Chimeric antigen receptors, mrna, nucleic acid molecules, macrophages, and nucleic acid drugs

Abstract

The application provides a chimeric antigen receptor, mRNA, a nucleic acid molecule, macrophages and a nucleic acid drug, and relates to the technical field of biotechnology.The chimeric antigen receptor provided by the application, wherein the chimeric antigen receptor targeting FAP comprises a △TGFβRII fragment, the △TGFβRII fragment can only bind with TGFβ but cannot transmit a signal, thereby blocking the immune inhibition of TGFβ on macrophages; the chimeric antigen receptor targeting GPC3 comprises a Super IL2 fragment, the Super IL2 fragment can selectively activate CD8 + T cells.The chimeric antigen receptor provided by the application is expressed in macrophages, which can not only kill tumor cells and tumor-associated fibroblasts at the same time, but also can relieve the immune inhibition of TGFβ, promote the antigen epitope extension to activate T cells, overcome the heterogeneity of solid tumors, and finally realize the synergistic anti-tumor effect.

Description

TECHNICAL FIELD

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

[0002] Cancer-associated fibroblasts (CAFs) are one of the important components of tumor microenvironment, activated CAFs can form a physical barrier by remodeling extracellular matrix (ECM) to inhibit the infiltration of drugs or immune cells, causing tumor treatment resistance. Fibroblast activation protein-alpha (FAP) is a marker for CAF activation, which is essential for the ECM remodeling function of CAF cells, and its expression level is positively correlated with poor prognosis of cancer. Currently, FAP-CAR T cells targeting CAF cells have been used for the treatment of solid tumors in clinic. 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 has been reported that FAP-CAR T cells can also cause serious side effects of bone marrow cell reduction and cachexia. In addition, this strategy still uses CAR T cells edited in vitro, which has a long preparation cycle and is very expensive. Since the number of T cells is very limited inside and outside the tumor, it is obvious that editing CAR T cells in vivo is not the first choice to break the physical barrier of solid tumors. Macrophages are the most numerous immune cells 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] Current research on CAR macrophages at home and abroad basically adopts the method of in vitro reprogramming and in vivo input for tumor treatment. In vitro editing of macrophages faces many problems such as low transfection efficiency, limited cell number, long preparation period, high price and so on, and in vivo input faces the problem of difficult effective infiltration of tumor sites due to retention in the liver. Moreover, the design of the previous CAR macrophages mainly aims to improve phagocytosis or M1 polarization, ignoring the influence 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 application 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 play an anti-tumor role. 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 negative for target antigens. This is easy to cause immune escape and recurrence of solid tumors. In vivo editing of CAR macrophages is expected to kill target antigen-negative tumor cells through antigen spreading, and is expected to overcome the heterogeneity of solid tumors.

[0004] In view of this, the present application is proposed. SUMMARY

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

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

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

[0008] The fourth object of the present application is to provide a macrophage.

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

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

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

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

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

[0014] From N-terminal to C-terminal, the chimeric antigen receptor targeting GPC3 comprises, in sequence, an extracellular domain, a transmembrane domain, an intracellular domain and a Super IL2 fragment; wherein the extracellular domain is an anti-GPC3 scFV, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5; the amino acid sequence of the Super IL2 fragment is shown as 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 △TGFβRII fragment are connected through a connecting peptide;

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

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

[0019] In a second aspect, the present application provides an mRNA, wherein the mRNA expresses the chimeric antigen receptor.

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

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

[0022] In a fifth aspect, the present application provides use of the mRNA in the preparation of a drug for treating a solid tumor.

[0023] In a sixth aspect, the present application provides a nucleic acid drug, comprising a carrier and the mRNA.

[0024] The carrier 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 application has the following beneficial effects:

[0027] The chimeric antigen receptor provided by the present application, wherein the chimeric antigen receptor targeting FAP comprises a △TGFβRII fragment, the △TGFβRII fragment can only bind to TGFβ but cannot transmit signals, thereby blocking the immune suppression of TGFβ on macrophages; the chimeric antigen receptor targeting GPC3 comprises a Super IL2 fragment, the Super IL2 fragment can selectively activate CD8 + T cells. The chimeric antigen receptor provided by the present application is expressed in macrophages, which can not only kill tumor cells and tumor-associated fibroblasts at the same time, but also can relieve the immune suppression of TGFβ, specifically activate CD8 + T cells, and ultimately achieve synergistic anti-tumor effect. In addition, the chimeric antigen receptor targeting GPC3 can also promote the antigen expansion of macrophages depending on the GPC3-CAR structure, present more extensive tumor antigens to T cells, and the Super IL2 can further promote the activation of CD8 + T cells, and the strategy can effectively overcome the heterogeneity of solid tumors.

[0028] The nucleic acid drug provided by the present application comprises a carrier and mRNA expressing the chimeric antigen receptor of the present application, wherein the carrier is preferably a liposome nanoparticle, and the CAR macrophage is obtained by editing the LNP-mRNA system in vivo, which is safe, has high editing efficiency, short preparation period and greatly reduced cost. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0030] Figure 1 In order to compare the expression rates of FAP and GPC3 CAR structures of macrophages after infection of two kinds of LNP;

[0031] Figure 2 In order to compare the phagocytic ability of macrophages to FAP positive 293T cells and GPC3 positive Hepa1-6 cells after infection of two kinds of LNP;

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

[0033] Figure 4 To compare the response of macrophages to CD8 + T cell proliferation after infection with the two LNPs;

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

[0035] Figure 6 To compare the ability of CAR macrophages edited in vivo to break the physical barrier of solid tumors;

[0036] Figure 7 To compare the ability of CAR macrophages edited in vivo to form an immunological memory to prevent tumor relapse;

[0037] Figure 8 To compare the antigenic broadening of CAR macrophages;

[0038] Figure 9 To compare the structure of a chimeric antigen receptor targeting FAP and a chimeric antigen receptor targeting GPC3. DETAILED DESCRIPTION

[0039] Embodiments of the present application will be described in detail with reference to the drawings and examples, but it will be understood that the following embodiments and examples are merely illustrative of the present application and should not be considered to limit the scope of the present application. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts are within the scope of the present application. Unless specific conditions are specified, the conventional conditions or the conditions recommended by the manufacturer are used. Unless the manufacturer of the reagent or instrument is specified, it is a conventional product that can be purchased on the market.

[0040] Macrophages (abbreviated as ) are a type of white blood cell found in tissues that develop from monocytes, which in turn are derived from precursor cells in the bone marrow. Macrophages and monocytes are phagocytes that participate in both nonspecific (innate) and specific (cell-mediated) immunity in vertebrates. Their main function is to engulf and digest cellular debris and pathogens, either as fixed cells or as free cells. They also activate lymphocytes and other immune cells that are involved in the immune response.

[0041] Chimeric Antigen Receptor (CAR): mainly composed of three parts of extracellular antigen binding region, transmembrane region and intracellular signal transduction region. The extracellular region is a single chain Fv domain (scFv) with the function of specific recognition and binding of tumor specific antigens. The transmembrane region is usually composed of immunoglobulin superfamily, such as CD8 or CD28. The intracellular signal transduction region is mainly composed of costimulatory factor (4-1BB or CD28) and signal activation region CD3ζ. After the immune cells loaded with chimeric antigen receptor bind to the surface antigens of tumor cells, the extracellular antigen binding region transmits signals to the intracellular signal activation region, and activates the immune cell response.

[0042] CAR macrophage: similar to CAR T cell, CAR macrophage is to load CAR structure on macrophage, which gives macrophage the ability to specifically bind to tumor cell surface antigens.

[0043] Lipid nanoparticle (LNP) is composed of four components: ionizable cationic lipid, phospholipid, cholesterol, PEG-lipid, which encapsulates mRNA by electrostatic adsorption, protects mRNA from nuclease degradation, and avoids clearance by blood mononuclear phagocyte system.

[0044] The inventors found in previous studies that exogenously input CAR macrophages cannot effectively infiltrate liver primary tumors, because the physical barrier composed of CAFs and ECM restricts the infiltration of exogenous cells. Spatial transcriptome data of liver cancer patients showed that between the tumor area and the healthy area, there is a dense fibrocyte band, and immune cells such as myeloid cells, B cells and T cells are restricted to the periphery of the tumor by fibrocytes. In addition, this layer of fibrocyte band in the periphery of the tumor is also an important factor for the resistance of solid tumors to chemotherapy and immunotherapy. 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 for 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 for preventing the recurrence of solid tumors. Based on this, the following technical solutions are proposed.

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

[0046] From N-terminal to C-terminal, the FAP-targeting chimeric antigen receptor comprises, in sequence, an extracellular domain, a transmembrane domain, an intracellular domain and a ΔTGFβRII fragment; wherein the extracellular domain is an anti-FAP scFV, the amino acid sequence of the heavy chain variable region of which is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of which is shown as 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 as SEQ ID NO. 3:

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

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

[0054] GPC3-VH:

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

[0056] GPC3-VL:

[0057] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK (SEQ ID NO. 5);

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

[0059] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNMINNYDNP KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNQAQSKNFHLDPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO. 6).

[0060] The inventors found that fibrocytes in the periphery of tumor highly express FAP protein. FAP is not only a marker of CAFs activation, but also essential for the ECM remodeling function of CAFs cells, and its expression is positively correlated with poor prognosis of cancer. Macrophages are the most abundant immune cells in and out of tumor, so the inventors edited these cells in vivo by LNP-mRNA system to break the physical barrier composed of CAFs. By editing CAR macrophages targeting FAP in vivo, CAFs cells are killed, but the activation of CAFs cannot be blocked from the source, because the CAR macrophages are easily exhausted by the immunosuppressive microenvironment. In the activation of CAFs, TGFβ is the core cytokine. Moreover, TGFβ is an immunosuppressive cytokine that can inhibit the phagocytic function of macrophages, antigen presentation function and expression of inflammatory factors. TGFβ mainly acts through TGFβRII on the cell surface. In cells, expression of intracellular domain-deleted TGFβRII receptor (△TGFβRII) can significantly inhibit TGFβ signal transduction. Therefore, the inventors propose that the intracellular domain-deleted TGFβRII receptor sequence is connected in series on the FAP-CAR structure (FAP-CAR-△TGFβRII), so that the CAR macrophages generated in vivo can target and kill CAFs cells while competitively binding TGFβ, and the effect of TGFβ on other cells in the tumor environment is also reduced while the CAR macrophages are tolerant to TGFβ signal.

[0061] With the breaking of the physical barrier of solid tumor, CD8 + T immune cells will infiltrate into the interior of solid tumor. Activated CD8 + T cells can maximize the anti-tumor effect. Therefore, the inventors envisage that CAR macrophages edited in vivo can activate CD8 + T cells through antigen presentation. Glypican-3 (GPC3) is specifically highly expressed in various solid tumors, including hepatocellular carcinoma, but the activation of CD8 + T cells requires stimulation of cytokines in addition to antigen presentation. IL2 is the most important cytokine for activating CD8 + T cells, which can promote the proliferation and activation of CD8 + T cells. IL2 is also the earliest cytokine product used for tumor treatment, but its systemic administration has serious side effects, which limits its clinical application. The LNP-mRNA system can target macrophages to limit the delivery range of drugs, so the inventors envisage connecting IL2 in series on the CAR structure to edit CAR macrophages that can secrete IL2 in situ. However, natural IL2 preferentially activates Treg cells that highly express CD25, rather than CD8 +T cells. Recent researchers based on synthetic biology to modify the IL2 not only has higher affinity to CD8 surface CD122 (IL2Rβ) receptor, and better stability, to obtain specific activation of CD8 + T cells. This IL2 is called Super IL2. Therefore, the inventors use the sequence of Super IL2 and GPC3-CAR structure in series (GPC3-CAR-Super IL2), and the macrophages edited in vivo can specifically activate CD8 + T cells. This IL2 is called Super IL2. Therefore, the inventors use the sequence of Super IL2 and GPC3-CAR structure in series (GPC3-CAR-Super IL2), and the macrophages edited in vivo can specifically activate CD8

[0062] In addition, the inventors found that the chimeric antigen receptor targeting GPC3 can also promote the antigen spreading of macrophages depending on the GPC3-CAR structure, and present more extensive tumor antigens to T cells. Super IL2 can further promote the activation of CD8 + T cells. This strategy can effectively overcome the heterogeneity of solid tumors.

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

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

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

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

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

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

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

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

[0071] In a second aspect, the present application provides an mRNA expressing the chimeric antigen receptor.

[0072] In some alternative 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] GGCCAGGGCATCAGCGTGACCGTGAGCAGCGGAGGCGGTGGTTCAGGGGGCGGA

[0082] GGGAGCGGCGGCGGTGGCTCTGACGTGCTGATGACCCAGACCCCCCTGTGGCTGC

[0083] GAGTGGTACCTGCAGAAGCCCGGCCAGAAGTGCGTGATCGCCCTGAGCGTGCGT

[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 application provides a nucleic acid molecule, which transcribes the mRNA.

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

[0148] The macrophage has better killing ability for solid tumors.

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

[0150] In a sixth aspect, the present application provides a nucleic acid drug, comprising a carrier and the mRNA.

[0151] The carrier 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 application obtains CAR macrophages through LNP-mRNA system in vivo editing, has high safety, high editing efficiency, short preparation period and greatly reduced cost.

[0154] The present application will be further described below through specific examples, but it should be understood that these examples are only for more detailed description and should not be understood as limiting the present application in any form.

[0155] Example 1

[0156] I. Obtain mRNA of two CAR structures by in vitro transcription method (such as Figure 9), respectively, 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 kinds of mRNA were mixed in equal proportions to prepare LNP, named LNP-GF CAR mRNA. The mRNA of GFP 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, and the expression levels of FAP CAR and GPC3 CAR were detected 24 hours later. Uninfected macrophages were used as Control group at the same time. The results showed that Figure 1 ), LNP-GF CAR mRNA can efficiently edit CAR macrophages, and the expression efficiency of the two CAR structures reaches more than 50%.

[0157] II. Next, it was verified whether the CAR macrophages edited by LNP-GF mRNA could achieve the expected function. LNP-GF CAR mRNA transfected macrophages were then co-incubated with Td-tomato fluorescently labeled GPC3 (human source) overexpressed Hepa1-6 cells and GFP fluorescently labeled FAP (murine source) overexpressed 293T cells, respectively. In the phagocytosis experiment of GPC3 overexpressed Hepa1-6 cells, mRNA of GFP was used as Control mRNA, and in the phagocytosis experiment of FAP overexpressed 293T cells, mRNA of luciferase was used as Control mRNA. After co-incubation for 48 hours, phagocytosis was detected by flow cytometry, in which F4 / 80 + Td-tomato + represents the phagocytosis of macrophages on GPC3 overexpressed Hepa1-6 cells, F4 / 80 + GFP + represents the phagocytosis of macrophages on FAP overexpressed 293T cells. The results showed that Figure 2 ), LNP-GF CAR mRNA edited CAR macrophages significantly increased the phagocytosis ability of GPC3 positive Hepa1-6 cells and FAP + 293T cells. This indicates that LNP-GF CAR mRNA edited CAR macrophages indeed acquire the dual targeting phagocytosis ability of GPC3 and FAP.

[0158] III. LNP-Control mRNA and LNP-GF CAR mRNA were used to infect macrophages. After 24 hours, TGFβ stimulation was added, and macrophages were collected 30 minutes later for WB detection. The results showed that Figure 3), compared with LNP-Control mRNA. This indicates that the macrophages edited by LNP-GF CAR mRNA achieved the expected function of resisting TGFp signal.

[0159] Four, co-incubate CAR macrophages edited by LNP-GF CAR mRNA with splenic T cells, and then detect the proliferation of T cells. Among them, the splenic T cells are stained with CFSE (5 uM) to mark the proliferation. The T cell culture group alone is used as the NC group (negative control group), the aCD3 / 28 group is stimulated by adding aCD3 (1 ug / ml) and aCD28 (5 ug / ml) in the T cell culture (positive control group), and the LNP-Control mRNA group and the LNP-GF CAR mRNA group are co-incubated with T cells by adding macrophages edited by different LNPs on the basis of aCD3 (1 ug / ml) and aCD28 (5 ug / ml) stimulation. After co-incubation for 72 hours, the proportion and proliferation of each T cell group are detected by flow cytometry. The results show that, compared with LNP-Control mRNA, CAR macrophages edited by LNP-GF CAR mRNA do not affect the proportion of CD4 + T cells in the T cell group (A in Figure 4 ), but significantly increase the proportion of CD8 + T cells in the CD3 T cell group (B in Figure 4 ), and promote the proliferation of CD8 + T cells (C in Figure 4 ). These results show that CAR macrophages edited by LNP-GF CAR mRNA can indeed specifically promote the proliferation of CD8 + T cells. This indicates that macrophages edited by LNP-CAR mRNA achieve the expected function of secreting Super IL2.

[0160] Five, next, establish an orthotopic liver cancer model to explore the anti-tumor effect of LNP-GF CAR mRNA. An orthotopic liver cancer model is established using a Hepa1-6 cell line overexpressing human GPC3 (Luciferase+Td-tomato+), and LNP-GF CAR mRNA is injected intravenously for treatment, twice a week (A in Figure 5 ). The results show that LNP-GF CAR mRNA has a significant anti-tumor effect, with a treatment efficiency of 100%, and 4 out of 5 mice are in a completely cured state (B in Figure 5B) in FIG. 6. Next, the in vivo anti-tumor experiment of orthotopic liver cancer model was performed again after deleting macrophages with Clodronate Liposomes ( Figure 5 C) in FIG. 6. The results showed that the anti-tumor effect of LNP-GF CAR mRNA was significantly reduced and the tumor could not be eradicated in the case of deleting macrophages ( Figure 5 D) in FIG. 6. This indicates that LNP-GF CAR mRNA depends on the editing of in vivo macrophages to exert anti-tumor effect.

[0161] Six, Next, the physical barrier constructed by fibrocytes in the tumor periphery was analyzed. The orthotopic liver cancer model was established according to the method of the fifth part, and LNP-GF CAR mRNA was injected intravenously for treatment. First, the inventors analyzed the activation of TGFβ signal in the treatment group. After 2 weeks of treatment, the liver was taken for F4 / 80 and p-SMAD2 / 3 immunofluorescence staining, and the results showed that the p-SMAD2 / 3 signal in the liver macrophages of the LNP-GF CAR mRNA treatment group was significantly reduced ( Figure 6 A) in FIG. 7. This indicates that the in vivo edited CAR macrophages indeed acquired the ability to resist TGFβ signal. In addition, p-SMAD2 / 3 immunohistochemical staining was performed on the 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 FIG. 7. This indicates that the in vivo edited CAR macrophages not only acquired the ability to resist TGFβ signal themselves, but also reduced the stimulation of TGFβ to other cells, prompting the overall level of the liver to reduce the conduction of TGFβ signal. Sirius red staining was performed on the liver tissue, and the results showed that the fibrosis area in the tumor of the LNP-GF CAR mRNA treatment group was significantly reduced, and the thickness of the fibrocyte belt in the tumor periphery was significantly thinned ( Figure 6 C, D) in FIG. 7. In addition, qRT-PCR detection 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) in FIG. 7. Through FAP immunofluorescence experiment, 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 belt composed of FAP positive cells in the tumor periphery of the treatment group ( Figure 6 F, G) in FIG. 7. This fully indicates that the LNP-GF CAR mRNA edited CAR macrophages broke the physical barrier constructed by CAF cells in the tumor periphery. Then the distribution of CD8 + T cells was analyzed, and the results showed that a large number of CD8 + T cells in the control group were blocked in the FAP positive cell area, while the treatment group significantly promoted the distribution of CD8+ T cell infiltration into the tumor ( Figure 6 (H, I in). Based on the above data, it is indicated that LNP-GF CAR mRNA-edited CAR macrophages can break through the physical barrier constructed by FAP-positive CAFs by killing them and promote CD8. + The activation and infiltration of T cells ultimately work in conjunction with adaptive immunity to exert an anti-tumor effect sufficient to eradicate solid tumors.

[0162] VII. The inventors analyzed the effect of LNP-GF CAR mRNA on immune memory in vivo editing of CAR macrophages. Mice cured by LNP-GF CAR mRNA in an orthotopic liver cancer model (denoted as Cured mice) were again subcutaneously inoculated with WTHepa1-6 cells (GPC3 negative) in ectopic locations. Figure 7 (A) Compared to naive mice, LNP-GF CAR mRNA-cured orthotopic hepatocellular carcinoma mice completely inhibited the growth of WT Hepa1-6 subcutaneous tumors. However, in LNP-GF CAR mRNA-cured orthotopic hepatocellular carcinoma mice treated with intraperitoneal injection of αCD3 antibody to delete T cells (denoted as Cured mice + αCD3), these 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 (B in the text). Correspondingly, after subcutaneous tumor inoculation with WT Hepa1-6 cells, mice cured of orthotopic liver cancer remained tumor-free and showed no mortality. However, all cured mice with T-cell deletion and naive mice died within two months. Figure 7 (C in the text). This indicates that in vivo editing of CAR macrophages with LNP-GF CAR mRNA not only forms T cell immune memory, but may also promote the formation of T cell 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 overexpressing full-length GPC3 and OVA (Ovalbumin) antigens. + GPC3 + Hepa1-6 cells were used, and experiments were conducted using spleen T cells from OT1 mice. Because OT1 mice have CD8... + T cells only recognize OVA antigens that have been presented. Therefore, only when GPC3-targeting CAR macrophages present OVA antigens on Hepa1-6 cells through antigen expansion can CD8 cells in OT1 mice be activated. +T cells. Furthermore, the inventors constructed a GPC3-CAR molecule (denoted as LNP-GPC3-CAR mRNA) without the Super IL2 sequence to differentiate the effects of GPC3-CAR and Super IL2 on T cell activation and antigen expansion. The inventors established two co-culture systems: CAR macrophages edited with different LNPs and OVA. + GPC3 + Hepa1-6 cells and spleen T cells derived from OT1 mice were co-incubated ( Figure 8 In A), CAR macrophages edited with different LNPs and OVA - GPC3 + Hepa1-6 cells and spleen T cells derived from OT1 mice were co-incubated ( Figure 8 (B) The group names in this experiment were: 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), and GPC3-CAR-Super IL2 (LNP-GPC3-CAR-Super IL2 mRNA-edited macrophages + tumor cells + T cells). After co-incubation for 72 hours, flow cytometry was used to detect CD8+ in both co-culture systems. + The proliferation of T cells. In relation to OVA... + GPC3 + In the Hepa1-6 cell co-culture system, compared with LNP-Control mRNA, CAR macrophages edited with LNP-GPC3 CAR mRNA significantly promoted CD8 cell growth. + T cell proliferation ( Figure 8 (A) This indicates that CAR macrophages can indeed exert antigen expansion through the GPC3-CAR structure, further presenting OVA antigen in Hepa1-6 cells, thereby promoting CD8 in OT1 mice. + T cell proliferation. Compared with LNP-GPC3 CAR mRNA, CAR macrophages edited with LNP-GPC3-CAR-Super IL2 mRNA further promoted CD8 cell proliferation. + T cell proliferation ( Figure 8 (A) This indicates that Super IL2 can further promote CD8 during antigen expansion based on the GPC3-CAR structure. + T cell proliferation. Conversely, in the case of OVA - GPC3 +In the Hepa1-6 cell co-culture system, because Hepa1-6 cells in this system do not express OVA antigen, macrophages in all treatment groups were unable to promote CD8 expression. + T cell proliferation. Only CAR macrophages edited with LNP-GPC3 CAR-Super IL2 mRNA benefit from Super IL2's own inhibition of CD8+. + The effect on T cell proliferation was a slight increase in CD8. + T cell proliferation ( Figure 8 (B) This further confirms the antigen expansion effect of CAR macrophages targeting GPC3. In conclusion, CAR macrophages targeting GPC3 can indeed present OVA antigens through an antigen expansion mechanism, promoting CD8 expression from OT1 mice. + SuperIL2 can further enhance the proliferation of T cells, and on this basis, CD8+ proliferation is enhanced. + T cell proliferation. In summary, these results demonstrate that the GPC3-CAR-Super IL2 structure can promote antigen expansion by macrophages in a GPC3-CAR-dependent manner 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 composition characterized in that, the chimeric antigen receptor targeting FAP and the chimeric antigen receptor targeting GPC3; from N-terminal to C-terminal, the chimeric antigen receptor targeting FAP comprises, in sequence, an extracellular domain, a transmembrane domain, an intracellular domain and a △TGFβRII fragment; wherein the extracellular domain is an anti-FAP scFV, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the △TGFβRII fragment is shown as SEQ ID NO. 3; from N-terminal to C-terminal, the chimeric antigen receptor targeting GPC3 comprises, in sequence, an extracellular domain, a transmembrane domain, an intracellular domain and a Super IL2 fragment; wherein the extracellular domain is an anti-GPC3 scFV, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5; the amino acid sequence of the Super IL2 fragment is shown as SEQ ID NO.

6.

2. The chimeric antigen receptor composition of claim 1, wherein, 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.

3. The chimeric antigen receptor composition of claim 1, wherein, the intracellular domain and the △TGFβRII fragment are connected by a connecting peptide; the intracellular domain and the Super IL2 fragment are connected by a connecting peptide; the amino acid sequence of the connecting peptide is shown as SEQ ID NO.

9.

4. mRNA, characterized in that the mRNA expresses the chimeric antigen receptor composition of any one of claims 1-3.

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 composition of any one of claims 1-3 or contains the mRNA of claim 4.

7. Use of the mRNA of claim 4 in the preparation of a medicament for treating liver cancer.

8. A nucleic acid drug, characterized by, the vector and the mRNA of claim 4; the vector is used for targeted delivery of the mRNA to macrophages.

9. The nucleic acid drug of claim 8, wherein the vector is a liposome nanoparticle.

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