CN115894697A

CN115894697A - Nano antibody for resisting guanylate cyclase 2C and application thereof

Info

Publication number: CN115894697A
Application number: CN202211582937.5A
Authority: CN
Inventors: 余学军; 狄升蒙; 范艳秋; 李照润; 马恒; 赵佐瞬; 周奚; 陈鑫; 冯冬歌
Original assignee: Huadao Shanghai Biopharma Co ltd
Current assignee: Huadao Shanghai Biopharma Co ltd
Priority date: 2022-12-09
Filing date: 2022-12-09
Publication date: 2023-04-04
Anticipated expiration: 2042-12-09
Also published as: CN116874602A; CN116751303A; CN115894697B; CN116751303B

Abstract

The invention discloses an anti-guanylate cyclase 2C nano antibody and application thereof. The amino acid sequence of CDR1 of the nano antibody comprises a sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, the amino acid sequence of CDR2 of the nano antibody comprises a sequence shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10, and the amino acid sequence of CDR3 of the nano antibody comprises a sequence shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO. 13. The anti-guanylate cyclase 2C nano antibody can be specifically combined with a guanylate cyclase 2C antigen, has good affinity, is used as an antigen combination structure to construct a chimeric antigen receptor and CAR-T cells, and has obvious killing activity on guanylate cyclase 2C positive tumor cells.

Description

Nano antibody for resisting guanylate cyclase 2C and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a nano antibody for resisting guanylate cyclase 2C (GUCY 2C) and application thereof.

Background

Guanylate cyclase 2C (GUCY 2C) is a transmembrane receptor protein expressed mainly in human intestinal epithelial cells, the gene of which is located on human chromosome 12, and the cDNA of GUCY2C comprises a 3219bp open reading frame and 27 exons in total, encodes a protein consisting of 1073 amino acids and having a relative molecular mass of about 120kDa, and belongs to a receptor guanylate cyclase family member. Its transmembrane and intracellular domains (kinase homeodomain, linker domain and guanylate cyclase domain) share homology with other membrane-bound guanylate cyclases, and its unique N-terminal extracellular domain (residues 1-430) determines its ligand specificity.

In the extracellular domain, STa binds to the amino acid microdomain close to the transmembrane region (87-393), while the mechanism by which Sta binding increases cGMP production is still unclear (see: john C Flickinger, jeffrey A Rappaport, joshua R Barton, et al, guanylyl cyclase C as a biomarker for immunology for the treatment of the molecular biology. Med. (2021) 15 (3), 201-217, doi. The current research shows that endogenous ligands guanylin and uroguanylin can activate GUCY2C, increase the intracellular cyclic guanosine monophosphate concentration through combining with GUCY2C, and further activate cGMP dependent protein kinase GII, thereby causing corresponding physiological changes. This physiological change plays a key role in maintaining intestinal homeostasis, improving visceral hyperalgesia, inhibiting intestinal tissue degeneration, and so on (Kuhn M. Molecular biology of membrane guanyl cyclemeters. Physiology Rev,2016,96 (2): 751-804, doi.

GUCY2C is an intestinal tissue-specific polypeptide expressed in normal intestinal mucosal epithelium and primary or secondary colorectal cancers (see: frick GS, pitari GM, weinberg DS, et al. Guanyl cycle C: a molecular marker for stabilizing and preserving biological activity of tissues with color cancer. Ext Rev Mol Diagn,2005,5 (5): 701-713, doi: 10.1586/14737159.5.5.701.). Immunohistochemistry detected the expression of GUCY2C in normal intestinal tissue, colorectal cancer tissue and extraintestinal tissue, and used carcinoembryonic antigen (CEA) and cytokeratin 20 (CK 20) as controls, and the results showed that GUCY2C has positive rates of 100% and 80%, CK20 of 94% and 100%, and CEA of 94% and 94% in normal intestinal tissue and colorectal cancer tissue, respectively. GUCY2C is mainly expressed in intestinal mucosal crypts and villi, with the most expression in the middle villi and little expression in extraintestinal tissues, while CEA and CK20 are expressed to different degrees in both esophageal and gastric cancer tissues. GUCY2C has higher specificity and sensitivity than CEA and CK20, and is an effective index for detecting colorectal cancer (see: buc E, vartanian MD, darcha C, et al. Guanyl cyclase C as a reusable immunological marker and its ligand and Escherichia coli heat-stable enzyme as a potential protein-derived vector for colon cancer cells. Eur Jcancer,2005,41 (11): 1618-1627, doi 10.1016/j. Ejca. 2005.02.031..

GUCY2C, originally called the STa receptor, was previously identified as a receptor for heat stable enterotoxin (STa) causing diarrhea-causing pathogens in travelers. Initial studies have shown that STa is secreted by enterotoxigenic e.coli (ETEC) and, in contrast to other diarrheagenic toxins, such as cholera toxin, it causes the accumulation of cyclic GMP (cGMP) in the intestinal tract. This suggests that STa signal activation may be by guanylate cyclase, one of the family members of proteins known to catalyze the conversion of GTP to cGMP and pyrophosphate, GUCY2C was first cloned from rat small intestine cDNA libraries using the highly conserved sequence of the guanylate cyclase catalytic domain, and demonstrated to be both the STa receptor and the source of cGMP accumulation. GUCY2C is more conserved in all vertebrates, and mutation of GUCY2C is its effect on secretion, loss of function can lead to reduced secretion and neonatal ileus, while GUCY2C gain of function will lead to increased secretion and secretory diarrhea.

Recent studies have now shown that GUCY2C may be a very potential immunotherapeutic target for the treatment of gastrointestinal cancer. As early as 2008, an immunotherapy method for preparing a vaccine by using targeting GUCY2C is available, researchers select the extracellular domain of GUCY2C as a vaccine target spot and clone the vaccine target spot into recombinant adenovirus serotype 5 (Ad 5) for immunogenicity and colorectal cancer treatment research, and compared with a control group, ad5-GUCY2C significantly reduces tumor burden of a liver and lung metastasis (two parts most common in colorectal cancer metastasis) mouse model and increases survival rate of the mouse. (see: john C Flickinger Jr, jeffrey A Rappaport, joshua R Barton, et al guanylyl cyclase C as a biomarker for immunology for the treatment of organic malignoids Biomark. Med. (2021) 15 (3), 201-217.).

Michael s. magee et al, using human specific GUCY2C single-chain variable fragments, construct CART cells targeting human GUCY2C expressing metastases, promote activation of antigen-dependent T cells, and can enable activation markers to upregulate cytokine production and kill GUCY2C expressing tumor cells. In a homograft mouse model, CAR-T cells against GUCY2C can have a long-term protective effect on a lung metastasis model of mouse colorectal cancer cells expressing human GUCY2C. Second, in the human xenograft model of immunodeficient miceIn type ii, CAR-T cells directed against GUCY2C recognize and kill human colorectal cancer cells endogenously expressing GUCY2C, thereby providing a model-long lasting survival. Investigators utilized CAR design encoding scFv specific for human GUCY2C, and dose studies showed that a single injection of the highest dose of 10 in the colorectal lung metastasis model ⁷ One CART cell, 60% of the animals were cured. And repeated injections of CAR-T cells at this dose increased the survival of the treatment to 80% 100 days after tumor transplantation. Furthermore, 75% of the animals initially treated with GUCY2C CAR-T cells survived more than 80 days after tumor re-transplantation, indicating that GUCY2C CAR cells remained present and functional within 6 months after the initial dose. In addition, in the CDX model of peritoneal metastasis, a tumor formation experiment was performed using the human colorectal cancer cell line T84, and CAR-T cells of human GUCY2C were constructed to have a curative effect on 100% of the tested animals. The above strongly supports the potential value of GUCY 2C-directed CAR-T cell therapy in clinical applications of gastrointestinal malignancies. (Magee MS, abraham TS, baybutt TR, et al, human GUCY2C-targeted polymeric antigen receptor (CAR) -expressing T cells antibiotic color cancer meters. Cancer Immunol. Res.6 (5), 509-516 (2018)).

In conclusion, GUCY2C can be used as a target spot for tumor treatment, provides an anti-GUCY2C antibody with high specificity and affinity, and has important significance for treating malignant tumors.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides the nano antibody for resisting guanylate cyclase 2C and the application thereof, the nano antibody for resisting GUCY2C has high affinity, can be used as an antigen binding domain of a chimeric antigen receptor molecule to prepare CAR-T cells, and the obtained CAR-T cells have better application prospect in the aspect of tumor treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an anti-guanylate cyclase 2C nanobody, wherein the amino acid sequence of CDR1 of the nanobody comprises the sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, the amino acid sequence of CDR2 of the nanobody comprises the sequences shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10, and the amino acid sequence of CDR3 of the nanobody comprises the sequences shown in SEQ ID No.11, SEQ ID No.12 and SEQ ID No. 13.

According to the invention, guanylate cyclase 2C recombinant protein is used for immunizing an unimmunized alpaca to construct a phage display nano antibody library, and anti-guanylate cyclase 2C antibodies are screened according to the phage display nano antibody library to obtain a monoclonal antibody which can be specifically combined with guanylate cyclase 2C antigens, wherein the antibody belongs to a single-domain antibody.

The GUCY 2C-resistant nano antibody provided by the invention has high affinity, can be used as an antigen binding domain of a chimeric antigen receptor molecule to prepare CAR-T cells, and the obtained CAR-T cells have good application prospects in the aspect of tumor treatment.

SEQ ID NO.1：GYTYSSYC。

SEQ ID NO.2：GINYRNYC。

SEQ ID NO.3：GYTRGTHA。

SEQ ID NO.4：IDSDGST。

SEQ ID NO.5：IYNGD。

SEQ ID NO.6：IYNGA。

SEQ ID NO.7：IYNAA。

SEQ ID NO.8：SYNGD。

SEQ ID NO.9：VETDGNT。

SEQ ID NO.10：MYNGD。

SEQ ID NO.11：AADGPGRCYGGYWYRSPKISFGI。

SEQ ID NO.12：AAGAQFLCANNNWYQHLYTY。

SEQ ID NO.13：AAGDTVASRWYARRTSPMAVRGYND。

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.4, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 11;

preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.5, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 12;

preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.6, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 12;

preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.7, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 12;

preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.8, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 12.

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.3, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.9, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 13.

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.10, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 12.

Preferably, the heavy chain variable region of the nanobody further comprises: the framework region 1 (FR 1) shown by SEQ ID NO.14 or SEQ ID NO.15, SEQ ID NO.16 or SEQ ID NO.17, the framework region 2 (FR 2) shown by SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20, the framework region 3 (FR 3) shown by SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 or SEQ ID NO.25, and the framework region 4 (FR 4) shown by SEQ ID NO.26 or SEQ ID NO. 27.

SEQ ID NO.14：QVKLVQSGGGLVQPGGSLRLSCAAS。

SEQ ID NO.15：QVKLVQSGGGSVQAGGSLRLSCAAS。

SEQ ID NO.16：EVKLVESGGGSVQAGGSLRLSCEAS。

SEQ ID NO.17：QVQLVESGGGSVQAGGSLRLSCAAS。

SEQ ID NO.18：MGWFRQAPGKEREGVAA。

SEQ ID NO.19：MAWFRQAPGKQREGVAR。

SEQ ID NO.20：MGWFRQAPGKEREGVAT。

SEQ ID NO.21：

SYADSVKGRFTISKDNAKNTLYLQMNSLEPEDTAMYYC。

SEQ ID NO.22：

SYADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYC。

SEQ ID NO.23：

SYADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAVYYC。

SEQ ID NO.24：

SYADSVKGRFTISQDNAKNLVYLQMNSLKPEDTAMYYC。

SEQ ID NO.25：

RYADSVKGRFTIIKDNAKNTLYLQMNSLKPDDTAMYYC。

SEQ ID NO.26：WGQGTQVTVSS。

SEQ ID NO.27：WGQGTQVTVST。

Preferably, the heavy chain variable region of the nanobody comprises the amino acid sequence shown in SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No.32, SEQ ID No.33 or SEQ ID No. 34.

SEQ ID NO.28：

QVKLVQSGGGLVQPGGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVAAIDSDGST SYADSVKGRFTISKDNAKNTLYLQMNSLEPEDTAMYYCAADGPGRCYGGYWYRSPKISFG IWGQGTQVTVSS。

SEQ ID NO.29：

QVKLVQSGGGSVQAGGSLRLSCAASGINYRNYCMAWFRQAPGKQREGVARIYNGDS YADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAAGAQFLCANNNWYQHLYTYW GQGTQVTVSS。

SEQ ID NO.30：

EVKLVESGGGSVQAGGSLRLSCEASGINYRNYCMAWFRQAPGKQREGVARIYNGASY ADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAVYYCAAGAQFLCANNNWYQHLYTYWG QGTQVTVST。

SEQ ID NO.31：

QVKLVQSGGGSVQAGGSLRLSCAASGINYRNYCMAWFRQAPGKQREGVARIYNAAS YADSVKGRFTISQDNAKNLVYLQMNSLKPEDTAMYYCAAGAQFLCANNNWYQHLYTYW GQGTQVTVSS。

SEQ ID NO.32：

QVKLVQSGGGSVQAGGSLRLSCAASGINYRNYCMAWFRQAPGKQREGVARSYNGDS YADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAAGAQFLCANNNWYQHLYTYW GQGTQVTVSS。

SEQ ID NO.33：

QVQLVESGGGSVQAGGSLRLSCAASGYTRGTHAMGWFRQAPGKEREGVATVETDGN TRYADSVKGRFTIIKDNAKNTLYLQMNSLKPDDTAMYYCAAGDTVASRWYARRTSPMAVR GYNDWGQGTQVTVSS。

SEQ ID NO.34：

QVKLVQSGGGSVQAGGSLRLSCAASGINYRNYCMAWFRQAPGKQREGVARMYNGD SYADSVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCAAGAQFLCANNNWYQHLYTY WGQGTQVTVSS。

In the invention, the phage display technology is utilized to screen the guanylate cyclase 2C immune camel VHH library, and the obtained nano antibody has high affinity and has important application prospect in the aspect of constructing the chimeric antigen receptor of the target guanylate cyclase 2C.

In a second aspect, the present invention provides a nucleic acid molecule comprising a gene encoding the anti-GUCY2C nanobody of the first aspect.

In a third aspect, the present invention provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain comprising the nanobody of the first aspect against guanylate cyclase 2C, a hinge region, a transmembrane region, and a signal transduction domain.

Preferably, the signal peptide comprises a CD8 α signal peptide.

Preferably, the hinge region comprises a CD8 a hinge region.

Preferably, the transmembrane region comprises any one of, or a combination of at least two of, a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region.

Preferably, the signaling domain comprises an immunoreceptor tyrosine activation motif (CD 3 ζ).

Preferably, the signaling domain further comprises a co-stimulatory molecule comprising any one of the 4-1BB, CD28 intracellular domain, OX40, ICOS or DAP10 intracellular domain or a combination of at least two thereof.

Preferably, the chimeric antigen receptor comprises a CD8 α signal peptide, the nanobody of the anti-guanylate cyclase 2C of the first aspect, a CD8 α hinge region, a CD8 α transmembrane region, and an immunoreceptor tyrosine activation motif.

In a fourth aspect, the present invention provides an expression vector comprising a gene encoding the chimeric antigen receptor of the third aspect.

Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to the third aspect, preferably a lentiviral vector.

In a fifth aspect, the present invention provides a recombinant lentivirus comprising the expression vector of the fourth aspect.

Preferably, the recombinant lentivirus is prepared from mammalian cells transfected with the expression vector and helper plasmid of the fourth aspect.

In a sixth aspect, the present invention provides a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the third aspect.

Preferably, the chimeric antigen receptor immune cell comprises the expression vector of the fourth aspect and/or the recombinant lentivirus of the fifth aspect.

Preferably, the chimeric antigen receptor immune cells include any one of T cells, B cells, NK cells, mast cells or macrophages or a combination of at least two thereof.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell of the sixth aspect and/or the nanobody of the first aspect.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a surfactant, a disintegrating agent, a coating material, an excipient, a solubilizer, a diluent, a pH regulator, a binder, a wetting agent, a colorant, an emulsifier, a bacteriostatic agent, a cosolvent, an osmotic pressure regulator, a filler, an antioxidant or a buffering agent.

In an eighth aspect, the present invention provides a use of the nanobody against guanylate cyclase 2C of the first aspect, the nucleic acid molecule of the second aspect, the chimeric antigen receptor of the third aspect, the expression vector of the fourth aspect, the recombinant lentivirus of the fifth aspect, the chimeric antigen receptor immune cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect, in the preparation of a medicament for treating tumor.

Preferably, the tumor comprises a tumor expressing guanylate cyclase 2C.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) According to the invention, GUCY2C recombinant protein is used for immunizing non-immunized alpacas to construct a phage display nano antibody library, anti-GUCY2C antibodies are screened according to the phage display nano antibody library, the obtained nano antibodies can be specifically combined with GUCY2C antigens, the affinity is good, and the KD (M) is respectively 2.75 multiplied by 10 as can be seen through the determination of the antibody affinity ^-11 、1.80×10 ^-8 、1.26×10 ^-8 、1.09×10 ^-8 、2.08×10 ^-8 、5.20×10 ^-8 And 1.82X 10 ^-8 ；

(2) The GUCY 2C-resistant nano antibody provided by the invention has better affinity, is used as an antigen binding domain to construct a chimeric antigen receptor, and is used for preparing T cells, the CAR-T cells have killing activity on GUCY 2C-positive tumor cells, and efficiently secrete a cytokine IFN-gamma after being co-cultured with the GUCY 2C-positive cells, and the nano antibody can be effectively applied to immunotherapy and has important significance for developing tumor treatment medicines.

Drawings

FIG. 1A is a graph of the affinity of the anti-GUCY2C nanobody VHH-A1 detected using Biacore in example 2;

FIG. 1B is a graph of the affinity of the anti-GUCY2C nanobody VHH-A2 detected using Biacore in example 2;

FIG. 1C is a graph of the affinity of the anti-GUCY2C nanobody VHH-A3 detected using Biacore in example 2;

FIG. 1D is a graph showing the affinity of anti-GUCY2C nanobody VHH-14 detected using Biacore in example 2;

FIG. 1E is a graph of the affinity of the anti-GUCY2C nanobody VHH-29 tested using Biacore in example 2;

FIG. 1F is a graph of the affinity of the anti-GUCY2C nanobody VHH-36 detected using Biacore in example 2;

FIG. 1G is a graph of the affinity of anti-GUCY2C nanobody VHH-43 detected using Biacore in example 2;

FIG. 2 is a diagram showing the result of FACS detection in example 3 in which an anti-GUCY2C nanobody recognizes a GUCY2C antigen;

FIG. 3 is a GUCY2C-targeted chimeric antigen receptor lentiviral vector plasmid map of example 4;

FIG. 4 is a schematic diagram of the structure of a chimeric antigen receptor expressing GUCY2C in example 4;

FIG. 5 is a graph showing the results of flow measurement of the expression rate of the chimeric antigen receptor of T lymphocytes in example 6;

FIG. 6 is a graph of the killing effect of CAR-T cells on 293T cells in example 7;

FIG. 7 is a graph showing the killing effect of CAR-T cells on colon cancer cells Colo205-GUCY2C in example 7;

FIG. 8 is a graph showing the killing effect of CAR-T cells on colon cancer cells SW620-GUCY2C in example 7;

FIG. 9 is a histogram of the secretion of IFN γ by CAR-T cells in example 8.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and that no limitation of the invention is intended.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1

In this example, phage nanobody library was constructed and panned, and preliminary screening was performed by ELISA, which specifically comprises the following steps:

(1) Construction of phage Nanobody libraries

Adopting GUCY2C (GUCY 2C) extracellular recombinant protein to immunize alpaca, and extracting peripheral blood after detecting serum titer by ELISA; separating lymphocytes, extracting total RNA, performing reverse transcription to obtain cDNA, and amplifying VHH gene by using nested PCR; the VHH gene was inserted into the pShort phagemid and electrotransformed

After the competent cells are amplified, the phage clones are separated and purified by a PEG8000/NaCI precipitation method to obtain an antibody library; adjusting concentration, subpackaging and freezing at-80 deg.C in refrigerator for use;

(2) Screening of phage Nanobody libraries

Firstly, incubating 293T cells and an antibody library for negative screening, then taking supernatant, and incubating the supernatant with 293T-GUCY2C cells (GUCY 2C positive) and 293T cells respectively; washing with pre-cooled PT buffer solution at 4 deg.C for 4 times; infecting NEB alpha 5F' cell, adding helper phage, and culturing overnight; coating a plate by a Drop method, and counting the enrichment degree the next day; separating and purifying phage by PEG8000/NaCI precipitation method, and performing the next round of screening; after enrichment, the obtained phage is used as a template, a VHH region is amplified, and secondary sequencing is carried out to obtain 7 kinds of anti-GUCY2C nano antibodies, wherein the amino acid sequences are respectively shown as SE1 ID NO.28, SE1 ID NO.29, SE1 ID NO.30, SE1 ID NO.31, SE1 ID NO.32, SE1 ID NO.33 and SE1 ID NO.34 and are respectively named as VHH-A1, VHH-A2, VHH-A3, VHH-14, VHH-29, VHH-36 and VHH-43.

Example 2

This example was performed for VHHFc nanobody expression, purification, and antibody affinity determination.

Screening various antibodies meeting the conditions, and screening seven candidate antibodies which are named as VHH-A1, VHH-A2, VHH-A3, VHH-14, VHH-29, VHH-36 and VHH-43. To further identify these antibodies, we constructed recombinant VHH antibodies with a mouse Fc tag (committed to third party implementation) and obtained antibody quality measurements as shown in table 1.

TABLE 1

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The 7 GUCY2C VHH antibodies described above were subjected to affinity determination by Biacore. Biacore is a bioanalytical sensing technology developed based on Surface Plasmon Resonance (SPR), which can detect and track the whole change process of the binding and dissociation of molecules in a solution and molecules fixed on the surface of a chip, record the change process in the form of a sensorgram and provide kinetic and affinity data, during the measurement process, antibodies are immobilized on the surface of the chip, the mobile phase is a solution containing antigen, the measurement results are shown in Table 2 and FIGS. 1A-1G, and the affinities of 7 antibodies reach subnanomolar levels, namely 2.75 × E-11, 1.80 × E-8, 1.26 × E-8, 1.09 × E-8, 2.08 × E-8, 5.20 × E-8 and 1.82 × E-8.

TABLE 2

Nanobodies	Ka(1/Ms)	Kd(1/s)	KD(M)
				VHH-A1(SEQ ID NO.28)	4.63E+04	1.27E-06	2.75E-11
VHH-A2(SEQ ID NO.29)	1.31E+05	2.36E-03	1.80E-8
				VHH-A3(SEQ ID NO.30)	1.44E+05	1.83E-03	1.26E-8
VHH-14(SEQ ID NO.31)	1.30E+05	1.41E-03	1.09E-8
				VHH-29(SEQ ID NO.32)	1.32E+05	2.74E-03	2.08E-8
VHH-36(SEQ ID NO.33)	1.16E+05	6.04E-04	5.20E-9
				VHH-43(SEQ ID NO.34)	1.38E+05	2.50E-03	1.82E-8

Example 3

This example was performed by flow assay of the nanobody against GUCY2C of the examples.

Jurkat (GUCY 2C-), jurkat-GUCY2C cells and purified anti-GUCY2C nano-antibodies are mixed, incubated for 30min under ice bath, then incubated for 30min with APC marked goat anti-mouse IgG antibody, and detected by a flow cytometer, the result is shown in figure 2, which shows that the anti-GUCY2C nano-antibodies of the invention can identify GUCY2C antigens on the cell surface.

Example 4

This example prepares lentiviral vectors expressing a chimeric antigen receptor targeting GUCY2C (GUCY 2C CAR).

First, a lentiviral vector pSIN03 GUCY2C CAR carrying a GUCY2C CAR chimeric antigen receptor was constructed, the vector map is shown in FIG. 3, the schematic diagram of the chimeric antigen receptor is shown in FIG. 4, and the lentiviral vector comprises a CD8 alpha signal peptide, an anti-GUCY2C nanobody (anti-GUCY 2C VHH), a CD8 alpha hinge region, a transmembrane region, and an immunoreceptor tyrosine activation motif (CD 3 zeta).

Wherein the amino acid sequence (SEQ ID NO. 35) of the signal peptide is:

MALPVTALLLPLALLLHAARP。

the amino acid sequence of anti-GUCY2C VHH is shown in SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ DNO.31, SEQ ID NO.32, SEQ ID NO.33 or SEQ ID NO. 34.

The amino acid sequence of the CD8 α hinge and transmembrane regions (SEQ ID NO. 36) is:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC。

the amino acid sequence of the 4-1BB intracellular domain (SEQ ID NO. 37) is:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

the CD3 zeta amino acid sequence (SEQ ID NO. 38) is:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

the preparation method comprises the following steps:

(1) A PCR reaction system was prepared according to table 3 (the reagent in table is from toyobo oinc.), each anti-GUCY2C nanobody fragment was amplified, and the reaction was performed according to the PCR procedure shown in table 4, with the primer sequences:

GU-A1-F(SEQ ID NO.39)：

ctgccgctggccttgctgctccacgccgccaggccgcaggtcaaactcgtcc。

GU-A3-F(SEQ ID NO.40)：

ctgccgctggccttgctgctccacgccgccaggccggaggtgaagttggtgg。

GU-36-F(SEQ ID NO.41)：

ctgccgctggccttgctgctccacgccgccaggccgcaggtccagctcgtcg。

GU-A1-R(SEQ ID NO.42)：gcgctggcgtcgtggtgctggacactgtcact。

GU-A2-R(SEQ ID NO.43)：gcgctggcgtcgtggtggagctgacggtcacc。

GU-A3-R(SEQ ID NO.44)：gcgctggcgtcgtggtggtggacacggtgacc。

GU-14-R(SEQ ID NO.45)：gcgctggcgtcgtggtggaggacacggtcacc。

GU-29-R(SEQ ID NO.46)：gcgctggcgtcgtggtgctggacacggtcacc。

GU-36-R(SEQ ID NO.47)：gcgctggcgtcgtggtgctagacacggtcacc。

GU-43-R(SEQ ID NO.48)：gcgctggcgtcgtggtgctggagacggtcacc。

TABLE 3

Reagent	Volume (mu L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 μ M upstream primer	1
		10 μ M downstream primer	1
Template DNA (cDNA clone)	1
		Sterile deionized water (PCR grade water)	33
KOD-Plus-Neo high fidelity PCR enzyme	1

TABLE 4

(2) A PCR reaction system was prepared according to table 5, and a CD8 α signal peptide was added before the amplification product obtained, and a reaction was performed according to the PCR procedure shown in table 4 using the primers:

BamH-CD8αsig-F(SEQ ID NO.49)：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC。

CD8H-R(SEQ ID NO.50)：GTCGCGGCGCTGGCGTCGTGGT。

TABLE 5

After the reaction is finished, carrying out 1% agarose gel electrophoresis on the PCR product, recovering 500bp fragments, and quantifying by an ultraviolet absorption method;

(3) PCR reaction systems were prepared according to Table 6, and after preparation, PCR reactions were carried out according to the PCR procedures shown in Table 4 to amplify the CD 8. Alpha. Hinge-TM-41BB-CD3Z fragment using the following primers:

CD8αH-F(SEQ ID NO.51)：ACCACGACGCCAGCGCCGCGAC。

Vector-R(SEQ ID NO.52)：TCGATAAGCTTGATATCG。

TABLE 6

Reagent	Volume (μ L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 μ M of upstream primer CD 8. Alpha.H-F	1
		10 μ M downstream primer Vector-R	1
Template DNA (HD CD19 CAR)	1
		Sterile deionized water (PCR grade water)	33
KOD-Plus-Neo high fidelity PCR enzyme	1

After the PCR is finished, carrying out 1% agarose gel electrophoresis, recovering fragments of about 680bp, and quantifying by an ultraviolet absorption method;

(4) Carrying out BamHI and EcoRI double enzyme digestion on 5 mu g of HD SIN03 CD19 CAR plasmid constructed in a laboratory, carrying out water bath reaction at 37 ℃ for 1h, and then recovering the vector;

the 3 fragments recovered above were ligated to the vector backbone with recombinase, the recombination system is shown in Table 7, and after the preparation, the reaction was carried out in a water bath at 37 ℃ for 0.5h, and then the resulting mixture was transformed into Escherichia coli stbl3 competent cells by a conventional method.

TABLE 7

Reagent	Amount of the composition used
		HD CD19 CAR scaffold	154.2ng
CD8αsingal GUCY2C VHH	10ng
		CD8αhinge-TM-41BB-CD3Z	14ng
5×CE buffer	2μL
		Exnase ^TM II	1.2μL
Sterile deionized water (PCRgradewater)	Make up to 12. Mu.L

Selecting monoclone from the solid culture medium, culturing overnight, performing PCR identification, preparing a PCR reaction system as shown in table 8, performing PCR program as shown in table 9, selecting positive clone after PCR is finished, further sequencing and identifying, and ensuring that the sequencing result is in line with expectation. The primer sequences are as follows:

LV-F2(SEQ ID NO.53)：TCTTGGTTCATTCTCAAGCCTC。

LV-R(SEQ ID NO.54)：GCAACATAGTTAAGAATACC。

TABLE 8

TABLE 9

Example 5

In this example, the lentiviral vector HDSIN03-GUCY2CCAR prepared in example 4 was subjected to lentiviral packaging, concentration and titer detection, and the method comprises the following steps:

(1) Lentiviral packages

At 6.0 × 10 ⁶ Cell number 293T cells plated in 10cm dishes at 37 ℃ C. And 5% CO ₂ Culturing overnight to prepare a packaged virus, wherein a culture medium is DMEM containing 10% fetal calf serum; dissolving 5.4 mu g of HDSIN03-GUCY2CCAR of the lentiviral vector, 6.2 mu g of the auxiliary plasmid pMDLg-RRE, 6.2 mu g of the auxiliary plasmid pRSV-REV and 2.4 mu g of the envelope plasmid VSVg into 0.8mL of serum-free DMEM culture solution, and uniformly mixing;

dissolve 60.6. Mu.g PEI (1. Mu.g/. Mu.L) in 0.8mL serum free DMEM medium, vortex at 1000rpm for 5 seconds, incubate at 25 ℃ for 5min; adding the PEI mixed solution into the DNA mixed solution, slightly mixing uniformly after adding, and incubating for 25min at 25 ℃ to form a transfection compound; then dripping 1.6mL of the transfection compound into 10mL of DMEM medium containing 293T cells, and replacing the fresh medium after 6 hours; collecting the virus liquid supernatant after 48 hours;

(2) Lentiviral concentration

Filtering the virus supernatant with a 0.45-micron filter membrane, collecting the filtrate in a 50mL centrifuge tube, adding 1/4 of PEG-NaCl virus concentrated solution, turning upside down, mixing uniformly, and standing at 4 ℃ overnight; centrifuging at 4 deg.C and 3500rpm for 30min; removing supernatant, adding RPMI1640 medium (containing 10% FBS), and dissolving the heavy suspension virus precipitate; subpackaging the concentrated lentivirus suspension into 50 μ L portions, storing in a finished product tube, and storing at-80 deg.C;

(3) Lentiviral titer detection

500 μ L Jurkat cells (2X 10) ⁵ Individual cells) were seeded into 24-well culture plates, and the concentrated lentivirus was added to the cell suspension in volumes of 1. Mu.L, 0.2. Mu.L, and 0.04. Mu.L, respectively, and polybrene was added to a final concentration of 8. Mu.g/mL, 37 ℃,5% ₂ After overnight culture, the fresh medium was replaced;

72h after infection, centrifugation at 500 Xg for 5min, discarding cells collected from the supernatant, adding 100. Mu.L PBS +2% FBS to resuspend the cells, adding 0.5. Mu.g Rabbitanti-Camelid VHHANtibody (iFluor 488) antibody, and incubating on ice for 30min; after washing 1 time with flow buffer (PBS containing 2% fbs), 300 μ L of flow buffer was added to resuspend the cells, and the infection efficiency was measured by flow cytometry; the titer was calculated as follows: titer (TU/mL) = number of cells x positive rate/virus volume (mL).

Example 6

Construction of overexpression cell lines

GUCY2C-GFP lentivirus was obtained by co-transfection of GUCY2C-GFP plasmid (purchased from Cloupia filiformis), pMGlg-RRE, pRSV-REV and VSVg plasmid, in the same manner as in example 5;

respectively taking 1 × 10 ⁶ The 293T cells (stored in this company), jurkat cells (stored in this company), SW620 cells (purchased from the cell bank of Chinese academy), and colo205 cells (purchased from ATCC) were inoculated into 6-well plates, and 1mL of the above-obtained GUCY2C-GFP lentivirus was added to obtain Jurkat-GUCY2C, SW620-GUCY2C, and colo205-GUCY2C overexpressing GUCY2C proteins.

Example 7

This example uses the lentiviruses prepared in example 5 to transduce T lymphocytes, comprising the following steps:

(1) Human PBMC are adjusted to a density of 1X 10 with T cell medium (X-VIVO +10% FBS + 300U/mLIL-2) ⁶ Activation by adding 1/100 volume of TCellTransAct (magnetic beads coupled with CD3 and CD 28) for 24h;

(2) Collecting activated T cells, adjusting cell density to 1 × 10 ⁶ mL, lentivirus was added at a multiplicity of infection (MOI) of 10, polybrene was added to a final concentration of 8 μ g/mL; at 37 ℃ C, 5% CO ₂ After overnight culture in the environment, replacing the fresh culture medium, and carrying out passage every 3 days;

(3) After 5 days of T cell infection, 3X 10 cells were taken ⁵ Centrifuging the T cells at the temperature of 4 ℃ for 5min at the speed of 500g, discarding the supernatant, and washing the T cells once by using a flow buffer; add 50 u L buffer heavy suspension cells, add 0.5 u g Rabbitanti-Camelid VHHANtibody (iFluor 488) antibody, ice incubation for 30min; adding a buffer solution for washing for 1 time, and then adding 300 mu L of the buffer solution for resuspending the cells;

the chimeric antigen receptor expression rate of the T lymphocytes was measured by flow cytometry, and the results are shown in fig. 5, where the infection efficiency of each set of CAR-T cells is: 71.6%, 44.1%, 63.9%, 43.3%, 52.3%, 59.9% and 40.7%, indicating successful construction of CAR-T cells.

Example 8

In this example, CAR-T cell in vitro toxicity experiments were performed, comprising the following steps:

(1) Target cell seeding

293-GPF-luci (GUCY 2C) ^- ) Colo205-GUCY2C-luci and SW620-GUCY2C-luci as target cells, and adjusting the concentration of the target cells to 1X 10 ⁵ mL, 100 μ L was inoculated into 96-well plates;

(2) Seeding of effector cells

GUCY2CCAR-T and control T cells are effector cells, and CAR-T cells and control T cells are added into a 96-well plate according to an effective target ratio of 0.3;

(3) Each group was provided with 3 replicate wells, the average of the 3 replicate wells was taken, where each experimental group and each control group were as follows:

experimental groups: a specific killing group (effector cells + target cells), and a non-specific killing control group (effector cells + non-specific target cells);

control group: an effector cell independent culture group, a target cell and culture medium natural release and maximum release group;

(4) After 18h of co-culture of effector cells and target cells, 20 μ L of lysate (10 ×) was added to the wells of the labeled maximal release group; the cell lysis condition is not observed regularly, so that the cell lysis is ensured to be complete;

(5) After blowing and sucking each hole for 8 times by using a discharging gun, centrifuging for 5min at 500g, and taking 50 mu L/hole supernatant to an enzyme label plate;

(6) The detection method comprises the following steps: adding 50 mu L of prepared substrate into an enzyme label plate, covering the plate, incubating for 30min at room temperature in a dark place, adding 50 mu L of stop solution (acetic acid) into each hole, and measuring the light absorption value at 490nm within 30 min.

(7) The CAR-T killing efficiency calculation formula is as follows:

specific killing rate (%) = (experimental pore LDH value-natural release pore LDH value)/(maximum release pore LDH value-natural release LDH value).

The results are shown in fig. 6-8, the CAR-T cells constructed by the invention have no killing effect on GUCY 2C-negative 293T cells and have killing activity on GUCY 2C-positive tumor cells, which indicates that the CAR-T cells constructed by the invention not only have high-efficiency tumor killing ability, but also have high specificity.

Example 9

In this example, the secretion of the CAR-T cytokine IFN-. Gamma.was detected using HumanIFN-. Gamma.ELISAKit (Union organism, cat # EK 180-96).

1. Cell culture supernatant

The cell culture with the effective target ratio of 1:1 is centrifuged at 400 Xg for 10min to remove precipitates, and the supernatant is taken and stored at-80 ℃ for detection.

2. Reagent preparation

Before the detection, all reagents and samples were returned to 25 ℃, and if the concentrated reagents appeared to crystallize, the samples were incubated at 37 ℃ until all crystals were dissolved, and 1 × washing solution, 1 × detection buffer, was prepared according to the instructions.

3. Standard substance and sample preparation

And (3) standard substance: the standard stock was diluted 2-fold using 5%1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample preparation: the samples were diluted proportionally using 5%1640 medium.

4. Detection step

(1) Soaking the enzyme label plate: adding 300 μ L of 1 × lotion, standing, soaking for 30s, discarding the lotion, and patting the microporous plate on absorbent paper;

(2) Adding a standard substance: adding 100 mu L of standard substance diluted by 2 times into the standard substance hole, and adding 100 mu L of standard substance diluent into the blank hole;

(3) Adding a sample: adding 100 mu L of cell culture supernatant into a sample hole;

(4) Adding a detection antibody: add 50 μ L of diluted detection antibody per well (1;

(5) And (3) incubation: incubating for 2h at 25 ℃ by using a sealing plate and a membrane sealing plate and oscillating at 300 rpm;

(6) Washing: discarding the liquid, adding 300 mu L of washing liquid into each hole to wash the plate, washing for 6 times, and patting the plate on absorbent paper for each time;

(7) Adding enzyme for incubation: add 100 μ L of diluted horseradish peroxidase-labeled streptavidin per well (1;

(8) And (3) incubation: sealing the plate with a new sealing plate membrane, oscillating at 300rpm, incubating at 25 ℃ for 45min, and washing;

(9) Adding a substrate for color development: adding 100 μ L chromogenic substrate TMB into each well, keeping out of the sun, and incubating for 20min at 25 ℃;

(10) Adding a stop solution: adding 100 mu L of stop solution into each hole;

(11) And (3) detection reading: within 30min, performing dual-wavelength detection by using an enzyme-labeling instrument, and determining OD values under the maximum absorption wavelength of 450nm and the reference wavelength; the OD value after calibration was the value measured at 450nm minus the value measured at the reference wavelength.

IFN-gamma factor secretion results are shown in FIG. 9, respectively, wherein spontaneous MOCK group is CAR-T cell group alone, and cytokine release is not substantially detected; CAR-T cells were co-cultured with 293T cells, and no cytokine was detected; CAR-T cells secrete IFN- γ in excess of 600pg/mL after co-culture with GUCY 2C-overexpressing target cells. The CAR-T cells constructed by the invention release cytokines for GUCY2C positive tumor cells, and have no obvious cytokine secretion for GUCY2C negative cells.

In conclusion, the GUCY 2C-resistant nano antibody with high affinity is screened and prepared, can be efficiently and specifically combined with GUCY2C, and is used as an antigen binding domain to construct a chimeric antigen receptor and a CAR-T cell, the obtained CAR-T cell has obvious killing activity and specificity on GUCY 2C-positive tumor cells, and can secrete cell factors for killing tumors, and the nano antibody can be effectively applied to immunotherapy and has important significance for developing tumor treatment medicines.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. The nano antibody for resisting guanylate cyclase 2C is characterized in that the amino acid sequence of CDR1 of the nano antibody comprises a sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;

the amino acid sequence of CDR2 of the nano antibody comprises a sequence shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10;

the amino acid sequence of CDR3 of the nano antibody comprises a sequence shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO. 13.

2. The nano-antibody against guanylate cyclase 2C according to claim 1, wherein the amino acid sequence of CDR1 of the nano-antibody comprises the sequence shown in SEQ ID No.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.4, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 11;

preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.8, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 12;

preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.3, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.9, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 13;

3. A nucleic acid molecule comprising a gene encoding the anti-GUCY2C nanobody of claim 1 or 2.

4. A chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, and a signaling domain;

the antigen binding domain comprises the nanobody against guanylate cyclase 2C of claim 1 or 2.

5. The chimeric antigen receptor according to claim 4, wherein the signal peptide comprises a CD8 a signal peptide;

preferably, the hinge region comprises a CD8 a hinge region;

preferably, the transmembrane region comprises any one of or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region;

preferably, the signaling domain comprises an immunoreceptor tyrosine activation motif;

preferably, the signaling domain further comprises a co-stimulatory molecule comprising any one of the 4-1BB, CD28 intracellular domain, OX40, ICOS or DAP10 intracellular domain or a combination of at least two thereof;

preferably, the chimeric antigen receptor comprises a CD8 a signal peptide, the nanobody of claim 1 or 2 against guanylate cyclase 2C, a CD8 a hinge region, a CD8 a transmembrane region, and an immunoreceptor tyrosine activation motif.

6. An expression vector comprising a gene encoding the chimeric antigen receptor of claim 4 or 5;

preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to claim 4 or 5, preferably a lentiviral vector.

7. A recombinant lentivirus comprising the expression vector of claim 6.

8. A chimeric antigen receptor immune cell, wherein said chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 4 or 5;

preferably, the chimeric antigen receptor immune cell comprises the expression vector of claim 6 and/or the recombinant lentivirus of claim 7;

9. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of claim 8 and/or the nanobody of claim 1 or 2;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients;

10. Use of the nanobody against guanylate cyclase 2C according to claim 1 or 2, the nucleic acid molecule of claim 3, the chimeric antigen receptor of claim 4 or 5, the expression vector of claim 6, the recombinant lentivirus of claim 7, the chimeric antigen receptor immune cell of claim 8 or the pharmaceutical composition of claim 9 for the preparation of a medicament for the treatment of tumors;

preferably, the tumor comprises a tumor expressing guanylate cyclase 2C.