阅读说明：本技术 EGFP-Wnt2融合蛋白抗原和Wnt2单克隆抗体以及Wnt2单克隆抗体的应用 (Application of EGFP-Wnt2 fusion protein antigen, Wnt2 monoclonal antibody and Wnt2 monoclonal antibody ) 是由 付利 黄土雄 范春雷 武虎 陈喆 匡红 刘美星 于 2019-04-30 设计创作，主要内容包括：本发明涉及生物医学技术领域,尤其涉及EGFP-Wnt2融合蛋白抗原和Wnt2单克隆抗体以及Wnt2单克隆抗体的应用,EGFP-Wnt2融合蛋白抗原主要是EGFP与人源Wnt2除信号肽的全长氨基酸序列的融合蛋白,所述EGFP-Wnt2融合蛋白抗原包括EGFP氨基酸序列、TEV酶切位点和FLAG标签序列、Wnt2除信号肽全长氨基酸序列、6×His标签序列,抗Wnt2单克隆抗体能够用于抑制肿瘤细胞的免疫逃逸和生长。本发明的Wnt2单克隆抗体结合细胞分泌的人Wnt2抗原时,拮抗细胞对肿瘤内抑制性免疫微环境形成的促进,抑制肿瘤细胞的免疫逃逸及生长。本发明提供的Wnt2单克隆抗体能够用于食管鳞癌、胃癌、胰腺癌、直肠结肠癌、肺癌、乳腺癌、胶质细胞瘤、肝癌等实体肿瘤的免疫治疗中。(The invention relates to the technical field of biomedicine, in particular to an EGFP-Wnt2 fusion protein antigen, a Wnt2 monoclonal antibody and an application of a Wnt2 monoclonal antibody, wherein the EGFP-Wnt2 fusion protein antigen is mainly a fusion protein of EGFP and human Wnt2 except a full-length amino acid sequence of a signal peptide, the EGFP-Wnt2 fusion protein antigen comprises an EGFP amino acid sequence, a TEV enzyme digestion site and a FLAG tag sequence, a Wnt2 except the full-length amino acid sequence of the signal peptide and a 6 XHis tag sequence, and an anti-Wnt 2 monoclonal antibody can be used for inhibiting immune escape and growth of tumor cells. When the Wnt2 monoclonal antibody is combined with a human Wnt2 antigen secreted by cells, the Wnt2 monoclonal antibody antagonizes the promotion of the formation of an inhibitory immune microenvironment in a tumor by the cells, and inhibits the immune escape and growth of the tumor cells. The Wnt2 monoclonal antibody provided by the invention can be used for immunotherapy of solid tumors such as esophageal squamous cell carcinoma, gastric cancer, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, glioma and liver cancer.)

The EGFP-Wnt2 fusion protein antigen is mainly a fusion protein of EGFP and human Wnt2 except a full-length amino acid sequence of a signal peptide, and the EGFP-Wnt2 fusion protein antigen comprises an EGFP amino acid sequence, a TEV-FLAG tag sequence, a Wnt2 except the full-length amino acid sequence of the signal peptide and a 6 × His tag sequence.

2. The EGFP-Wnt2 fusion protein antigen of claim 1, wherein the amino acid sequence of the EGFP-Wnt2 fusion protein antigen is shown in SEQ ID No. 2.

3. A nucleotide encoding the EGFP-Wnt2 fusion protein antigen of claim 2, the nucleotide sequence being set forth in SEQ ID No. 1.

The preparation method of the EGFP-Wnt2 fusion protein antigen is characterized by comprising the following steps:

s1, fusing an EGFP gene and a human wild type Wnt2 gene from which ER signal peptides are removed, inserting a TEV enzyme cutting site and a FLAG label sequence between the two genes, adding an ER signal peptide of rat growth hormone at an amino end for secretory expression, connecting a 6 xHis label to a carboxyl terminal of Wnt2, adding a pair of restriction enzymes BamH I and SexA I at the front end and the rear end of the generated sequence, constructing the downstream of a CMV promoter of a lentiviral expression vector pLV-Puro, and removing the original Puro gene to obtain a plasmid pCMV-EGFP-Wnt 2;

s2, co-transfecting pCMV-EGFP-Wnt2 and plasmids of pH1 and pH2 to a lentivirus packaging line cell 293V, culturing for 5 generations or more than 20 days to obtain stably expressed protein cells, then under a fluorescence microscope, using a 50-microliter pipette to pick 25-30 EGFP-Wnt2/293 cell clones with different expression levels, respectively amplifying and culturing the cell clones, collecting supernatant, performing Wnt2 protein function verification for promoting cell migration, amplifying and culturing functional clones, collecting supernatant, purifying by a nickel ion affinity chromatography gel column, and obtaining the EGFP-Wnt2 fusion protein.

The Wnt2 monoclonal antibody, wherein the Wnt2 monoclonal antibody is an antibody capable of specifically binding to the EGFP-Wnt2 fusion protein antigen of claim 1 or 2 and Wnt2 protein obtained by removing EGFP with TEV enzyme or EK enzyme, and the Wnt2 monoclonal antibody is used for inhibiting immune escape and growth of tumor cells.

6. The Wnt2 monoclonal antibody of claim 5, wherein the Wnt2 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity determining regions having amino acid sequences as follows:

H-CDR1：GYTFTDFD，

H-CDR2：HPESDYT，

and H-CDR 3: TGDR;

the light chain variable region comprises three complementarity determining regions having amino acid sequences as follows:

L-CDR1：KSVSTSGYSY，

L-CDR2：LVS，

L-CDR3：QHIRELTR。

7. the Wnt2 monoclonal antibody according to claim 6, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4 of the sequence Listing, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6 of the sequence Listing.

8. An isolated nucleotide molecule, wherein the nucleotide sequence encoding the variable region of the Wnt2 monoclonal antibody heavy chain as claimed in claim 7 is shown in SEQ ID NO. 3, and the nucleotide sequence encoding the variable region of the Wnt2 monoclonal antibody light chain as claimed in claim 7 is shown in SEQ ID NO. 5.

9. A pharmaceutical composition comprising (1) an effective amount of the Wnt2 monoclonal antibody, the Wnt2 monoclonal antibody-based binding protein or antibody fragment of claim 7; and (2) an effective amount of the Wnt2 monoclonal antibody, Wnt2 monoclonal antibody-based binding protein, or antibody fragment of claim 7 in combination with a PD-1 antibody; and (3) one or more pharmaceutically acceptable pharmaceutical excipients and/or pharmaceutically acceptable carriers.

10. The use of the Wnt2 monoclonal antibody according to claim 6 or 7 in the immunotherapy of solid tumors with high Wnt2 expression, including esophageal squamous carcinoma, gastric cancer, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, glioma and liver cancer.

Technical Field

The invention relates to the technical field of biomedicine, in particular to an EGFP-Wnt2 fusion protein antigen, a Wnt2 monoclonal antibody and an application of the Wnt2 monoclonal antibody.

Background

The immune escape of solid tumor cells is one of the hot spots and difficulties in the basic research of clinical tumor immunotherapy and tumor immunology. Currently, PD-1/PD-L1 blocking antibodies have good potential in early clinical trials of solid tumor treatment, however, immunotherapy is often limited by the immune microenvironment of solid tumors and complex immune escape regulation thereof, resulting in low overall response rate and even drug resistance. Deeply discussing the tumor immune escape molecular mechanism, searching for a new immunotherapy target point and providing very important guiding significance for developing novel and efficient tumor immunotherapy.

At present, biomedical researchers are more and more paying attention to the role of tumor microenvironment in tumor progression and treatment, among which, cancer-associated fibroblasts (CAFs) are in the central position. At present, the CAFs are considered to play a role in promoting the evolution of tumors, have important influence in various processes such as tumorigenesis, tumor progression and metastasis, and can promote the evolution of tumors through tumor and non-tumor components. The research finds that: targeted FAP⁺The CXCL12 secreted by the CAFs can promote the tumor infiltration of T cells, thereby effectively enhancing the immunotherapy effect of PD-L1, and the FAP is shown⁺CAFs can modulate the immunosuppressive microenvironment by releasing CXCL12 (Feig C et al, pnas.2013). In addition, FAP protein released by CAFs can promote MDSC swelling through STAT3-CCL2 signaling pathwayTumors infiltrate, inhibiting the anti-tumor immune response (Yang Xet al, Cancer res.2016). Although CAFs have been shown to regulate the immune escape of tumor cells, due to the complex mechanisms of immune tolerance of tumor cells, the molecular regulatory networks in which CAFs are involved remain to be further explored and elucidated.

CAFs and corresponding Normal Fibroblasts (NFs) are successfully separated from cancer tissues and tissues beside the cancer of esophageal cancer patients respectively, and the gene expression difference of CAF and NF is analyzed by using gene chip comparison. The results showed that 126 genes were functionally related to cell proliferation (61 genes), extracellular matrix remodeling (40 genes) and immune response (25 genes), respectively. Previous researches show that the expression of Wnt2 gene is significantly up-regulated in esophageal squamous cell carcinoma CAFs (Zhang C et al, Clin Cancer Res.2009), and previous researches report that Wnt/beta-catenin signal path can promote immune escape, which suggests that Wnt2 protein secreted by CAFs may participate in regulation and control of solid tumor immune suppression microenvironment. Furthermore, Wnt2 has also been reported to be highly expressed in a variety of tumor tissues, including esophageal squamous carcinoma (Fu et al, gut.2010) pancreatic Cancer (Yu M et al, nature.2012), colorectal Cancer (Kramer N et al, oncogene.2017), gastric Cancer (Katoh M, Int joncol.2001), lung Cancer (Huang C et al, Am J Cancer res.2015), breast Cancer (Dale TC et al, Cancer res.1996), and the like. In conclusion, the deep understanding of the immune regulation mechanism of the secreted Wnt2 protein has important clinical significance for developing novel immunotherapy targeting Wnt2 high-expression tumors, and establishing effective measures for tumor risk assessment and prognosis judgment.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel Wnt2 monoclonal antibody, which, when binding to human Wnt2 antigen secreted by cells, antagonizes promotion of inhibitory immune microenvironment formation in tumors by cells, and inhibits immune escape and growth of tumor cells. The Wnt2 monoclonal antibody provided by the invention can be used for immunotherapy of solid tumors such as esophageal squamous cell carcinoma, gastric cancer, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, glioma and liver cancer.

The invention solves the technical problems by the following technical means:

in a first aspect of the invention, an EGFP-Wnt2 fusion protein antigen is provided, the EGFP-Wnt2 fusion protein antigen is mainly a fusion protein of EGFP and a full-length amino acid sequence of a human Wnt2 signal-removing peptide, and the EGFP-Wnt2 fusion protein antigen comprises an EGFP amino acid sequence, a TEV enzyme cutting site and a FLAG tag sequence, a Wnt2 signal-removing peptide full-length amino acid sequence and a 6 × His tag sequence.

Furthermore, the amino acid sequence of the EGFP-Wnt2 fusion protein antigen is shown in SEQ ID NO. 2.

In a second aspect of the invention, there is provided a nucleotide encoding the EGFP-Wnt2 fusion protein antigen according to the first aspect, wherein the nucleotide sequence is shown in SEQ ID No. 1.

In a third aspect of the invention, there is provided an expression vector comprising a nucleotide as described in the second aspect above.

In the fourth aspect of the invention, the preparation method of the EGFP-Wnt2 fusion protein antigen is provided, which comprises the following steps:

s1, fusing an EGFP gene and a human wild type Wnt2 gene from which ER signal peptides are removed, inserting a TEV enzyme cutting site and a FLAG label sequence between the two genes, adding an ER signal peptide of rat growth hormone at an amino terminal for secretory expression, connecting a 6 xHis label to a carboxyl terminal of Wnt2, adding a pair of restriction enzymes BamHI and SexA I at the front and rear ends of the generated sequence, constructing a CMV promoter downstream of a lentivirus expression vector pLV-Puro, and removing the original Puro gene to obtain a plasmid pCMV-EGFP-Wnt 2;

s2, co-transfecting pCMV-EGFP-Wnt2 and plasmids of pH1 and pH2 to a lentivirus packaging line cell 293V, culturing for 5 generations or more than 20 days to obtain cells stably expressing target proteins, under a fluorescence microscope, using a 50-microliter pipette to pick 25-30 EGFP-Wnt2/293 cell clones with different expression levels, respectively amplifying and culturing the cell clones, collecting supernatant, performing Wnt2 protein functional verification for promoting cell migration, amplifying and culturing functional clones, collecting supernatant, purifying by a nickel ion affinity chromatography gel column, and obtaining the fusion protein EGFP-Wnt2 fusion protein.

In the fifth aspect of the invention, a Wnt2 monoclonal antibody is provided, wherein the Wnt2 monoclonal antibody is an antibody capable of specifically binding to the EGFP-Wnt2 fusion protein antigen of the first aspect and the Wnt2 protein obtained by removing EGFP with TEV enzyme or EK enzyme, and the Wnt2 monoclonal antibody is used for inhibiting immune escape and growth of tumor cells.

The Wnt2 gene and the coding protein thereof provided by the invention have the functions of inducing DC precursor cells to express IDO1 protein, promoting the development and differentiation of inhibitory DC (IDO1+ CD11c +) cells, further reducing the immunocompetence of the DC cells, promoting the formation of Treg cells and inhibiting CD8+ T cells, and the Wnt2 monoclonal antibody prepared aiming at human Wnt2 can effectively reduce the application of the process of reversing.

Further, the Wnt2 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions with amino acid sequences as follows:

H-CDR1：GYTFTDFD，

H-CDR2：HPESDYT，

and H-CDR 3: TGDR;

the light chain variable region comprises three complementarity determining regions having amino acid sequences as follows:

L-CDR1：KSVSTSGYSY，

L-CDR2：LVS，

L-CDR3：QHIRELTR。

further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6 in the sequence table.

In the sixth aspect of the present invention, there is provided an isolated nucleotide molecule, wherein the nucleotide sequence encoding the variable region of the Wnt2 monoclonal antibody heavy chain as described in the fifth aspect is shown in SEQ ID NO. 3, and the nucleotide sequence encoding the variable region of the Wnt2 monoclonal antibody light chain as described in the fifth aspect is shown in SEQ ID NO. 5.

In a seventh aspect of the invention, there is provided a vector comprising a nucleotide molecule as described in the above sixth aspect.

In an eighth aspect of the present invention, there is provided a host cell comprising a vector according to the seventh aspect above or incorporating a nucleotide molecule as described in the sixth aspect above.

In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising (1) an effective amount of the Wnt2 monoclonal antibody, the Wnt2 monoclonal antibody-based binding protein or the antibody fragment as described in the above sixth aspect; and (2) an effective amount of a Wnt2 monoclonal antibody, a Wnt2 monoclonal antibody-based binding protein or antibody fragment in combination with a PD-1 antibody as described in the sixth aspect above; and (3) one or more pharmaceutically acceptable pharmaceutical excipients and/or pharmaceutically acceptable carriers.

In a tenth aspect of the present invention, there is provided an application of the Wnt2 monoclonal antibody in solid tumor immunotherapy, wherein the solid tumor is a tumor with high Wnt2 expression, and includes esophageal squamous carcinoma, gastric cancer, pancreatic cancer, colorectal cancer, lung cancer, breast cancer, glioma and liver cancer.

The Wnt2 gene and the encoded protein thereof provided by the invention are positively correlated with the inhibitory component of a tumor immune microenvironment and negatively correlated with the activating component, the Wnt2 monoclonal antibody blocks the secretion of the Wnt2 gene and the encoded protein thereof in cancer cells and tissues, and the proportion of Tregs is further reduced by reducing the formation of IDO1 positive inhibitory DC; in addition, the Wnt2 antibody pharmaceutical composition released in the bodies of the animals with the diseases can more effectively eliminate cancer tissues in the bodies of the animals, and has obvious treatment effect on the esophageal cancer. In addition, comparative experiments prove that compared with a control group, the Wnt2 antibody tissue can enhance the anti-tumor immune response in animals, has obvious anti-tumor immunotherapy effect, obviously increases the proportion of activated DC cells in the tumor tissue of C57 mice of a treatment group, and has obvious killing capacity of spleen lymphocytes of C57 mice of a Wnt2 antibody treatment group on tumor cells. The examples of the invention are sufficient to demonstrate: the Wnt2 antibody pharmaceutical composition prepared by aiming at Wnt2 protein secreted in a tumor microenvironment has the functions of relieving the immunosuppression state of the tumor microenvironment in vivo and reactivating the anti-tumor immune effect of an organism, thereby preventing or treating solid tumors and providing a new medicine and a method for the effective immunotherapy of the solid tumors.

Drawings

FIG. 1 shows the distribution of CAFs, FoxP3 and CD4 (FoxP3+/CD4+) which are both Wnt2 and Vimentin positive (Wnt2+/Vimentin +), and T cells, IFN-gamma and CD8+ positive (IFN-gamma + CD8+), in the tumor tissues of patients with esophageal squamous carcinoma;

FIG. 2 shows that in 50 cases of tumor tissues of esophageal cancer patients, the amount of Wnt2+/Vimentin + CAFs is positively correlated with the proportion of Tregs relative to CD4+ T cells (Treg/CD4+ T);

FIG. 3 is a graph showing that the amount of Wnt2+/Vimentin + CAFs is inversely related to the ratio of IFN-. gamma. + CD8+ T cells to CD8+ T cells (IFN-. gamma. + CD8+ T/CD8+ T) in the tumor tissues of 50 patients with esophageal cancer;

FIG. 4 is a diagram showing the identification result of the EGFP-Wnt2 fusion protein as a purified antigen;

FIG. 5 is a graph showing the results of measurement of serum titer of mice;

FIG. 6 is a SDS-PAGE identification of purified Wnt2 monoclonal antibody;

FIG. 7 is a schematic diagram showing the PCR result of the heavy chain light chain variable region gene of Wnt2 monoclonal antibody;

FIG. 8 shows the expression of Wnt2 in mCAFs and mEC-1 cells;

FIG. 9 shows that Wnt2 protein induces the expression of the immune negative regulator IDO1 in mouse DC precursor cells;

FIG. 10 is a graph showing the effect of Wnt2 antibody drug in inhibiting the formation of mouse inhibitory DC cells induced by secretory Wnt2 in vitro;

FIG. 11 is a graph showing the inhibitory effect of Wnt2 antibody drug on the activation function of mouse DC cells by alleviating secreted Wnt2 in vitro;

figure 12 is a graph of the effect of Wnt2 protein treated mouse DC cells on inducing Treg cell formation;

FIG. 13 is a graph showing the effect of Wnt2 protein-treated mouse DC cells on inhibiting the activation of CD8+ T cells;

figure 14 is a graph of the effect of Wnt2 antibody in vitro on inhibiting the formation of mouse suppressor T cells induced by secreted Wnt2 protein;

FIG. 15 is a graph of the in vivo treatment of Wnt2 antibody pharmaceutical composition increasing the proportion of killer T lymphocytes (IFN-. gamma. + CD8+ T) relative to CD8+ T cells in the spleen of mice;

figure 16 is an in vivo treatment of Wnt2 antibody pharmaceutical compositions to enhance the ability of mouse spleen lymphocytes to kill tumor cells.

Detailed Description

The invention will be described in detail below with reference to the following figures and specific examples:

the invention discloses an EGFP-Wnt2 fusion protein antigen, a Wnt2 monoclonal antibody and an application of the Wnt2 monoclonal antibody, and a person skilled in the art can realize the fusion protein antigen by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention. While the methods of making and using the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods of making and using the techniques described herein may be made and used without departing from the spirit and scope of the invention.

Some terms and abbreviations illustrate:

CAFs: tumor-associated fibroblasts (cancer-associated fibroblasts)

MDSC: bone marrow-derived suppressor cells (Myeloid-derived suppressor cells)

And (3) PCR: polymerase Chain Reaction (Polymerase Chain Reaction)

ELISA: enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay)

EGFP: enhanced Green Fluorescent Protein (Enhanced Green Fluorescent Protein)

Treg: regulatory T cells (Regulatory cells)

The molecular biological experimental techniques used in the following examples, including PCR amplification, plasmid extraction, plasmid transformation, DNA fragment ligation, enzyme digestion, gel electrophoresis, etc., are generally performed according to conventional methods, unless otherwise specified, and specifically refer to molecular cloning instruction (third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peyer et al, 2002, Beijing: science Press), or according to the conditions recommended by the manufacturers. The starting reagents and materials used in the following examples are commercially available. Wherein:

the following animals were used for the experiments: immunodeficient BALB/C4-5 week old nude mice (purchased from Experimental animals center, Guangdong province); normal immunity C57 mice (purchased from experimental animals center, guangdong province).

The following antibodies were used for the experiments: wnt2 monoclonal antibody, CD4 antibody, FoxP3 antibody, CD8 antibody, CD3 antibody, CD28 antibody, CD11c antibody (Abcam, UK), Vimentin antibody (Cell Signaling technology, USA), IFN-gamma antibody, TNF-alpha antibody (R & D, USA), PD-1 antibody (BioX Cell, USA), fluorescent mouse secondary antibody (Life Technologies, USA), fluorescent rabbit secondary antibody (Life Technologies, USA).

The following cell lines were used for the experiments: murine esophageal squamous carcinoma cell line mEC-1 (owned by itself), murine primary esophageal tumor fibroblast mCAFs (owned by itself).