the CD19-targeting 28Z CAR, E28Z CAR, and B6I-28Z CAR was replaced with GC33 scFv (anti-GPC3) to target GPC328Z CAR, E28Z CAR, and B6I-28Z CAR, respectively.

[0169] The nucleotide sequence of Anti-GPC3 B6I-28Z CAR is shown in SEQ ID NO: 9. Specifically:

[0170] Flow cytometry was used to detect the CAR levels on the cell membrane surfaces of 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T using anti-HA-rabbit primary antibody and anti-rabbit-AF647 secondary antibody. The flow cytometry results are shown in Figure 2:b, indicating that B6I-28Z CAR compensated for the decrease in CAR molecular membrane levels caused by E28Z CAR.

[0171] 28Z CAR-T, E28Z CAR-T, and B6I-28Z CAR-T cells were detected by flow cytometry using anti-human CD4-PB and anti-human CD8-PE antibodies. The flow cytometric results are shown in Figure 2:c, indicating that the ratios of CD4+ CAR-T cells and CD8+ CAR-T cells in the three cells were similar.

[0172] CAR-T cells were cultured for a period of time without antigen stimulation and counted every 3-4 days. The results are shown in Figure 2: d. The growth ability of B6I-28Z CAR-T and E28Z CAR-T cells was better than that of 28Z CAR-T cells.

[0173] Comparative Example 1

[0174] As shown in Figure 3:a and Figure 3:d, the FMC63 scFv (anti-CD19) sequence in the CD1928Z CAR, E28Z CAR, and B6I-28Z CAR was replaced with SS1 scFv (anti-mesothelin) and anti-BCMA, respectively, to form a CAR targeting mesothelin and a CAR targeting BCMA. In the CAR targeting mesothelin, the linker sequence of the CD3ε intracellular region is 0, 3, 6, and 9 Gs, and the resulting CARs are named Anti-Meso B0I-28Z, Anti-Meso B3I-28Z, Anti-Meso B6I-28Z, and Anti-Meso B09I-28Z; in the CAR targeting BCMA, the linker sequence of the CD3ε intracellular region is 4, 5, 6, 7, and 8 Gs, and the resulting CARs are named Anti-BCMAB4I-28Z, Anti-BCMAB5I-28Z, Anti-BCMAB6I-28Z, Anti-BCMAB7I-28Z, and Anti-BCMAB8I-28Z; the sequences of the chimeric antigen receptors are as follows:

[0175] The nucleotide sequence of Anti-Meso B0I-28Z is shown in SEQ ID NO: 12, specifically:

[0176] The nucleotide sequence of Anti-Meso B3I-28Z is shown in SEQ ID NO: 13, specifically:

[0177] The amino acid sequence structure of Anti-BCMAB4I-28Z is: BCMA single-chain antibody (see patent CN109134665B) + CD28TM (SEQ ID NO: 5) + truncated CD3ε intracellular region (KNRKAKAKGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 14) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4).

[0178] The amino acid sequence structure of Anti-BCMAB5I-28Z is: BCMA single-chain antibody (see patent CN109134665B) + CD28TM (SEQ ID NO: 5) + truncated CD3ε intracellular region (KNRKAKAKGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 15) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4).

[0179] The amino acid sequence structure of Anti-BCMAB6I-28Z is: BCMA single-chain antibody (see patent CN109134665B) + CD28TM (SEQ ID NO: 5) + truncated CD3ε intracellular region (SEQ ID NO: 1) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4).

[0180] The nucleotide sequence of Anti-Meso B6I-28Z is shown in SEQ ID NO: 19, specifically:

[0181] The amino acid sequence structure of Anti-BCMAB7I-28Z is: BCMA single-chain antibody (see patent CN109134665B) + CD28TM (SEQ ID NO: 5) + truncated CD3ε intracellular region (KNRKAKAKGGGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 16) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4).

[0182] The amino acid sequence structure of Anti-BCMAB8I-28Z is: BCMA single-chain antibody (see patent CN109134665B) + CD28TM (SEQ ID NO: 5) + truncated CD3ε intracellular region (KNRKAKAKGGGGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 17) + CD28 intracellular region (SEQ ID NO: 2) + CD3ζ intracellular region (SEQ ID NO: 4).

[0183] The nucleotide sequence of Anti-Meso B9I-28Z is shown in SEQ ID NO: 18, specifically:

[0184] The results are shown in Figure 3 . The structures of Mesothelin 28Z CAR, E28Z CAR, B0I-28Z CAR, B3I-28Z CAR, B6I-28Z CAR, and B9I-28Z CAR targeting are shown in Figure 3: a.

[0185] Flow cytometry was used to detect CAR levels on the cell membranes 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 using anti-HA-rabbit primary antibody and anti-rabbit-AF647 secondary antibody. The flow cytometric results are shown in Figure 3b, and the statistical results are shown in Figure 3c. The results showed that B0I-28Z, B3I-28Z, B6I-28Z, and B9I-28Z CARs were able to partially replenish the levels of CAR molecules on the cell membrane compared to E28Z, with B6I-28Z CAR being the most effective.

[0186] The structures of 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 3: d.

[0187] Flow cytometry was used to detect the CAR levels on the cell membrane surfaces of B4I-28Z CAR-T, B5I-28Z CAR-T, B6I-28Z CAR-T, B7I-28Z CAR-T, and B8I-28Z CAR-T using anti-HA-rabbit primary antibody and anti-rabbit-AF647 secondary antibody. The flow cytometry results are shown in Figure 3:e, indicating that the level of CAR molecules on the cell membrane surface of B6I-28Z CAR-T was the highest.

[0188] Using the mesothelin antigen-expressing solid tumor HCT116 as the target cell, the aforementioned in vitro repeated killing experiment was performed with the above-mentioned mesothelin-targeting B6I 28Z CAR, and the changes in the number of CAR-T cells were monitored. As shown in Figure 3:f, it was shown that compared with the traditional 28Z CAR, the Anti-Mesothelin B6I-28Z CAR-T cells showed a better long-term proliferation effect.

[0189] HCT116 tumor cells were injected subcutaneously into mice to establish a subcutaneous tumor model. Five days later, CAR-T cells were reinfused, as shown in Figure 3:g. The results showed that the Anti-Mesothelin B6I-28Z CAR-T cells produced better tumor control effects and prolonged the survival of the mice compared to 28Z CAR-T cells.

[0190] Comparative Example 2

[0191] As shown in Figure 4: a, 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 named Anti-CD19 B6I-BBZ CAR. The amino acid sequence structure of Anti-CD19 B6I-BBZ is:

[0192] CD19 single-chain antibody (SEQ ID NO: 10) + CD8TM (SEQ ID NO: 5) + truncated CD3ε intracellular region (KNRKAKAKGGGGGGGDYEPIRKGQRDLYSGLNQRRI, SEQ ID NO: 16) + 4-1BB intracellular region (SEQ ID NO: 4) + CD3ζ intracellular region (SEQ ID NO: 4).

[0193] The amino acid sequence is shown in SEQ ID NO: 22:

[0194] The nucleotide sequence is shown in SEQ ID NO: 23:

[0195] Stimulating CAR-T cells with K562 cells expressing low CD19 revealed stronger downstream signaling with the Anti-CD19 B6I-BBZ CAR, including pERK and pS6, demonstrating enhanced antigen sensitivity (Figure 4: b). Using these CAR-T cells to kill K562 cells expressing low or high CD19, cell counts were taken at 24, 48, and 72 hours, revealing that the Anti-CD19 B6I-BBZ CAR was more effective in eliminating tumor cells (Figure 4: c).

[0196] Using Raji cells naturally expressing CD19 as target cells, the designed cells were used to perform continuous tumor killing in vitro, and the proliferation of CAR-T cells was monitored during the killing process. The CD19 B6I BBZ CAR demonstrated stronger sustained proliferation, as shown in Figure 4(d). After eight rounds of killing, CAR-T cells were collected and assayed for the NK activation marker CD56, demonstrating that B6I BBZ cells exhibited weaker NK activation and better sustained function, as shown in Figure 4(e).

[0197] To simulate the heterogeneity of tumor antigen expression in vivo, equal amounts of K562 tumor cells expressing high and low CD19 were mixed and injected subcutaneously into mice to establish a tumor model. Five days later, CAR-T cells were reinfused. The B6I BBZ group demonstrated superior tumor control (Figure 4f) and significantly prolonged mouse survival (Figure 4g).

[0198] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A chimeric antigen receptor, comprising: The extracellular domain, transmembrane domain and intracellular domain are connected in sequence; The extracellular domain includes an antigen recognition region and a hinge region; The intracellular domain includes a truncated CD3ε intracellular region, a co-stimulatory signal transduction region and a CD3ζ intracellular region which are sequentially connected.

2. The chimeric antigen receptor according to claim 1, wherein Compared with the CD3ε intracellular region, the truncated form of the CD3ε intracellular region only retains the BRS sequence and the ITAM sequence connected in sequence, and also includes a linker sequence between the BRS sequence and the ITAM sequence.

3. The chimeric antigen receptor according to claim 2, characterized in that The connecting sequence includes several amino acid residues; the amino acid residues of the connecting sequence include glycine and / or serine; further, the number of amino acid residues in the connecting sequence is 0 to 9; further, the amino acid sequence of the truncation of the CD3ε intracellular region is shown in SEQ ID NO:

1.

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

5. The chimeric antigen receptor according to claim 4, wherein Also includes one or more of the following characteristics: (1) The amino acid sequence of the intracellular region of CD28 is shown in SEQ ID NO: 2; (2) the amino acid sequence of the 4-1BB intracellular region is shown in SEQ ID NO: 3; (3) The amino acid sequence of the CD3ζ intracellular region is shown in SEQ ID NO:

4.

6. The chimeric antigen receptor according to claim 1, wherein Also includes one or more of the following characteristics: a. The antigen recognition region is selected from a single-chain antibody against a tumor surface antigen, and the single-chain antibody is selected from one or more of 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 selected from CD28TM, CD4, CD8α, OX40 or H2-Kb.

7. The chimeric antigen receptor according to claim 6, wherein Also includes one or more of the following characteristics: c. The amino acid sequence of the single-chain antibody is shown in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 20; d. The transmembrane domain is selected from CD28TM, and the amino acid sequence of the CD28TM is shown in SEQ 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. A polynucleotide sequence selected from: (1) a polynucleotide sequence encoding the chimeric antigen receptor according to any one of claims 1 to 8; and / or (2) A complementary sequence to the polynucleotide sequence described in (1).

10. The polynucleotide sequence according to claim 9, characterized in that The polynucleotide sequence is shown as 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 according to 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 initiation site, a 3'LTR, a 5'LTR and the polynucleotide sequence of any one of claims 9-10.

12. A lentiviral vector system, comprising the nucleic acid construct of claim 11 and lentiviral vector auxiliary components.

13. A genetically modified T cell, characterized in that: The T cell contains the polynucleotide sequence of any one of claims 9-10, or contains the nucleic acid construct of claim 11, or is infected with the lentiviral vector system of claim 12.

14. Use of the chimeric antigen receptor according to any one of claims 1 to 8, or the polynucleotide sequence according to any one of claims 9 to 10, or the nucleic acid construct according to claim 11, or the lentiviral vector system according to claim 12 in the preparation of any one or more of the following products: (1) preparing CAR-T cells; (2) increasing the level of CAR molecules on the membrane surface of CAR-T cells; (3) improving the antigen sensitivity of CAR-T cells; (4) inhibiting the secretion of cytokines IFN-γ, IL-2, and TNF by CAR-T cells; (5) promoting the natural growth of CAR-T cells in the resting state; (6) enhancing the sustained proliferation ability of CAR-T cells after stimulation by target cells; (7) improving the repeated killing ability of CAR-T cells; (8) reducing NK cell transformation and cell exhaustion of CAR-T cells; (9) improving the stemness and memory cell subsets of CAR-T.

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

16. A method for treating tumors, characterized in that: The method of treatment comprises administering a therapeutic amount of the genetically modified T cells of claim 13 to a subject in need thereof.

17. The method of claim 16, wherein the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute myeloid leukemia, hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, or lung squamous cell carcinoma.

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