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 application relates to the field of targeted inhibitors, in particular to neutralizing antibodies targeting FZD6 which can effectively inhibit the progression of gallbladder cancer.

[0002] The present application claims priority to the Chinese patent application with application number 2024116855287, filed on November 23, 2024, and entitled "A neutralizing antibody targeting FZD6 which can effectively inhibit the progression of gallbladder cancer", the entire contents of which are incorporated herein by reference. BACKGROUND

[0003] Gallbladder cancer is a tumor originating from the mucosa of the gallbladder and cystic duct, and is the most common malignant tumor of the biliary system, ranking sixth in the world in terms of gastrointestinal tumors, and is highly prevalent in East Asia. The occurrence of gallbladder cancer is highly related to chronic inflammation of the biliary tract and biliary tract-related diseases, among which biliary stones, gallbladder polypoid lesions, gallbladder cysts, liver flukes, and Clostridium sphenoides infection are high-risk factors for the occurrence of gallbladder cancer. Gallbladder cancer has an initial insidious onset, strong heterogeneity, high malignancy, strong invasiveness, and easy metastasis and diffusion, and only about 10% to 20% of patients can be diagnosed at an early stage. The 5-year survival rate of patients with advanced gallbladder cancer is only 5% to 15%.

[0004] Currently, surgical resection is the most effective strategy for treating gallbladder cancer, but the vast majority of patients have metastasis at the time of diagnosis, losing the opportunity for surgery. Radiotherapy has poor therapeutic effect on patients with distant metastasis and is only suitable for some locally advanced patients. Whether combined radiotherapy and chemotherapy benefit patients with advanced gallbladder cancer is still controversial. Gemcitabine combined with platinum is a first-line chemotherapy regimen for gallbladder cancer, which can improve patient prognosis, prolong overall survival, and reduce recurrence to some extent, but is prone to drug resistance. Non-specific targeted inhibitors targeting VEGF and PD1 / PDL1 can inhibit tumors to some extent, but the overall treatment is not ideal. Therefore, it is urgent to study specific targeted inhibitors for the progression and chemotherapy resistance of gallbladder cancer through the biological behavior and molecular mechanisms of tumor occurrence of gallbladder cancer. Wnt pathway plays an important role in regulating stem cell self-renewal and cell fate. Under normal circumstances, the activation of Wnt pathway is strictly controlled by some tumor suppressor genes. The occurrence of most tumors is often accompanied by inactivation of tumor suppressor genes. Inactivation of tumor suppressor genes leads to persistent activation of Wnt pathway, which confers self-renewal ability to tumors and also leads to the occurrence of treatment resistance.

[0005] Currently, previous studies have shown that 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

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

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

[0008] SEQ ID NO: 1: DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVYYCSQSYNLWTFGGGTKLEIK SEQ ID NO: 2: EVQLQESGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGRSLYWIGGVNPNYGGSTYNQKFKGRATFTVDKSSSTAYMELRSLTSEDSAVYYCARWTPLRLYFDYWGQGTTLTVSS The neutralizing antibody is a neutralizing antibody targeting the wnt pathway receptor FZD6.

[0009] The administration amount of the neutralizing antibody is 6-10 mg / kg of animal body weight.

[0010] The neutralizing antibody targeting FDZ6 is Anti-FZD6 mAb.

[0011] The present application also relates to the use of a targeted inhibitor that can effectively inhibit the progression of gallbladder cancer in the preparation of a drug for preventing and treating tumors.

[0012] The dosage form of the drug is any pharmaceutically acceptable dosage form.

[0013] The dosage of the drug is any pharmaceutically acceptable dosage.

[0014] A polynucleotide, the nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2.

[0015] A drug for preventing or treating the progression of gallbladder cancer, comprising a targeted inhibitor and a pharmaceutically acceptable carrier.

[0016] The excellent effect of the present application is: Researchers of this invention have discovered that FZD6 neutralizing antibodies have the effect of inhibiting the progression of gallbladder cancer. This application provides a method for preparing and using FZD6 neutralizing antibodies, offering a novel drug for the treatment of gallbladder cancer. Studies have found that FZD6 neutralizing antibodies are significantly effective in treating advanced gallbladder cancer, showing broad application prospects and significant potential for widespread application. Attached Figure Description

[0017] Figure 1 Statistical graph of FZD1-FZD10 gene expression (RT-qPCR) and expression levels of key proteins in 3 gallbladder cancer cell lines and 4 gallbladder cancer organoids; Figure 2 Immunofluorescence detection of FZD6 expression levels in gallbladder cancer and adjacent non-cancerous tissues; Figure 3 Photographs and statistical graphs showing the changes in cell proliferation capacity before and after knockout of the FZD6 gallbladder cancer cell line; Figure 4 Photographs and statistical graphs showing the changes in cell migration ability before and after knockout of the FZD6 gallbladder cancer cell line; Figure 5 Photographs, statistical graphs, and IHC staining results of PDOX model of gallbladder carcinoma before and after FZD6 knockout; Figure 6 The figure shows the results of the in vitro co-culture experiment, illustrating the interaction between Wnt5a-FZD6 and CAF and gallbladder tumor cells; Figure 7 Photographs and statistical graphs showing the changes in cell migration ability of gallbladder cancer cell lines after treatment with neutralizing antibodies; Figure 8 The figure shows the results of in vitro experiments, demonstrating that the neutralizing antibody targeting FZD6 inhibits the growth of GBC organoids in vitro. Figure 9 These are photographs and statistical graphs showing the changes in tumor size in an in vivo drug efficacy experiment based on the PDOX model. Detailed Implementation

[0018] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0019] In this embodiment, the FZD6 neutralizing antibody is an inhibitor that targets the Wnt pathway ligand FZD6.

[0020] FZD6 is the gene encoding the Wnt pathway receptor. It plays an important role in the activation of the non-canonical Wnt pathway. Tumorigenesis is often accompanied by overactivation of the Wnt pathway.

[0021] Antibodies are proteins produced by the immune system that can specifically bind to target molecules (antigens). Monoclonal antibodies (mAbs) are antibodies produced from a single cell clone and have high specificity and consistency.

[0022] Researchers of this invention have discovered that Anti-FZD6 mAb is effective in treating advanced gallbladder cancer. This application provides a new use for Anti-FZD6 mAb and offers a novel drug for treating advanced gallbladder cancer. Studies have shown that Anti-FZD6 mAb has significant efficacy in treating advanced gallbladder cancer, demonstrating broad application prospects and considerable value for widespread application.

[0023] The technical research scheme and effects of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. Experimental methods without specific conditions are generally performed according to conventional experimental protocols, such as those described in textbooks, experimental guides, and product manuals, or according to the conditions recommended by the manufacturer, which are readily available or understood by researchers in the art. The following embodiments are merely preferred embodiments of the present invention and do not limit the invention. Researchers in the art can make various choices and optimizations regarding conditions and schemes. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0024] The following is the experimental section. RNA was extracted and cDNA was reverse transcribed from three gallbladder cancer cell lines and four gallbladder cancer organoids. The mRNA expression levels of the ten FZD proteins (FZD1-FZD10) were analyzed. According to the RT-qPCR results, FZD6 was relatively highly expressed in all three gallbladder cancer cell lines and four gallbladder cancer organoids. Figure 1 As shown. To further detect the protein expression level of FZD6 in gallbladder cancer, immunofluorescence was performed on gallbladder cancer tissue and corresponding adjacent normal tissue. The immunofluorescence results showed that gallbladder cancer tissue had higher FZD6 expression than adjacent normal tissue, such as... Figure 2 As shown in the figure, the above experimental results indicate that FZD6 is relatively highly expressed in gallbladder tumors. Therefore, FZD6 may be associated with poor prognosis in gallbladder cancer.

[0025] 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 assays were 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 Edu proliferation assay results showed that the mutant gallbladder cancer cell lines (NOZ-sg1, NOZ-sg2, OCUG-sg1, OCUG-sg2) had significantly reduced proliferation capacity compared to wild-type. This phenotype was also relatively stable in Wnt-conditioned medium. Figure 3 As shown.

[0026] To investigate the differences 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 assays were performed on both cell lines. The results showed that the mutant gallbladder cancer cell lines (NOZ-sg1, NOZ-sg2, OCUG-sg1, OCUG-sg2) exhibited significantly reduced migration ability compared to wild-type cells. This phenotype was also relatively stable in Wnt-conditioned medium. Figure 4 As shown.

[0027] Subcutaneous injection models have the advantages of simple modeling methods and rapid tumor formation, and can effectively simulate clinical characteristics. This animal model is widely used in tumor establishment and drug screening and detection. This experiment used subcutaneous injection to establish a tumor model. To investigate whether the FZD6 knockout phenotype is stable in vivo, eight 8-week-old M-NSG female mice were randomly divided into two groups (WT group and KO group). Each mouse in the WT group received a subcutaneous injection of 1.5 × 10⁻⁶ ppm. 6 GBC17 wild-type organoids with a cell count of 1.5 × 10⁶ cells per organoid; KO group: subcutaneous injection of 1.5 × 10⁶ cells per organoid. 6 A PDOX model was established using GBC17 mutant organoids with a cell count of [number missing] cells, and the growth of subcutaneous tumors in mice was observed. The results of the subcutaneous tumorigenesis experiment showed that the tumor volume in the KO group was significantly smaller than that in the WT group, and the difference was statistically significant. Figure 5 As shown in the figure. IHC staining results showed that the Ki67 expression level in the WT group was significantly higher than that in the KO group, as shown in the figure. Figure 5 As shown above, the results indicate that FZD6 knockout effectively inhibits gallbladder tumor growth in both in vitro and in vivo experiments.

[0028] To investigate the interaction between Wnt5a and FZD6 in vitro, we conducted co-culture experiments with primary isolated CAF and gallbladder cancer organoids GBC46-WT and GBC46-KO. The organoids were cultured alone in three groups: a complete culture medium group, an rWnt5a recombinant protein group, and a CAF condition medium group. The organoids were co-cultured with CAF in two groups: organoids co-cultured with CAF-WT and organoids co-cultured with CAF-shWnt5a. After six days of in vitro co-culture, the results showed that adding recombinant Wnt5a or CAF to the conditioned medium promoted organoid growth when culturing organoids alone. When WT CAF was co-cultured with WT organoids, WT-CAF promoted the growth of gallbladder cancer organoids (compared to the organoid-only culture group). However, regardless of whether Wnt5a was knocked down by CAF or FZD6 was knocked out by organoids, this effect on promoting gallbladder cancer organoid growth was significantly weakened upon re-co-culture. In vitro co-culture experiments strongly demonstrate that Wnt5a-FZD6 exhibits a strong interaction signaling between CAF and gallbladder tumor cells, and this interaction can significantly promote gallbladder cancer progression. See [link to relevant documentation]. Figure 6Adding rWnt5a or CAF CM to WT organoids promoted organoid growth. Co-culturing WT organoids with WT CAF also promoted organoid growth. However, the growth-promoting effect of CAF disappeared when WT organoids were co-cultured with CAF-shWnt5a. FZD6-KO organoids were insensitive to rWnt5a and CAF CM; when co-cultured with WT CAF, CAF slightly promoted organoid growth. However, the phenotype of FZD6-KO organoids co-cultured with CAF-shWnt5a was not significantly different from that of FZD6-KO organoids co-cultured with WT CAF.

[0029] To explore whether FZD6 can serve 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, and activate immune responses. They can also be used in combination with other treatment methods to improve overall efficacy. Therefore, neutralizing antibodies show promising application prospects in tumor treatment and are gradually becoming an important means of cancer therapy. Mice were immunized with an FZD6-enhancing plasmid to induce the production of antibodies with high affinity for the FZD6 protein. After fusing B cells with myeloma cells to form hybridoma cells, highly specific hybridoma clones were screened and amplified using two rounds of ELISA experiments. Sixteen monoclonal antibody supernatants were ultimately obtained. The binding ability of the 16 antibodies was detected by flow cytometry, and five monoclonal antibody supernatants were selected for functional experiments. NOZ and OCUG wild-type gallbladder cancer cell lines were incubated with the five monoclonal antibody supernatants, and Transwell migration assays were performed. Migration assay results showed that after treatment with the supernatants of the five monoclonal antibodies, the migration ability of both gallbladder cancer cell lines was significantly reduced, with the most significant difference observed after treatment with supernatant No. 8. Figure 7 As shown. Therefore, we finally amplified the supernatant of monoclonal antibody 8 to obtain a recombinant monoclonal antibody against FZD6.

[0030] The complete core sequence of the recombinant monoclonal antibody FZD6 was determined by testing and is as follows: The amino acid sequence of the light chain variable region is SEQ ID NO:1: DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVYYCSQSYNLWTFGGGTKLEIK Heavy chain variable region amino acid sequence SEQ ID NO:2: EVQLQESGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGRSLYWIGGVNPNYGGSTYNQKFKGRATFTVDKSSSTAYMELRSLTSEDSAVYYCARWTPLRLYFDYWGQGTTLTVSS To clarify whether FZD6-targeting neutralizing antibodies have a therapeutic effect on GBC, we first conducted in vitro experiments. We added different concentrations of neutralizing antibodies to two GBC organoids and supplemented the organoid culture medium with 30% Wnt5a conditioned medium to provide exogenous Wnt5a stimulation. On day six of organoid culture, we performed CTG analysis to determine the inhibitory effect and extent of the neutralizing antibodies. CTG results showed that 10 µM of neutralizing antibody inhibited the growth of both GBC organoids to varying degrees, and the inhibitory effect decreased with decreasing antibody concentration. This experiment confirms that FZD6-targeting neutralizing antibodies have a certain inhibitory effect on organoid growth in vitro. See [link to CTG analysis]. Figure 8 Different concentration gradients of neutralizing antibody and IgG control antibody were added to two GBC organoids, respectively. 30% Wnt5a conditioned medium was added to the organoid culture supernatant, and after six days of culture, CTG detection was performed. The FZD6-targeting neutralizing antibody inhibited the growth of GBC organoids to a certain extent.

[0031] To investigate the in vivo efficacy of the recombinant FZD6 monoclonal antibody, 21 eight-week-old female huPBMC-(M-NSG) mice were randomly divided into seven groups of three. The groups were: sh control-CAF+IgG (Group 1), sh control-CAF+FZD6 (Group 2), sh wnt5a-CAF+IgG (Group 3), sh wnt5a-CAF+FZD6 (Group 4), sh control-CAF+FZD6+PD1 (Group 5), sh control-CAF+FZD6+PKC (Group 6), and sh control-CAF+FZD6+PKC+PD1 (Group 7). On day 0, GBC17 WT PDOX was inoculated into tumors, and 5 × 10⁵ ppm was injected peritumorally. 4 Tumor-associated fibroblasts (CAFs) of a certain number of cells were injected, with groups 3 and 4 receiving Wnt5a-knockdown CAFs, and the remaining groups receiving control group CAFs. On day 7, when the tumor volume reached approximately 70 mm... 3At approximately 3:30 PM, all mice were treated with intraperitoneal injection. Group 1 received 8 mg / kg IgG; Group 2 received 8 mg / kg FZD6 neutralizing antibody; Group 3 received 8 mg / kg IgG; Group 4 received 8 mg / kg FZD6 neutralizing antibody; Group 5 received 8 mg / kg FZD6 neutralizing antibody + 8 mg / kg toripalimab; Group 6 received 8 mg / kg FZD6 neutralizing antibody + 5 mg / kg PKC inhibitor; and Group 7 received 8 mg / kg FZD6 neutralizing antibody + 5 mg / kg PKC inhibitor + 8 mg / kg toripalimab. The administration was repeated three times, every three days. In vivo efficacy studies showed that blocking Wnt5a expression in CAF or blocking FZD6 expression in tumors with neutralizing antibodies significantly reduced tumor volume compared to the control group. The combined use of FZD6 neutralizing antibody, downstream molecule PKC inhibitor, and toripalimab significantly reduced tumor volume, with statistically significant differences. Experimental results are as follows Figure 9 As shown above, the experimental results indicate that neutralizing antibodies against FZD6 can specifically block Wnt5a-FZD6 signaling and inhibit gallbladder tumor growth; the therapeutic effect is more significant when FZD6 is used in combination with PD1 inhibitors and downstream pathway inhibitors.

[0032] The FZD6 neutralizing antibody of this 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. It can be used to prepare targeted drugs specifically for gallbladder tumors.

[0033] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal neutralizing antibody against FZD6 or its antigen-binding fragment that can effectively inhibit the progression of gallbladder cancer, characterized in that, The monoclonal neutralizing antibody or its antigen-binding fragment includes 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. A monoclonal neutralizing antibody against FZD6 or its antigen-binding fragment that can effectively inhibit the progression of gallbladder cancer, characterized in that, The neutralizing antibody is a neutralizing antibody targeting the Wnt pathway receptor FZD6.

3. The neutralizing antibody or its antigen-binding fragment as described in claim 1, wherein the dosage of the neutralizing antibody targeting FZD6 is 6-10 mg / kg animal body weight.

4. The neutralizing antibody or its antigen-binding fragment as described in claim 1, wherein the neutralizing antibody targeting FZD6 is Anti-FZD6 mAb.

5. The use of the targeted inhibitor that can effectively inhibit the progression of gallbladder cancer as described in any one of claims 1-4 in the preparation of drugs for the prevention and treatment of tumors.

6. The application as described in claim 5, characterized in that, The dosage form of the drug is any pharmacologically acceptable dosage form.

7. The application as described in claim 5, characterized in that, The dosage of the drug is any pharmacologically acceptable dosage.

8. A polynucleotide, wherein the nucleic acid molecule encodes the monoclonal antibody or antigen-binding fragment thereof as described in 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 its antigen-binding fragment as described in any one of claims 1-4, and a pharmaceutically acceptable vector.

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