Chimeric antigen receptor targeting c-MET, CAR-M and use thereof

Abstract

The present disclosure relates to a chimeric antigen receptor targeting c-MET, CAR-M and its use. The present disclosure provides a construct targeting c-MET, and verifies in vitro the specific binding and killing ability of CAR-M cells targeting c-MET to solid tumors with high expression of c-MET.

Description

Technical Field

[0001] The present disclosure relates to the fields of clinical, biological, and oncology. Background Art

[0002] The incidence of pancreatic cancer is showing a clear upward trend, and its prognosis is extremely poor, with an overall 5-year survival rate of only about 10%. By 2030, pancreatic cancer is likely to become the second leading cause of cancer death. Adoptive immune cell therapy represented by chimeric antigen receptor-T cells (CAR-T) and immune checkpoint inhibition therapy represented by PD-L1 and CTLA4 are milestones in the success of tumor immunotherapy, but these two treatments have extremely limited effects in pancreatic cancer. The application of CAR-T in solid tumors faces many difficulties that have not yet been effectively resolved.

[0003] Compared with CAR-T, macrophages (CAR-M) have stronger tumor tissue infiltration, phagocytosis and killing ability, antigen presentation ability and microenvironment remodeling ability, and are at the forefront of cellular immunotherapy. Macrophages have a strong ability to migrate into tumor tissues, thus solving the problem that effector cells cannot enter tumor tissues. CAR-M has both specific and non-specific extensive phagocytosis and killing capabilities. CAR-M cells can act as efficient antigen presenting cells and then activate effector T cells. Finally, as a key cell in the regulation of the tumor microenvironment, CAR-M can improve the immunosuppressive state of the tumor microenvironment.

[0004] At present, there are only a few reports on CAR-M preclinical studies. So far, there is only one registered clinical study on Clinicalltrial.org. Klichinsky et al. developed human CAR-PBCM-HER2 and found that it can significantly prolong the survival of HER2-positive mice and reduce the metastatic tumor load. So far, there are no reports on the research of CAR-M in pancreatic cancer at home and abroad. Summary of the invention

[0005] c-MET is known as the mesenchymal epithelial transition factor, which is a member of the receptor tyrosine kinase family. It is abnormally activated in a variety of tumors and promotes tumor growth. The inventors found that c-MET is widely and highly expressed in pancreatic cancer, promotes the progression of pancreatic cancer, and is negatively correlated with the patient's prognosis. It can become a potential target for CAR-M therapy.

[0006] In view of this, according to some embodiments of the present application, a construct targeting c-MET is provided, and the specific binding and killing ability of CAR-M cells targeting c-MET (hereinafter referred to as CAR-Mc-MET cells) on solid tumors with high expression of c-MET (such as pancreatic cancer) are verified in vitro.

[0007] According to some embodiments of the present application, a construct targeting human c-MET is provided.

[0008] The target refers to the object to which the construct of the present disclosure is directed; the target can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isoform, variant, etc.). In the present disclosure, the target particularly refers to the human c-MET gene or its expression product.

[0009] Human c-MET in the present disclosure should be interpreted broadly, referring to the human c-MET gene itself and its expression products in various forms at various stages, such as but not limited to molecules produced during gene amplification, replication, transcription, splicing, processing, translation, and modification, such as cDNA, mRNA, precursor protein, mature protein, natural variants, modified forms, and fragments thereof.

[0010] As a specific example, the target in the present disclosure is human c-MET protein or c-MET expressed on the surface of tumor cells.

[0011] The nucleotide or amino acid information of human c-MET is well known in the art, and can be obtained from literature or databases, for example but not limited to. The technician should understand that human c-MET is not limited to the accession number in a specific database, and is also intended to cover equivalent references in any literature, books, and databases in the prior art.

[0012] According to some embodiments of the present application, the construct targeting human c-MET is shown in Formula 1, in order from the amino terminus to the carboxyl terminus:

[0013] Signal peptide-scFv-hinge region-transmembrane domain-intracellular activation signal domain (Formula 1).

[0014] Transmembrane proteins can be classified according to the location of their N- and C-terminal domains. Types I, II, and III are single-spanning proteins, while type IV is multi-spanning. Type I transmembrane proteins are anchored to the lipid membrane using a stop-transit anchor sequence, and their N-terminal domain is targeted to the lumen of the endoplasmic reticulum during synthesis, or to the extracellular space if the mature form is located on the plasma membrane.

[0015] In view of the above, in principle, the signal peptide of any type I transmembrane protein is suitable for the signal peptide of the present application. As a non-limiting example, the signal peptide is a CD8 signal peptide or an igk signal peptide. In some specific embodiments, the CD8 signal peptide is shown in SEQ ID No: 11.

[0016] In some embodiments, the scFv (single chain antibody fragment) specifically binds to human c-MET. ScFv is a single-chain antibody formed by connecting the antibody heavy chain variable region VH and the light chain variable region VL through a 15-20 amino acid linker. Structurally, the N-terminus of VH can be connected to the C-terminus of VL, or the N-terminus of VL can be connected to the C-terminus of VH. Although the sequence of the linker varies greatly, the linker commonly used in the CAR field contains repeated glycine and serine residues to provide the flexibility required for the antigen binding site to change conformation and maintain good stability in aqueous solution.

[0017] The length of the linker affects various structural features of scFv, such as size, flexibility and valency. Linkers greater than 12 residues help the variable domains to fold in a natural orientation and form a monovalent antigen binding site. In contrast, shorter linkers limit the flexibility of the variable domains, prevent intramolecular variable chain pairing, and facilitate intermolecular oligomerization and the formation of scFv multimers. In some embodiments, an exemplary linker is shown in SEQ ID No: 23.

[0018] In some embodiments, the construct targeting human c-MET is as shown in Formula 1-1, in order from the amino terminus to the carboxyl terminus:

[0019] Signal peptide-VH-linker-VL-hinge region-transmembrane domain-intracellular activation signal domain

[0020] (Formula 1-1).

[0021] In other embodiments, the construct targeting human c-MET is as shown in Formula 1-2, in order from the amino terminus to the carboxyl terminus:

[0022] Signal peptide-VL-linker-VH-hinge region-transmembrane domain-intracellular activation signal domain

[0023] (Formula 1-2).

[0024] In some specific embodiments, the heavy chain variable region comprises: HCDR1 shown in SEQ ID No: 3, HCDR2 shown in SEQ ID No: 4, and HCDR3 shown in SEQ ID No: 5; the light chain variable region comprises: LCDR1 shown in SEQ ID No: 6, LCDR2 shown in SEQ ID No: 7, and LCDR3 shown in SEQ ID No: 8.

[0025] In some specific embodiments, the scFv comprises a heavy chain variable region shown in SEQ ID No: 1 and a light chain variable region shown in SEQ ID No: 2.

[0026] In some specific embodiments, the scFv is shown in SEQ ID No:9.

[0027] In some embodiments, the hinge region is selected from any one of the following: the hinge region of CD8, the hinge region of CD28, the hinge region of IgG1, and the hinge region of IgG4.

[0028] In some specific embodiments, the hinge region is the hinge region of CD8.

[0029] In some specific embodiments, the hinge region is shown in SEQ ID No:21.

[0030] The function of the transmembrane domain is to anchor the construct disclosed in the present invention on the macrophage cell membrane. In theory, the transmembrane domains of CD8 (such as CD8a), CD3ζ, CD4, and CD28 are all applicable. In some specific embodiments, the CD8 transmembrane domain is shown in SEQ ID No: 13.

[0031] In some embodiments, the intracellular activation signal domain comprises: the immunoreceptor tyrosine activation domain of FCER1G and the Tyr-XX-Met domain of CD19 (abbreviated as YXXM, such as YEDM, YENM).

[0032] The reason for selecting the immunoreceptor tyrosine activation domain of FCER1G (such as FCER1G 45-86aa) is that the present application unexpectedly found that CAR introduced into this region can trigger the activation of SYK, CARD9 and NF-kb, thereby mediating the activation of macrophages.

[0033] CD19 expressed on the surface of B cells contains a Tyr-XX-Met domain that can bind to PI3K kinase (Phosphatidylinositol 3-Kinase). The present application unexpectedly found that the CAR introduced into this region promotes the recruitment of PI3K kinase in CAR-M cells.

[0034] In some specific embodiments, the immunoreceptor tyrosine activation domain of FCER1G is shown in SEQ ID No:15.

[0035] In some specific embodiments, the Tyr-XX-Met domain is shown in SEQ ID No:17.

[0036] The immunoreceptor tyrosine-based activation domain of FCER1G and the Tyr-XX-Met domain of CD19 can be interchanged in order.

[0037] In some embodiments, the construct targeting human c-MET is as shown in Formula 1-3, in order from the amino terminus to the carboxyl terminus:

[0038] Signal peptide-VH-linker-VL-hinge region-transmembrane domain-FCER1G immunoreceptor tyrosine activation domain-YXXM domain

[0039] (Formula 1-3).

[0040] In other embodiments, the construct targeting human c-MET is as shown in Formula 1-4, in order from the amino terminus to the carboxyl terminus:

[0041] Signal peptide-VL-linker-VH-hinge region-transmembrane domain-immunoreceptor tyrosine activation domain of FCER1G-YXXM domain (Formula 1-4).

[0042] In other embodiments, the construct targeting human c-MET is shown in Formula 1-5, in order from the amino terminus to the carboxyl terminus:

[0043] Signal peptide-VH-linker-VL-hinge region-transmembrane domain-YXXM domain-immunoreceptor tyrosine activation domain of FCER1G

[0044] (Formula 1-5).

[0045] In other embodiments, the construct targeting human c-MET is as shown in Formula 1-6, in order from the amino terminus to the carboxyl terminus:

[0046] Signal peptide-VL-linker-VH-hinge region-transmembrane domain-YXXM domain-immunoreceptor tyrosine activation domain of FCER1G (Formula 1-6).

[0047] In a specific embodiment, the construct targeting human c-MET is shown in SEQ ID No:19.

[0048] According to some embodiments of the present application, a polynucleotide is provided, which encodes any of the aforementioned constructs targeting human c-MET.

[0049] In a specific embodiment, the polynucleotide is shown as SEQ ID No:20.

[0050] Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences.

[0051] According to some embodiments of the present application, an expression vector is provided.

[0052] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a plasmid, which refers to a circular double-stranded DNA loop, to which additional DNA segments can be connected. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication).

[0053] The term "expression vector" refers to a vector that can transform a host cell and contains a nucleic acid sequence that directs and / or controls (along with the host cell) the expression of one or more heterologous coding regions operably linked thereto.

[0054] In principle, all overexpression plasmids can be used to express the constructs disclosed herein. As an example, for example but not limited to GV401, pET, pGEX, pMAL, pT7, CRISPR / Cas9 system.

[0055] In some embodiments, an expression vector comprises the aforementioned polynucleotide.

[0056] In a specific embodiment, the expression vector is a viral vector. As an example, the viral vector is selected from any one of the following: a lentiviral vector, a retrovirus, and an adenoviral vector.

[0057] According to some embodiments of the present application, a host cell is provided, which expresses the aforementioned construct targeting human c-MET.

[0058] In a specific embodiment, the host cell is a macrophage. As an example, the macrophage is obtained from the subject to be treated, or an allogeneic subject.

[0059] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. In specific embodiments, the individual or subject is a human.

[0060] According to some embodiments of the present application, provided is the use of the aforementioned construct targeting human c-MET in the preparation of CAR-M.

[0061] According to some embodiments of the present application, provided is the use of the aforementioned polynucleotide in preparing CAR-M.

[0062] According to some embodiments of the present application, use of the aforementioned expression vector in preparing CAR-M is provided.

[0063] According to some embodiments of the present application, there is provided a use of the aforementioned host cell in the preparation of a drug for treating solid tumors (such as pancreatic cancer) that highly express c-MET. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 : Transfection efficiency of CAR-M cells.

[0065] Figure 2 :The binding ability with PE-labeled c-MET recombinant protein was detected by flow cytometry, confirming that the CAR molecules on the cell surface can stably and efficiently bind to the target protein c-MET.

[0066] Figure 3 : Detection of c-MET expression levels in multiple pancreatic cancer cell lines (AsPC-1, PaTu8988t, PANC-1, Bxpc-3, MIA PaCa2).

[0067] FIG. 4A to FIG. 4G : Phagocytic events of CAR-Mc-MET cells. DETAILED DESCRIPTION

[0068] Example 1. c-MET is a poor prognostic factor for pancreatic ductal adenocarcinoma

[0069] c-MET is a poor prognostic factor for patients with pancreatic ductal adenocarcinoma (also known as PDAC), and CD133 is a marker for cancer stem cells and is associated with the recurrence of pancreatic cancer.

[0070] Bioinformatics data from different databases (TIME, OncoLnc, GEPIA, Kaplan-Meier plot) showed that the expression level of c-MET in pancreatic ductal adenocarcinoma tissue was significantly positively correlated with the overall survival and disease-free survival of pancreatic ductal adenocarcinoma patients. It was also found that the expression of c-MET in pancreatic ductal adenocarcinoma tissue was significantly higher than that in adjacent tissues (p<0.0001), which was a poor prognostic factor for patients with pancreatic ductal adenocarcinoma. Bioinformatics data from different databases showed that the expression level of CD133 in pancreatic ductal adenocarcinoma tissue was positively correlated with the expression level of c-MET. It was also found that the expression of CD133 in pancreatic ductal adenocarcinoma tissue was significantly higher than that in adjacent tissues (p<0.0001), and was significantly correlated with the recurrence of pancreatic cancer (data not shown).

[0071] Example 2. Design of constructs targeting c-MET

[0072] 1. Design of scFv targeting c-MET

[0073] (1) Amino acid sequence of scFv:

[0074] Amino acid sequence of VH (SEQ ID No: 1):

[0075] QVQLVQSGAEVKKPGASVKVSCEASGYTFTSYGFSWVRQAPGQGLEWMGWISASNGNTYYAQKLQGRVTMTTDTSTSAYMELRSLRSDDTAVYYCARVYADYADYWGQGTLVTVSS;

[0076] The amino acid sequence of VL (SEQ ID No: 2):

[0077] DIQMTQSPSSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK;

[0078] Amino acid sequence of HCDR1: GYTFTSYG (SEQ ID No: 3)

[0079] Amino acid sequence of HCDR2: ISASNGNT (SEQ ID No: 4)

[0080] Amino acid sequence of HCDR3: ARVYADYADY (SEQ ID No: 5)

[0081] Amino acid sequence of LCDR1: QGINTW (SEQ ID No: 6)

[0082] Amino acid sequence of LCDR2: AAS (SEQ ID No: 7)

[0083] Amino acid sequence of LCDR3: QQANSFPLT (SEQ ID No: 8)

[0084] (2) Amino acid sequence of scFv (from N to C):

[0085]

[0086] Bold: VH; Underline: VL;

[0087] The italicized portion GSTSGSGKPGSGEGSTKG (SEQ ID No: 23) is the linker.

[0088] (3) Nucleotide sequence encoding scFv (from 5' to 3'):

[0089] (SEQ ID No: 10).

[0090] 2. Design of CAR-M

[0091] CAR-modified macrophages (CAR-Macrophage, or CAR-M) are considered to be a promising cell type.

[0092] Similar to CAR-T and CAR-NK cells, CAR-M cells contain: an extracellular domain that recognizes antigens (such as scFv), a transmembrane domain, and an intracellular activation signal domain.

[0093] (1) Signal peptide

[0094] Amino acid sequence of CD8 signal peptide (from N to C) (SEQ ID No: 11):

[0095] MALPVTALLLPLALLLHAARP;

[0096] The coding nucleotide sequence of CD8 signal peptide (from 5' to 3') (SEQ ID No: 12):

[0097] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg.

[0098] (2) Transmembrane domain

[0099] The amino acid sequence of the CD8 transmembrane domain (from N to C) (SEQ ID No: 13):

[0100] IYIWAPLAGTCGVLLLSLVITLYC.

[0101] The encoding nucleotide sequence of CD8 transmembrane domain (from 5' to 3') (SEQ ID No: 14):

[0102] atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc.

[0103] (3) FCER1G immunoreceptor tyrosine activation domain

[0104] The amino acid sequence of the immunoreceptor tyrosine-based activation domain of FCER1G (from N to C) (SEQ ID No: 15):

[0105] RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ.

[0106] Nucleotide sequence encoding the immunoreceptor tyrosine-based activation domain of FCER1G (from 5' to 3') (SEQ ID No: 16):

[0107] cgactgaagatccaagtgcgaaaggcagctataaccagctatgagaaatcagatggtgtttacacgggcctgagcaccaggaaccaggagacttacgagactctgaagcatgagaaaccaccacag.

[0108] (4) Tyr-XX-Met domain

[0109] The amino acid sequence of YXXM (from N to C) (SEQ ID No: 17):

[0110] YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM.

[0111] The coding nucleotide sequence of YXXM (from 5' to 3') (SEQ ID No: 18):

[0112] tatgaggatatgagaggaatcctgtatgcagccccccagctccgctccattcggggccagcctggacccaatcatgaggaagatgcagactctttatgagaacatg.

[0113] (5) Hinge

[0114] Amino acid sequence of hinge (from N to C) (SEQ ID No: 21):

[0115] Nucleotide sequence encoding the hinge (from 5' to 3') of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID No: 22):

[0116] accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat.

[0117] 3. Design of constructs

[0118] Amino acid sequence of Scfv-CD8TM-FCER1G-CD19 (from N to C) (SEQ ID No: 19):

[0119]

[0120] Scfv-CD8TM-FCER1G-CD19 from 5' to 3' end (SEQ ID No: 20):

[0121]

[0122] Underline: signal peptide;

[0123] bold: scfv;

[0124] Dot-dash line: hinge;

[0125] Italics: transmembrane domain;

[0126] Wavy line: immunoreceptor tyrosine-based activation domain of FCER1G (NP_004097.1 45-86aa);

[0127] Regular font: YXXM domain of CD19;

[0128] Box: stop codon.

[0129] Example 3. Design and synthesis of plasmids

[0130] The Scfv-CD8TM-FCER1G-CD19 construct prepared in Example 2 was inserted into an appropriate position (eg, restriction site) of the GV401 vector (commercially available).

[0131] Example 4. Design and transfection of viral vectors

[0132] CAR-M cells (hereinafter referred to as CAR-Mc-MET cells) that stably express CAR molecules were obtained by lentiviral transfection of THP-1 cell lines, and the transfection efficiency could be stably maintained at 20%-25% ( Figure 1 ).

[0133] Example 5. Test of the binding ability of CAR-Mc-MET cells and c-MET

[0134] Flow cytometry was used to detect the binding ability of PE-labeled c-MET recombinant protein, confirming that the CAR molecules on the cell surface can stably and efficiently bind to the target protein c-MET ( Figure 2 ).

[0135] By subculturing, CAR-M cells that stably express CAR were obtained. The expression of c-MET in various pancreatic cancer cell lines (AsPC-1, PaTu8988t, PANC-1, Bxpc-3, MIA PaCa2) was detected. It was found that the Bxpc-3 cell line had the highest expression of c-MET, while the MIA PaCa2 cell line hardly expressed c-MET ( Figure 3 ), and these two cell lines were used as tool cells for subsequent experiments.

[0136] Example 6. Phagocytic ability of CAR-Mc-MET cells on pancreatic cancer cells with high expression of c-MET

[0137] In order to detect the specific phagocytic ability of CAR-Mc-MET cells on tumor cells with high levels of c-MET expression in vitro, the effector-target ratio of tumor cells and CAR-Mc-MET cells was set to 5:1. Flow cytometry was used to analyze the cells at different time points, and the double-positive cell population was counted as CAR-MET cells that successfully phagocytosed tumor cells.

[0138] It was found that CAR-Mc-MET cells had higher phagocytic efficiency at different time points compared with control macrophages (abbreviated as NC-M). After 24 hours of co-culture at an effector-target ratio of 5:1, the number of remaining tumor cells was detected by flow cytometry, and it was found that CAR-Mc-MET cells had fewer remaining tumor cells than NC-M.

[0139] Similarly, different effector-target ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 were used for co-culture for different time periods, and the tumor cell killing was evaluated by detecting the release of LDH in the culture system, and the number of remaining tumor cells was detected by bioluminescence. It was found that CAR-Mc-MET cells had stronger killing ability than NC-M in co-culture systems with different effector-target ratios and culture times. Moreover, this gap gradually widened with the extension of culture time and the increase of effector-target ratio.

[0140] The co-culture system was photographed under a confocal microscope. Five fields of view were randomly photographed in each culture well. The number of cells with superimposed red and green fluorescence represented the phagocytic events, and the number of red fluorescence alone represented the remaining tumor cells. The two indicators were counted and it was found that CAR-Mc-MET cells had more phagocytic events and fewer remaining tumor cells than NC-M cells ( FIG. 4A to FIG. 4G ).

[0141] In summary, using flow cytometry, immunofluorescence, dynamic imaging and other technologies, the present application has clarified that CAR-Mc-MET can efficiently phagocytose and kill pancreatic cancer cells with high c-MET expression by specifically binding to c-MET.

Claims

1. A construct targeting human c-MET, which is represented by the following formula, in order from the amino terminus to the carboxyl terminus: Signal peptide-scFv-hinge region-transmembrane domain-immunoreceptor tyrosine activation domain of FCER1G-YXXM domain; in: The signal peptide is a signal peptide of a type I transmembrane protein; The scFv specifically binds to human c-MET, and the scFv comprises a heavy chain variable region (VH), a linker, and a light chain variable region (VL); in order from the amino terminus to the carboxyl terminus, the scFv is shown in the structure of VL-linker-VH; The VH comprises HCDR1 shown in SEQ ID No: 3, HCDR2 shown in SEQ ID No: 4, and HCDR3 shown in SEQ ID No: 5; The VL comprises LCDR1 shown in SEQ ID No: 6, LCDR2 shown in SEQ ID No: 7, and LCDR3 shown in SEQ ID No: 8; The amino acid sequence of the immunoreceptor tyrosine activation domain of FCER1G is shown in SEQ ID No: 15; The amino acid sequence of the YXXM domain is shown in SEQ ID No:

17.

2. The construct targeting human c-MET according to claim 1, in: The signal peptide is a CD8 signal peptide or an igk signal peptide.

3. The construct targeting human c-MET according to claim 1, in: The hinge region is selected from any one of the following: the hinge region of CD8, the hinge region of CD28, the hinge region of IgG1, and the hinge region of IgG4.

4. The construct targeting human c-MET according to claim 1, in: The transmembrane domain is selected from any one of the following: CD8 transmembrane domain, CD3ζ transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain.

5. The construct targeting human c-MET according to claim 1, in: The amino acid sequence of the linker is shown in SEQ ID No:

23.

6. The construct targeting human c-MET according to claim 1, in: The scFv comprises VH shown in SEQ ID No: 1 and VL shown in SEQ ID No:

2.

7. The construct targeting human c-MET according to claim 1, in: The amino acid sequence of the scFv is shown in SEQ ID No:

9.

8. The construct targeting human c-MET according to claim 1, in: The amino acid sequence of the hinge region is shown in SEQ ID No:

21.

9. The construct targeting human c-MET according to claim 2, in: The amino acid sequence of the CD8 signal peptide is shown in SEQ ID No:

11.

10. The construct targeting human c-MET according to claim 4, in: The amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID No:

13. The construct targeting human c-MET according to claim 1 , wherein the amino acid sequence thereof is shown as SEQ ID No:

19. 12 . A polynucleotide encoding the construct targeting human c-MET according to any one of claims 1 to 11 .

13. The polynucleotide according to claim 12, whose nucleotide sequence is shown as SEQ ID No:

20. An expression vector comprising the polynucleotide according to claim 12 . The expression vector according to claim 14 , which is a viral vector. The expression vector according to claim 15 , which is selected from any one of the following: a lentiviral vector, a retrovirus, or an adenoviral vector. 17 . A host cell, which expresses the construct targeting human c-MET according to any one of claims 1 to 11. The host cell according to claim 17 , which is a macrophage.

19. The host cell according to claim 18, wherein the macrophage is obtained from a subject to be treated.

20. Use of the construct targeting human c-MET according to any one of claims 1 to 11 in preparing CAR-M.

21. Use of the polynucleotide according to claim 12 in preparing CAR-M.

22. Use of the expression vector according to claim 14 in preparing CAR-M.

23. Use of the host cell according to claim 17 in preparing a drug for treating pancreatic cancer with high expression of c-MET.

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