Chimeric antigen receptor, nucleic acid molecule, CAR-Ms and application thereof

Abstract

The invention provides a chimeric antigen receptor, a nucleic acid molecule, CAR-Ms and application thereof, and belongs to the field of biomedical engineering and peptides. The chimeric antigen receptor provided by the invention comprises a CD8 alpha leading signal peptide, a single-chain variable fragment, a CD8 alpha hinge, a CD8 transmembrane structural domain and an intracellular costimulatory signal transduction structural domain. Wherein the single-chain variable fragment can target any known antigen; the intracellular costimulatory signal transduction structural domain is any one or a combination of TLR2 (Toll-Like Receptor 2), TLR4 (Toll-Like Receptor 4), TLR7 (Toll-Like Receptor 7), Mincle, Dectin-1, NOD2 (Nitride Oxidase 2), CD40, 4-1BB (Beta), OX40 and CD3zeta. The invention also provides the macrophage targeted nanogel for RNA delivery, and the macrophage transfected by the nanogel system expresses the chimeric antigen receptor, so that the specific targeting and phagocytosis of known antigen positive (such as MUC1) tumor cells can be realized.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical engineering technology and peptides, and relates to a chimeric antigen receptor, a nucleic acid molecule, CAR-Ms (chimeric antigen receptor - macrophages), and their applications. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Malignant tumors are a major health challenge globally, with their incidence and mortality rates continuously rising. According to the latest data, common cancers such as breast cancer pose a serious threat to women's health, while lung cancer is the main cause of cancer deaths. Traditional treatment methods such as surgery, radiotherapy, and chemotherapy are still the main means, but emerging methods such as targeted therapy and immunotherapy are gradually being taken seriously. CAR-T therapy (chimeric antigen receptor T cell immunotherapy) has shown significant efficacy in the treatment of leukemia and multiple myeloma, and several CAR-T therapies have been approved for marketing globally. However, immunotherapy still faces challenges. Some tumors may develop tolerance to CAR-T therapy, leading to a decline in treatment efficacy. At the same time, CAR-T therapy may cause serious side effects such as cytokine release syndrome.

[0004] Macrophages are core members of the immune system, with functions including phagocytosing pathogens (such as bacteria and viruses), clearing senescent cells, antigen presentation, and immune regulation. Their plasticity enables them to differentiate into pro-inflammatory (M1 type) or anti-inflammatory (M2 type) phenotypes according to microenvironmental signals, which is particularly crucial in tumor immunity. Traditional CAR-T cells have limited effects on solid tumors because solid tumors have a dense stroma and an immunosuppressive microenvironment, such as the infiltration of MDSC (myeloid-derived suppressor cells) and Treg (regulatory T cells), which hinder the infiltration of immune cells. Macrophages naturally have the ability to migrate towards tumors (the proportion of macrophages in tumors can reach 40% - 50%), making them an ideal carrier for breaking through the solid tumor barrier. By using gene editing techniques (such as adenovirus vectors, lipid nanoparticles), introducing chimeric antigen receptors (CARs) into macrophages not only endows them with tumor targeting ability, specifically recognizing tumor-associated antigens, but also can induce macrophages to polarize towards the M1 type through co-stimulatory signals, enhancing phagocytic activity and the secretion of pro-inflammatory factors. In addition, CAR-Ms can also present tumor antigens to T cells, activating the adaptive immune response, forming a "kill three birds with one stone" effect. However, the structural design of CAR-Ms is a complex system that requires coordination among multiple modules, and a balance needs to be achieved among targeting, activation signals, safety, and scalability. There is still an urgent need in the industry to develop CAR-Ms with better anti-tumor effects. Summary of the Invention

[0005] To solve the above problems, the present invention provides a chimeric antigen receptor, a nucleic acid molecule, CAR-Ms and their applications. The CAR-Ms obtained by using the nucleic acid molecule and its nano-gel preparation provided by the present invention can express CAR proteins targeting known antigens and have the effects of recognizing and phagocytosing tumor cells of known antigens.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, a chimeric antigen receptor is provided, including: a CD8α leader signal peptide, a single-chain variable fragment, a CD8α hinge region, a CD8 transmembrane region, and an intracellular co-stimulatory signal transduction domain; Wherein, the single-chain variable fragment can target any known antigen; The intracellular co-stimulatory signal transduction domain is selected from at least one of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, CD3ζ; The amino acid sequence of the CD8α leader signal peptide is as shown in SEQ ID NO:1, the amino acid sequence of the CD8α hinge region is as shown in SEQ ID NO:2, the amino acid sequence of the CD8 transmembrane region is as shown in SEQ ID NO:3, and the amino acid sequences of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, CD3ζ are as shown in SEQ ID NO:4-SEQ ID NO:13.

[0007] Preferably, the single-chain variable fragment is derived from a single-chain antibody of MUC1 and includes a light-chain variable region and a heavy-chain variable region, which are connected by a linker sequence; The intracellular co-stimulatory signal transduction domain is selected from one of the combinations of Mincle and CD40, Mincle and 4-1BB, Mincle and OX40, Dectin-1 and CD40, Dectin-1 and 4-1BB, Dectin-1 and OX40, TLR2 and CD40, TLR2 and 4-1BB, TLR2 and OX40, TLR4 and CD40, TLR4 and 4-1BB, TLR4 and OX40, TLR7 and CD40, TLR7 and 4-1BB, TLR7 and OX40, NOD2 and CD40, NOD2 and 4-1BB, NOD2 and OX40.

[0008] In the second aspect of the present invention, a nucleic acid molecule is provided, and the nucleic acid molecule includes: the coding gene of the above chimeric antigen receptor; The nucleotide sequence of the CD8α leader signal peptide is as shown in SEQ ID NO:14, the nucleotide sequence of the CD8α hinge region is as shown in SEQ ID NO:15, the nucleotide sequence of the CD8 transmembrane region is as shown in SEQ ID NO:16, and the nucleotide sequences of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ are as shown in SEQ ID NO:17-SEQ ID NO:26.

[0009] In the third aspect of the present invention, a kind of CAR-Ms is provided, and the CAR-Ms expresses the above-mentioned chimeric antigen receptor; Or, the CAR-Ms contains the above-mentioned nucleic acid molecule.

[0010] Preferably, the CAR-Ms is constructed by the following method: Mix natural calreticulin, a crosslinking agent, and the above-mentioned nucleic acid molecule evenly in a solvent, carry out a reaction, and then perform surface modification with 4-(2-aminoethyl)benzenesulfonamide to obtain a nano-gel preparation; Transfect macrophages with the nano-gel preparation to obtain CAR-Ms.

[0011] In the fourth aspect of the present invention, the application of the above-mentioned nucleic acid molecule in the preparation of a nano-gel preparation is provided, including: Mix natural calreticulin, a crosslinking agent, and the above-mentioned nucleic acid molecule evenly in a solvent, carry out a reaction, and then perform surface modification with 4-(2-aminoethyl)benzenesulfonamide to obtain a nano-gel preparation.

[0012] In the fifth aspect of the present invention, the application of the above-mentioned chimeric antigen receptor in the preparation of an anti-tumor drug composition is provided.

[0013] Preferably, the tumor is selected from at least one of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, nervous system cancer, leukemia, and cervical cancer.

[0014] Preferably, the anti-tumor drug composition includes: immune cells, and the immune cells express the above-mentioned chimeric antigen receptor.

[0015] More preferably, the immune cells are selected from at least one of T cells, γδ T cells, macrophages, NK cells, NKT cells, regulatory T cells, and neutrophils.

[0016] Advantages of the present invention (1)The CAR-Ms obtained from the nucleic acid molecule and its nanogel preparation provided by the present invention can express CAR proteins targeting known antigens and have the functions of recognizing and phagocytosing tumor cells with known antigens.

[0017] (2)The present invention has strong practicability and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0019] Figure 1 It is a schematic diagram of the CAR gene.

[0020] Figure 2 It is a transmission electron micrograph of the nanogel preparation.

[0021] Figure 3 It is the expression rate of the CAR protein in CAR-Ms.

[0022] Figure 4 It is the phagocytosis result of CAR-Ms on tumor cells.

[0023] Figure 5 It is the effect of CAR-Ms in treating orthotopic pancreatic cancer in mice. DETAILED DESCRIPTION OF THE INVENTION

[0024] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0025] In the first aspect of the present invention, a chimeric antigen receptor is provided, and the amino acid sequences of each part are shown in SEQ ID NO: 1-SEQ ID NO: 13. The scFv (single-chain variable fragment) used in the present invention can be derived from various monoclonal antibodies well known in the art, including but not limited to monoclonal antibodies targeting HER2, MUC1, GPC3, EGFRvIII, ROR1, CD171, B7-H3, integrin αvβ3, GPC1, Claudin18.2, CD19, BCMA, etc. In one embodiment verified to be feasible by the present invention, the scFv is derived from the tumor-specific MUC1 monoclonal antibody 5E5.

[0026] According to the general understanding in the art, the MUC1-CAR protein includes an extracellular domain, a transmembrane domain, and an intracellular domain. Among them, the extracellular domain includes a single-chain variable fragment (scFv) of a monoclonal antibody and a hinge region (Hinge) for connection; in a preferred embodiment of the present invention, the single-chain variable fragment is a single-chain antibody against MUC1, including a light-chain variable region (5E5 VL) and a heavy-chain variable region (5E5 VH), which are connected by a linker sequence; the hinge region is selected from CD8α.

[0027] Preferably, the transmembrane domain is derived from one of CD4, CD8, CD28, or CD3ζ; further, the transmembrane structure is selected from CD8.

[0028] Preferably, the intracellular domain is a signal transduction domain; further, the signal transduction domain is the intracellular region of CD3ζ.

[0029] Preferably, the co-stimulatory domain is TLR7 and CD40.

[0030] In one embodiment of the above preferred technical solution, the CAR protein is a fusion protein sequentially connected with a CD8α signal peptide, an anti-MUC1 single-chain antibody, a myc-tag labeling gene, a CD8α hinge region, a CD8 transmembrane region, a CD3ζ intracellular region, a TLR7 and CD40 co-stimulatory region, P2A, and EGFP.

[0031] In the above-described embodiments, the various parts of the fusion protein, such as the CD8α signal peptide, the light and heavy chain variable regions of the single-chain antibody, the myc-tag labeling gene, the CD8α hinge region, the CD8 transmembrane region, the CD3ζ intracellular region, the co-stimulatory region, P2A, and EGFP, etc., can be directly connected to each other or can be connected through a linker sequence. The linker sequence can be a linker sequence well-known in the art applicable to antibodies, such as a linker sequence containing G and S. Generally, the linker contains one or more motifs repeated before and after. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motif is adjacent in the linker sequence and there is no insertion of amino acid residues between the repetitions. The linker sequence can be composed of 1, 2, 3, 4, or 5 repeating motifs. The length of the linker can be 3 - 25 amino acid residues, such as 3 - 15, 5 - 15, 10 - 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited and is usually 2 - 20, such as 2 - 15, 2 - 10, 2 - 8. In addition to glycine and serine, the linker 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 certain embodiments, the light and heavy chain variable regions of the anti-MUC1 single-chain antibody of the present invention are connected by (GGGGS)n, where n is an integer from 1 to 5.

[0032] It should also be understood that in gene cloning operations, it is often necessary to design appropriate restriction enzyme sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, but do not affect the activity of the target sequence. Additionally, in order to construct a fusion protein, promote the expression of the 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, such as suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Based on the above design, the amino or carboxyl terminus of the fusion protein of the present invention can also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in the present invention. The tags can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, and Ty1. These tags can be used for protein purification.

[0033] The present invention also includes mutants of the amino acid sequences shown in SEQ ID NO: 1 - SEQ ID NO: 13. The mutants include: amino acid sequences having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence similarity with the CAR and retaining the biological activity of the chimeric antigen receptor. The sequence similarity of the mutant sequences can be calculated by BLAST such as that of NCBI.

[0034] The above mutants also include: amino acid sequences having one or several mutations (insertions, deletions or substitutions) in the amino acid sequences shown in SEQ ID NO: 1 - SEQ ID NO: 13 and still retaining the biological activity of the CAR; the several mutations generally refer to within 1 - 10, such as 1 - 8, 1 - 5 or 1 - 3. The substitutions are preferably conservative substitutions. For example, in the art, conservative substitutions are made with amino acids having similar or close properties. When making conservative substitutions, the function of the protein or polypeptide is generally not changed. "Amino acids having similar or close properties" include, for example, families of amino acid residues having similar side chains, and these families include amino acids having basic side chains (such as lysine, arginine, histidine), amino acids having acidic side chains (such as aspartic acid, glutamic acid), amino acids having uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or several sites with another amino acid residue from the same side chain class in the polypeptide of the present invention will not substantially affect its activity.

[0035] The second aspect of the present invention provides a nucleic acid molecule, including the nucleotide sequences shown in SEQ ID NO: 14 - SEQ ID NO: 16 and any one of the nucleotide sequences shown in SEQ ID NO: 17 - SEQ ID NO: 26 or their combinations.

[0036] In some embodiments, the nucleic acid molecule of the nucleotide sequences shown in SEQ ID NO: 14 - SEQ ID NO: 16 and any one of the nucleotide sequences shown in SEQ ID NO: 17 - SEQ ID NO: 26 or their combinations includes DNA and RNA. Further, the DNA and RNA include plasmid DNA, mRNA, circular RNA, etc.

[0037] In some embodiments, the RNA comprises at least one chemical modification selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0038] In some aspects, the mRNA comprises at least one chemically modified nucleoside, wherein the at least one chemically modified nucleoside is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some aspects, the at least one chemically modified nucleoside is N1-methylpseudouridine. In some aspects, the polynucleotide is a fully modified N1-methylpseudouridine mRNA.

[0039] In some embodiments, the nucleic acid molecule may comprise the nucleic acid sequences of one or more of the following marker genes: EGFP, NLS-EGFP, sfGFP, sfGFP, TurboGFP, hrGFP, d2EGFP, ZsGreen1, EGFP(S65T), mNeonGreen, Venus, EYFP, YPet, Cerulean, CyPet, EBFP, TagBFP, TagBFP2, NLS_TagBFP2, dTomato, tdTomato, NLS_tdTomato, DsRed_Express2, TurboRFP, mRFP1, mCherry, NLS_mCherry, mApple, mKate2, Neo, Puro, Hygro, Bsd, Bar, Neo / Kana, Hygro, FKBP / Casp8, FKBP / Casp8, deltaTK, CodA, DTA, DTR, Luciferase, Luc2, MetLuc, Rluc, Nluc, Aequorin, hRluc, LacZ, SEAP, GUSPlus, pHluorin2, and Superecliptic-pHluorin and other reporter genes.

[0040] Preferably, EGFP or Luciferase is used as the reporter gene.

[0041] In a third aspect of the present invention, there is provided a CAR-Ms, which expresses the chimeric antigen receptor as described in the first aspect.

[0042] In a fourth aspect of the present invention, there is provided a gel material, which is prepared from natural calreticulin by a crosslinking agent and surface-modified with 4-(2-aminoethyl)benzenesulfonamide.

[0043] In some embodiments, the natural calreticulin can be from different species, including human, mouse, rat, chicken, bovine, sheep, rabbit, etc.

[0044] In a fifth aspect of the present invention, there is provided a nano-gel preparation, wherein each nanoparticle contains the nucleic acid molecule as described in the second aspect and the gel material as described in the fourth aspect. In some embodiments, each nanoparticle contains one or more excipients.

[0045] In a sixth aspect of the present invention, there is provided a method for constructing CAR-Ms, comprising the following steps: transfecting macrophages with the nucleic acid molecule provided in the second aspect of the present invention, or the gel material provided in the fourth aspect, or the nano-gel preparation provided in the fifth aspect to obtain CAR-Ms.

[0046] Methods for introducing genes into cells and expressing genes in cells are known in the art. The vector can be easily introduced into host cells by any method in the art, for example, mammalian, bacterial, yeast or insect cells. In some embodiments, the nucleic acid molecule provided in the second aspect of the present invention can be delivered by technical means such as viral vectors (including but not limited to lentivirus, adenovirus, retrovirus, etc.), lipid nanoparticles, cationic polymers (including but not limited to chitosan, hyaluronic acid, acid, dextran, cyclodextrin, polyethyleneimine, polylysine, polyamide and poly-β-aminoester, etc.), inorganic polymers (including but not limited to calcium phosphate, mesoporous silica, gold nanoparticles and quantum dots, etc.) and exosomes.

[0047] Preferably, macrophages are transfected with the nano-gel preparation provided in the fifth aspect of the present invention.

[0048] In a seventh aspect of the present invention, there is provided a CAR-Ms or a pharmaceutical combination containing the CAR-Ms.

[0049] The CAR-Ms of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as relevant cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention comprises the above-mentioned CAR-Ms, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Since the chimeric antigen receptor-modified macrophages described in the present invention are used as the active substance, in order to maintain the activity of such macrophages, the composition may also include one or more of 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 (for example, aluminum hydroxide) or preservatives.

[0050] The eighth aspect of the present invention provides the administration mode of the seventh aspect of the present invention, including by spraying, injection, swallowing, infusion, implantation or transplantation; further, the composition can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally. The dose of the active ingredient in the pharmaceutical composition can be determined by conventional research means in the art. The quantity and frequency of administration of the pharmaceutical composition will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0051] Preferably, the mode of use of the seventh aspect of the present invention is intravenous injection.

[0052] The ninth aspect of the present invention provides the application of the chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the CAR-Ms described in the third aspect, the nanogel preparation described in the fifth aspect, the construction method of the CAR-Ms described in the sixth aspect, the CAR-Ms described in the seventh aspect or the pharmaceutical combination containing the CAR-Ms in the preparation of anti-tumor products, and the anti-tumor products include but are not limited to drugs or model drugs.

[0053] The tenth aspect of the present invention provides an immunocyte therapy method for tumors, and the treatment method includes using the nanogel preparation described in the fifth aspect for a patient in need of treatment, or reinfusing the CAR-Ms described in the seventh aspect or the pharmaceutical combination containing the CAR-Ms.

[0054] Preferably, the tumors include but are not limited to one of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, nervous system cancer, leukemia, cervical cancer.

[0055] Preferably, the treatment method further includes obtaining macrophages of the patient and transferring the nucleic acid molecule described in the second aspect or the nanogel preparation described in the fifth aspect into the macrophages.

[0056] The present invention also includes a class of cell therapies, in which immune cells are genetically modified in vivo to express the fusion protein described in the first aspect of the present invention. In some embodiments, the immune cells can be T cells, γδ T cells, macrophages, NK cells, NKT cells, regulatory T cells, neutrophils, etc.

[0057] Preferably, the immune cells are macrophages.

[0058] In some embodiments, the CAR-Ms or pharmaceutical compositions containing the CAR-Ms of the present invention can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants.

[0059] The following is a further detailed description of the present invention in conjunction with specific examples. It should be noted that the specific examples are interpretations rather than limitations of the present invention.

[0060] Example 1. CAR Structure Design (1) The CAR protein structure is as Figure 1 shown, which is a CD8α signal peptide, an scFv fragment, a CD8α hinge region, a CD8 transmembrane region, and an intracellular co-stimulatory signal transduction domain (co-stimulatory region and CD3ζ intracellular region) connected in sequence. The scFv fragment is derived from the MUC1 monoclonal antibody 5E5 and includes a light chain variable region (5E5 VL) and a heavy chain variable region (5E5 VH), which are connected by (GGGGS)3. The co-stimulatory region in the intracellular co-stimulatory signal transduction domain is designed as shown in Table 1.

[0061] Table 1. Design and numbering of co-stimulatory regions in the intracellular structure of CAR protein

[0062] Construction of CAR gene mRNA Construct a CAR gene expression vector. In this example, the scFv segment is derived from the 5E5 antibody targeting MUC1, and EGFP is used as a reporter gene. Add the restriction enzyme digestion system, mix well by centrifugation and incubate overnight; use the QIAquick column to purify and recover the linear plasmid, and then use agarose gel electrophoresis to identify the purity of the linear plasmid; use the mRNA transcription system to perform in vitro transcription of the linear plasmid, and again use agarose gel electrophoresis to identify the purity of the mRNA; configure the capping system to cap the 5' end of the mRNA, and use the RNeasy Mini centrifugal column to purify the capped mRNA; collect the mRNA and verify the purity again for subsequent use.

[0063] Example 2. Preparation of Nanogel Preparation N-Hydroxysuccinimide-disulfide-N-hydroxysuccinimide (NHS-SS-NHS, 93.5 μg) dissolved in 9.35 μL of dimethyl sulfoxide (DMSO) was added to phosphate buffered saline (PBS, pH 7.4, 132 μL) containing calreticulin (1,320 μg), and mRNA was added. The mixture was incubated with rotation at 25 °C for 30 minutes, and then diluted by adding 1,188 μL of PBS (phosphate) buffer. The resulting nanogel product was subjected to buffer replacement through an ultrafiltration tube: centrifugally washed 3 times with 1.5 mL of PBS repeatedly to remove unreacted small molecules, obtaining nanogel (NG). Subsequently, 4-(2-aminoethyl)benzenesulfonamide (ABS) was introduced by Michael addition reaction. NG and ABS were dissolved in methanol and reacted in the dark for 24 hours. Impurities were removed using a dialysis membrane (MWCO = 500), and then freeze-dried to obtain nanogel (ANG). The morphology of the nanogel is as Figure 2 shown, and the average particle size is about 120 nm.

[0064] Example 3. Detection of transfection efficiency of nanogel preparation Human peripheral blood-derived macrophages (HPBDMs) were seeded in 6-well plates (1×10 6 cells per well) and cultured for 24 h. The DMEM medium containing nanogel was filtered through a 0.22 μm microporous membrane, and then added to the 6-well plates containing HPBDMs (1 mL per well). Incubated in a cell culture incubator at 37°C and 5% CO2 for 4 h, and then the cells were washed 3 times with PBS to remove unphagocytosed nanogel. Fresh DMEM medium was replaced - 10% FBS (fetal bovine serum), 10 ng / ml M-CSF (macrophage colony-stimulating factor), and continued to incubate in a cell culture incubator at 37°C and 5% CO2 for 24 h. Using PBS as the control group and the samples corresponding to CAR proteins No. 1-18 in Table 1 as the treatment groups, flow cytometry was used to detect the transfection efficiency of each group of nanogel preparations with EGFP fluorescence signal. The transfection results are as Figure 3 shown, and the nanogel preparation can achieve efficient transfection of macrophages.

[0065] Example 4. Specific phagocytosis detection of CAR-Ms on tumor cells MUC1 + PANC-1 cells were pre-labeled with calcein AM (2 μM), and then seeded in 12-well plates (5×10 4 cells per well). Different groups of CAR-Ms were added to the tumor cells (5×10 4The cells were further incubated in an incubator at 37°C and 5% CO2 for 4 h. Then the cells were collected, labeled with Anti-hCD68-PerCP-Cy5.5, and the phagocytosis was analyzed using a flow cytometer. Among them, PBS was used as the control group, and the samples corresponding to the CAR proteins numbered 1-18 in Table 1 were used as the treatment groups. The experimental results are as Figure 4 shown. Macrophages transfected with the nanogel formulation showed specific recognition and phagocytosis of MUC1 + tumor cells, and the 5th CAR-Ms showed the strongest phagocytosis effect.

[0066] Example 5. Detection of the anti-tumor effect of the nanogel Seven days after inoculating in situ pancreatic cancer tumor mice, CAR-Ms were injected for treatment, once every 7 days, for a total of 2 doses. The normal saline group was used as the control group, and the samples corresponding to the CAR proteins numbered 1-18 in Table 1 were used as the treatment groups. On the 15th day after tumor inoculation, the mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (0.5 mL / mouse), and then 0.1 mL of a physiological saline solution of potassium D-luciferin (30 mg / mL) was injected intraperitoneally. The fluorescence signal intensity of the tumor tissue was recorded using an IVIS detection system to evaluate the tumor growth. The experimental results are as Figure 5 shown. CAR-Ms transfected with the nanogel formulation could effectively inhibit tumor growth, and the 5th CAR-Ms showed the strongest tumor inhibition effect.

[0067] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that include: CD8α leader signal peptide, single-chain variable fragment, CD8α hinge region, CD8 transmembrane region and intracellular co-stimulatory signaling domain; Wherein, the single-chain variable fragment can target any known antigen; The intracellular co-stimulatory signal transduction domain is selected from at least one of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ; The amino acid sequence of the CD8α leader signal peptide is shown in SEQ ID NO:1, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO:2, the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:3, and the amino acid sequences of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ are shown in SEQ ID NO:4-SEQ ID NO:

13.

2. The chimeric antigen receptor according to claim 1, characterized in that The single-chain variable fragment is derived from a single-chain antibody of MUC1, including a light chain variable region and a heavy chain variable region, connected by a linker sequence; The intracellular co-stimulatory signaling domain is selected from the combination of Mincle and CD40, the combination of Mincle and 4-1BB, the combination of Mincle and OX40, the combination of Dectin-1 and CD40, the combination of Dectin-1 and 4-1BB, the combination of Dectin-1 and OX40, the combination of TLR2 and CD40, the combination of TLR2 and 4-1BB, the combination of TLR2 and OX40, the combination of TLR4 and CD40, the combination of TLR4 and 4-1BB, the combination of TLR4 and OX40, the combination of TLR7 and CD40, the combination of TLR7 and 4-1BB, the combination of TLR7 and OX40, the combination of NOD2 and CD40, the combination of NOD2 and 4-1BB, and the combination of NOD2 and OX40.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises: a gene encoding the chimeric antigen receptor according to claim 1 or 2; The CD8α leader signal peptide nucleotide is shown in SEQ ID NO:14, the CD8α hinge region nucleotide sequence is shown in SEQ ID NO:15, the CD8 transmembrane region nucleotide sequence is shown in SEQ ID NO:16, and the TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ nucleotide sequences are shown in SEQ ID NO:17-SEQ ID NO:

26.

4. A CAR-Ms, characterized in that: The CAR-Ms expresses the chimeric antigen receptor according to claim 1 or 2; Or, the CAR-Ms contains the nucleic acid molecule described in claim 3.

5. The CAR-Ms according to claim 4, characterized in that The following method is used to construct: The natural calreticulin, the cross-linking agent, and the nucleic acid molecule according to claim 3 are uniformly mixed in a solvent, reacted, and then surface modified with 4-(2-aminoethyl)benzenesulfonamide to obtain a nanogel preparation; The nanogel preparation is used to transfect macrophages to obtain CAR-Ms.

6. The use of the nucleic acid molecule according to claim 3 in the preparation of a nanogel preparation, characterized in that: include: The natural calreticulin, the cross-linking agent and the nucleic acid molecule according to claim 3 are uniformly mixed in a solvent, reacted, and then surface modified with 4-(2-aminoethyl)benzenesulfonamide to obtain a nanogel preparation.

7. Use of the chimeric antigen receptor according to claim 1 or 2 in the preparation of an anti-tumor pharmaceutical composition.

8. Use of the chimeric antigen receptor according to claim 7 in the preparation of an anti-tumor pharmaceutical composition, characterized in that: The tumor is selected from at least one of lung cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, nervous system cancer, leukemia, and cervical cancer.

9. Use of the chimeric antigen receptor according to claim 7 in the preparation of an anti-tumor pharmaceutical composition, characterized in that: The anti-tumor pharmaceutical composition comprises: immune cells, which are genetically modified in vivo to express the chimeric antigen receptor according to claim 1 or 2.

10. Use of the chimeric antigen receptor according to claim 9 in preparing an anti-tumor pharmaceutical composition, characterized in that: The immune cells are selected from at least one of T cells, γδ T cells, macrophages, NK cells, NKT cells, regulatory T cells, and neutrophils.

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