Chimeric antigen receptor targeting c-met, car-m and use thereof

Abstract

The present disclosure relates to a chimeric antigen receptor targeting c-MET, a CAR-M, and a use thereof. 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 having high c-MET expression.

Description

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

[0001] This disclosure claims priority to Chinese patent application 202311259738.5 filed on September 27, 2023. Technical Field

[0002] The present disclosure relates to the fields of clinical, biological, and oncology. Background Art

[0003] The incidence of pancreatic cancer is showing a significant upward trend, and its prognosis is extremely poor, with an overall 5-year survival rate of only approximately 10%. By 2030, pancreatic cancer is expected 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, represented by PD-L1 and CTLA4, are milestones in the success of tumor immunotherapy. However, these two treatment approaches have shown extremely limited effectiveness in pancreatic cancer. The application of CAR-T in solid tumors faces numerous challenges that remain unresolved.

[0004] Compared to CAR-T cells, CAR-M cells possess greater tumor tissue infiltration, phagocytosis, killing, antigen presentation, and microenvironment remodeling capabilities, making them a cutting-edge area of ​​cellular immunotherapy. Macrophages possess a powerful ability to migrate into tumor tissue, thus resolving the challenge of preventing effector cells from entering the tumor tissue. CAR-M cells possess both specific and nonspecific broad phagocytic and killing capabilities. CAR-M cells can act as highly efficient antigen-presenting cells, subsequently activating effector T cells. Finally, as key cells in regulating the tumor microenvironment, CAR-M cells can ameliorate the immunosuppressive state of the tumor microenvironment.

[0005] Currently, only a limited number of preclinical studies of CAR-Ms have been reported. To date, only one clinical study has been registered on Clinicalltrial.org. Klichinsky et al. developed a human CAR-PBCM-HER2 and found that it significantly prolonged the survival of HER2-positive mice and reduced metastatic tumor burden. To date, no studies of CAR-Ms in pancreatic cancer have been reported, either domestically or internationally.

[0006] Summary of the Invention

[0007] c-MET, also known as mesenchymal-to-epithelial transition factor, is a member of the receptor tyrosine kinase family that is abnormally activated in various tumors, promoting tumor growth. The inventors discovered that c-MET is widely overexpressed in pancreatic cancer, promoting its progression and negatively correlated with patient prognosis, making it a potential target for CAR-M therapy.

[0008] 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.

[0009] According to some embodiments of the present application, a construct targeting human c-MET is provided.

[0010] Target refers to the object to which the constructs of the present disclosure are 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.

[0011] Human c-MET in this disclosure should be interpreted broadly to refer to the human c-MET gene itself and its various expression products 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.

[0012] 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.

[0013] Nucleotide or amino acid information of human c-MET is well known in the art and can be obtained, for example, but not limited to, from literature or databases. A skilled artisan will appreciate that the term "human c-MET" is not limited to accession numbers in a particular database and is intended to encompass equivalent references in any prior art literature, books, or databases.

[0014] According to some embodiments of the present application, the construct targeting human c-MET is represented by Formula 1, in order from the amino terminus to the carboxyl terminus:

[0015] Signal peptide-scFv-hinge region-transmembrane domain-intracellular activation signal domain (Formula 1).

[0016] Transmembrane proteins can be classified based on the location of their N- and C-terminal domains. Types I, II, and III span the membrane once, while type IV spans the membrane multiple times. Type I transmembrane proteins are anchored to the lipid membrane using a stop-transit anchor sequence. Their N-terminal domain is targeted to the endoplasmic reticulum lumen during synthesis, while if the mature form is localized to the plasma membrane, the N-terminal domain is targeted to the extracellular space.

[0017] 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.

[0018] 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.

[0019] Linker length influences various structural characteristics of scFvs, such as size, flexibility, and valency. Linkers greater than 12 residues facilitate folding of the variable domains in their natural orientation and formation of a monovalent antigen-binding site. Conversely, shorter linkers restrict variable domain flexibility, preventing intramolecular variable chain pairing and favoring intermolecular oligomerization and scFv multimer formation. In some embodiments, an exemplary linker is set forth in SEQ ID No: 23.

[0020] In some embodiments, the construct targeting human c-MET is represented by Formula 1-1, in order from the amino terminus to the carboxyl terminus:

[0021] Signal peptide-VH-linker-VL-hinge region-transmembrane domain-intracellular activation signal domain

[0022] (Formula 1-1).

[0023] In other embodiments, the construct targeting human c-MET is represented by Formula 1-2, in order from the amino terminus to the carboxyl terminus:

[0024] Signal peptide-VL-linker-VH-hinge region-transmembrane domain-intracellular activation signal domain

[0025] (Formula 1-2).

[0026] 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.

[0027] In some specific embodiments, the scFv comprises the heavy chain variable region shown in SEQ ID No: 1 and the light chain variable region shown in SEQ ID No: 2.

[0028] In some specific embodiments, the scFv is represented by SEQ ID No:9.

[0029] 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.

[0030] In some specific embodiments, the hinge region is the hinge region of CD8.

[0031] In some specific embodiments, the hinge region is represented by SEQ ID No: 21.

[0032] The function of the transmembrane domain is to anchor the construct of the present disclosure 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.

[0033] In some embodiments, the intracellular activation signaling domain comprises: the immunoreceptor tyrosine activation domain of FCER1G and the Tyr-XX-Met domain of CD19 (abbreviated as YXXM, such as YEDM, YENM).

[0034] 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.

[0035] 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 discovered that CAR introduced into this region promotes the recruitment of PI3K kinase in CAR-M cells.

[0036] In some specific embodiments, the immunoreceptor tyrosine-based activation domain of FCER1G is represented by SEQ ID No: 15.

[0037] In some specific embodiments, the Tyr-XX-Met domain is represented by SEQ ID No: 17.

[0038] The immunoreceptor tyrosine-based activation domain of FCER1G and the Tyr-XX-Met domain of CD19 can be interchanged in order.

[0039] In some embodiments, the construct targeting human c-MET is represented by Formula 1-3, in order from the amino terminus to the carboxyl terminus:

[0040] Signal peptide-VH-linker-VL-hinge region-transmembrane domain-FCER1G immunoreceptor tyrosine-based activation domain-YXXM domain

[0041] (Formula 1-3).

[0042] In other embodiments, the construct targeting human c-MET is represented by Formula 1-4, in order from the amino terminus to the carboxyl terminus:

[0043] Signal peptide-VL-linker-VH-hinge region-transmembrane domain-immunoreceptor tyrosine activation domain of FCER1G-YXXM domain (Formula 1-4).

[0044] In other embodiments, the construct targeting human c-MET is represented by Formula 1-5, in order from the amino terminus to the carboxyl terminus:

[0045] Signal peptide-VH-linker-VL-hinge region-transmembrane domain-YXXM domain-FCER1G immunoreceptor tyrosine-based activation domain

[0046] (Formula 1-5).

[0047] In other embodiments, the construct targeting human c-MET is represented by Formula 1-6, in order from the amino terminus to the carboxyl terminus:

[0048] Signal peptide-VL-linker-VH-hinge region-transmembrane domain-YXXM domain-immunoreceptor tyrosine activation domain of FCER1G (Formula 1-6).

[0049] In a specific embodiment, the construct targeting human c-MET is represented by SEQ ID No:19.

[0050] According to some embodiments of the present application, a polynucleotide is provided, which encodes any of the aforementioned constructs targeting human c-MET.

[0051] In a specific embodiment, the polynucleotide is represented by SEQ ID No: 20.

[0052] Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences.

[0053] According to some embodiments of the present application, an expression vector is provided.

[0054] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is attached. One type of vector is a plasmid, which refers to a circular double-stranded DNA loop to which additional DNA segments can be attached. 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 attached 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 having bacterial origins of replication).

[0055] 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 operatively linked thereto.

[0056] In principle, all overexpression plasmids can be used to express the constructs of the present disclosure. As an example, such as but not limited to GV401, pET, pGEX, pMAL, pT7, CRISPR / Cas9 system.

[0057] In some embodiments, an expression vector comprises the aforementioned polynucleotide.

[0058] 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 retroviral vector, and an adenoviral vector.

[0059] As an example, the vector is introduced into cells by electroporation.

[0060] According to some embodiments of the present application, a host cell is provided, which expresses the aforementioned construct targeting human c-MET.

[0061] 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.

[0062] 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.

[0063] According to some embodiments of the present application, provided is the use of the aforementioned construct targeting human c-MET in preparing CAR-M.

[0064] According to some embodiments of the present application, provided is the use of the aforementioned polynucleotide in preparing CAR-M.

[0065] According to some embodiments of the present application, use of the aforementioned expression vector in preparing CAR-M is provided.

[0066] According to some embodiments of the present application, there is provided a use of the aforementioned host cell in preparing a drug for treating solid tumors (such as pancreatic cancer) that highly express c-MET. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1: Transfection efficiency of CAR-M cells.

[0068] Figure 2: Flow cytometry was used to detect the binding ability of CAR molecules to 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.

[0069] Figure 3: Detection of c-MET expression levels in various pancreatic cancer cell lines (AsPC-1, PaTu8988t, PANC-1, Bxpc-3, MIA PaCa2).

[0070] Figures 4A to 4G show phagocytic events in CAR-Mc-MET cells. Figure 4B shows LDH levels in the system after 24 hours of co-culture. Figure 4C shows fluorescence intensity after 24 hours of co-culture. Figure 4D shows fluorescence intensity after 48 hours of co-culture. Figure 4E shows fluorescein detection.

[0071] Figure 5A: The survival time of mice in the CAR-Mc-MET cell treatment group was prolonged.

[0072] FIG5B : CAR-Mc-MET cells reduce tumor burden in mice.

[0073] FIG5C : HE staining showed that no micrometastasis was observed in the liver of mice in the CAR-Mc-MET group.

[0074] Figure 5D: HE and TUNEL staining showed that the pancreatic tumors in the CAR-Mc-MET group showed larger necrotic areas.

[0075] Figure 5E: No evidence of organ damage was observed in vital organs such as the lungs, stomach, and intestines in the CAR-Mc-MET group.

[0076] Figure 5F: Compared with control macrophages, the CAR-Mc-MET group showed specific intratumoral migration ability.

[0077] FIG6A : Phagocytic efficiency of the hMDMC-CAR-c-MET group.

[0078] FIG6B : Changes in phagocytic efficiency of the hMDMC-CAR-c-MET group over time. DETAILED DESCRIPTION

[0079] Example 1. c-MET is a poor prognostic factor for pancreatic ductal adenocarcinoma

[0080] 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.

[0081] Bioinformatics data from various databases (TIME, OncoLnc, GEPIA, and Kaplan-Meier plot) showed that c-MET expression levels in pancreatic ductal adenocarcinoma tissues were significantly positively correlated with overall survival and disease-free survival in patients with pancreatic ductal adenocarcinoma. Furthermore, c-MET expression was found to be significantly higher in pancreatic ductal adenocarcinoma tissues than in adjacent tissues (p < 0.0001), suggesting it is a poor prognostic factor for patients with pancreatic ductal adenocarcinoma. Bioinformatics data from various databases also showed that CD133 expression levels in pancreatic ductal adenocarcinoma tissues were positively correlated with c-MET expression levels. Furthermore, CD133 expression was found to be significantly higher in pancreatic ductal adenocarcinoma tissues than in adjacent tissues (p < 0.0001) and was significantly correlated with pancreatic cancer recurrence (data not shown).

[0082] Example 2. Design of constructs targeting c-MET

[0083] 1. Design of scFv targeting c-MET

[0084] (1) Amino acid sequence of scFv:

[0085] Amino acid sequence of VH (SEQ ID No: 1):

[0086] Amino acid sequence of VL (SEQ ID No: 2):

[0087] Amino acid sequence of HCDR1: GYTFTSYG (SEQ ID No: 3)

[0088] Amino acid sequence of HCDR2: ISASNGNT (SEQ ID No: 4)

[0089] Amino acid sequence of HCDR3: ARVYADYADY (SEQ ID No: 5)

[0090] Amino acid sequence of LCDR1: QGINTW (SEQ ID No: 6)

[0091] Amino acid sequence of LCDR2: AAS (SEQ ID No: 7)

[0092] Amino acid sequence of LCDR3: QQANSFPLT (SEQ ID No: 8)

[0093] (2) Amino acid sequence of scFv (from N to C):

[0094] Bold: VH; Underline: VL;

[0095] The italicized portion GSTSGSGKPGSGEGSTKG (SEQ ID No: 23) is a linker.

[0096] (3) Nucleotide sequence encoding scFv (from 5' to 3'):

[0097] 2. CAR-M Design

[0098] CAR-modified macrophages (CAR-Macrophage, or CAR-M) are considered to be a promising cell type.

[0099] 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 signaling domain.

[0100] (1) Signal peptide

[0101] Amino acid sequence of CD8 signal peptide (from N to C) (SEQ ID No: 11):

[0102] Nucleotide sequence encoding CD8 signal peptide (from 5' to 3') (SEQ ID No: 12):

[0103] (2) Transmembrane domain

[0104] Amino acid sequence of CD8 transmembrane domain (from N to C) (SEQ ID No: 13):

[0105] Nucleotide sequence encoding the CD8 transmembrane domain (from 5' to 3') (SEQ ID No: 14):

[0106] (3) FCER1G immunoreceptor tyrosine activation domain

[0107] Amino acid sequence of the immunoreceptor tyrosine-based activation domain of FCER1G (from N to C) (SEQ ID No: 15):

[0108] Nucleotide sequence encoding the immunoreceptor tyrosine-based activation domain of FCER1G (from 5' to 3') (SEQ ID No: 16):

[0109] (4) Tyr-XX-Met domain

[0110] The amino acid sequence of YXXM (from N to C) (SEQ ID No: 17):

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

[0112] (5) Hinge

[0113] Hinge amino acid sequence (from N to C) (SEQ ID No: 21):

[0114] Hinge encoding nucleotide sequence (from 5' to 3') (SEQ ID No: 22):

[0115] 3. Construct Design

[0116] Amino acid sequence of Scfv-CD8TM-FCER1G-CD19 (from N to C) (SEQ ID No: 19):

[0117] Scfv-CD8TM-FCER1G-CD19 from 5' to 3' end (SEQ ID No: 20):

[0118] Underline: signal peptide;

[0119] bold: scfv;

[0120] Dotted line: hinge;

[0121] Italics: transmembrane domain;

[0122] Wavy line: immunoreceptor tyrosine-based activation domain of FCER1G (NP_004097.1 aa 45-86);

[0123] Regular font: YXXM domain of CD19;

[0124] Box: stop codon.

[0125] Example 3. Design and synthesis of plasmids

[0126] The Scfv-CD8TM-FCER1G-CD19 construct prepared in Example 2 was inserted into an appropriate position (such as an enzyme cleavage site) of the GV401 vector (commercially available).

[0127] Example 4. Design and transfection of viral vectors

[0128] CAR-M cells stably expressing CAR molecules (hereinafter referred to as CAR-Mc-MET cells) were obtained by lentiviral transfection of the THP-1 cell line, and the transfection efficiency was stably maintained at 20%-25% (Figure 1).

[0129] Example 5. Test of the binding ability of CAR-Mc-MET cells and c-MET

[0130] Flow cytometry was used to detect the binding ability to 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).

[0131] By passage, CAR-M cells that stably express CAR were obtained. By detecting the expression of c-MET in a variety of pancreatic cancer cell lines (AsPC-1, PaTu8988t, PANC-1, Bxpc-3, MIA PaCa2). 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). These two cell lines were used as tool cells for subsequent experiments.

[0132] Example 6. Phagocytic ability of CAR-Mc-MET cells on pancreatic cancer cells with high expression of c-MET

[0133] 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 the CAR-Mc-MET cells that successfully phagocytosed tumor cells.

[0134] The researchers found that CAR-Mc-MET cells had higher phagocytic efficiency than control macrophages (NC-M) at different time points. After 24 hours of co-culture at an effector-target ratio of 5:1, flow cytometry was used to measure the number of remaining tumor cells, and found that CAR-Mc-MET cells had fewer remaining tumor cells than NC-M cells.

[0135] Similarly, co-cultures were performed at different effector-target ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 for different periods of time. Tumor cell killing was assessed by measuring LDH release in the culture system, and the number of remaining tumor cells was measured by bioluminescence. It was found that CAR-Mc-MET cells had a stronger killing ability than NC-M cells in co-culture systems with different effector-target ratios and culture times. Furthermore, this difference gradually widened with longer culture time and increased effector-target ratio.

[0136] The co-culture system was photographed under a confocal microscope, and 5 fields of view were randomly photographed in each culture well. The number of cells with superimposed red and green fluorescence represented phagocytic events, and the number of red fluorescence alone represented the remaining tumor cells. These two indicators were counted and it was found that CAR-Mc-MET cells had more phagocytic events and fewer remaining tumor cells compared with NC-M (Figures 4A to 4G).

[0137] Example 7. Anti-tumor ability of CAR-Mc-MET cells in a mouse pancreatic orthotopic implantation model (pancreatic cancer cells highly expressing c-MET)

[0138] To further validate the therapeutic efficacy of CAR-M, an orthotopic pancreatic cancer tumor model was established in immunodeficient mice using BxPc-3luci+ cells. Five days after surgery, CAR-Mc-MET cells were administered intraperitoneally once a week, and tumor burden was monitored weekly using a small animal imaging device.

[0139] The results showed that compared with the control macrophage group (abbreviated as NC-M), the survival time of mice in the CAR-Mc-MET cell treatment group was prolonged (Figure 5A), and the tumor burden was significantly reduced. Consistent results were obtained by quantitative analysis of tumor burden using bioluminescence imaging (Figure 5B).

[0140] HE staining of specific organ sections of mice revealed numerous micrometastases in the livers of mice in the PBS and NC-M groups, whereas no micrometastases were found in the livers of mice in the CAR-Mc-MET group (Figure 5C). HE and TUNEL staining revealed large necrotic areas in pancreatic tumors in the CAR-Mc-MET group (Figure 5D). There was no evidence of organ damage in important organs such as the lungs, stomach, and intestines (Figure 5E).

[0141] Furthermore, after intraperitoneal injection, the CAR-Mc-MET group showed specific intratumoral migration ability compared with control macrophages ( Figure 5F ).

[0142] Example 8. Construction of hMDMC-CAR-c-MET cells and verification of their ability to kill pancreatic cancer cells that highly express c-MET

[0143] Considering the potential risks of using immortalized tumor cell lines (such as the THP-1 cell line used in Example 4) and lentiviral vectors in clinical translational research, the inventors adopted a strategy of obtaining primary macrophages using human PBMCs through cytokine induction in vitro.

[0144] Subsequently, the above CAR sequence was transfected into these macrophages using an adenoviral vector to produce human monocyte-derived CAR-M (hereinafter referred to as hMDMC-CAR-c-MET). The challenges of macrophage transfection were overcome, and a high transfection efficiency was achieved at an optimal multiplicity of infection (MOI) of 3000.

[0145] In addition, at 3h, 6h, and 12h of co-culture with BxPc-3 cells, the phagocytic efficiency of hMDMC-CAR-c-MET was significantly higher than that of untreated macrophages (UTD) and macrophages transfected with empty adenovirus vectors (Figure 6A). Bioluminescence detection further confirmed this finding. At 12h, 24h, and 48h after co-culture, the phagocytic efficiency of the hMDMC-CAR-c-MET group was significantly better than that of the control adenovirus group, and the difference gradually expanded over time (Figure 6B).

[0146] hMDMC-CAR-c-MET cells exhibited an immune activation state, which was consistent with the results of RNA sequencing and cytokine array.

[0147] In summary, using flow cytometry, immunofluorescence, dynamic imaging and other technologies, this application has clarified that CAR-Mc-MET can effectively phagocytose and kill pancreatic cancer cells that highly express c-MET by specifically binding to c-MET.

Claims

1. A construct targeting human c-MET, which is shown in Formula 1, in the order from the amino terminus to the carboxyl terminus: signal peptide-scFv-hinge region-transmembrane domain-intracellular activation signal domain (Formula 1); in: The signal peptide is a signal peptide of a type I transmembrane protein, preferably a CD8 signal peptide or an igk signal peptide; 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); 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; The heavy chain variable region and the light chain variable region can be interchanged in order; Preferably, 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; Preferably, the transmembrane domain is selected from any one of the following: CD8 transmembrane domain, CD3ζ transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain; The intracellular activation signal domain comprises: the immunoreceptor tyrosine activation domain of FCER1G and the YXXM domain of CD19; The immunoreceptor tyrosine-based activation domain of FCER1G and the YXXM domain of CD19 can be interchanged in order.

2. The construct targeting human c-MET according to claim 1, which is selected from any one of the following: as shown in Formula 1-1, Formula 1-2, Formula 1-3, Formula 1-4, Formula 1-5, and Formula 1-6, in order from the amino terminus to the carboxyl terminus: Signal peptide-VH-linker-VL-hinge region-transmembrane domain-intracellular activation signal domain (Formula 1-1); Signal peptide-VL-linker-VH-hinge region-transmembrane domain-intracellular activation signal domain (Formula 1-2); Signal peptide-VH-linker-VL-hinge region-transmembrane domain-immunoreceptor tyrosine-based activation domain of FCER1G-YXXM domain (Formula 1-3); Signal peptide-VL-linker-VH-hinge region-transmembrane domain-immunoreceptor tyrosine-based activation domain of FCER1G-YXXM domain (Formula 1-4); Signal peptide-VH-linker-VL-hinge region-transmembrane domain-YXXM domain-FCER1G immunoreceptor tyrosine-based activation domain (Formula 1-5); or signal peptide-VL-linker-VH-hinge region-transmembrane domain-YXXM domain-FCER1G immunoreceptor tyrosine-based activation domain (Formula 1-6).

3. The construct targeting human c-MET according to claim 1 or 2, wherein: Preferably, the linker is shown in SEQ ID No: 23; Preferably, the scFv comprises the heavy chain variable region shown in SEQ ID No: 1 and the light chain variable region shown in SEQ ID No: 2; more preferably, the scFv is shown in SEQ ID No: 9; Preferably, the hinge region is shown in SEQ ID No: 21; Preferably, the CD8 signal peptide is shown in SEQ ID No: 11; Preferably, the CD8 transmembrane domain is shown in SEQ ID No: 13; Preferably, the immunoreceptor tyrosine activation domain of FCER1G is shown in SEQ ID No: 15; Preferably, the YXXM domain is shown in SEQ ID No:

17. 4 . The construct targeting human c-MET according to any one of claims 1 to 3 , which is shown in SEQ ID No:

19.

5. A polynucleotide encoding the construct targeting human c-MET according to any one of claims 1 to 4; Preferably, it is represented by SEQ ID No:

20.

6. An expression vector comprising the polynucleotide according to claim 5; Preferably, it is a viral vector; More preferably, the viral vector is selected from any one of the following: a lentiviral vector, a retrovirus, and an adenoviral vector.

7. A host cell expressing the construct targeting human c-MET according to any one of claims 1 to 4; Preferably, the host cell is a macrophage; More preferably, said macrophages are obtained from the subject to be treated.

8. Use of the construct targeting human c-MET according to any one of claims 1 to 4 in the preparation of CAR-M.

9. Use of the polynucleotide according to claim 5 in preparing CAR-M.

10. Use of the expression vector according to claim 6 in preparing CAR-M.

11. Use of the host cell according to claim 7 in preparing a drug for treating solid tumors with high expression of c-MET, preferably pancreatic cancer.

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