Chimeric antigen receptor, mRNA, nucleic acid molecule, macrophage and nucleic acid drug

Abstract

The invention provides a chimeric antigen receptor, mRNA, a nucleic acid molecule, a macrophage and a nucleic acid drug, and relates to the technical field of biology. According to the chimeric antigen receptor provided by the invention, the FAP-targeted chimeric antigen receptor comprises a TGF [beta] RII fragment, and the TGF [beta] RII fragment only can be combined with TGF [beta] but cannot transmit a signal, so that the immunosuppression effect of the TGF [beta] on macrophages is blocked; the chimeric antigen receptor targeting the GPC3 comprises a Super IL2 fragment, and the Super IL2 fragment can be used for selectively activating CD8 < + > T cells. The chimeric antigen receptor provided by the invention is expressed in macrophages, so that not only can tumor cells and tumor-related fiber cells be killed at the same time, but also the immunosuppression effect of TGF beta can be relieved, and epitope extension is promoted, so that T cells are activated, solid tumor heterogeneity is overcome, and finally a synergistic anti-tumor effect is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to chimeric antigen receptors, mRNA, nucleic acid molecules, macrophages and nucleic acid drugs. Background Art

[0002] Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment. Activated CAFs can form a physical barrier by remodeling the extracellular matrix (ECM), inhibiting the infiltration of drugs or immune cells and causing resistance to tumor treatment. Fibroblast activation protein-α (FAP) is a marker for CAFs activation, which is essential for the ECM remodeling function of CAFs cells, and its expression level is positively correlated with the poor prognosis of cancer. Currently, FAP-CAR T cells that target and kill CAFs cells are used in the clinic for the treatment of solid tumors. However, CAR T cell therapy is easily affected by the immunosuppressive tumor microenvironment in the treatment of solid tumors, causing T cell functional exhaustion. It is reported that FAP-CAR T cells can also cause severe side effects of bone marrow cell reduction and cachexia. In addition, this strategy still uses in vitro edited CAR T cells, which has a long preparation cycle and is very expensive. Since the number of T cells inside and outside the tumor is very limited, it is obvious that in vivo editing of CAR T cells is not the first choice for breaking the physical barrier of solid tumors. Macrophages are the most numerous of all immune cells, both inside and outside the tumor, so editing these cells in vivo may be the best choice to break the physical barrier composed of CAFs.

[0003] At present, the research on CAR macrophages at home and abroad basically adopts in vitro reprogramming and in vivo input methods for tumor treatment. Macrophage editing in vitro faces many problems such as low transfection efficiency, limited cell number, long preparation cycle, and high price. In vivo input faces the problem of being retained in the liver and difficult to effectively infiltrate the tumor site. In addition, the design of CAR macrophages in the past was mainly aimed at improving phagocytosis or M1 polarization, ignoring the effect of the tumor microenvironment on macrophages. The tumor microenvironment contains a large number of immunosuppressive cytokines, which can stimulate immune cells to cause immunosuppression. The present invention designs two new CAR molecules from the perspective of the tumor microenvironment, and uses the LNP-mRNA system to achieve in vivo editing of CAR macrophages to exert anti-tumor effects. In addition, the heterogeneity of solid tumors is also an important reason for the failure of immunotherapy. CAR T cell therapy can only kill tumor cells expressing target antigens, and cannot eliminate tumor cells with negative target antigens. This easily causes immune escape and recurrence of solid tumors. In vivo editing of CAR macrophages is expected to form killing of target antigen-negative tumor cells through antigen expansion, which is expected to overcome the heterogeneity of solid tumors.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first object of the present invention is to provide a chimeric antigen receptor to solve the above technical problems.

[0006] The second object of the present invention is to provide mRNA.

[0007] The third object of the present invention is to provide a nucleic acid molecule.

[0008] A fourth object of the present invention is to provide a macrophage.

[0009] The fifth object of the present invention is to provide the use of the above mRNA in the preparation of drugs for treating solid tumors.

[0010] The sixth object of the present invention is to provide a nucleic acid drug.

[0011] In order to achieve the above objectives, the following technical solutions are adopted:

[0012] In a first aspect, the present invention provides chimeric antigen receptors, including a chimeric antigen receptor targeting FAP and a chimeric antigen receptor targeting GPC3;

[0013] From the N-terminus to the C-terminus, the chimeric antigen receptor targeting FAP includes an extracellular domain, a transmembrane domain, an intracellular domain and a ΔTGFβRII fragment in sequence; wherein the extracellular domain is an anti-FAP scFV, the amino acid sequence of the heavy chain variable region thereof is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the ΔTGFβRII fragment is shown in SEQ ID NO.3;

[0014] From N-terminus to C-terminus, the chimeric antigen receptor targeting GPC3 includes an extracellular domain, a transmembrane domain, an intracellular domain and a Super IL2 fragment in sequence; wherein the extracellular domain is an anti-GPC3 scFV, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.5; the amino acid sequence of the Super IL2 fragment is shown in SEQ ID NO.6.

[0015] As a further technical solution, the amino acid sequence of the transmembrane domain of the chimeric antigen receptor targeting FAP or the chimeric antigen receptor targeting GPC3 is shown as SEQ ID NO.7, and the amino acid sequence of the intracellular domain is shown as SEQ ID NO.8.

[0016] As a further technical solution, the intracellular domain and the ΔTGFβRII fragment are connected via a connecting peptide;

[0017] The intracellular domain and the Super IL2 fragment are connected via a connecting peptide;

[0018] The amino acid sequence of the connecting peptide is shown in SEQ ID NO.9.

[0019] In a second aspect, the present invention provides mRNA, which expresses the chimeric antigen receptor.

[0020] In a third aspect, the present invention provides a nucleic acid molecule that transcribes the mRNA.

[0021] In a fourth aspect, the present invention provides a macrophage, wherein the macrophage expresses the chimeric antigen receptor or contains the mRNA.

[0022] In a fifth aspect, the present invention provides the use of the mRNA in the preparation of drugs for treating solid tumors.

[0023] In a sixth aspect, the present invention provides a nucleic acid drug, comprising a vector and the mRNA;

[0024] The vector is used for targeted delivery of the mRNA to macrophages.

[0025] As a further technical solution, the carrier is a liposome nanoparticle.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The chimeric antigen receptor provided by the present invention includes a △TGFβRII fragment targeting FAP, which can only bind to TGFβ but cannot transmit signals, thereby blocking the immunosuppressive effect of TGFβ on macrophages; the chimeric antigen receptor targeting GPC3 includes a Super IL2 fragment, which can selectively activate CD8 + The chimeric antigen receptor provided by the present invention is expressed in macrophages, which can not only kill tumor cells and tumor-associated fibroblasts at the same time, but also relieve the immunosuppressive effect of TGFβ and specifically activate CD8 + In addition, the chimeric antigen receptor targeting GPC3 can also prompt macrophages to rely on the GPC3-CAR structure to play an antigen expansion role and present a wider range of tumor antigens to T cells. Super IL2 can further promote CD8 + Activation of T cells is a strategy that can effectively overcome the heterogeneity of solid tumors.

[0028] The nucleic acid drug provided by the present invention includes a vector and mRNA expressing the chimeric antigen receptor of the present invention, wherein the vector is preferably a liposome nanoparticle, and CAR macrophages are obtained by in vivo editing through the LNP-mRNA system, which has high safety, high editing efficiency, short preparation cycle, and greatly reduced cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 To compare the expression rates of two CAR structures, FAP and GPC3, in macrophages after macrophages were infected with two types of LNPs;

[0031] Figure 2 To compare the phagocytic ability of macrophages on FAP-positive 293T cells and GPC3-positive Hepa1-6 cells after macrophages were infected with two types of LNPs;

[0032] Figure 3To compare the response of macrophages to TGFβ after infection of macrophages with two types of LNPs;

[0033] Figure 4 To compare the effects of two types of LNP on the expression of CD8 + Effects on T cell proliferation;

[0034] Figure 5 To edit the anti-tumor effect of CAR macrophages in vivo;

[0035] Figure 6 To edit CAR macrophages in vivo to break the physical barriers of solid tumors;

[0036] Figure 7 Editing CAR macrophages in vivo can form immune memory to prevent tumor recurrence;

[0037] Figure 8 The antigen expansion effect of CAR macrophages;

[0038] Fig. 9 The structures of the chimeric antigen receptor targeting FAP and the chimeric antigen receptor targeting GPC3. DETAILED DESCRIPTION

[0039] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0040] Macrophages (abbreviated as ) is a type of white blood cell located in tissues, derived from monocytes, which in turn originate from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytes that participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates. Their main function is to phagocytose (i.e., engulf and digest) cell debris and pathogens in the form of fixed or free cells, and to activate lymphocytes or other immune cells to respond to pathogens.

[0041] Chimeric Antigen Receptor (CAR): It is mainly composed of three parts: the extracellular antigen binding region, the transmembrane region, and the intracellular signal transduction region. The extracellular region is a single chain variable region domain (single chain Fvdomain, scFv), which has the function of specifically recognizing and binding to tumor-specific antigens. The transmembrane region is usually composed of the immunoglobulin superfamily, such as CD8 or CD28. The intracellular signal transduction region is mainly composed of co-stimulatory factors (4-1BB or CD28) and the signal activation region CD3ζ. After the immune cells loaded with chimeric antigen receptors bind to the surface antigens of tumor cells, the extracellular antigen binding region transmits the signal to the intracellular signal activation region, initiating the immune cell activation response.

[0042] CAR macrophages: Similar to CAR T cells, CAR macrophages load the CAR structure on macrophages, giving macrophages the ability to specifically bind to tumor cell surface antigens.

[0043] Lipid nanoparticles (LNP) are composed of four components: ionizable cationic lipids, phospholipids, cholesterol, and PEG-lipids. They use electrostatic adsorption to encapsulate mRNA, protecting it from degradation by nucleases and avoiding clearance by the blood mononuclear phagocyte system.

[0044] The inventor's previous studies found that exogenously introduced CAR macrophages cannot effectively infiltrate liver in situ tumors because the physical barrier composed of CAFs and the ECM constructed by them limits the infiltration of exogenous cells. Spatial transcriptome data of patients with liver cancer showed that there is a dense fibroblast band between the tumor area and the healthy area, and immune cells such as myeloid cells, B cells, and T cells are restricted to the periphery of the tumor by fibroblasts. In addition, this layer of fibroblast bands around the tumor is also an important factor in causing chemotherapy tolerance and immunotherapy tolerance of solid tumors. Therefore, breaking the physical barrier composed of CAFs is particularly important for the treatment of solid tumors. In addition, the heterogeneity of solid tumors is an important factor in tumor immune escape and recurrence. How to overcome the heterogeneity of solid tumors and form long-term immune memory against a wide range of tumor antigens in vivo is crucial to preventing the recurrence of solid tumors. Based on this, the following technical solution is proposed.

[0045] In a first aspect, the present invention provides chimeric antigen receptors, including a chimeric antigen receptor targeting FAP and a chimeric antigen receptor targeting GPC3;

[0046] From the N-terminus to the C-terminus, the chimeric antigen receptor targeting FAP includes an extracellular domain, a transmembrane domain, an intracellular domain and a ΔTGFβRII fragment in sequence; wherein the extracellular domain is an anti-FAP scFV, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2:

[0047] FAP-VH:

[0048] QVQLKESGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTADKRLELVATTNNN GGVTYYPDSVKGRFTISRDNAKNTLYLQMSSLQSEDTAMYYCARYGYYAMDYWGQ GISVTVSS(SEQ ID NO.1);

[0049] FAP-VL:

[0050] DVLMTQTPLWLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTVKISRVEAEDLGVYYCFGGSHVPYTFGGGTKLEIK(SEQ ID NO. 2);

[0051] The amino acid sequence of the ΔTGFβRII fragment is shown in SEQ ID NO.3:

[0052] MGRGLLRGLWPLHIVLWTRIASTIPPHVPKSVNSDVMASDNGGAVKLPQLCKFCDVRLSTCDNQKSCMSNCSITAICEKPHEVCVAVWRKNDKNITLETVCHDPKLTYHGFTLEDAASPKCVMKEKKRAGETFFMCACNMEECNDYIIFSEEYTTSSPDLLLVIIQVTGVSLLPPLGIAIAVIIIFYCYRVHRQQKLSP(SEQ ID NO.3);

[0053] From the N-terminus to the C-terminus, the chimeric antigen receptor targeting GPC3 includes an extracellular domain, a transmembrane domain, an intracellular domain and a Super IL2 fragment in sequence; wherein the extracellular domain is an anti-GPC3 scFV, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5:

[0054] GPC3-VH:

[0055] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQG TLVTVSS (SEQ ID NO.4);

[0056] GPC3-VL:

[0057] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK (SEQ ID NO.5);

[0058] The amino acid sequence of the Super IL2 fragment is shown in SEQ ID NO.6:

[0059] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNMINNYDNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNQAQSKNFHLDPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO.6).

[0060] The inventors found that tumor peripheral fibroblasts highly expressed FAP protein. FAP is not only a marker for CAFs activation, but also essential for the ECM remodeling function of CAFs cells, and its expression level is positively correlated with poor prognosis of cancer. Whether inside or outside the tumor, macrophages are the most numerous of all immune cells, so the inventors edited such cells in vivo through the LNP-mRNA system to break the physical barrier composed of CAFs. By editing CAR macrophages targeting FAP in vivo to kill CAFs cells, in addition to being easily exhausted by the immunosuppressive microenvironment, they cannot block the activation of CAFs from the source. TGFβ is the core cytokine that plays a role in the activation of CAFs. Not only that, TGFβ is also an immunosuppressive cytokine that can inhibit the phagocytic function of macrophages, antigen presentation function, and the expression of inflammatory factors. TGFβ mainly acts through TGFβRII on the cell surface. The expression of TGFβRII receptors (△TGFβRII) with intracellular domain deletion in cells can significantly inhibit the conduction of TGFβ signals. Therefore, the present invention proposes: to connect the intracellular domain-deficient TGFβRII receptor sequence (FAP-CAR-△TGFβRII) in series to the FAP-CAR structure, so that the CAR macrophages generated in vivo can target and kill CAFs cells while competitively binding to TGFβ, and also reduce the effect of TGFβ on other cells in the tumor environment when the cells are tolerant to TGFβ signals.

[0061] As the physical barriers of solid tumors are broken, CD8 + T immune cells will infiltrate the solid tumor in large numbers. Activating CD8 + T cells can maximize the anti-tumor effect. Therefore, the inventors envision that in vivo edited CAR macrophages can activate CD8 through antigen presentation + T cells. Glypican-3 (GPC3) is highly expressed in a variety of solid tumors including hepatocellular carcinoma, but CD8 + In addition to antigen presentation, T activation also requires cytokine stimulation. IL2 is the key factor that activates CD8 + The most important cytokine for T cells, which can promote CD8 + Proliferation and activation of T cells. IL2 is also the earliest cytokine product used in tumor treatment. However, its severe side effects caused by systemic administration limit its clinical application. The LNP-mRNA system can target macrophages and limit the delivery range of drugs. Therefore, the inventors envisioned cascading IL2 on the CAR structure and editing CAR macrophages that can secrete IL2 in situ. However, natural IL2 preferentially activates Treg cells that highly express CD25, rather than CD8 +T cells. Recently, researchers have modified IL2 based on synthetic biology to not only have a higher affinity for the CD122 (IL2Rβ) receptor on the surface of CD8, but also have better stability, thereby achieving specific activation of CD8 + The ability of T cells, this IL2 is called Super IL2. Therefore, the inventors used the sequence of Super IL2 and the GPC3-CAR structure in series (GPC3-CAR-Super IL2), and the macrophages obtained by in vivo editing can specifically activate CD8 T cells while targeting and killing solid tumors. + CAR macrophages for T cells.

[0062] In addition, the inventors have found that the chimeric antigen receptor targeting GPC3 can also prompt macrophages to rely on the GPC3-CAR structure to play an antigen expansion role and present a wider range of tumor antigens to T cells. Super IL2 can further promote the CD8 + Activation of T cells is a strategy that can effectively overcome the heterogeneity of solid tumors.

[0063] In some optional embodiments, the amino acid sequence of the transmembrane domain of the chimeric antigen receptor targeting FAP or the chimeric antigen receptor targeting GPC3 is shown in SEQ ID NO.7:

[0064] TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICV ALLLSLIITLIC(SEQ ID NO.7);

[0065] The amino acid sequence of the intracellular domain is shown in SEQ ID NO.8:

[0066] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRR RNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHM QTL (SEQ ID NO. 8).

[0067] In some optional embodiments, the intracellular domain and the ΔTGFβRII fragment are connected via a connecting peptide;

[0068] The intracellular domain and the Super IL2 fragment are connected via a connecting peptide;

[0069] The amino acid sequence of the connecting peptide is shown in SEQ ID NO.9:

[0070] GSGEGRGSLLTCGDVEENPGP (SEQ ID NO. 9).

[0071] In a second aspect, the present invention provides mRNA, which expresses the chimeric antigen receptor.

[0072] In some optional embodiments, the nucleotide sequence of the mRNA expressing the chimeric antigen receptor targeting FAP is as follows:

[0073] The nucleotide sequence of the mRNA expressing the chimeric antigen receptor targeting FAP is as follows:

[0074] ATGGCTCTCCCTGTCACCGCCCTGCTTCTCCCTTTGGCGCTGCTCCTCCATGC

[0075] TGCCCGCCCACAGGTGCAGCTGAAGGAGAGCGGCGGCCTGGTGCAGCCCGGCGG

[0076] CAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGGCATG

[0077] AGCTGGGTGAGGCAGACCGCCGACAAGAGGCTGGAGCTGGTGGCCACCACCAAC

[0078] AACAACGGCGGCGTGACCTACTACCCCGACAGCGTGAAGGGCAGGTTCACCATCA

[0079] GCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGCAGAGCG

[0080] AGGACACCGCCATGTACTACTGCGCCAGGTACGGCTACTACGCCATGGACTACTGG

[0081] GGCCAGGGCATCAGCGTGACCGTGAGCAGCGGAGGCCGGTGGTTCAGGGGGCGGA

[0082] GGGAGCGGCGGCGGTGGCTCTGACGTGCTGATGACCCAGACCCCCCTGTGGCTGC

[0083] CCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAGCATCG

[0084] TGCACAGCAACGGCAACACCTACCTGGAGTGGTACCTGCAGAAGCCCGGCCAGA

[0085] GCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAG

[0086] GTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCGTGAAGATCAGCAGGGTGGA

[0087] GGCCGAGGACCTGGGCGTGTACTACTGCTTCGGCGGCAGCCACGTGCCCTACACC

[0088] TTCGGCGGCGGCACCAAGCTGGAGATCAAGACCACGACAAAACCCGTGCTCCGA

[0089] ACCCCTTCCCCTGTCCATCCTACTGGGACAAGCCAGCCTCAGAGGCCTGAGGACT

[0090] GCAGGCCAAGAGGCAGTGTCAAAGGAACCGGACTTGACTTTGCCTGTGACATCTA

[0091] CATCTGGGCTCCTCTGGCCGGTATATGTGTGGCGCTGCTGTTATCCCTCATTATAAC

[0092] GCTTATCTGTCGAGCAAAGTTCTCTCGTTCAGCAGAGACAGCTGCAAACCTGCAG

[0093] GACCCCAACCAGTTATATAATGAACTAAATCTTGGCCGCCGGGAAGAGTATGATGT

[0094] ACTGGAAAAGAAGAGGGCCAGAGACCCTGAAATGGGTGGAAAGCAACAGCGGC

[0095] GGAGGAACCCACAAGAAGGTGTGTATAATGCACTCCAGAAAGACAAGATGGCTGA

[0096] GGCCTACTCAGAAATTGGAACCAAGGGAGAACGTCGCAGGGGGAAGGGGCACGA

[0097] CGGCCTTTATCAGGGACTTAGCACGGCCACAAAGGACACATACGATGCCCTCCACA

[0098] TGCAGACTCTGGCACCTCGGGGAAGCGGCgagggcagaggcagcctgctgacatgtggcgacgtggaa

[0099] gagaaccctggccccATGGGCAGGGGCCTGCTGAGGGGCCTGTGGCCCCTGCACATCGTG

[0100] CTGTGGACCAGGATCGCCAGCACCATCCCCCCCCACGTGCCCAAGAGCGTGAACA

[0101] GCGACGTGATGGCCAGCGACAACGGCGGCGCCGTGAAGCTGCCCCAGCTGTGCA

[0102] AGTTCTGCGACGTGAGGCTGAGCACCTGCGACAACCAGAAGTCCTGCATGAGCAA

[0103] CTGCAGCATCACCGCCATCTGCGAGAAGCCCCACGAGGTGTGCGTGGCCGTGTGG

[0104] AGGAAGAACGACAAGAACATCACCCTGGAGACCGTGTGCCACGACCCCAAGCTG

[0105] ACCTACCACGGCTTCACCCTGGAGGACGCCGCCAGCCCCAAGTGCGTGATGAAGG

[0106] AGAAGAAGAGGGCCGGCGAGACCTTCTTCATGTGCGCCTGCAACATGGAGGAGT

[0107] GCAACGACTACATCATCTTCAGCGAGGAGTACACCACCAGCAGCCCCGACCTGCT

[0108] GCTGGTGATCATCCAGGTGACCGGCGTGAGCCTGCTGCCCCCCCTGGGCATCGCCA

[0109] TCGCCGTGATCATCATCTTCTACTGCTACAGGGTGCACAGGCAGCAGAAGCTGAGC

[0110] CCC(SEQ ID NO.10).

[0111] The nucleotide sequence of the mRNA expressing the chimeric antigen receptor targeting GPC3 is as follows:

[0112] ATGGCTCTCCCTGTCACCGCCCTGCTTCTCCCTTTGGCGCTGCTCCTCCATGC

[0113] TGCCCGCCCACAAGTTCAACTCGTGCAATCTGGCGCTGAGGTGAAAAAGCCCGGG

[0114] GCCTCAGTAAAAGTGAGCTGCAAGGCCTCTGGTTACACTTTTACAGACTATGAGAT

[0115] GCACTGGGTGAGGCAGGCCCCAGGCCAGGGGCTGGAATGGATGGGAGCACTGGA

[0116] CCCTAAGACAGGCGATACCGCCTACAGTCAGAAGTTTAAGGGGCGAGTTACACTT

[0117] ACTGCTGATGAGTCTACTTCAACCGCGTACATGGAGTTGTCATCGCTCCGGTCTGA

[0118] GGATACTGCTGTGTACTACTGTACAAGGTTCTACAGTTACACCTACTGGGGACAGG

[0119] GCACTTTGGTGACAGTATCCTCTGGAGGCGGTGGTTCAGGGGGCGGAGGGAGCGG

[0120] CGGCGGTGGCTCTGATGTAGTGATGACCCAGAGCCCACTGAGTTTACCAGTGACTC

[0121] CCGGAGAGCCAGCAAGCATCTCTTGCAGGTCTAGCCAATCTCTGGTGCACTCGAA

[0122] CGGAAACACATACCTGCACTGGTACCTACAGAAACCAGGGCAAAGTCCTCAGCTG

[0123] CTTATCTACAAGGTGAGCAATAGATTCAGTGGAGTCCCTGACAGATTTTCTGGTTC

[0124] CGGAAGTGGCACCGACTTCACGCTAAAGATCAGCCGGGTAGAAGCGGAAGATGTT

[0125] GGAGTATATTACTGCAGCCAGAACACCCATGTGCCTCCCACCTTTGGACAAGGTAC

[0126] AAAGCTGGAGATCAAGACCACGACAAAACCCGTGCTCCGAACCCCTTCCCCTGTC

[0127] CATCCTACTGGGACAAGCCAGCCTCAGAGGCCTGAGGACTGCAGGCCAAGAGGC

[0128] AGTGTCAAAGGAACCGGACTTGACTTTGCCTGTGACATCTACATCTGGGCTCCTCT

[0129] GGCCGGTATATGTGTGGCGCTGCTGTTATCCCTCATTATAACGCTTATCTGTCGAGC

[0130] AAAGTTCTCTCGTTCAGCAGAGACAGCTGCAAACCTGCAGGACCCCAACCAGTTA

[0131] TATAATGAACTAAATCTTGGCCGCCGGGAAGAGTATGATGTACTGGAAAAGAAGAG

[0132] GGCCAGAGACCCTGAAATGGGTGGAAAGCAACAGCGGCGGAGGAACCCACAAG

[0133] AAGGTGTGTATAATGCACTCCAGAAAGACAAGATGGCTGAGGCCTACTCAGAAAT

[0134] TGGAACCAAGGGAGAACGTCGCAGGGGGAAGGGGCACGACGGCCTTTATCAGGG

[0135] ACTTAGCACGGCCACAAAGGACACATACGATGCCCTCCACATGCAGACTCTGGCA

[0136] CCTCGGGGAAGCGGCgagggcagaggcagcctgctgacatgtggcgacgtggaagagaaccctggccccATGT

[0137] ACAGGATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAG

[0138] CGCCCCCACCAGCAGCAGCACCAAGAAGACCCAGCTGCAGCTGGAGCACCTGCT

[0139] GCTGGACCTGCAGATGATCCTGAACATGATCAACAACTACGACAACCCCAAGCTG

[0140] ACCAGGATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGC

[0141] ACCTGCAGTGCCTGGAGGAGGAGCTGAAGCCCCTGGAGGAGGTGCTGAACCAGG

[0142] CCCAGAGCAAGAACTTCCACCTGGACCCCAGGGACCTGATCAGCAACATCAACGT

[0143] GATCGTGCTGGAGCTGAAGGGCAGCGAGACCACCTTCATGTGCGAGTACGCCGAC

[0144] GAGACCGCCACCATCGTGGAGTTCCTGAACAGGTGGATCACCTTCTGCCAGAGCA

[0145] TCATCAGCACCCTGACC (SEQ ID NO. 11).

[0146] In a third aspect, the present invention provides a nucleic acid molecule that transcribes the mRNA.

[0147] In a fourth aspect, the present invention provides a macrophage, wherein the macrophage expresses the chimeric antigen receptor or contains the mRNA.

[0148] The macrophages have better ability to kill solid tumors.

[0149] In a fifth aspect, the present invention provides the use of the mRNA in the preparation of drugs for treating solid tumors.

[0150] In a sixth aspect, the present invention provides a nucleic acid drug, comprising a vector and the mRNA;

[0151] The vector is used for targeted delivery of the mRNA to macrophages.

[0152] In some optional embodiments, the carrier is a liposome nanoparticle.

[0153] The nucleic acid drug provided by the present invention is edited in vivo through the LNP-mRNA system to obtain CAR macrophages, which has high safety, high editing efficiency, short preparation cycle and greatly reduced cost.

[0154] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0155] Example 1

[0156] 1. The mRNA of two CAR structures was obtained by in vitro transcription (such as Fig. 9), GPC3-CAR-Super IL2 (sequence as shown in SEQ ID NO.11) and FAP-CAR-△TGFβRII (sequence as shown in SEQ ID NO.10). Then the two mRNAs were mixed in equal proportions to prepare LNP, named LNP-GF CAR mRNA. GFP mRNA was used as Control mRNA to prepare LNP, named LNP-Control mRNA. Bone marrow-derived macrophages were induced in vitro and infected with LNP-GFCAR mRNA. The expression levels of FAP CAR and GPC3 CAR were detected after 24 hours. At the same time, uninfected macrophages were used as the Control group. The results showed ( Figure 1 ), LNP-GF CAR mRNA was able to efficiently edit CAR macrophages, and the expression efficiency of both CAR structures reached more than 50%.

[0157] 2. Next, we verified whether the LNP-GF mRNA-edited CAR macrophages could achieve the expected function. LNP-GF CAR mRNA was transfected into macrophages, and then co-incubated with Td-tomato fluorescently labeled GPC3 (human) overexpressing Hepa1-6 cells and GFP fluorescently labeled FAP (mouse) overexpressing 293T cells. In the phagocytosis experiment of Hepa1-6 cells overexpressing GPC3, GFP mRNA was used as the Control mRNA, and in the phagocytosis experiment of 293T cells overexpressing FAP, luciferase mRNA was used as the Control mRNA. After 48 hours of co-incubation, phagocytosis was detected by flow cytometry, and F4 / 80 + Td-tomato + Indicates the phagocytosis of Hepa1-6 cells overexpressing GPC3 by macrophages, F4 / 80 + GFP + Indicates the phagocytic effect of macrophages on 293T cells overexpressing FAP. The results show ( Figure 2 ), LNP-GF CAR mRNA edited CAR macrophages significantly increased the proliferation of GPC3-positive Hepa1-6 cells and FAP + Phagocytic ability of 293T cells. This indicates that LNP-GF CAR mRNA-edited CAR macrophages do acquire the dual-targeting phagocytic ability of GPC3 and FAP.

[0158] 3. LNP-Control mRNA and LNP-GF CAR mRNA were used to infect macrophages. TGFβ was added for stimulation 24 hours later, and macrophages were collected for WB detection 30 minutes later. The results showed ( Figure 3), compared with LNP-Control mRNA, LNP-GFCAR mRNA significantly inhibited the activation of TGFβ downstream signal p-SMAD2 / 3 in macrophages. This indicates that macrophages edited with LNP-GF CAR mRNA achieved the expected function of resisting TGFβ signaling.

[0159] 4. The CAR macrophages edited by LNP-GF CAR mRNA were co-incubated with spleen T cells, and then the proliferation of T cells was detected. The spleen T cells were stained with CFSE (5uM) to mark proliferation. The T cell culture group alone was used as the NC group (negative control group), the αCD3 / 28 group was stimulated by adding αCD3 (1ug / ml) and αCD28 (5ug / ml) to the T cell culture (positive control group), and the LNP-Control mRNA group and the LNP-GF CAR mRNA group were stimulated by αCD3 (1ug / ml) and αCD28 (5ug / ml). Macrophages edited by different LNPs were added and co-incubated with T cells. After 72 hours of co-incubation, the proportion and proliferation of each T cell population were detected by flow cytometry. The results showed that compared with LNP-Control mRNA, CAR macrophages edited by LNP-GF CAR mRNA did not affect CD4 + The proportion of Treg cells in the T cell population Figure 4 A), but significantly increased CD8 in the CD3 T cell population + The proportion of T cells ( Figure 4 B) and promoted CD8 + T cell proliferation ( Figure 4 These results indicate that LNP-GF CAR mRNA-edited CAR macrophages can indeed specifically promote CD8 + Proliferation of T cells. This indicates that LNP-CAR mRNA edited macrophages achieve the expected effect of secreting Super IL2.

[0160] 5. Next, an orthotopic liver cancer model was established to explore the anti-tumor effect of LNP-GF CAR mRNA. The orthotopic liver cancer model was established using the Hepa1-6 cell line (Luciferase+Td-tomato+) overexpressing human GPC3, and LNP-GFCAR mRNA was injected intravenously for treatment twice a week ( Figure 5 The results showed that LNP-GF CAR mRNA had a significant anti-tumor effect, with a therapeutic efficiency of 100%, and 4 out of 5 mice were completely cured ( Figure 5Next, Clodronate Liposomes were used to remove macrophages in vivo, and the anti-tumor experiment of orthotopic liver cancer model was performed again ( Figure 5 The results showed that when macrophages were deleted, the anti-tumor effect of LNP-GF CAR mRNA was significantly reduced and the tumor could not be eradicated ( Figure 5 D). This suggests that LNP-GF CAR mRNA relies on editing by macrophages in vivo to exert its anti-tumor effect.

[0161] 6. Next, we will focus on analyzing whether the physical barrier constructed by fibroblasts around the tumor is broken. According to the method of Part 5, an in situ liver cancer model was established, and LNP-GF CAR mRNA was injected intravenously for treatment. First, the inventors analyzed the activation of TGFβ signaling in the treatment group. After 2 weeks of treatment, the liver was taken for immunofluorescence staining of F4 / 80 and p-SMAD2 / 3. The results showed that the p-SMAD2 / 3 signal in the liver macrophages in the LNP-GF CAR mRNA treatment group was significantly reduced ( Figure 6 A in the figure). This indicates that the in vivo edited CAR macrophages have indeed acquired the ability to tolerate TGFβ signals. In addition, immunohistochemical staining of p-SMAD2 / 3 was performed on liver tissue, and the results showed that the overall level of p-SMAD2 / 3 signal in the liver of the LNP-GF CAR mRNA treatment group was significantly reduced ( Figure 6 B in the figure). This indicates that the in vivo edited CAR macrophages not only acquire the ability to tolerate TGFβ signals, but also reduce the stimulation of TGFβ on other cells, prompting the liver to reduce the conduction of TGFβ signals at the overall level. Sirius red staining of liver tissue showed that the fibrosis area in the tumor of the LNP-GF CAR mRNA treatment group was significantly reduced, and the thickness of the fibroblast band around the tumor was significantly thinner ( Figure 6 In addition, qRT-PCR was performed on the single cell suspension of the liver, and the results showed that the LNP-GF CAR mRNA treatment group significantly reduced the mRNA expression level of FAP ( Figure 6 E). Through the immunofluorescence experiment of FAP, it was observed that the positive area of ​​FAP in the LNP-GF CAR mRNA treatment group was significantly reduced, and there was almost no cell band composed of FAP-positive cells around the tumor of the treatment group ( Figure 6 F, G in Figure 3). This fully demonstrates that LNP-GFCAR mRNA edited CAR macrophages broke the physical barrier constructed by CAFs cells outside the tumor. Then, CD8 + The distribution of T cells showed that the CD8 + A large number of T cells were blocked in the FAP-positive cell area, while the treatment group significantly promoted CD8+ T cell infiltration into the tumor Figure 6 H, I in Figure 3). The above data indicate that LNP-GF CARmRNA-edited CAR macrophages can break the physical barrier constructed by FAP-positive CAFs cells by killing them and promote CD8 + The activation and infiltration of T cells ultimately synergize with adaptive immunity to exert an anti-tumor effect sufficient to regress solid tumors.

[0162] VII. The inventors analyzed the effect of LNP-GF CAR mRNA in vivo editing of CAR macrophages on immune memory. For mice cured by LNP-GF CAR mRNA in the orthotopic liver cancer model (referred to as Cured mice), WTHepa1-6 cells (GPC3 negative) were subcutaneously inoculated again ( Figure 7 A in the figure). Compared with naive mice, LNP-GF CAR mRNA cured orthotopic liver cancer mice completely prevented the growth of WT Hepa1-6 subcutaneous tumors. However, in the case of intraperitoneal injection of αCD3 antibody to delete T cells in LNP-GF CAR mRNA cured orthotopic liver cancer mice (denoted as Cured mice+αCD3), the cured mice showed the growth of WT Hepa1-6 cell subcutaneous tumors, and the growth trend was consistent with that of naive mice ( Figure 7 In contrast, after subcutaneous tumor inoculation with WT Hepa1-6 cells, the mice cured of orthotopic liver cancer remained tumor-free and there was no death. However, the cured mice with T cell deletion and the naive mice all died within 2 months ( Figure 7 C in Figure 1). This suggests that LNP-GF CAR mRNA editing of CAR macrophages in vivo not only forms T cell immune memory, but also promotes T cells to form immune memory against target antigen-negative tumor cells through antigen expansion, thereby overcoming tumor heterogeneity.

[0163] 8. To further confirm the antigen expansion effect of CAR macrophages, the inventors constructed a Hepa1-6 cell line (OVA + GPC3 + Hepa1-6 cells) and spleen T cells from OT1 mice were used for the experiment. + T cells only recognize OVA antigens that have been presented by antigens, so only when CAR macrophages targeting GPC3 present OVA antigens on Hepa1-6 cells through antigen expansion can they activate the CD8 +In addition, the inventors constructed a GPC3-CAR molecule without the Super IL2 sequence (denoted as LNP-GPC3-CAR mRNA) to distinguish the effects of GPC3-CAR and Super IL2 on T cell activation and antigen expansion. The inventors set up two co-culture systems: CAR macrophages edited with different LNPs and OVA + GPC3 + Hepa1-6 cells were co-cultured with spleen T cells from OT1 mice ( Figure 8 A), CAR macrophages edited with different LNPs and OVA - GPC3 + Hepa1-6 cells were co-cultured with spleen T cells from OT1 mice ( Figure 8 B in the experiment). The names of the groups in this experiment are: NC (tumor cells + T cells), Mac (untransfected macrophages + tumor cells + T cells), Control (LNP-Control mRNA edited macrophages + tumor cells + T cells), GPC3-CAR (LNP-GPC3-CAR mRNA edited macrophages + tumor cells + T cells), GPC3-CAR-Super IL2 (LNP-GPC3-CAR-Super IL2 mRNA edited macrophages + tumor cells + T cells). After 72 hours of co-incubation, flow cytometry was used to detect the CD8 + Proliferation of T. + GPC3 + In the Hepa1-6 cell co-culture system, LNP-GPC3 CAR mRNA-edited CAR macrophages significantly promoted CD8 + T cell proliferation ( Figure 8 This indicates that CAR macrophages can indeed play an antigen expansion role through the GPC3-CAR structure, further presenting OVA antigens in Hepa1-6 cells, thereby promoting CD8 + Compared with LNP-GPC3 CAR mRNA, LNP-GPC3-CAR-Super IL2 mRNA edited CAR macrophages further promoted CD8 + T cell proliferation ( Figure 8 This indicates that Super IL2 can further promote CD8 antigen expansion in the process based on the GPC3-CAR structure. + In contrast, in the presence of OVA - GPC3 +In the Hepa1-6 cell co-culture system, since Hepa1-6 in this system does not express OVA antigen, macrophages in each treatment group could not promote CD8 + Only LNP-GPC3 CAR-Super IL2 mRNA edited CAR macrophages can promote the proliferation of T cells due to Super IL2 itself. + The effect on T cell proliferation was slight, increasing CD8 + T cell proliferation ( Figure 8 This further confirms the antigen expansion effect of CAR macrophages targeting GPC3. In summary, CAR macrophages targeting GPC3 can indeed present OVA antigens through the antigen expansion mechanism and promote CD8 + Super IL2 can further enhance the proliferation of CD8 T cells on this basis. + In summary, these results indicate that the GPC3-CAR-Super IL2 structure can induce macrophages to rely on the GPC3-CAR structure to exert antigen expansion and further activate CD8 + T cells.

[0164] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that Including chimeric antigen receptor targeting FAP and chimeric antigen receptor targeting GPC3; From the N-terminus to the C-terminus, the chimeric antigen receptor targeting FAP includes an extracellular domain, a transmembrane domain, an intracellular domain and a ΔTGFβRII fragment in sequence; wherein the extracellular domain is an anti-FAP scFV, the amino acid sequence of the heavy chain variable region thereof is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the ΔTGFβRII fragment is shown in SEQ ID NO.3; From N-terminus to C-terminus, the chimeric antigen receptor targeting GPC3 includes an extracellular domain, a transmembrane domain, an intracellular domain and a Super IL2 fragment in sequence; wherein the extracellular domain is an anti-GPC3 scFV, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.5; the amino acid sequence of the Super IL2 fragment is shown in SEQ ID NO.

6.

2. The chimeric antigen receptor according to claim 1, characterized in that The amino acid sequence of the transmembrane domain of the chimeric antigen receptor targeting FAP or the chimeric antigen receptor targeting GPC3 is shown in SEQ ID NO.7, and the amino acid sequence of the intracellular domain is shown in SEQ ID NO.

8.

3. The chimeric antigen receptor according to claim 1, characterized in that The intracellular domain and the ΔTGFβRII fragment are connected via a connecting peptide; The intracellular domain and the Super IL2 fragment are connected via a connecting peptide; The amino acid sequence of the connecting peptide is shown in SEQ ID NO.

9.

4. mRNA, characterized in that The mRNA expresses the chimeric antigen receptor according to any one of claims 1 to 3.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule transcribes the mRNA of claim 4.

6. A macrophage, characterized in that: The macrophage expresses the chimeric antigen receptor according to any one of claims 1 to 3 or contains the mRNA according to claim 4.

7. Use of the mRNA according to claim 4 in the preparation of drugs for treating solid tumors.

8. A nucleic acid drug, characterized in that: Comprising a vector and the mRNA according to claim 4; The vector is used for targeted delivery of the mRNA to macrophages.

9. The nucleic acid drug according to claim 8, characterized in that The carrier is liposome nanoparticles.

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