FZD6-targeting neutralizing antibody capable of effectively inhibiting progress of gallbladder cancer

Abstract

The invention relates to the field of biological medicines, and discloses a preparation method and application of a neutralizing antibody capable of effectively inhibiting dryness of gallbladder cancer cells and progress of gallbladder cancer and remarkably improving the lifetime and prognosis of a patient. A gene sequence for coding the humanized anti-FZD6 monoclonal neutralizing antibody comprises a sequence of a heavy chain variable region and a sequence of a light chain variable region. The gene sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the gene sequence of the light chain variable region is as shown in SEQ ID NO: 2. The neutralizing antibody targets a non-classical Wnt pathway receptor FZD6, so that the activation of a Wnt / Ca < 2 + > signal pathway can be effectively inhibited, and the migration and growth of gallbladder tumor cells can be inhibited. Experimental detection proves that the FZD6 neutralizing antibody can effectively block the interaction between Wnt5a-FZD6-mediated tumor-related fibroblasts and gallbladder cancer cells, remodel the gallbladder cancer immunosuppressive tumor microenvironment and inhibit the dryness of gallbladder tumor cells and the migration and growth of gallbladder cancer cells, and can be applied to preparation of gallbladder cancer specific targeting drugs.

Description

Technical Field

[0001] The present invention relates to the field of targeted inhibitors, and in particular to a targeted inhibitor capable of effectively inhibiting the progression of gallbladder cancer, and its application and medicine. Background Art

[0002] Gallbladder cancer is a tumor originating from the mucosal epithelium of the gallbladder and cystic duct. It is the most common malignant tumor of the biliary system, ranking sixth among gastrointestinal tumors worldwide, and is highly prevalent in East Asia. The occurrence of gallbladder cancer is highly correlated with chronic inflammation of the bile duct and bile duct-related diseases. Among them, bile duct stones, gallbladder polypoid lesions, gallcysts, liver flukes, and Clostridium difficile infection are high-risk factors for the occurrence of gallbladder cancer. Gallbladder cancer has an insidious initial onset, strong heterogeneity, high malignancy, strong invasiveness, and is easy to metastasize and spread. Only about 10% to 20% of patients can be diagnosed early, and the 5-year survival rate of patients with advanced gallbladder cancer is only 5% to 15%.

[0003] At present, surgical resection is the most effective strategy for the treatment of gallbladder cancer, but the vast majority of patients have already developed metastasis when they seek medical treatment and lose the opportunity for surgery. Radiotherapy has a poor therapeutic effect on patients with distant metastasis and is only suitable for some patients with local advanced disease. Whether combined radiotherapy and chemotherapy are beneficial for patients with advanced gallbladder cancer remains controversial. Gemcitabine combined with platinum is the first-line chemotherapy regimen for the treatment of gallbladder cancer. Although it can improve patient prognosis, prolong overall survival and reduce recurrence to a certain extent, it is very easy to develop drug resistance. Although non-specific targeted inhibitors for VEGF and PD1 / PDL1 can inhibit tumors to a certain extent, the overall treatment situation is not ideal. Therefore, it is urgently necessary to study specific targeted inhibitors for gallbladder cancer progression and chemotherapy resistance through the biological behavior of gallbladder cancer and the molecular mechanism of tumor occurrence.

[0004] The Wnt pathway plays an important role in regulating stem cell self-renewal and cell fate. Under normal circumstances, the activation of the Wnt pathway is strictly controlled by some tumor suppressor genes. However, the occurrence of most tumors is often accompanied by the inactivation of tumor suppressor genes. The inactivation of tumor suppressor genes leads to the continuous activation of the Wnt pathway, which gives the tumor the ability to self-renew, but also leads to the occurrence of treatment resistance.

[0005] At present, preliminary studies have shown that the Wnt pathway plays a certain regulatory role in the development of gallbladder cancer, so FZD6 inhibitors are expected to become specific targeted inhibitors for the treatment of gallbladder cancer. Summary of the invention

[0006] The main purpose of the present invention is to solve the problem that the drugs for treating gallbladder cancer progression are limited in the prior art, resulting in short survival and poor prognosis of patients.

[0007] A neutralizing antibody against FZD6 that can effectively inhibit the progression of gallbladder cancer, the gene sequence of the antibody comprising a heavy chain variable region sequence and a light chain variable region sequence, the gene sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the gene sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0008] SEQ ID NO: 1:

[0009] YTMHWVKQSHGRSLYWIGGVNPNYGGSTYNQKFKGRATFTVDKSSSTAYMELRSLTSEDSAVYYCARWTPLRL

[0010] SEQ ID NO:2:

[0011] KVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVY

[0012] The neutralizing antibody is a neutralizing antibody targeting the wnt pathway receptor FZD6.

[0013] The neutralizing antibody is administered in an amount of 6-10 mg / kg animal body weight.

[0014] The neutralizing antibody targeting FDZ6 is Anti-FZD6 mAb.

[0015] The present invention also relates to the use of a targeted inhibitor that can effectively inhibit the progress of gallbladder cancer in the preparation of drugs for preventing and treating tumors.

[0016] The dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

[0017] The dosage of the drug is any therapeutically acceptable dosage.

[0018] A polynucleotide, wherein the nucleic acid molecule encodes the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

[0019] A drug for preventing or treating the progression of gallbladder cancer, comprising a targeted inhibitor and a pharmaceutically acceptable carrier.

[0020] The excellent effects of the present invention are:

[0021] The researchers of the present invention have found through research that FZD6 neutralizing antibodies have the effect of inhibiting the progression of gallbladder cancer. The application provides a preparation method and application of FZD6 neutralizing antibodies, and provides a new drug for treating gallbladder cancer. The study found that neutralizing antibodies against FZD6 have obvious efficacy in treating advanced gallbladder cancer, have broad application prospects, and have great promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The statistical graphs of FZD1-FZD10 gene expression (RT-qPCR) in gallbladder cancer cell lines and organoids;

[0023] Figure 2 Immunofluorescence detection of FZD6 expression in gallbladder cancer and adjacent tissues;

[0024] Figure 3 Photos and statistical graphs showing the changes in cell proliferation ability before and after FZD6 knockout in gallbladder cancer cell lines;

[0025] Figure 4 Photos and statistical graphs showing the changes in cell migration ability before and after FZD6 knockout in gallbladder cancer cell lines;

[0026] Figure 5 Photos, statistical graphs, and IHC staining results of the changes in tumor size in the PDOX model before and after FZD6 knockout in gallbladder cancer organoids;

[0027] Figure 6 The photos and statistical graphs show the changes in cell migration ability after treatment with neutralizing antibodies in gallbladder cancer cell lines;

[0028] Figure 7 The photos and statistical graphs of the changes in tumor size based on the in vivo efficacy experiment of the PDOX model; DETAILED DESCRIPTION

[0029] The terms "first", "second", "third", "fourth", etc. (if any) in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "including" or "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0030] In this embodiment, the FZD6 neutralizing antibody is an inhibitor targeting the Wnt pathway ligand FZD6.

[0031] FZD6 is the gene encoding the Wnt pathway receptor. It plays an important role in the activation of the non-classical Wnt pathway. The occurrence of tumors is often accompanied by excessive activation of the Wnt pathway.

[0032] Antibodies are proteins produced by the immune system that can specifically bind to a target molecule (antigen). A monoclonal antibody (mAb) is an antibody produced by a single cell clone and is highly specific and consistent.

[0033] The researchers of the present invention have found through research that Anti-FZD6 mAb has the efficacy of treating advanced gallbladder cancer. The application provides a new use for Anti-FZD6 mAb and provides a new drug for treating advanced gallbladder cancer. The study found that Anti-FZD6 mAb has obvious efficacy in treating advanced gallbladder cancer, has broad application prospects, and has great promotion and application value.

[0034] The technical research scheme and effect of the present invention are described in detail below in combination with the specific embodiments and the accompanying drawings. Experimental methods without specific conditions are usually based on conventional experimental scheme conditions, such as the conditions described in textbooks, experimental guides, and product manuals, or according to the conditions recommended by the manufacturer, which are mastered or easily obtained by researchers of related technologies in this field. The following embodiments are only preferred embodiments of the present invention and do not limit the present invention. For researchers of related technologies in this field, the present invention can have various conditions and schemes. Selection and optimization, any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0035] The following is the experimental part. RNA extraction and cDNA reverse transcription were performed on three gallbladder cancer cell lines and four gallbladder cancer organoids, and the expression of 10 Fzd proteins from FZD1 to FZD10 at the mRNA level was analyzed. According to the results of RT-qPCR experiments, FZD6 was relatively highly expressed in the three gallbladder cancer cell lines and four gallbladder cancer organoids, such as Figure 1 To further detect the protein expression of FZD6 in gallbladder cancer, immunofluorescence detection was performed on gallbladder cancer tissues and corresponding adjacent tissues. The results of immunofluorescence detection showed that gallbladder cancer tissues had higher FZD6 expression than adjacent tissues, as shown in Figure 2. Figure 2 The above experimental results show that FZD6 is relatively highly expressed in gallbladder tumors. Therefore, FZD6 may be associated with the poor prognosis of gallbladder cancer.

[0036] To further verify the above experimental conclusions, the FZD6 gene was knocked out in gallbladder cancer cell lines (NOZ, OCUG) and gallbladder cancer organoids (GBC17, GBC46) using CRISPR-Cas9 technology, and the knockout efficiency was verified by western blot and Sanger sequencing. Edu proliferation detection was performed on wild-type gallbladder cancer cell lines (NOZ-WT, OCUG-WT) and mutant gallbladder cancer cell lines (NOZ-sg1, NOZ-sg2, OCUG-sg1, OCUG-sg2). The results of the Edu proliferation experiment showed that the proliferation ability of mutant gallbladder cancer cell lines (NOZ-sg1, NOZ-sg2, OCUG-sg1, OCUG-sg2) was significantly reduced compared with the wild type. And this phenotype is also relatively stable in the Wnt conditioned medium group, such as Figure 3 shown.

[0037] In order to explore the difference in migration ability between wild-type gallbladder cancer cell lines (NOZ-WT, OCUG-WT) and mutant gallbladder cancer cell lines (NOZ-sg1, NOZ-sg2, OCUG-sg1, OCUG-sg2), Transwell migration experiments were performed on wild-type cell lines and mutant cell lines. The results of the migration experiment showed that the migration ability of mutant gallbladder cancer cell lines (NOZ-sg1, NOZ-sg2, OCUG-sg1, OCUG-sg2) was significantly reduced compared with the wild type. And this phenotype is also relatively stable in the Wnt conditioned medium group, such as Figure 4 shown.

[0038] The subcutaneous injection model has the advantages of simple modeling method and fast tumor formation, which can better simulate clinical characteristics. This animal model is widely used in tumor establishment and drug screening and testing. In this experiment, subcutaneous injection was used to establish a tumor model. To explore whether the knockout phenotype of FZD6 is stable in in vivo experiments, 8 8-week-old M-NSG female mice were randomly divided into two groups (WT group and KO group). Each mouse in the WT group was subcutaneously injected with 1.5×10 6 GBC17 wild-type organoids; KO group subcutaneously injected 1.5×10 6 GBC17 mutant organoids with a cell count were used to establish a PDOX model and observe the growth of subcutaneous tumors in mice. The results of the subcutaneous tumor formation experiment showed that the tumor volume in the KO group was significantly lower than that in the WT group, and the difference was statistically significant. Figure 5 As shown in Figure 2. IHC staining results showed that the expression of Ki67 in the WT group was significantly higher than that in the KO group. Figure 5 The above results show that FZD6 knockout can effectively inhibit gallbladder tumor growth both in vitro and in vivo experiments.

[0039] In order to explore whether FZD6 can be used as a specific drug target for gallbladder cancer in the field of tumor treatment, we prepared neutralizing antibodies against FZD6. Neutralizing antibodies can specifically recognize tumor cells, block tumor signaling pathways, activate immune responses, and can also be used in combination with other treatments to improve the overall efficacy. Therefore, neutralizing antibodies show good application prospects in tumor treatment and are gradually becoming an important means of cancer treatment. Mice were immunized with FZD6 immune enhancement plasmids to produce antibodies with high affinity for FZD6 protein. After B cells and myeloma cells were fused into hybridoma cells, hybridoma clones with high specificity were screened and amplified by two rounds of ELISA experiments. Finally, 16 monoclonal antibody supernatants were obtained. The binding ability of 16 antibodies was detected by flow cytometry, and 5 monoclonal antibody supernatants were finally selected for functional experiments. NOZ and OCUG wild-type gallbladder cancer cell lines were incubated with 5 monoclonal antibody supernatants, and Transwell migration experiments were performed. The results of the migration experiment showed that the migration ability of the two gallbladder cancer cell lines was significantly reduced after treatment with the supernatant of the five monoclonal antibodies, among which the difference was most obvious after treatment with supernatant No. 8. Figure 6 Therefore, we finally amplified the supernatant of monoclonal antibody No. 8 to obtain a recombinant monoclonal antibody against FZD6.

[0040] After testing, the amino acid sequence of the light chain variable region of the recombinant monoclonal antibody of FZD6 is SEQ ID NO: 1:

[0041] YTMHWVKQSHGRSLYWIGGVNPNYGGSTYNQKFKGRATFTVDKSSSTAYMELRSLTSEDSAVYYCARWTPLRL

[0042] Heavy chain variable region amino acid sequence SEQ ID NO:2:

[0043] KVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVY

[0044] To investigate the in vivo efficacy of FZD6 recombinant monoclonal antibody, 21 8-week-old female huPBMC-(M-NSG) mice were randomly divided into 7 groups, 3 mice in each group. They were divided into sh control-CAF+IgG group (group 1), sh control-CAF+FZD6 group (group 2), sh wnt5a-CAF+IgG group (group 3), sh wnt5a-CAF+FZD6 group (group 4), sh control-CAF+FZD6+PD1 group (group 5), sh control-CAF+FZD6+PKC group (group 6), and sh control-CAF+FZD6+PKC+PD1 group (group 7). GBC17 WT PDOX tumors were inoculated on day 0, and 5×10 4 The third and fourth groups were injected with wnt5a knockdown CAFs, and the rest of the groups were injected with control group CAFs. On the seventh day, when the tumor volume grew to about 70 mm 3 At about 14:00 p.m., all mice were treated by intraperitoneal injection. The first group of mice was given 8 mg / kg IgG; the second group of mice was given 8 mg / kg FZD6 neutralizing antibody; the third group of mice was given 8 mg / kg IgG; the fourth group of mice was given 8 mg / kg FZD6 neutralizing antibody; the fifth group of mice was given 8 mg / kg FZD6 neutralizing antibody + 8 mg / kg Teprexa; the sixth group of mice was given 8 mg / kg FZD6 neutralizing antibody + 5 mg / kg PKC inhibitor; the seventh group of mice was given 8 mg / kg FZD6 neutralizing antibody + 5 mg / kg PKC inhibitor + 8 mg / kg Teprexa. The administration was three times, once every three days. The results of the in vivo efficacy experiment showed that blocking the expression of Wnt5a in CAF or blocking the expression of tumor FZD6 by neutralizing antibody significantly reduced the tumor volume compared with the control group. When FZD6 neutralizing antibody + downstream molecule PKC inhibitor + Teprexa was used in combination, the tumor volume was significantly reduced, and the difference was statistically significant. The experimental results are as follows Figure 7 The above experimental results show that neutralizing antibodies against FZD6 can specifically block Wnt5a-FZD6 signals and inhibit the growth of gallbladder tumors; when FZD6 is used in combination with PD1 inhibitors and downstream pathway inhibitors, the therapeutic effect is more significant.

[0045] The FZD6 neutralizing antibody of the present invention can effectively inhibit the growth of gallbladder tumors. Experimental tests have shown that the inhibition of FZD6 can block the Wnt5a-FZD6-PKC signaling axis, thereby effectively inhibiting the growth of gallbladder cancer cells in vitro and in vivo, and can be used to prepare targeted drugs specific to gallbladder tumors.

[0046] As described above, the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-FZD6 monoclonal neutralizing antibody or an antigen-binding fragment thereof, characterized in that: The monoclonal neutralizing antibody or its antigen-binding fragment comprises a heavy chain variable region sequence and a light chain variable region sequence. The gene sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the gene sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. An anti-FZD6 monoclonal neutralizing antibody or an antigen-binding fragment thereof that can effectively inhibit the progression of gallbladder cancer, characterized in that: The neutralizing antibody is a neutralizing antibody against the Wnt pathway receptor FZD6.

3. The neutralizing antibody or antigen-binding fragment thereof according to claim 1, wherein the neutralizing antibody targeting FZD6 is administered at a dose of 6-10 mg / kg animal body weight.

4. The neutralizing antibody or antigen-binding fragment thereof according to claim 1, wherein the neutralizing antibody targeting FZD6 is Anti-FZD6 mAb.

5. Use of the targeted inhibitor that can effectively inhibit the progression of gallbladder cancer according to any one of claims 1 to 4 in the preparation of drugs for preventing and treating tumors.

6. The use according to claim 5, characterized in that The dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.

7. The use according to claim 5, characterized in that The dosage of the drug is any therapeutically acceptable dosage.

8. A polynucleotide, wherein the nucleic acid molecule encodes the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

9. A drug for preventing or treating the progression of gallbladder cancer, characterized in that: It comprises the monoclonal neutralizing antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

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