CN115894697B

CN115894697B - Nanometer antibody for resisting guanylate cyclase 2C and application thereof

Info

Publication number: CN115894697B
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: 2023-08-29
Anticipated expiration: 2042-12-09
Also published as: CN116751303A; CN116751303B; CN115894697A; CN116874602A

Abstract

The invention discloses a nano antibody for resisting guanylate cyclase 2C and application thereof. The amino acid sequence of the CDR1 of the nano antibody comprises a sequence shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, the amino acid sequence of the CDR2 of the nano antibody comprises a sequence shown as 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 the CDR3 of the nano antibody comprises a sequence shown as SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO. 13. The nano antibody for resisting guanylate cyclase 2C can specifically bind to guanylate cyclase 2C antigen, has better affinity, is used as an antigen binding domain to construct chimeric antigen receptor and CAR-T cell, and has obvious killing activity on guanylate cyclase 2C positive tumor cells.

Description

Nanometer 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, its gene is located on human chromosome 12, the cDNA of GUCY2C contains an open reading frame of 3219bp, and it contains 27 exons in total, codes for a protein consisting of 1073 amino acids with a relative molecular mass of about 120kDa, belonging to the family of receptor ornithine cyclases. Its transmembrane and intracellular domains (kinase homology, junction and guanylate cyclase domains) share homology with other membrane-bound guanylate cyclases, while its unique N-terminal extracellular domain (residues 1-430) determines its ligand specificity.

In the extracellular domain, STa binds to the amino acid microdomains adjacent to the transmembrane region (87-393), while the mechanism by which Sta binding increases cGMP production is still unknown (see: john C Flickinger Jr, jeffrey A Rappaport, joshua R Barton, et al, guanyyl cyclase C as a biomarker for immunotherapies for the treatment of gastrointestinal, magnancies. Biomark.Med. (2021) 15 (3), 201-217, doi: 10.2217/bmm-2020-0359). Current studies indicate that the endogenous ligands guanosine and uridine can activate GUCY2C, which, by binding to GUCY2C, causes an increase in intracellular cyclic guanosine phosphate concentration, further activating cGMP-dependent protein kinase gii, thereby causing a corresponding physiological change. This physiological change plays a key role in maintaining intestinal homeostasis, improving visceral pain sensitivity, inhibiting intestinal tissue degeneration, etc. (Kuhn M. Molecular physiology of membrane guanylyl cyclereceptors. Physiol Rev,2016,96 (2): 751-804, doi: 10.1152/Physrev.00022.2015.).

GUCY2C is an intestinal tissue-specific polypeptide that is expressed in normal intestinal mucosal epithelium and primary or secondary colorectal cancer (see: frick GS, pitari GM, weinberg DS, et al, guanyyl cyclasecC: a molecularmarker for staging and postoperative surveillance of patients with colorectal cancer. Expert Rev Mol Diagn,2005,5 (5): 701-713, doi: 10.1586/14737159.5.5.701.). Immunohistochemistry was performed to examine the expression of GUCY2C in normal intestinal tissue, colorectal cancer tissue and parenteral tissue, and carcinoembryonic antigen (CEA) and cytokeratin 20 (CK 20) were used as controls, and the results showed that the positive rate of GUCY2C in normal intestinal tissue and colorectal cancer tissue was 100% and 80%, CK20 was 94% and 100%, and CEA was 94% and 94%, respectively. GUCY2C is expressed predominantly in the mucosa crypt and villus, with the most expressed in the mid-villus but almost not in extra-intestinal tissue, while CEA and CK20 are expressed to different extents in esophageal and gastric cancer tissues. GUCY2C is more specific and sensitive than CEA and CK20, and is an effective index for detecting colorectal cancer (see: buc E, vartanian MD, darcha C, et al, guanyyl cyclase C as a reliable immunohistochemical marker and its ligand Escherichia coli heat-stable enterotoxin as a potential protein-delivering vehicle for colorectal cancer cells Eur JCancer,2005,41 (11): 1618-1627, doi: 10.1016/j.ejca.2005.02.031.).

GUCY2C was originally termed the STa receptor and was previously identified as a receptor for thermostable enterotoxins (STa) that cause diarrhea pathogenic bacteria in travelers. Initial studies showed that STa is secreted by enterotoxigenic escherichia coli (ETEC) and can lead to cyclic GMP (cGMP) accumulation in the gut compared to other diarrheal toxins such as cholera toxin. This suggests that STa signal activation is possible by guanylate cyclase, one of the known protein family members catalyzing the conversion of GTP to cGMP and pyrophosphoric acid, and that GUCY2C was first cloned from a rat small intestine cDNA library using a highly conserved sequence of guanylate cyclase catalytic domains and demonstrated to be a source of both STa receptor and cGMP accumulation. GUCY2C is more conserved in all vertebrates, whereas a mutation in GUCY2C is its effect on secretion, whose loss of function can lead to decreased secretion and neonatal ileus, whereas GUCY2C gain of function will lead to increased secretion and secretory diarrhea.

Recent studies indicate that GUCY2C may be an extremely potential immunotherapeutic target for the treatment of gastrointestinal cancers. As early as 2008, there was an immunotherapeutic approach to making a vaccine using targeted GUCY2C, and researchers selected the extracellular domain of GUCY2C as a vaccine target, cloned into recombinant adenovirus serotype 5 (Ad 5), for immunogenicity and colorectal cancer treatment studies, ad5-GUCY2C significantly reduced tumor burden in mice models of liver and lung metastasis (two sites most common for colorectal cancer metastasis) and increased survival rate of mice compared to control. (see: john C Flickinger Jr, jeffrey A Rappaport, joshua R Barton, et al, guanylyl cyclase C as a biomarker for immunotherapies for the treatment of gastrointestinal magnanices. Biomark. Med. (2021) 15 (3), 201-217.).

Michael S.Magee et al constructed CART cells targeting human GUCY 2C-expressing metastases using human specific single-chain variable fragments, promoted activation of antigen-dependent T cells, and could upregulate activation markers and cytokine production, and killed GUCY 2C-expressing tumor cells. In an allograft mouse model, CAR-T cells directed against GUCY2C may have long-term protective effects on lung metastasis models of mouse colorectal cancer cells expressing human GUCY2C. Secondly in the human xenograft model of immunodeficient mice, CAR-T cells directed against GUCY2C recognize and kill human colorectal cancer cells endogenously expressing GUCY2C, thereby providing a long-lasting survival of the model. Researchers have shown that a single injection of the highest dose of 10 in colorectal lung metastasis model using CAR design encoding scFv specific for human GUCY2C ⁷ With each CART cell, 60% of animals could be cured. And CAR-T cells were repeatedly injected at this dose, the survival rate of treatment was increased to 80% 100 days after tumor implantation. In addition, 75% of animals initially treated with GUCY2C CAR-T cells survived more than 80 days after tumor re-transplantation, indicating that GUCY2C CART cells persisted and acted within 6 months after initial dosing. In addition, in the CDX model of peritoneal metastasis, a tumor-forming experiment was performed using the human colorectal cancer cell line T84, and the constructed CAR-T cells of human GUCY2C had a curative effect on 100% of test animals. The above strongly supports the potential value of GUCY 2C-directed CAR-T cell therapies in clinical applications of gastrointestinal malignancies. (Magee MS, abraham TS, baybutt TR, et al human GUCY2C-targeted Chimeric Antigen Receptor (CAR) -expressing T cells eliminate colorectal cancer meta-acids.cancer immunol.Res.6 (5), 509-516 (2018)).

In conclusion, GUCY2C can be used as a target spot of 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 actual demands of the prior art, the invention provides the anti-guanylate cyclase 2C nanobody and the application thereof, and the anti-GUCY2C nanobody has high affinity, can be used as an antigen binding domain of a chimeric antigen receptor molecule to prepare the CAR-T cell, and has good application prospect in the aspect of tumor treatment.

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

in a first aspect, the present invention provides a nanobody against guanylate cyclase 2C, 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, and 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, a non-immunized alpaca is immunized by guanylate cyclase 2C recombinant protein, a phage display nano antibody library is constructed, and screening is carried out on anti-guanylate cyclase 2C antibodies according to the phage display nano antibody library to obtain monoclonal antibodies capable of specifically binding guanylate cyclase 2C antigens, wherein the antibodies belong to single domain antibodies.

The anti-GUCY2C 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 the CAR-T cell, and has a good application prospect 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 in SEQ ID NO.14 or 15, SEQ ID NO.16 or 17, the framework region 2 (FR 2) shown in SEQ ID NO.18, SEQ ID NO.19 or 20, the framework region 3 (FR 3) shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 or 25 and the framework region 4 (FR 4) shown in SEQ ID NO.26 or 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 as 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, a phage display technology is utilized to screen a 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 chimeric antigen receptor of targeted guanylate cyclase 2C.

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

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

Preferably, 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 or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region or a DAP10 transmembrane region.

Preferably, the signal transduction domain comprises an immunoreceptor tyrosine activation motif (cd3ζ).

Preferably, the signal transduction domain further comprises a co-stimulatory molecule comprising any one or a combination of at least two of 4-1BB, the intracellular domain of CD28, OX40, ICOS or the intracellular domain of DAP 10.

Preferably, the chimeric antigen receptor comprises a CD8 a signal peptide, a nanobody against guanylate cyclase 2C according to the first aspect, a CD8 a hinge region, a CD8 a transmembrane region and an immune receptor 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 comprising a 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 comprise any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages.

In a seventh aspect, the invention provides a pharmaceutical composition comprising a chimeric antigen receptor immune cell according to the sixth aspect and/or a nanobody according to 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 carriers, surfactants, disintegrants, coating materials, excipients, solubilizers, diluents, pH regulators, binders, wetting agents, colorants, emulsifiers, bacteriostats, cosolvents, osmotic pressure regulators, fillers, antioxidants or buffers.

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

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

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

(1) According to the invention, the non-immunized alpaca is immunized by using the GUCY2C recombinant protein, a phage display nanobody library is constructed, the anti-GUCY2C antibody is screened according to the phage display nanobody library, the obtained nanobody can specifically bind to the GUCY2C antigen and has better affinity, and the KD (M) is respectively 2.75X10 as shown by the determination of the affinity of the antibody ^-11 、1.80×10 ^-8 、1.26×10 ^-8 、1.09×10 ^-8 、2.08×10 ^-8 、5.20×10 ^-8 And 1.82×10 ^-8 ；

(2) The nanometer antibody for resisting GUCY2C 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, wherein the CAR-T cells have killing activity on GUCY2C positive tumor cells, and after being co-cultured with the GUCY2C positive cells, the nanometer antibody can efficiently secrete cell factor IFN-gamma, can be effectively applied to immunotherapy, and has important significance for developing tumor therapeutic drugs.

Drawings

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

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

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

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

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

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

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

FIG. 2 is a graph showing the results of FACS detection of GUCY2C antigen by anti-GUCY2C nanobodies in example 3;

FIG. 3 is a map of a chimeric antigen receptor lentiviral vector targeting GUCY2C in example 4;

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

FIG. 5 is a graph showing the results of flow assay of the expression rate of 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 as described 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 bar graph of IFNγ secretion by CAR-T cells in example 8.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1

In this example, phage nanobody libraries were constructed and panning were performed and primary screening was performed using ELISA, as follows:

(1) Construction of phage nanobody library

Immunizing alpaca with GUCY2C (GUCY 2C) extracellular recombinant protein, detecting serum titer by ELISA, and extracting peripheral blood; separating lymphocytes, extracting total RNA, performing reverse transcription to obtain cDNA, and amplifying VHH genes by nested PCR; VHH gene was inserted into pShort phagemid and electrotransformedSeparating and purifying the phage by PEG8000/NaCI precipitation method after the competent cells are amplified to obtain an antibody library; adjusting concentration, packaging, and freezing in refrigerator at-80deg.C;

(2) Screening of phage nanobody libraries

Firstly, incubating 293T cells with an antibody library for negative screening, and then incubating supernatant with 293T-GUCY2C cells (GUCY 2C positive) and 293T cells respectively; washing for 4 times by adopting a pre-cooled PT buffer solution at 4 ℃; infecting NEB alpha 5F' cells, adding helper phage, and culturing overnight; coating a plate by a Drop method, and counting the enrichment degree the next day; separating and purifying the phage by PEG8000/NaCI precipitation method, and then carrying out the next round of screening; after enrichment, the VHH region was amplified using the obtained phage as a template, and subjected to second generation sequencing to obtain 7 anti-GUCY2C nanobodies, the amino acid sequences of which were respectively designated as VHH-A1, VHH-A2, VHH-A3, VHH-14, VHH-29, VHH-36 and VHH-43, as shown in 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.

Example 2

The present example performs VHHFc nanobody expression, purification, and antibody affinity assay.

The various antibodies that met the conditions were screened for a total of seven candidate antibodies designated 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 mouse Fc tags (which were done by a third party) and the antibody quality test results obtained are shown in table 1.

TABLE 1

The above 7 GUCY2C VHH antibodies were subjected to affinity assay by Biacore. Biacore is a bioanalytical sensing technology developed based on surface plasmon resonance (surface Plasmon resonance, SPR), and can detect and track the whole change process of binding and dissociating molecules in a solution and molecules fixed on the surface of a chip, record the whole change process in the form of a sensor graph, provide kinetic and affinity data, solidify antibodies on the surface of the chip in the measuring process, enable a mobile phase to be a solution containing antigens, and enable the affinity of 7 antibodies to reach sub-nanomolar levels as shown in a table 2 and a graph 1A-1G, wherein the affinities of the 7 antibodies are respectively 2.75 xE-11, 1.80 xE-8, 1.26 xE-8, 1.09 xE-8, 2.08 xE-8, 5.20 xE-8 and 1.82 xE-8.

TABLE 2

Nanobody	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 describes a flow assay for the nanobodies of example anti-GUCY 2C.

Jurkat (GUCY 2C-), jurkat-GUCY2C cells were mixed with purified anti-GUCY2C nanobodies, incubated for 30min in an ice bath, then incubated with APC-labeled goat anti-mouse IgG antibodies for 30min, and detected by flow cytometry, as shown in FIG. 2, indicating that the anti-GUCY2C nanobodies of the invention recognize 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, and the schematic of the chimeric antigen receptor is shown in FIG. 4, comprising a CD8 alpha signal peptide, a nanobody against GUCY2C (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 of the signal peptide (SEQ ID NO. 35) is:

MALPVTALLLPLALLLHAARP。

the amino acid sequence of the anti-GUCY2C VHH is 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.

The amino acid sequences of the CD 8. Alpha. Hinge region and the transmembrane region (SEQ ID NO. 36) are:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC。

the amino acid sequence of the 4-1BB intracellular region (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) PCR reaction systems were prepared according to Table 3 (reagents in Table were derived from TOYOBOINC.) and nanobody fragments of each anti-GUCY2C were amplified and reacted according to the PCR procedure shown in Table 4, with primer sequences of:

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 Table 3

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

TABLE 4 Table 4

(2) A PCR reaction system was prepared according to Table 5, and CD 8. Alpha. Signal peptide was added before the obtained amplified product, and the 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, the PCR product is subjected to 1% agarose gel electrophoresis, a 500bp fragment is recovered, and the quantity is fixed by an ultraviolet absorption method;

(3) A PCR reaction system was prepared according to Table 6, and after the completion of the preparation, a PCR reaction was performed according to the PCR procedure shown in Table 4 to amplify the CD 8. Alpha. Range-TM-41 BB-CD3Z fragment, using the primers as follows:

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

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

TABLE 6

Reagent(s)	Volume (mu L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 mu M upstream primer CD8 alpha H-F	1
		10 mu 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, 1% agarose gel electrophoresis is carried out, fragments about 680bp are recovered, and the quantification is carried out by an ultraviolet absorption method;

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

the 3 fragments recovered above and the vector backbone were linked by recombinase, the recombination reaction system is shown in Table 7, and after completion of the preparation, the mixture was subjected to a water bath reaction at 37℃for 0.5h, and transformed into E.coli stbl3 competent cells by a conventional method.

TABLE 7

Reagent(s)	Usage amount
		HD CD19 CAR skeleton	154.2ng
CD8αsingal GUCY2C VHH	10ng
		CD8αhinge-TM-41BB-CD3Z	14ng
5×CE buffer	2μL
		Exnase ^TM II	1.2μL
Sterile deionized water (PCRgrader)	Make up to 12 mu L

And selecting a monoclonal from the solid culture medium, culturing overnight, carrying out PCR identification, preparing a PCR reaction system as shown in table 8, carrying out a PCR procedure as shown in table 9, selecting a positive clone after the PCR is finished, and further carrying out sequencing identification, wherein the sequencing result meets the expectations. The primer sequences were 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, lentiviral vector HDSIN03-GUCY2CCAR prepared in example 4 was subjected to lentiviral packaging, concentration and titer detection, comprising the steps of:

(1) Lentivirus package

At 6.0X10 ⁶ Cell count 293T cells were seeded in 10cm dishes at 37℃with 5% CO ₂ The virus is prepared for packaging after overnight culture, and the culture medium is DMEM containing 10% of fetal calf serum; 5.4 mu g of HDSIN03-GUCY2CCAR, 6.2 mu g of helper plasmid pMDLg-RRE, 6.2 mu g of helper plasmid pRSV-REV and 2.4 mu g of envelope plasmid VSVg of lentiviral vector are dissolved in 0.8mL of serum-free DMEM culture solution and evenly mixed;

60.6. Mu.g PEI (1. Mu.g/. Mu.L) was dissolved in 0.8mL serum-free DMEM medium and vortexed at 1000rpm for 5 seconds and incubated at 25℃for 5min; adding PEI mixed solution into the DNA mixed solution, gently mixing after adding, and incubating at 25 ℃ for 25min to form a transfection complex; 1.6mL of the transfection complex is added into 10mL of DMEM medium containing 293T cells in a dropwise manner, and after 6 hours, the fresh medium is replaced; after 48 hours, collecting the virus liquid supernatant;

(2) Lentivirus concentration

Filtering the virus supernatant with a 0.45 μm filter membrane, collecting the filtered virus supernatant into a 50mL centrifuge tube, adding 1/4 PEG-NaCl virus concentrate, mixing the mixture upside down, and standing the mixture at 4 ℃ overnight; centrifuging at 4 ℃ at 3500rpm for 30min; removing supernatant, adding RPMI1640 medium (containing 10% FBS), and dissolving the resuspended viral pellet; split charging 50 μl of the concentrated lentiviral suspension into each portion, storing in a finished tube, and storing at-80deg.C;

(3) Lentivirus titer detection

mu.L Jurkat cells (2X 10) ⁵ Individual cells) were inoculated into 24-well plates, and the concentrated lentiviruses were 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℃at 5% CO ₂ After overnight incubation, fresh medium was changed;

after 72h infection, 500 Xg was centrifuged for 5min, the supernatant was discarded to collect cells, 100. Mu.LPBS+2% FBS was added to resuspend the cells, 0.5. Mu.g of Rabbit anti-CamellidVHH hantyy (iFluor 488) antibody was added and incubated on ice for 30min; after washing 1 time with streaming buffer (PBS containing 2% FBS), 300. Mu.L of streaming buffer was added to resuspend 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 overexpressing cell lines

GUCY2C-GFP lentiviruses were obtained by cotransfection of GUCY2C-GFP plasmid (purchased from Crohn's disease), pMGlg-RRE, pRSV-REV and VSVg plasmid, in the same manner as in example 5;

taking 1×10 pieces respectively ⁶ 293T cells (stored by the company), jurkat cells (stored by the company), SW620 cells (purchased from the cell bank of the department of Chinese sciences), and colo205 cells (purchased from ATCC) were inoculated into a 6-well plate, and 1mL of the above-obtained GUCY2C-GFP lentivirus was added to thereby obtain Jurkat-GUCY2C, SW620-GUCY2C, colo-GUCY 2C over-expressing the GUCY2C protein.

Example 7

This example uses lentivirus transduced T lymphocytes prepared in example 5, comprising the steps of:

(1) Human PBMC were conditioned to a density of 1X 10 with T cell culture medium (X-VIVO+10% FBS+300U/mLIL-2) ⁶ 1/100 volume of tcelltranact (magnetic beads commercially available to couple CD3 and CD 28) was added to activate for 24h;

(2) The activated T cells were collected and the cell density was adjusted to 1X 10 ⁶ Lentivirus was added per mL at a multiplicity of infection (multiplicityof infection, MOI) of 10, polybrene was added to a final concentration of 8 μg/mL; at 37℃with 5% CO ₂ Replacing fresh culture medium after overnight culture in the environment, and carrying out passage every 3 days;

(3) 5 days after T cell infection, 3X 10 cells were taken ⁵ Centrifuging at 4 ℃ for 5min at 500g, discarding the supernatant, and washing once with streaming buffer; add 50. Mu.L buffer to resuspend cells, add 0.5. Mu.g Rabbit antibody-CamellidVHH hanntibody (iFluor 488) and incubate on ice for 30min; after washing 1 time with buffer solution, 300. Mu.L of buffer solution is added to resuspend the cells;

the expression rate of chimeric antigen receptor of T lymphocytes was measured by flow cytometry, and as a result, as shown in fig. 5, the infection efficiency of each group of CAR-T cells was: 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, an in vitro toxicity test of CAR-T cells was performed, comprising the steps of:

(1) Target cell inoculation

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 ⁵ 100. Mu.L of the solution is inoculated into a 96-well plate;

(2) Effector cell seeding

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 the effective target ratio of 0.3:1, 1:1 and 3:1;

(3) Each group was set with 3 duplicate wells and the average of 3 duplicate wells was taken, wherein each experimental group and each control group were as follows:

experimental group: specific killing group (effector cell + target cell), non-specific killing control group (effector cell + non-specific target cell);

control group: effector cells alone in culture, target cells and medium in natural and maximum release groups;

(4) After 18h co-culture of effector cells with target cells, 20 μl of lysate (10×) was added to the wells of the labeled maximum 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 by a row gun for 8 times, centrifuging 500g for 5min, and taking 50 mu L/hole supernatant into an ELISA plate;

(6) The detection method comprises the following steps: the prepared substrate was added to the ELISA plate at a concentration of 50. Mu.L/well, the plate was covered, incubated at room temperature for 30min in the absence of light, 50. Mu.L of stop solution (acetic acid) was added to each well, and absorbance at 490nm was measured within 30 min.

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

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

The results are shown in figures 6-8, the CAR-T cells constructed by the invention have no killing effect on GUCY2C negative 293T cells and have killing activity on GUCY2C positive tumor cells, and the CAR-T cells constructed by the invention have high-efficiency tumor killing capacity and high specificity.

Example 9

In this example, the secretion of IFN-. Gamma.by CAR-T cytokines was detected using the respective kit HumanIFN-. Gamma.ELISAkit (Bigella, cat. Number: EK 180-96).

1. Cell culture supernatant

Cell cultures with an effective target ratio of 1:1 were centrifuged at 400 Xg for 10min to remove sediment, and the supernatant was stored at-80℃for examination.

2. Reagent preparation

All reagents, samples were returned to 25℃before testing, and if crystallization occurred in the concentrated reagents, the incubation was performed at 37℃until the crystals were completely dissolved, and 1 Xof wash solution and 1 Xof assay buffer were prepared according to the instructions of use.

3. Standard substance and sample preparation

Standard substance: standard stock was 2-fold diluted with 5%1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample: samples were diluted in ratio using 5%1640 medium.

4. Detection step

(1) Soaking the ELISA plate: adding 300 mu L of 1 Xwashing liquid, standing and soaking for 30s, discarding the washing liquid, and beating the micro-pore plate on water-absorbing paper;

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

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

(4) Adding a detection antibody: 50. Mu.L of diluted detection antibody (1:100 dilution) was added to each well;

(5) Incubation: sealing plates by using sealing plates, vibrating at 300rpm, and incubating at 25 ℃ for 2 hours;

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

(7) And (3) enzyme adding and incubation: mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) was added to each well;

(8) Incubation: using a new sealing plate membrane sealing plate, oscillating at 300rpm, incubating at 25 ℃ for 45min, and washing;

(9) And (3) color development of the substrate: 100 mu L of chromogenic substrate TMB is added into each hole, and incubated for 20min at 25 ℃ in the dark;

(10) Adding a stop solution: 100 mu L of stop solution is added to each well;

(11) Detecting and reading: within 30min, performing dual-wavelength detection by using an enzyme-labeled instrument, and measuring the OD value at the maximum absorption wavelength of 450nm and the reference wavelength; the OD value after calibration was 450nm minus the measurement at the reference wavelength.

The IFN-gamma factor secretion results are shown in FIG. 9, respectively, wherein spontaneous MOCK groups are independent CAR-T cell groups, and cytokine release is not basically detected; no cytokines were detected in the CAR-T cell co-cultured group with 293T cells; after co-culture with target cells that overexpress GUCY2C, the IFN-gamma secreted by the CAR-T cells exceeds 600pg/mL. The CAR-T cells constructed by the invention release cytokines to GUCY2C positive tumor cells, and have no obvious cytokine secretion to GUCY2C negative cells.

In conclusion, the nano antibody with high affinity for resisting GUCY2C can be screened and prepared, can be combined with GUCY2C in a high-efficiency mode, is used as an antigen binding domain to construct a chimeric antigen receptor and CAR-T cells, and the obtained CAR-T cells have obvious killing activity and specificity on GUCY2C positive tumor cells and can secrete cytokines for killing tumors, so that the nano antibody can be effectively applied to immunotherapy, and has important significance in developing tumor therapeutic drugs.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. The nanometer antibody for resisting guanylate cyclase 2C is characterized in that the amino acid sequence of CDR1 of the nanometer antibody is shown as SEQ ID NO.2, the amino acid sequence of CDR2 is any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.10, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 12.

2. A nucleic acid molecule comprising a gene encoding the nanobody of guanylate cyclase 2C according to claim 1.

3. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, and a signal transduction domain;

the antigen binding domain comprises the nanobody of anti-guanylate cyclase 2C of claim 1;

the signal peptide comprises a CD8 a signal peptide;

the hinge region comprises a CD8 a hinge region;

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

the signal transduction domain includes an immunoreceptor tyrosine activation motif.

4. The chimeric antigen receptor according to claim 3, wherein the signaling domain further comprises a co-stimulatory molecule comprising any one or a combination of at least two of 4-1BB, CD28 intracellular region, OX40, ICOS or DAP10 intracellular region.

5. An expression vector comprising a gene encoding the chimeric antigen receptor of claim 3.

6. The expression vector according to claim 5, wherein the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector comprising a gene encoding the chimeric antigen receptor according to claim 3.

7. The expression vector according to claim 5, wherein the expression vector is a lentiviral vector comprising a gene encoding the chimeric antigen receptor according to claim 3.

8. A recombinant lentivirus comprising the expression vector of any one of claims 5-7.

9. A chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 3 or 4.

10. The chimeric antigen receptor immune cell according to claim 9, comprising the expression vector of any one of claims 5-7 and/or the recombinant lentivirus of claim 8.

11. The chimeric antigen receptor-immune cell according to claim 9, wherein the chimeric antigen receptor-immune cell comprises any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages.

12. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of any one of claims 9-11 or the nanobody of claim 1 against guanylate cyclase 2C.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

14. The pharmaceutical composition of claim 13, wherein the adjuvant comprises any one or a combination of at least two of a carrier, a surfactant, a disintegrant, a coating material, an excipient, a solubilizing agent, a diluent, a pH adjuster, a binder, a wetting agent, a colorant, an emulsifier, a bacteriostatic agent, a co-solvent, an osmotic pressure adjuster, a filler, an antioxidant, or a buffer.

15. Use of the nanobody against guanylate cyclase 2C according to claim 1, the nucleic acid molecule according to claim 2, the chimeric antigen receptor according to claim 3 or4, the expression vector according to any one of claims 5-7, the recombinant lentivirus according to claim 8, the chimeric antigen receptor immune cell according to any one of claims 9-11 or the pharmaceutical composition according to any one of claims 12-14 for the preparation of a medicament for the treatment of a tumor; the tumor includes a tumor expressing guanylate cyclase 2C.