A chimeric antigen receptor promoting macrophage m2-like polarization, nucleic acid molecules and antigen-dependent m2-like polarized macrophages

Abstract

The application provides a chimeric antigen receptor for promoting macrophage M2-like polarization, a nucleic acid molecule and an antigen-dependent M2-like polarization macrophage, and relates to the technical field of biotechnology.The chimeric antigen receptor provided by the application comprises an extracellular domain, a transmembrane domain and an intracellular domain; the extracellular domain is an anti-CD19 scFV; the amino acid sequence of the anti-CD19 scFV is shown in SEQ ID NO.1 or SEQ ID NO.2; the intracellular domain is IL4Ra; the amino acid sequence of the IL4Ra is shown in SEQ ID NO.3 or SEQ ID NO.4.The chimeric antigen receptor replaces the intracellular domain of the CAR molecule with the intracellular domain IL4Ra related to macrophage M2 polarization, so that the macrophage can be polarized to M2-like in addition to phagocytizing and eliminating pathogenic B cells, and can play the roles of anti-inflammation, immunosuppression, tissue repair and improvement of fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a chimeric antigen receptor for promoting M2-like polarization of macrophages, a nucleic acid molecule and an antigen-dependent M2-like polarized macrophage. BACKGROUND

[0002] Chimeric antigen receptor (CAR) T cell therapy is a breakthrough cell therapy that has been applied in clinic. CARs are a class of artificially synthesized receptors that can bind to specific antigens and activate T cells to kill target cells. The structure of CAR usually contains an antibody-derived single-chain variable region (scFv) for recognizing specific antigens of target cells, and the intracellular domain is derived from T cell receptor-related signaling molecules (such as CD3ζ) and costimulatory receptors (such as CD28 and 4-1BB) for activating T cells. CAR-modified T cells (CAR-T cells) have achieved remarkable results in treating refractory advanced B cell malignancies, indicating that immune cells guided by CARs have great potential in tumor immunotherapy. However, CAR-T therapy has side effects such as cytokine release syndrome, neurotoxicity, and B cell aplasia.

[0003] Macrophages are an important part of the innate immune system and play a core role in inflammation and host defense. In response to various environmental factors (e.g., microbial products, damaged cells, activated lymphocytes) or under different pathophysiological conditions, macrophages are transformed into different functional phenotypes. Among them, M2-type macrophages are activated by factors such as IL-4 and IL-13, mainly have the potential to inhibit immune response, promote angiogenesis, tissue repair and promote tumor growth, and more secrete factors such as IL-10, TGF-β, VEGF, and relatively high expression of ARG1, CD163 and CD206. The imbalance and abnormal activation of macrophage polarization are one of the foundations of the occurrence and development of autoimmune diseases. For example, the onset of systemic lupus erythematosus (SLE) is associated with an increase in the ratio of M1 / M2 macrophages, and the frequency of inflammatory monocytes in SLE patients also increases, and the concentration of serum cytokines (such as CSF-1 and MCP-1) that promote macrophage differentiation is related to the severity of the disease. In addition, defects in the dead cell clearance function of macrophages also promote inflammation and the production of autoantibodies in lupus patients, thereby exacerbating disease symptoms. Studies in mouse models have shown that adoptive transfer of M2-like macrophages, or the use of drugs to increase M2-like macrophages in vivo to normalize the M1 / M2 ratio is beneficial for the treatment of lupus. These all reveal the prospect of M2-like macrophages for its polarization to M2-like in the treatment of autoimmune diseases. In addition, M2-like macrophages can also participate in the inhibition of inflammatory response, promotion of tissue damage repair and improvement of fibrosis, etc.

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

[0005] A first object of the present application is to provide a chimeric antigen receptor capable of polarizing macrophages to M2-like in addition to phagocytosis of B cells, and inhibiting immune or inflammatory responses.

[0006] A second object of the present application is to provide a nucleic acid molecule.

[0007] A third object of the present application is to provide a macrophage.

[0008] A fourth object of the present application is to provide use of the above macrophage in the preparation of a medicament for treating autoimmune diseases.

[0009] To achieve the above objects, the following technical solutions are adopted:

[0010] In a first aspect, the present application provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain.

[0011] The extracellular domain is an anti-CD19 scFV.

[0012] The amino acid sequence of the anti-CD19 scFV is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0013] The intracellular domain is IL4Ra.

[0014] The amino acid sequence of the IL4Ra is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0015] As a further technical solution, the chimeric antigen receptor further comprises a signal peptide.

[0016] The amino acid sequence of the signal peptide is shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0017] As a further technical solution, the extracellular domain and the transmembrane domain are connected by a hinge region, and the amino acid sequence of the hinge region is shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0018] As a further technical solution, the amino acid sequence of the transmembrane domain is shown in SEQ ID NO. 9 or SEQ ID NO. 10.

[0019] As a further technical solution, the chimeric antigen receptor further comprises a fluorescent label.

[0020] The fluorescent tag comprises GFP.

[0021] As a further technical solution, the fluorescent tag is linked to the intracellular domain through a connecting peptide.

[0022] The amino acid sequence of the connecting peptide is shown in SEQ ID NO. 11.

[0023] As a further technical solution, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 13 or SEQ ID NO. 14.

[0024] In a second aspect, the present application provides a nucleic acid molecule encoding the chimeric antigen receptor.

[0025] In a third aspect, the present application provides a macrophage expressing the chimeric antigen receptor.

[0026] In a fourth aspect, the present application provides potential applications of the above-mentioned macrophage in inhibiting immune and inflammatory responses and promoting tissue repair.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application provides a new chimeric antigen receptor, which replaces the intracellular domain of the CAR molecule with the intracellular domain IL4Ra related to the M2 polarization of macrophages, so that the macrophages can not only phagocytose and eliminate pathogenic B cells, but also can be polarized to M2-like, thereby inhibiting immune and inflammatory responses or promoting tissue and organ repair.

[0029] The macrophage provided by the present application can specifically recognize B cells, phagocytose them after contacting with the B cells, reduce the level of autoantibodies, and at the same time, the macrophage presents an M2-like state, secretes cytokines, inhibits the over-activated immune response, and promotes tissue repair. The macrophage can be used for the treatment of autoimmune diseases. BRIEF DESCRIPTION OF DRAWINGS

[0030] 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.

[0031] Figure 1 M2-CAR action schematic diagram;

[0032] Figure 2 M2-CAR vector structure schematic diagram;

[0033] Figure 3 The expression of the M2 marker gene Arg1 was investigated after macrophages overexpressing different CAR vectors were exposed to B cells (a, direct PCR detection of whether the M2 marker gene ARG1 was activated and expressed, with IL-4 stimulation as a positive control; b, Q-PCR detection of the relative expression level of ARG1).

[0034] Figure 4 The expression of CD163, an M2 polarization marker, on the cell surface of macrophages overexpressing the M2-CAR vector, after contact with B cells;

[0035] Figure 5 This refers to the phagocytic activity of CAR-macrophages on target cells.

[0036] Figure 6 The effect of M2-CAR-macrophages on inhibiting inflammation is shown in the figure. "+" and "-" indicate presence and absence, respectively, that is, whether or not there is LPS and IFN-γ co-stimulation, and whether or not there is CD19 target cell co-culture. Detailed Implementation

[0037] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0038] Macrophages (abbreviated as macrophages) Macrophages are white blood cells located within tissues, originating from monocytes, which in turn originate from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytic cells that participate in non-specific defense (innate immunity) and specific defense (cell-mediated immunity) in vertebrates. Their main function is to phagocytose cellular debris and pathogens in the form of fixed or free cells (i.e., phagocytosis and digestion), and to activate lymphocytes or other immune cells to respond to pathogens.

[0039] Macrophage polarization is an important part of innate immunity, and plays a key role in inflammation and host defense. In response to various environmental factors (e.g., microbial products, damaged cells, activated lymphocytes) or under different pathophysiological conditions, macrophages are transformed into different functional phenotypes, i.e., classically activated macrophages (M1) and alternatively activated macrophages (M2). Mature macrophages appear phenotypic and morphological differentiation under various factors, i.e., the polarization phenomenon of macrophages. According to the different responses to environmental stimuli, macrophages are mainly activated into two phenotypes of M1 and M2. M1 is activated by signals such as IFN-γ and LPS, mainly has the effects of anti-tumor and enhancing immunity, can secrete inflammatory factors, chemokines, effector molecules and TNF-α, etc., among which CD80, surface marker CD64, etc. are represented. M2 is activated by factors such as IL-4 and IL-13, mainly has the potential of inhibiting immune response, promoting angiogenesis, tissue repair and promoting tumor growth, and more secrete IL-10, TGF-β, VEGF, etc., and ARG1, CD163 and CD206 are relatively highly expressed.

[0040] Autoimmune disease: a disease caused by abnormal function of the body's immune system, in which the body attacks its own tissues. It includes systemic lupus erythematosus, rheumatoid arthritis, psoriasis, myasthenia gravis, multiple sclerosis, type 1 diabetes, ulcerative colitis, etc.

[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 initiates the activation reaction of immune cells.

[0042] In a first aspect, the present application provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain;

[0043] The extracellular domain is an anti-CD19 scFV;

[0044] The amino acid sequence of the anti-CD19 scFV is shown in SEQ ID NO. 1 or SEQ ID NO. 2:

[0045] For mouse:

[0046] CDIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLELKGGGGSGGGGSGGGGSGGGGSEVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSS (SEQ ID NO. 1);

[0047] For human:

[0048] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO. 2);

[0049] The intracellular domain is IL4Ra;

[0050] The amino acid sequence of the IL4Ra is shown in SEQ ID NO. 3 or SEQ ID NO. 4:

[0051] For mouse:

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

[0053] For humans:

[0054] KIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDFQEGREGIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS (SEQ ID NO. 4).

[0055] As shown in Figure 1 The chimeric antigen receptor contains the extracellular domain of CD19 and the intracellular domain of IL-4 receptor, which is expressed on macrophages by constructing a macrophage chimeric antigen receptor. First, the chimeric antigen receptor can enable macrophages to specifically phagocytize pathogenic B cells and reduce the secretion of autoantibodies. Second, when the chimeric antigen receptor macrophage binds to B cells, it also induces macrophages to function as M2 macrophages, thereby exerting the immune regulatory function of macrophages, including: secreting cytokines, inhibiting inflammatory immune responses, promoting the repair of damaged tissues, and achieving the effect of treating autoimmune diseases.

[0056] In some alternative embodiments, the chimeric antigen receptor further comprises a signal peptide for guiding the expression of the chimeric antigen receptor on the cell membrane;

[0057] The amino acid sequence of the signal peptide is shown in SEQ ID NO. 5 or SEQ ID NO. 6:

[0058] For mice:

[0059] MGVPTQLLGLLLLWITDAI (SEQ ID NO. 5);

[0060] For human:

[0061] MALPVTALLLPLALLLHAARP (SEQ ID NO. 6).

[0062] In some alternative embodiments, the extracellular domain and the transmembrane domain are connected by a hinge region, the amino acid sequence of the hinge region is set forth in SEQ ID NO. 7 or SEQ ID NO. 8:

[0063] For mouse:

[0064] IEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKL (SEQ ID NO. 7);

[0065] For human:

[0066] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO. 8).

[0067] In some alternative embodiments, the amino acid sequence of the transmembrane domain is set forth in SEQ ID NO. 9 or SEQ ID NO. 10:

[0068] For mouse:

[0069] FWALVVVAGVLFCYGLLVTVALCVIWT (SEQ ID NO. 9);

[0070] For human:

[0071] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 10).

[0072] In some alternative embodiments, the chimeric antigen receptor further comprises a fluorescent tag.

[0073] The fluorescent tag includes but is not limited to GFP.

[0074] The amino acid sequence of GFP is set forth in SEQ ID NO. 12:

[0075] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO. 12)

[0076] In some alternative embodiments, the fluorescent tag is linked to the intracellular domain by a linker peptide;

[0077] The amino acid sequence of the linker peptide is set forth in SEQ ID NO. 11:

[0078] GSGEGRGSLLTCGDVEENPGP (SEQ ID NO. 11);

[0079] In some alternative embodiments, the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO. 13 or SEQ ID NO. 14:

[0080] For mice:

[0081]

[0082] For humans:

[0083]

[0084] In a second aspect, the present application provides a nucleic acid molecule encoding the chimeric antigen receptor.

[0085] The nucleic acid molecule encodes the chimeric antigen receptor, and can be used for in vitro preparation of the chimeric antigen receptor.

[0086] In a third aspect, the present application provides a macrophage expressing the chimeric antigen receptor.

[0087] The macrophage provided by the present application can specifically recognize B cells, and after contacting with the B cells, the macrophage can phagocytose the B cells, thereby reducing the level of autoantibodies. Meanwhile, the macrophage presents an M2-like state, secretes cytokines, inhibits the over-activated immune response, and promotes tissue repair. The macrophage can be used for treating autoimmune diseases, and plays the roles of anti-inflammation, immune suppression, tissue repair, and improvement of fibrosis.

[0088] In a fourth aspect, the present application provides use of the macrophage in preparation of a medicament for treating autoimmune diseases.

[0089] In some optional embodiments, the autoimmune disease includes systemic lupus erythematosus.

[0090] The present application is further illustrated by specific examples below, but it should be understood that the examples are only used for more detailed illustration, and should not be understood as limiting the present application in any form.

[0091] Example 1

[0092] I. A series of M2-CARs capable of targeting B cells were constructed. The specific structure is as follows: Figure 2As shown, the promoters thereof are EF1a promoters; the extracellular domains are anti-CD19 scFV, which is used to recognize the surface antigen of B cells; the hinge regions and transmembrane regions are on the CD28 molecule; in order to enable macrophages to be specifically polarized, the intracellular domains are M2 polarization signal-related intracellular domains, which are the intracellular domains of IL-4Ra, IL-10a, IL-10b and IL-13Ra1, and a CAR without an intracellular domain (Truncated-CAR) is used as a control (the difference between the CARs with the intracellular domains of IL-4Ra, IL-10a, IL-10b and IL-13Ra1 lies only in the intracellular domains); the T2A peptide-linked GFP is used for the convenience of observing and detecting the expression of the CAR. The above CARs differ only in the intracellular domains, wherein the amino acid sequence of the CAR with the intracellular domain of IL-4Ra is shown as SEQ ID NO. 13; the amino acid sequence of the CAR with the intracellular domain of IL-10a is shown as SEQ ID NO. 15; the amino acid sequence of the CAR with the intracellular domain of IL-10b is shown as SEQ ID NO. 16; and the amino acid sequence of the CAR with the intracellular domain of IL-13Ra1 is shown as SEQ ID NO. 17:

[0093] RHPGKLPTVLVFKKPHDFFPANPLCPETPDAIHIVDLEVFPKVSLELRDSVLHGSTDSGFGSGKPSLQTEESQFLLPGSHPQIQGTLGKEESPGLQATCGDNTDSGICLQEPGLHSSMGPAWKQQLGYTHQDQDDSDVNLVQNSPGQPKYTQDASALGHVCLLEPKAPEEKDQVMVTFQGYQKQTRWKAEAAGPAECLDEEIPLTDAFDPELGVHLQDDLAWPPPALAAGYLKQESQGMASAPPGTPSRQWNQLTEEWSLLGVVSCEDLSIESWRFAHKLDPLDCGAAPGGLLDSLGSNLVTLPLISSLQVEE (SEQ ID NO. 15);

[0094] FVVLWLIYKKTKHTFRSGTSLPQHLKEFLGHPHHSTFLLFSFPPPEEA EVFDKLSIISEESEGSKQSPEDNCASEPPSDPGPRELESKDEAPSPPHDDPK LLTSTSEV (SEQ ID NO. 16);

[0095] KRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQSKEETDS VVLIENLKKAAP (SEQ ID NO. 17).

[0096] Secondly, the M2-CARs vector, the CAR vector with CD3ζ as the intracellular domain and the Truncated-CAR were transfected into RAW264.7 cells (a monocyte macrophage cell line) respectively, and the expression of M2 marker gene Arg1 was detected after the cells were co-cultured with mouse B cells for 48 hours, and the results are shown in Figure 3 The results show that only when IL4Ra is used as the intracellular domain, the Arg1 gene is activated and expressed. Q-PCR also shows that the expression of ARG1 gene is significantly up-regulated. The CAR with IL4Ra as the intracellular domain is selected for subsequent experiments, and it is designated as M2-CAR.

[0097] Thirdly, the M2-CAR-expressing macrophages were co-cultured with B cells for 48 hours, and the M2 polarization marker CD163 on the cell surface was detected by flow cytometry, and the results are shown in Figure 4 The results show that M2-CARs are obviously biased towards M2-like polarization compared with Truncated-CARs.

[0098] Fourthly, the CAR-macrophages have phagocytosis on target cells

[0099] After the M2-CAR-expressing macrophages (green, CAR-Ms) were co-cultured with K562 cells overexpressing murine CD19 (red, target cells, Murine CD19+K562), the interaction between the CAR-macrophages and the target cells was observed under a microscope. The results are shown in Figure 5 The results show that the CAR-expressing macrophages have obvious phagocytosis on K562 cells.

[0100] Fifthly, the M2-CAR-macrophages inhibit inflammation

[0101] Under normal conditions or inflammatory conditions (LPS and IFN-γ stimulation), the M2-CAR-expressing macrophages were co-cultured with target cells expressing murine CD19, and a non-co-culture control group (i.e. not co-cultured with target cells expressing murine CD19) was set. After 48 hours of culture, the activity of inflammatory indicator nitric oxide synthase of the macrophages was detected, and the results are shown in Figure 6 (LPS & IFN-γ stimulation refers to LPS and IFN-γ stimulation, CD19 co-culture refers to co-culture with target cells expressing murine CD19, and the vertical coordinate is the activity of endothelial nitric oxide synthase) The results show that under normal conditions and inflammatory conditions (LPS and IFN-γ stimulation), the activity of eNOS (endothelial nitric oxide synthase) significantly decreases after the two kinds of cells are co-cultured.

[0102] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A macrophage, characterized in that, The macrophage expresses a chimeric antigen receptor; The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain; The extracellular domain is an anti-CD19 scFv; The amino acid sequence of the anti-CD19 scFv is shown in SEQ ID NO. 1 or SEQ ID NO. 2; The intracellular domain is IL4Ra; The amino acid sequence of the IL4Ra is shown in SEQ ID NO. 3 or SEQ ID NO.

4.

2. The macrophage of claim 1, wherein, The chimeric antigen receptor further comprises a signal peptide; The amino acid sequence of the signal peptide is shown in SEQ ID NO. 5 or SEQ ID NO.

6.

3. The macrophage of claim 1, wherein, The extracellular domain and the transmembrane domain are connected by a hinge region, and the amino acid sequence of the hinge region is shown in SEQ ID NO. 7 or SEQ ID NO.

8.

4. The macrophage of claim 1, wherein, The amino acid sequence of the transmembrane domain is shown in SEQ ID NO. 9 or SEQ ID NO.

10.

5. The macrophage of claim 1, wherein, The chimeric antigen receptor further comprises a fluorescent tag; The fluorescent tag comprises GFP.

6. The macrophage of claim 5, wherein, The fluorescent tag is linked to the intracellular domain through a connecting peptide; The amino acid sequence of the connecting peptide is shown in SEQ ID NO.

11.

7. The macrophage of claim 1, wherein, The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 13 or SEQ ID NO. 14.

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