Method for obtaining engineered recombinant mammalian cells and application thereof

Abstract

The invention provides a method for obtaining engineered recombinant mammalian cells and application of the engineered recombinant mammalian cells, and relates to the technical field of biology. The method comprises introducing into a mammalian cell a minicircle DNA comprising a transposon and a polynucleotide encoding a transposase, the transposon comprising a fragment encoding an antigen binding domain. The method has the advantages of shortening the preparation period of recombinant mammalian cells, simplifying the process and reducing the cost.

Description

[0001] Priority Statement

[0002] This application claims the priority of a Chinese patent application with the application number 202311847990.8, the filing date of December 28, 2023, and the invention title of "Method for Obtaining Engineered Recombinant Mammalian Cells and Its Application", the entire content of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biotechnology, and particularly to a method for obtaining engineered recombinant mammalian cells and its application. Background Art

[0004] CAR-T cell therapy (Chimeric antigen receptor-T cell therapy) is a tumor immunotherapy that enables T cells to specifically recognize and eliminate target cells expressing the corresponding antigen by introducing the CAR gene into T cells. CAR is a recombinant receptor that can bind to antigens and activate T cells simultaneously. It can recognize tumor cells expressing specific antigens, leading to T cell activation and thus enabling CAR-T to exert its anti-tumor effect.

[0005] Although CAR-T cell therapy has significant effects and good development prospects, it still has the defects of high production costs and huge upfront investments, resulting in high costs for CAR-T therapy. Moreover, the preparation of CAR-T cells mostly relies on viral infection. The viral system can effectively transduce the CAR gene and achieve long-term expression after random gene integration. This system has been verified to be safe and effective in many CAR-T treatment projects. However, due to the influence of the virus capsid size, the size of the gene loaded by the virus is limited. For example, the diameter of lentivirus is 80-100 nm, and the loading capacity is 8 kb, making it difficult to apply to large-sized CAR genes. In addition, double-stranded DNA transduction is prone to cause T cell apoptosis. Furthermore, clinical-grade viruses need to be produced by trained employees in an environment with a biosafety level of 2 and meeting good manufacturing practice (GMP). And the preparation of lentivirus requires plasmid construction, plasmid extraction, culture of lentivirus packaging cells, lentivirus packaging, lentivirus collection and concentration, lentivirus titer detection, and various quality control index detections, including double-stranded DNA quantitative detection, BSA residue detection, etc. Generally speaking, the process of preparing CAR-T with lentivirus is cumbersome and has a long cycle. The entire process takes 2-3 weeks and has high production costs. Therefore, how to improve the above defects, reduce production costs, and increase patient accessibility is one of the urgent problems to be solved currently.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide a method for obtaining engineered recombinant mammalian cells and its application.

[0008] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0009] In a first aspect, a method for obtaining engineered recombinant mammalian cells is provided, the method comprising introducing a minicircle DNA containing a transposon and a polynucleotide encoding a transposase into a mammalian cell; the transposon contains DNA encoding an antigen-binding domain;

[0010] The antigen-binding domain specifically binds to at least one of MSLN, CD47, GUCY2C, GPC3, MUC16, MICA, HER2, CD70, FSHR, BAFFR, EPHA1, EGFRvIII and CEA.

[0011] In a second aspect, a recombinant mammalian cell is further provided, the recombinant mammalian cell being prepared by the method described in the first aspect.

[0012] In a third aspect, a system for preparing engineered recombinant mammalian cells is further provided, the system comprising the minicircle DNA containing a transposon and the polynucleotide encoding a transposase in the first aspect.

[0013] In a fourth aspect, the application of the method for obtaining engineered recombinant mammalian cells described in the first aspect, or the recombinant mammalian cell in the second aspect, or the system in the third aspect in immunotherapy or the preparation of a pharmaceutical composition for immunotherapy is further provided. Description of the Drawings

[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0015] Figure 1 It is the DNA map of the minicircle plasmid pSB containing the transposase SB100X in Example 1;

[0016] Figure 2 It is the DNA map of the transposon minicircle plasmid pT2-MSLN-EGFP containing the MSLN CAR gene in Example 1;

[0017] Figure 3Expression of MSLN-EGFP after transfection of T cells from different donors with pSB and pT2-MSLN-EGFP minicircle DNA in Example 2;

[0018] Figure 4 Proliferation curve of CAR-T after secondary stimulation with IL7 / IL15 or CD3 / CD28 magnetic beads in Example 3;

[0019] Figure 5 Results of detecting the in vitro killing ability of CAR-T cells prepared using lentivirus (Lenti-T) and CAR-T cells prepared by transfection with pSB and pT2-MSLN-EGFP minicircle DNA (MN-T) against human ovarian cancer cell line OVCAR-3 using real-time label-free dynamic cell analysis technology in Example 4;

[0020] Figure 6 Results of detecting the in vitro killing ability of CAR-T cells prepared using lentivirus (Lenti-T) and CAR-T cells prepared by transfection with pSB and pT2-MSLN-EGFP minicircle DNA (MN-T) against human metastatic pancreatic adenocarcinoma cell line ASPC-1 using real-time label-free dynamic cell analysis technology in Example 4;

[0021] Figure 7 Results of detecting the content of IFN-γ in the cell supernatant in the in vitro killing experiment of human ovarian cancer cell line OVCAR-3 using ELISA method in Example 5;

[0022] Figure 8 Results of detecting the content of Granzyme B in the cell supernatant in the in vitro killing experiment of human ovarian cancer cell line OVCAR-3 using ELISA method in Example 5;

[0023] Figure 9 Results of detecting the content of IFN-γ in the cell supernatant in the in vitro killing experiment of human metastatic pancreatic adenocarcinoma cell line ASPC-1 using ELISA method in Example 5;

[0024] Figure 10 Results of detecting the content of Granzyme B in the cell supernatant in the in vitro killing experiment of human metastatic pancreatic adenocarcinoma cell line ASPC-1 using ELISA method in Example 5;

[0025] Figure 11 Tumor volume change curve after treating ASPC-1 tumor-bearing mice with CAR-T cells prepared using lentivirus (Lenti-T) and CAR-T cells prepared by transfection with pSB and pT2-MSLN-EGFP minicircle DNA (MN-T) in Example 6. Detailed implementation mode

[0026] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] In the present invention, the term "minicircle DNA" or "minicircle plasmid" refers to a novel class of supercoiled expression cassettes, which are generally miniaturized plasmids obtained by site-specific recombination and lack resistance selection markers, replicons, unmethylated CG sequences, etc. on the plasmid backbone. It greatly reduces the DNA size and improves the transfection and transcription efficiency in vitro and in vivo. Compared with ordinary plasmids, minicircle DNA has higher transfection efficiency and more persistent and efficient expression ability.

[0028] In the present invention, the term "transposase" refers to an enzyme that can perform transposition function. The transposase acts on the transposon, recognizes the specific sequences at both ends of the transposon, and can detach the transposon from the adjacent sequence and integrate it into another DNA molecule.

[0029] In the present invention, the term "transposon" refers to a segment of DNA sequence that can autonomously transpose through the catalysis of transposase in DNA. It can insert into a new DNA molecule by forming or not forming a new copy. The transposon contains specific sequences at both ends recognized by the transposase, such as the terminal inverted repeats (TIRs). The DNA between the two transposon ends is transposed together with the transposon ends by the transposase. In the present invention, the DNA between the two transposon ends contains the DNA encoding the antigen-binding domain, which is integrated into the recombinant mammalian cell by the transposase as part of the transposon.

[0030] In the present invention, the term "transgenic expression cassette" refers to an expression cassette carried by the transposon and integrated into the recombinant mammalian cell, which contains a target gene, such as the antigen-binding domain of any embodiment of the present invention, or a T cell receptor or chimeric antigen receptor containing the antigen-binding domain, and one or more promoters and / or minimal regulatory elements. After the transgenic expression cassette is integrated into the host recombinant mammalian cell, it can independently regulate expression.

[0031] In the present invention, the term "antigen-binding domain" refers to a substance containing antibody CDRs, which can be a full antibody or a protein or polypeptide lacking some amino acids present in the full-length chain but still capable of specifically binding to an antigen. The antigen-binding domain has biological activity, capable of binding to a target antigen and competing with other antigen-binding molecules (including full antibodies) for binding to a given epitope.

[0032] Typically, a natural full antibody comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a variable region (VH) and first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, CH4, respectively). Each light chain consists of a variable region (VL) and a constant region (CL). The variable region of each chain usually contains three hypervariable regions, called "complementary determining regions (CDRs)", where the light-chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy-chain CDRs include HCDR1, HCDR2, and HCDR3. The variable regions (VH and VL) each consist of three complementary determining regions connected by 4 framework regions (FRs). Usually, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the CDRs and FRs numbered as follows in the following combination arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0033] In the present invention, CDR boundaries can be defined or identified according to the IMGT, Kabat, Chothia, AbM, Contact definition methods, and CDRs defined in other acceptable ways in the art also fall within the protection scope of the present invention (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); R.M. MacCallum et al.,. Antibody–antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, A.C.R. Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Developmental and Comparative Immunology 27(2003)55–77).

[0034] In the present invention, the term "single-chain antibody" or "scFv" refers to an antibody fragment formed by directly connecting the variable region of the light chain and the variable region of the heavy chain or by connecting them through a peptide linker sequence.

[0035] In the present invention, the term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids are, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Amino acid analogs typically have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds that have a structure different from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.

[0036] In the present invention, the percentage of "identity" refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. Alignment of the percentage of amino acid sequence identity can be performed in various ways in the art, such as by using software well known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment of the full length of the comparison sequences.

[0037] In the present invention, the term "specifically binds" or "specifically binds to" refers to a non-random binding reaction between two molecules, for example, the reaction between an antibody and an antigen. In some specific embodiments, for example, it is determined according to fluorescence-activated cell sorting technology.

[0038] In the present invention, the term "chimeric antigen receptor-modified immune effector cell (or simply chimeric antigen receptor immune effector cell)" is well known in the art, and it is an immune effector cell that uses genetic engineering techniques to express an antigen (such as a tumor antigen)-specific chimeric receptor and can exert a killing effect in a targeted manner.

[0039] In the present invention, the term "immune effector cell" generally refers to a cell that participates in an immune response, for example, a cell that promotes an immune effector response. Exemplary immune effector cells include, but are not limited to, T cells (e.g., α / β T cells and γ / δ T cells), natural killer cells (NK cells), monocytes, macrophages, NKT cells (Natural killer T cell), dendritic cells, granulocytes, B cells, lymphocytes, white blood cells, and / or peripheral blood mononuclear cells.

[0040] In the present invention, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, and polynucleotides include ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemical, or biochemical modifications, unnatural, or derivatized nucleobases. When a nucleic acid molecule encodes a protein or polypeptide, the encoding optionally encodes the sense strand or the antisense strand. The nucleic acid molecule can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" can be used interchangeably.

[0041] In the present invention, the term "recombinant mammalian cell" refers to a cell that can or has been introduced with an exogenous polynucleotide and / or vector.

[0042] In the present invention, the term "pharmaceutical composition" is present in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered.

[0043] In the present invention, "pharmaceutically acceptable carrier" can include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are physiologically compatible.

[0044] In the present invention, the term "subject" or "patient" refers to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0045] In a first aspect, a method for obtaining engineered recombinant mammalian cells is provided. The method includes introducing a minicircle DNA containing a transposon and a polynucleotide encoding a transposase into mammalian cells; the transposon contains DNA encoding an antigen-binding domain. The present invention uses a non-viral vector - the transposon system to prepare recombinant mammalian cells. The plasmid or mRNA production process used for non-viral vectors is simplified and easy to perform quality control, and has significant cost advantages. Among them, the transposon system is simple to operate, can carry large gene fragments, and does not require packaging of lentiviruses, which is an alternative to the expensive cost of viral vectors and can reduce the cost of immunocyte therapy.

[0046] The present invention uses minicircle DNA as a transposon vector, and using minicircle DNA as a vector also has the advantage of reducing T cell apoptosis. To prepare CAR-T cells, plasmid DNA needs to be electroporated into T cells, but this process will stimulate the activation of the cGAS-STING-mediated innate immune pathway, resulting in T cell apoptosis. Experiments have found that reducing the size of the plasmid can significantly reduce T cell apoptosis.

[0047] In an alternative embodiment, the transposase includes, but is not limited to, Sleeping Beauty, PiggyBac, Tn7, Tn5, Frog Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR or Hsmarl.

[0048] In an alternative embodiment, the transposase includes Sleeping Beauty.

[0049] In an alternative embodiment, the transposase includes SB100X.

[0050] In an alternative embodiment, the expression of the transposase is regulated by a CMV promoter.

[0051] In an alternative embodiment, the polynucleotide of the transposase is selected from minicircle DNA, plasmid DNA, linear DNA or mRNA.

[0052] In an alternative embodiment, the length of the minicircle DNA encoding the transposase does not exceed 3 kb, such as not exceeding 2.9 kb, not exceeding 2.8 kb, not exceeding 2.7 kb, not exceeding 2.6 kb, not exceeding 2.5 kb, not exceeding 2.4 kb, not exceeding 2.3 kb, not exceeding 2.2 kb, not exceeding 2.1 kb or not exceeding 2 kb. In an alternative embodiment, the length of the minicircle DNA encoding the transposase is 2.4 kb. In an alternative embodiment, the minicircle DNA encoding the transposase encodes the SB100X transposase and the length of the minicircle DNA is 2.4 kb.

[0053]

[0054] In an alternative embodiment, the minicircle DNA containing the transposon and the minicircle DNA encoding the transposase are located on the same or different minicircle DNAs.

[0055] In an alternative embodiment, the transposon has the following structure: 5'-terminal repeat sequence - transgenic expression cassette - 3'-terminal repeat sequence;

[0056] In an alternative embodiment, the nucleotide sequence of the 5'-terminal repeat sequence is as shown in SEQ ID NO.43; the nucleotide sequence of the 3'-terminal repeat sequence is as shown in SEQ ID NO.44:

[0057] cagttgaagtcggaagtttacatacacttaagttggagtcattaaaactcgtttttcaactactccacaaatttcttgttaacaaacaatagttttggcaagtcagttaggacatctactttgtgcatgacacaagtcatttttcca acaattgtttacagacagattatttcacttataattcactgtatcacaattccagtgggtcagaagtttacatacactaa(SEQ ID NO.43);

[0058] ttgagtgtatgtaaacttctgacccactgggaatgtgatgaaagaaataaaagctgaaatgaatcattctctctactattattctgatatttcacattcttaaaataaagtggtgatcctaactgacctaagacagggaattttta ctaggattaaatgtcaggaattgtgaaaaagtgagtttaaatgtatttggctaaggtgtatgtaaacttccgacttcaactg(SEQ ID NO.44).

[0059] In an alternative embodiment, the transposon contains a transgenic expression cassette that expresses the antigen-binding domain. After the transgenic expression cassette is integrated into the recombinant mammalian cell along with the transposon, it regulates the expression of the antigen-binding domain.

[0060] In an alternative embodiment, the expression cassette has the following structure: promoter - chimeric antigen receptor - PolyA.

[0061] In an alternative embodiment, the promoter is EF1α; the transgenic expression cassette is regulated and expressed by the EF1α promoter.

[0062] In an alternative embodiment, the nucleotide sequence of the EF1α is as shown in SEQ ID NO. 47:

[0063]

[0064] In alternative embodiments, the transgenic expression cassette further contains DNA encoding a marker protein, which is any conventional and available marker protein known to those skilled in the art, including but not limited to EGFP, mCherry, EYFP, Puro, Hygro or Neo.

[0065] In alternative embodiments, the transgenic expression cassette has the following structure: promoter - chimeric antigen receptor - cleavable linker - marker protein - PolyA.

[0066] In alternative embodiments, the marker protein is EGFP.

[0067] In alternative embodiments, the nucleotide sequence of the EGFP is as shown in SEQ ID NO.10:

[0068] gccactaacttctccctgttgaaacaagcaggggatgtcgaagagaatcccgggccaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa(SEQ ID NO.10).

[0069] In alternative embodiments, the tag protein is linked to the C-terminus of the chimeric antigen receptor, and the tag protein and the chimeric antigen receptor are linked by a cleavable linker, and the cleavable linker includes but is not limited to the F2A linker (foot-and-mouth disease virus (FMDV) 2A peptide), the P2A linker (porcine teschovirus-1 2A peptide), the T2A linker (Plutella xylostella virus 2A peptide or the E2A linker (equine rhinitis A virus 2A peptide).

[0070] In an alternative embodiment, the cleavable linker is P2A, and its nucleotide sequence is as shown in SEQ ID NO.45:

[0071] gccactaacttctccctgttgaaacaagcaggggatgtcgaagagaatcccgggcca(SEQ IDNO.45).

[0072] In an alternative embodiment, the nucleotide sequence of the PolyA is as shown in SEQ ID NO.46:

[0073] caacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctta(SEQ ID NO.46).

[0074] In an alternative embodiment, the transgenic expression cassette further contains DNA encoding a signal peptide, and the signal peptide is located at the N-terminus of the chimeric antigen receptor.

[0075] In an alternative embodiment, the length of the DNA encoding the transgenic expression cassette does not exceed 4 kb, for example, does not exceed 3.9 kb, does not exceed 3.8 kb, does not exceed 3.7 kb, does not exceed 3.6 kb, does not exceed 3.5 kb, does not exceed 3.4 kb, does not exceed 3.3 kb, does not exceed 3.2 kb, does not exceed 3.1 kb or does not exceed 3 kb. In an alternative embodiment, the length of the transgenic expression cassette is 3.6 kb.

[0076] In an alternative embodiment, the total length of the microcircular DNA encoding the transposon does not exceed 5 kb, for example, does not exceed 4.9 kb, does not exceed 4.8 kb, does not exceed 4.7 kb, does not exceed 4.6 kb, does not exceed 4.5 kb, does not exceed 4.4 kb, does not exceed 4.3 kb, does not exceed 4.2 kb, does not exceed 4.1 kb or does not exceed 4 kb. In an alternative embodiment, the total length of the microcircular DNA encoding the transposon is 4.8 kb.

[0077] In an alternative embodiment, the total length of the microcircular DNA encoding the transposon is not more than 1.5 kb longer than the length of the DNA encoding the transgenic expression cassette, for example, does not exceed 1.4 kb, does not exceed 1.3 kb, does not exceed 1.2 kb, does not exceed 1.1 kb or does not exceed 1 kb. In an alternative embodiment, the total length of the microcircular DNA encoding the transposon is 1.2 kb longer than the length of the DNA encoding the transgenic expression cassette.

[0078] In an alternative embodiment, the molar ratio of the minicircle DNA encoding the transposon to the polynucleotide encoding the transposase is (1 to 10):1, and can be, for example, but not limited to, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1.

[0079] In an alternative embodiment, the molar ratio of the minicircle DNA encoding the transposon to the polynucleotide encoding the transposase is 1:1.

[0080] In an alternative embodiment, in the genome of the recombinant mammalian cell, the copy number of the transposon is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10.

[0081] In the method provided in the first aspect, the antigen-binding domain is used to specifically bind to at least one of MSLN (mesothelin), CD47 (integrin-associated protein, IAP), GUCY2C (guanylate cyclase 2C), GPC3 (glypican-3), MUC16 (also known as CA-125, a highly glycosylated multi-domain type I transmembrane protein), MICA (class I human major histocompatibility complex-related protein A), HER2 (human epidermal growth factor receptor 2), CD70 (tumor necrosis factor receptor superfamily TNFSF factor, type II transmembrane protein), FSHR (follicle-stimulating hormone receptor), BAFFR (B cell-activating factor receptor), EGFRvIII (epidermal growth factor receptor type III mutant) and CEA (carcinoembryonic antigen).

[0082] In an alternative embodiment, the minicircle DNA encoding the transposon contains DNA encoding a T cell receptor or DNA encoding a chimeric antigen receptor; the above antigen-binding domain is the antigen-binding domain of a T cell receptor or the antigen-binding domain of a chimeric antigen receptor.

[0083] In an alternative embodiment, the minicircle DNA encoding the transposon contains DNA encoding a chimeric antigen receptor. A chimeric antigen receptor (CAR) is a recombinant transmembrane molecule mainly composed of an extracellular domain, a transmembrane domain, and an intracellular domain, and is an artificially constructed receptor that can recognize specific antigens. After the tumor cell antigen binds to the antigen-binding domain in the extracellular domain, the signal is transmitted through the transmembrane domain and the intracellular domain to the cell interior and converted into an activation signal to activate effector cells, causing immune cells to produce cytokines or perforins that kill tumor cells, and at the same time proliferating to further expand the killing effect. The chimeric antigen receptor encoded by the minicircle DNA encoding the transposon can have any chimeric antigen receptor that is conventional and known in the art.

[0084] Exemplary structures of the chimeric antigen receptor include an extracellular domain, a transmembrane domain, and an intracellular domain; the extracellular domain includes an antigen-binding domain and a stem domain. The intracellular domain includes a signal transduction domain, or the intracellular domain includes a co-stimulatory signal transduction domain and a signal transduction domain.

[0085] Exemplary antigen-binding domains contain at least one set of complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region, and / or at least one set of complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light-chain variable region.

[0086] In an alternative embodiment, the above antigen-binding domain contains a heavy-chain framework region and / or a light-chain framework region, and the above heavy-chain framework region and / or light-chain framework region are derived from at least one of murine antibodies, human antibodies, primate antibodies, or their mutants.

[0087] In an alternative embodiment, the three CDRs of the above antigen-binding domain are separated by flanking portions called framework regions (FR, the light-chain FR includes LFR1, LFR2, LFR3, and LFR4, and the heavy-chain FR includes HFR1, HFR2, HFR3, and HFR4).

[0088] In an alternative embodiment, the antigen-binding domain contains a single-chain antibody scFV.

[0089] In some specific embodiments, the antigen-binding domain is a single-chain antibody that specifically binds to MSLN. The heavy-chain variable region of the single-chain antibody has HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region as set forth in SEQ ID NO.1; the light-chain variable region of the single-chain antibody has LCDR1, LCDR2, and LCDR3 of the light-chain variable region as set forth in SEQ ID NO.2. The CDR regions are optionally determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, the IMGT definition, or any acceptable manner known in the art. The CDR regions determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, or the IMGT definition are shown in Table 1.

[0090] Table 1

[0091]

[0092]

[0093] In an alternative embodiment, 1, 2, 3, or 4 of HFR1, HFR2, HFR3, and HFR4 of the single-chain antibody that specifically binds to MSLN are selected from the framework regions of the heavy-chain variable region having the amino acid sequence as set forth in SEQ ID NO.1. The CDR regions are determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, or the IMGT definition in Table 1, and the FR regions in the corresponding definition manner are obtained according to the structure of the heavy-chain variable region. Taking the IMGT definition as an example, HFR1, HFR2, HFR3, and HFR4 respectively comprise the amino acid sequences as set forth in SEQ ID NOs. 33, 34, 35, and 36.

[0094] In an alternative embodiment, 1, 2, 3, or 4 of LFR1, LFR2, LFR3, and LFR4 are selected from the framework regions of the light-chain variable region having the amino acid sequence as set forth in SEQ ID NO.2. The CDR regions are determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, or the IMGT definition in Table 1, and the FR regions in the corresponding definition manner are obtained according to the structure of the light-chain variable region. Taking the IMGT definition as an example, LFR1, LFR2, LFR3, and LFR4 respectively comprise the amino acid sequences as set forth in SEQ ID NOs. 37, 38, 39, and 40.

[0095] In an alternative embodiment, the amino acid sequence of the heavy-chain variable region of the single-chain antibody is as set forth in SEQ ID NO.1, and the amino acid sequence of the light-chain variable region is as set forth in SEQ ID NO.2.

[0096] In an alternative embodiment, the heavy chain variable region and the light chain variable region in the scFv that specifically binds to MSLN are linked by a flexible linker peptide.

[0097] In an alternative embodiment, the scFv has the following structure from the N-terminus to the C-terminus: VH-linker-VL or VL-Linker-VH; the VH is the heavy chain variable region, the VL is the light chain variable region, and the linker is a flexible linker peptide; in an alternative embodiment, the amino acid sequence of the flexible linker peptide is (GGGGS)n, where n = 1 to 10 and n is a positive integer, and its amino acid sequence is SEQ ID NO.41.

[0098] In an alternative embodiment, the amino acid sequence of the flexible linker peptide is as shown in SEQ ID NO.42.

[0099] In an alternative embodiment, the amino acid sequence of the scFv is as shown in SEQ ID NO.3.

[0100] The stem domain is the part that connects the antigen-binding domain and the transmembrane domain, and it generally maintains the stability required for robust chimeric antigen receptor expression and activity in immune effector cells. In an alternative embodiment, the stem domain is derived from the extracellular region of CD8 or CD28 or the hinge of IgG.

[0101] The transmembrane domain connects the extracellular domain of the CAR to the intracellular signal transduction domain. The transmembrane domain can be any sequence derived from a natural molecule, an artificial sequence, or a combination thereof that helps insert the CAR into the cell membrane. Examples of transmembrane domains include, but are not limited to, the α, β, or ζ chains derived from the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), the transmembrane domains of Toll-like receptors 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9, or any derivatives, variants, or fragments thereof, any synthetic sequence with the same function, and any combination thereof.

[0102] Alternatively, the transmembrane domain can be synthetic and can contain hydrophobic residues such as leucine and valine. In an exemplary embodiment, one or both ends of the synthetic transmembrane domain are a triplet of phenylalanine, tryptophan, and valine.

[0103] Costimulatory signaling domains contribute to CAR-T cell expansion, function, persistence, and anti-tumor activity. Examples of costimulatory signaling domains include, but are not limited to, CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, costimulatory molecules that specifically bind to CD83, or any fragment thereof.

[0104] The signaling domain is responsible for activating at least one effector function of the cell expressing the CAR. The intracellular signaling domain transduces effector function signals and directs the cell (e.g., an immune effector cell) to perform its specialized function, such as injuring and / or destroying target cells. Examples of intracellular signaling domains include, but are not limited to, fragments or domains from one or more molecules or receptors, including but not limited to TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, FcRγ, FcRβ (FcεRib), CD79a, CD79b, FcγRlla, DAP 10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-l, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand specifically binding to CD83, CDS, ICAM-l, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1Id, ITGAE, CD103, ITGAL, CD11 a, LFA-l, ITGAM, CD lib, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD 96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and any derivatives, variants or fragments thereof, any synthetic sequences of signaling domains having the same functional capabilities, and any combinations thereof.

[0105] In an alternative embodiment, the chimeric antigen receptor comprises an antigen-binding domain, a stalk domain, a transmembrane domain, a co-stimulatory signaling domain, and a signaling domain; the antigen-binding domain includes, by way of example, a single-chain antibody, the stalk domain is derived, by way of example, from the hinge region of CD8, the transmembrane domain is derived, by way of example, from CD8, the co-stimulatory signaling domain includes, by way of example, 4-1BB, and the signaling domain includes, by way of example, CD3ζ.

[0106] The stalk domain derived from the extracellular region of CD8 includes, by way of example, the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.4.

[0107] Amino acid sequence of the stalk domain derived from the extracellular region of CD8 shown in SEQ ID NO.4:

[0108] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0109] The transmembrane domain derived from CD8 includes, by way of example, the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.5.

[0110] Amino acid sequence of the transmembrane domain derived from CD8 shown in SEQ ID NO.5:

[0111] IYIWAPLAGTCGVLLLSLVITLYC.

[0112] 4-1BB includes, by way of example, the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.6.

[0113] Amino acid sequence of 4-1BB shown in SEQ ID NO.6:

[0114] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0115] CD3ζ exemplarily includes the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in SEQ ID NO.7.

[0116] The amino acid sequence of CD3ζ shown in SEQ ID NO.7:

[0117] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR.

[0118] In an optional embodiment, the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.8.

[0119] The amino acid sequence of the chimeric antigen receptor shown in SEQ ID NO.8:

[0120] MALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0121] In an alternative embodiment, the nucleotide sequence of the transposon is as shown in SEQ ID NO. 11:

[0122]

[0123] In alternative embodiments, the mammalian cells include immune effector cells, and the immune effector cells include, but are not limited to, T cells (e.g., α / β T cells and γ / δ T cells), natural killer cells (NK cells), monocytes, macrophages, natural killer T cells (NKT cells), dendritic cells, granulocytes, B cells, lymphocytes, white blood cells, and / or peripheral blood mononuclear cells.

[0124] In alternative embodiments, the immune effector cells include T cells.

[0125] In alternative embodiments, the mammalian cells are human, murine, or primate-derived mammalian cells.

[0126] In alternative embodiments, known and conventional methods in the art can be used to introduce the minicircle DNA containing the transposon and the polynucleotide encoding the transposase into mammalian cells, including but not limited to electroporation, nucleofection, chemical transfection (such as calcium phosphate transfection), liposome pouring, nanoparticle delivery, gene gun, or microinjection to introduce the minicircle DNA containing the transposon and the polynucleotide encoding the transposase into mammalian cells.

[0127] The method for obtaining engineered recombinant mammalian cells provided in the first aspect above has the following beneficial effects:

[0128] The method for obtaining recombinant mammalian cells provided in the first aspect above uses the transposon system to prepare recombinant mammalian cells, which has the advantages of simple process, short preparation period, and low cost. Using the transposon system to prepare recombinant mammalian cells does not require virus packaging. Overall, the period is shorter, the process is simpler, and the later quality control links are more streamlined, thus greatly reducing the production cost of recombinant mammalian cells and helping to improve the accessibility of immunotherapy for patients. Compared with the maximum loading capacity of 8 kb of the lentiviral vector, the transposon system can load genes exceeding 100 kb, which is more suitable for gene modification due to complex protocols. Moreover, the transposon uses minicircle DNA as a vector, reducing apoptosis of T cells caused by electroporation of double-stranded DNA. In a preferred embodiment, the CAR-T targeting MSLN prepared by this method has a strong tumor-killing function.

[0129] In a second aspect, a recombinant mammalian cell is also provided, and the recombinant mammalian cell is prepared by the method for obtaining engineered recombinant mammalian cells in the first aspect.

[0130] In alternative embodiments, the recombinant mammalian cell is an immune effector cell modified with a chimeric antigen receptor.

[0131] In alternative embodiments, the source of the immune effector cells may include allogeneic and autologous sources.

[0132] In alternative embodiments, the immune effector cells may be differentiated from stem cells or induced pluripotent stem cells (iPSCs).

[0133] In alternative embodiments, the immune effector cells may be isolated from umbilical cord blood, peripheral blood, human embryonic stem cells or iPSCs.

[0134] In alternative embodiments, the immune effector cells are primary cells, such as T cells or NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0135] In alternative embodiments, the immune effector cells are cells derived from humans.

[0136] In a third aspect, there is also provided a system for preparing engineered recombinant mammalian cells, the system comprising the minicircle DNA containing a transposon and a polynucleotide encoding a transposase in the method of the first aspect, i.e., comprising the minicircle DNA encoding a transposon and a polynucleotide encoding a transposase.

[0137] In a fourth aspect, there is also provided the use of the method for obtaining engineered recombinant mammalian cells of the first aspect, or the recombinant mammalian cells of the second aspect, or the system of the third aspect in immunotherapy or in the preparation of a pharmaceutical composition for immunotherapy;

[0138] The immunotherapy includes administering the recombinant mammalian cells of the second aspect to a subject to target a target cell or tissue through the antigen-binding domain therein to treat, alleviate or relieve the target symptoms.

[0139] In alternative embodiments, the purposes of immunotherapy include treating, preventing or alleviating tumors.

[0140] In alternative embodiments, the pharmaceutical composition for immunotherapy includes a pharmaceutical composition for treating, preventing or alleviating tumors.

[0141] In alternative embodiments, the recombinant mammalian cells for immunotherapy include immune effector cells modified with chimeric antigen receptors.

[0142] In alternative embodiments, the recombinant mammalian cells for immunotherapy are allogeneic or autologous.

[0143] In alternative embodiments, the immunotherapy includes obtaining autologous cells and then obtaining recombinant mammalian cells for immunotherapy by the method provided in the first aspect.

[0144] In alternative embodiments, the dosage administered may vary over the course of treatment. For example, in some embodiments, the initial dosage administered may be higher than subsequent dosages. In some embodiments, the dosage administered is adjusted during the course of treatment based on the response of the subject being dosed.

[0145] In alternative embodiments, the recombinant mammalian cells may be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anti-cancer drug, a radiotherapy agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapeutic agent, a gene therapeutic agent, a hormone therapeutic agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine).

[0146] In alternative embodiments, when the recombinant mammalian cells are used in combination with one or more additional therapeutic agents, they may be administered simultaneously with the one or more additional therapeutic agents. In certain such embodiments, the recombinant mammalian cells and the additional therapeutic agents may be administered simultaneously as part of the same pharmaceutical composition. However, recombinant mammalian cells "used in combination" with other therapeutic agents do not need to be administered simultaneously or in the same composition as the therapeutic agent. The meaning of "used in combination" in the present invention also includes that recombinant mammalian cells administered before or after another therapeutic agent are also considered to be "used in combination" with that therapeutic agent, i.e., the recombinant mammalian cells and the second substance are administered by different routes of administration.

[0147] In alternative embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipients may be any carrier and / or excipient known and conventional in the art. Examples of carriers include but are not limited to any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc.; examples of excipients include but are not limited to fillers, disintegrants, preservatives, solubilizers, and emulsifiers, etc.

[0148] In alternative embodiments, the pharmaceutical composition further comprises one or several pharmaceutically active ingredients having other therapeutic effects, including but not limited to one or several combinations of a chemotherapeutic agent, an anti-cancer drug, a radiotherapy agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapeutic agent, a gene therapeutic agent, a hormone therapeutic agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, and a cytokine.

[0149] In alternative embodiments, the tumors include, but are not limited to, mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, cholangiocarcinoma, gastric cancer, colon cancer, thymic carcinoma, esophageal cancer, breast cancer, endometrial cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, non-Hodgkin lymphoma, bladder cancer, glioblastoma, cervical cancer, chondrosarcoma, thyroid cancer, renal cancer, osteosarcoma, bladder cancer, meningioma, multiple squamous cell carcinoma, small cell lung cancer, colorectal cancer, medulloblastoma, acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or diffuse large B-cell lymphoma (DLBCL).

[0150] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0151] Example 1: Construction of SB transposon system microcircular DNA

[0152] A nucleotide sequence containing a recombinase recognition site was synthesized by gene synthesis and recombined in the competent state of a specific recombinase to obtain the corresponding microcircular DNA. The constructed microcircular DNA includes the pSB microcircular DNA containing the transposase SB100X (SEQ ID NO.9) and the transposon microcircular DNA pT2-MSLN-EGFP (SEQ ID NO.11) containing the MSLN CAR gene (SEQ ID NO.8) and the enhanced green fluorescent protein EGFP (SEQ ID NO.10) in the gene expression cassette. The correctness of the constructed vector was verified by sequencing results. The construction of the microcircular DNA was entrusted to Nanjing Genscript Biotech Co., Ltd. The schematic diagram of the vector map is shown in Figure 1 and Figure 2 .

[0153] The transposon has the following structure: 5'-terminal repeat sequence - transgenic expression cassette - 3'-terminal repeat sequence; wherein the nucleotide sequence of the 5'-terminal repeat sequence is as shown in SEQ ID NO.43; the nucleotide sequence of the 3'-terminal repeat sequence is as shown in SEQ ID NO.44:

[0154] cagttgaagtcggaagtttacatacacttaagttggagtcattaaaactcgtttttcaactactccacaaatttcttgttaacaaacaatagttttggcaagtcagttaggacatctactttgtgcatgacacaagtcatttttcca acaattgtttacagacagattatttcacttataattcactgtatcacaattccagtgggtcagaagtttacatacactaa(SEQ ID NO.43),

[0155] ttgagtgtatgtaaacttctgacccactgggaatgtgatgaaagaaataaaagctgaaatgaatcattctctctactattattctgatatttcacattcttaaaataaagtggtgatcctaactgacctaagacagggaattttta ctaggattaaatgtcaggaattgtgaaaaagtgagtttaaatgtatttggctaaggtgtatgtaaacttccgacttcaactg(SEQ ID NO.44);

[0156] The transposon contains a transgenic expression cassette that expresses the antigen-binding domain. After the transgenic expression cassette is integrated into the recombinant mammalian cell along with the transposon, it regulates the expression of the antigen-binding domain.

[0157] In this example, the expression cassette has the following structure: promoter - chimeric antigen receptor - cleavable linker - marker protein - PolyA; the transgenic expression cassette is regulated by the EF1α promoter for expression. The nucleotide sequence of EF1α is shown in SEQ ID NO.47:

[0158]

[0159] The transgenic expression cassette also contains DNA encoding a marker protein. In this example, the marker protein is EGFP, and its nucleotide sequence is shown in SEQ ID NO.10:

[0160] gccactaacttctccctgttgaaacaagcaggggatgtcgaagagaatcccgggccaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa(SEQ ID NO.10).

[0161] In this example, the cleavable linker is P2A; its nucleotide sequence is shown in SEQ ID NO.45:

[0162] gccactaacttctccctgttgaaacaagcaggggatgtcgaagagaatcccgggcca(SEQ ID NO.45).

[0163] In this embodiment, the nucleotide sequence of PolyA is shown in SEQ ID NO.46:

[0164] caacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctta(SEQ ID NO.46).

[0165] In this embodiment, the transgenic expression cassette further contains DNA encoding a signal peptide, and the signal peptide is located at the N-terminus of the chimeric antigen receptor.

[0166] In this embodiment, the nucleotide sequence of the transposon is shown in SEQ ID NO.11:

[0167]

[0168] The pSB minicircle DNA nucleotides containing the transposase SB100X are as shown in SEQ ID NO.9:

[0169]

[0170] Example 2: Preparation of MSLN CAR-T cells

[0171] Resuscitate peripheral blood mononuclear cells (PBMC) from 1×10 7 healthy individuals, and purify T cells by positive selection: Resuspend 1×10 7 PBMC in 4 mL of basal medium (X-vivo-15, Lonza), add CD3 / CD28 magnetic beads at a cell:magnetic bead ratio of 2:1, gently invert and mix for 30 minutes, adsorb with a magnetic stand for 5 minutes, remove the supernatant, and resuspend the purified T cells at a density of 1×10 6 / mL in a medium containing 5% human AB serum and 300 IU / ml IL-2, and culture in an incubator at 37°C and 5% CO2.

[0172] After co-culturing the T cells obtained by positive selection with CD3 / CD28 magnetic beads for 36 - 48 hours, perform electroporation. First, demagnetize the T cells, take 1×10 6 cells, resuspend them in 20 μL of electroporation buffer (P3 buffer, Lonza), and then add pSB and pT2-MSLN-EGFP double microcircular DNA (at a molar ratio of 1:1, 0.67 μg pSB + 1.33 μg pT2-MSLN-EGFP); place the system in a Lonza 4D-Nucleofector electroporator and perform electroporation under condition EO-115. After electroporation, add 100 μL of pre-warmed medium and incubate for 10 minutes; transfer the electroporation system to the medium and culture in an incubator at 37°C and 5% CO2. Detect the electroporation efficiency 24 hours after electroporation, and then detect the expression of MSLN-EGFP every 2 - 3 days from the 5th day to the 15th day after electroporation. The results are shown in Figure 3 . The results show that there are differences in electroporation efficiency among different donors. On the 1st day after electroporation, the expression level of MSLN-EGFP with good results can reach more than 45%. From the 5th day to the 15th day, the expression levels of the inserted genes in most experimental groups are gradually increasing, and among them, the positive rate of about 70% (5 / 7) can reach more than 30% without enrichment.

[0173] Example 3: Detection of MSLN CAR-T proliferation

[0174] To optimize the proliferation of CAR-T prepared by electroporation, add IL7 / IL15 to the medium or perform secondary stimulation with CD3 / CD28 magnetic beads after electroporation, and then count the proliferation curves of CAR-T under different conditions. The results are shown inFigure 4 The results showed that the proliferation of the experimental group with magnetic beads added for secondary stimulation after electroporation was the best.

[0175] Example 4: Detection of in vitro killing function of MSLN CAR-T

[0176] The in vitro killing function was detected by Real Time Cellular Analysis (RTCA). By co-culturing effector cells with tumor target cells, the in vitro tumor-killing effect of effector cells was indicated according to the change in the number of target cells. Human ovarian cancer cells OVCAR-3 and human metastatic pancreatic adenocarcinoma cells ASPC-1 were used as target cells. First, medium was added to the E-Plate detection plate and the background impedance value was measured; the cells were digested with trypsin, counted, and 2×10 5 cells were resuspended in 1 mL of medium. 100 μL of the cell suspension was added to each well for plating, and then placed on the detection platform for real-time monitoring; after 24 hours, according to different effector-to-target ratios, the corresponding number of effector cells was taken, resuspended in 50 μL of target cell medium, added to the corresponding target cells, and co-cultured until the cell proliferation curve was stable; the detection was terminated, and the cell supernatant was collected for detecting the cytokine secretion. The in vitro killing results are shown in Figure 5 and Figure 6 . The results showed that the in vitro killing functions of Lenti-T (CAR-T cells prepared by lentivirus) and MN-T (CAR-T cells prepared by microcircular DNA) were comparable, and both had good tumor-killing effects.

[0177] Example 5: Detection of in vitro factor secretion of MSLN CAR-T

[0178] The secretion levels of IFN-γ and granzyme B in the cell supernatant collected in the RTCA experiment were detected by ELISA. First, the supernatant was centrifuged at 5000 rpm for 5 minutes to remove particulate matter and other impurities, and then the detection was carried out according to the kit instructions. The kits used were: Human IFN Gamma Uncoated ELISA Kit (Invitrogen) and Human Granzyme B Precoated ELISA Kit (Youda). The results of the cell supernatant of human ovarian cancer cells OVCAR-3 are shown in Figure 7 and Figure 8 . The results of the cell supernatant of human metastatic pancreatic adenocarcinoma cells ASPC-1 are shown in Figure 9 and Figure 10 . Both Lenti-T and MN-T secreted high levels of IFN-γ and granzyme B, which was consistent with the results of the function experiment.

[0179] Example 6: Detection of in vivo killing function of MSLN CAR-T

[0180] Establishment of immunodeficient mouse models: Take 5×10 6 Individuals transferred pancreatic adenocarcinoma cells ASPC-1 cells were subcutaneously inoculated into immunodeficient mice (NSG mice). After 15 - 20 days, the tumors grew to 350 - 400 mm 3 , and the model establishment was successful. The model mice were randomly divided into 3 groups, with 5 mice in each group. They were respectively intravenously injected with 5×10 6 T cells, Lenti-T (prepared by lentivirus, containing 30% CAR-T), and MN-T (prepared by minicircle DNA, containing 30% CAR-T), and the growth conditions, tumor sizes, adverse reactions, etc. of the mice were continuously observed. The experimental results are as Figure 11 shown. The results show that according to the tumor volume change curve, Lenti-T and MN-T have comparable in vivo tumor-killing abilities, and no obvious adverse reactions were observed during the experiment.

[0181] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: They can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for obtaining engineered recombinant mammalian cells, characterized in that: The method comprises introducing a minicircle DNA containing a transposon and a polynucleotide encoding a transposase into a mammalian cell; the transposon contains a DNA encoding an antigen binding domain; The antigen binding domain specifically binds to at least one of MSLN, GUCY2C, GPC3, MUC16, MICA, CD47, HER2, CD70, FSHR, BAFFR, EPHA1, EGFR and CEA.

2. The method according to claim 1, characterized in that The transposon contains DNA encoding a T cell receptor or a chimeric antigen receptor; the T cell receptor and the chimeric antigen receptor contain the antigen binding domain; Optionally, the transposase is selected from Sleeping Beauty, PiggyBac, Tn7, Tn5, Frog Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR or Hsmarl, or a derivative thereof having transposition activity; Optionally, the transposase is selected from Sleeping Beauty; Optionally, the transposase is selected from SB100X.

3. The method according to claim 1 or 2, characterized in that: The transposon has the following structure: 5' terminal repeat sequence-transgene expression cassette-3' terminal repeat sequence; Optionally, the nucleotide sequence of the 5' terminal repeat sequence is shown as SEQ ID NO.43; the nucleotide sequence of the 3' terminal repeat sequence is shown as SEQ ID NO.

44.

4. The method according to claim 3, characterized in that: The transgenic expression cassette has the following structure: promoter-chimeric antigen receptor-PolyA; Optionally, the promoter is EF1α; Optionally, the nucleotide sequence of EF1α is shown in SEQ ID NO.47; Optionally, the nucleotide sequence of PolyA is shown in SEQ ID NO.

46.

5. The method according to any one of claims 1 to 4, characterized in that: The antigen binding domain comprises a single-chain antibody that specifically binds to MSLN, wherein the single-chain antibody comprises complementary determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region, and / or complementary determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region; the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of a heavy chain variable region as shown in SEQ ID NO.1, and the LCDR1, LCDR2 and LCDR3 comprise amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of a light chain variable region as shown in SEQ ID NO.2; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the variable region is defined by any one system or a combination of multiple definition systems of Kabat, Chothia, IMGT, ABM or Contact; Optionally, the heavy chain variable region and the light chain variable region in the single-chain antibody that specifically binds to MSLN are connected by a flexible linker peptide; Optionally, the single-chain antibody has the following structure from N-terminus to C-terminus: VH-linker-VL or VL-Linker-VH; the VH is the heavy chain variable region, the VL is the light chain variable region, and the linker is a flexible connecting peptide; optionally, the amino acid sequence of the flexible connecting peptide is (GGGGS)n, n=1-10, n is a positive integer, and its amino acid sequence is SEQ ID NO.41; Optionally, the amino acid sequence of the flexible connecting peptide is shown in SEQ ID NO.42; Optionally, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

3.

6. The method according to any one of claims 1 to 5, characterized in that: The chimeric antigen receptor has the antigen binding domain, the stalk domain, the transmembrane domain, the co-stimulatory signaling domain and the signaling domain; Optionally, the stem domain is derived from the hinge region of CD8; Optionally, the stem domain comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.4; Optionally, the transmembrane domain is selected from the transmembrane domains of CD8, α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD7, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX-40, 4-1BB, CD154, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9, or any derivative, variant or fragment thereof, any synthetic sequence having the same function, and any combination thereof; Optionally, the transmembrane domain is selected from CD8; Optionally, the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.5; Optionally, the co-stimulatory signaling domain is selected from 4-1BB, CD3, CD4, CD8, T cell receptor, CD27, CD28, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, or a co-stimulatory molecule of any fragment thereof; Optionally, the co-stimulatory signaling domain is selected from 4-1BB; Optionally, the 4-1BB comprises an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.6; Optionally, the signaling domain comprises CD3ζ; Optionally, the CD3ζ comprises an amino acid sequence as shown in SEQ ID NO.7 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.7; In an optional embodiment, the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence having at least 70% sequence identity with the amino acid sequence as shown in SEQ ID NO.

8.

7. The method according to any one of claims 1 to 6, characterized in that: The nucleotide sequence of the transposon is shown in SEQ ID NO.11; Optionally, in the genome of the recombinant mammalian cell, the copy number of the transposon is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10; Optionally, the polynucleotide encoding the transposase is selected from minicircle DNA, plasmid DNA, linear DNA or mRNA; Optionally, the polynucleotide encoding the transposase is a minicircle DNA; Optionally, the length of the minicircle DNA encoding the transposase does not exceed 3 kb, preferably 2 to 3 kb; Optionally, the minicircle DNA encoding the transposase has a nucleotide sequence as shown in SEQ ID NO.9; Optionally, the mammalian cells include immune effector cells; Optionally, the immune effector cells include T cells, natural killer cells, monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes, leukocytes or peripheral blood mononuclear cells; Optionally, the immune effector cells include T cells; Optionally, the mammalian cell is a primate, mouse or mammalian cell; Optionally, the minicircle DNA containing the transposon and the polynucleotide encoding the transposase are introduced into mammalian cells by transfection; the transfection is selected from electrotransfection, nuclear transfection, chemical transfection, liposome introduction, nanoparticle delivery, gene gun or microinjection; Optionally, the molar ratio of the minicircle DNA encoding the transposon to the polynucleotide encoding the transposase is (1-10):1; Optionally, the molar ratio of the minicircle DNA encoding the transposon to the polynucleotide encoding the transposase is 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:

1.

8. A recombinant mammalian cell, characterized in that It is prepared by the method according to any one of claims 1 to 7.

9. A system for preparing engineered recombinant mammalian cells, characterized in that: The invention comprises the minicircle DNA containing a transposon according to any one of claims 1 to 7 and a polynucleotide encoding a transposase.

10. Use of the method for obtaining engineered recombinant mammalian cells according to any one of claims 1 to 7, or the recombinant mammalian cells according to claim 8, or the system according to claim 9 in immunotherapy or in the preparation of a pharmaceutical composition for immunotherapy.

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