An mRNA encoding HTRA1 protein antibody and its application

Abstract

The present invention relates to the field of biopharmaceuticals, and in particular to an mRNA encoding an HTRA1 protein antibody and its application. The present invention provides an mRNA encoding an HTRA1 protein antibody, comprising an mRNA encoding the heavy chain of the HTRA1 protein antibody as shown in SEQ ID No. 1 and an mRNA encoding the light chain of the HTRA1 protein antibody as shown in SEQ ID No. 2. Specific embodiments of the present invention used mRNA encoding the HTRA1 protein antibody to conduct in vitro experiments, demonstrating that the mRNA can inhibit the expression of HTRA1 protein in pancreatic cells, while limiting the proliferation, invasion, adhesion and migration of PANC-1 and SW1990 cells, and promoting the apoptosis of PANC-1 and SW1990 cells. It can be seen that the mRNA encoding the HTRA1 protein antibody provided by the present invention can be used for the treatment of cancer.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to mRNA encoding an HTRA1 protein antibody and applications thereof. Background Art

[0002] Pancreatic cancer (PC) is a malignant tumor with an extremely poor prognosis. Its 5-year survival rate is only 12%, so it is also called the king of cancer. It is estimated that in 2030, pancreatic cancer may become the second leading cause of cancer-related deaths. The pancreas is hidden in its location and has no specific symptoms in the early stages, making it easy to be overlooked. This increases the difficulty of early diagnosis of pancreatic cancer, and only 15%-20% of patients can undergo surgical resection at the initial diagnosis. However, due to the high malignancy of pancreatic cancer, the recurrence rate of the disease after surgery is as high as 85%, and these recurrent patients often show high resistance to radiotherapy and chemotherapy drugs. Therefore, standardized and reasonable drug treatment for pancreatic cancer patients undergoing surgical treatment is of great significance in improving the effectiveness of surgical treatment, prolonging patient survival, and improving quality of life.

[0003] HTRA1 (High Temperature Requirement Factor A1) is a heat-shock-induced, envelope-associated serine protease with dual chaperone and protease activities, capable of recognizing and degrading misfolded proteins in the cytoplasm. As a secreted protein, HTRA1 is involved in the degradation of the extracellular matrix. In addition to serving as a tumor marker and / or prognostic factor, HTRA1 has been implicated in tumorigenesis by regulating tumor cell proliferation, migration, apoptosis, and differentiation.

[0004] Antibody-based drugs have made rapid progress in the biopharmaceutical field, but the fragility of monoclonal antibodies and the high costs of production, storage, transportation, and distribution have limited their global application. Nucleic acid-encoded monoclonal antibodies, particularly those based on mRNA, hold great promise for improving the efficacy of antibody-based therapies. mRNA (messenger RNA) therapy harnesses the natural process of protein synthesis within cells for therapeutic purposes. By encoding immunostimulatory molecules or antigens, mRNA drugs can activate the immune system to target and destroy pathogens or cancer cells, thereby preventing and treating disease. mRNA therapy is a new type of gene therapy in which synthetic mRNA provides a template for the synthesis of any given protein, protein fragment, or peptide, facilitating a wide range of drug applications, including various modalities of cancer immunotherapy. mRNA therapy not only offers improved transfection efficiency and prolonged protein expression, but also offers significant advantages over DNA. Currently, there are no studies on mRNA drugs targeting HTRA1 for the treatment of pancreatic cancer. Summary of the Invention

[0005] The present invention aims to provide an mRNA encoding an HTRA1 protein antibody and its application to solve the problems existing in the above-mentioned prior art. The mRNA encoding an HTRA1 protein antibody provided by the present invention can inhibit the occurrence and development of pancreatic cancer by targeting the expression of HTRA1 in cells.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an mRNA encoding an HTRA1 protein antibody, wherein the mRNA includes an mRNA encoding an HTRA1 protein antibody heavy chain and an mRNA encoding an HTRA1 protein antibody light chain;

[0008] The sequence of the mRNA encoding the heavy chain of the HTRA1 protein antibody is shown as SEQ ID No. 1; the sequence of the mRNA encoding the light chain of the HTRA1 protein antibody is shown as SEQ ID No. 2.

[0009] The present invention provides a pharmaceutical composition comprising the above-mentioned mRNA and a delivery vector.

[0010] Preferably, the delivery vehicle comprises lipid nanoparticles.

[0011] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients;

[0012] The pharmaceutically acceptable excipients include one or more of fillers, diluents, binders, disintegrants, emulsifiers and drug carriers with no toxic or side effects.

[0013] Preferably, the dosage form of the pharmaceutical composition includes an injection preparation

[0014] Further preferably, the present invention provides use of the above-mentioned mRNA or the above-mentioned pharmaceutical composition in the preparation of mRNA drugs.

[0015] Further preferably, the present invention provides an mRNA drug, which comprises the above-mentioned mRNA.

[0016] Further preferably, the drug further comprises pharmaceutically acceptable excipients;

[0017] The pharmaceutically acceptable excipients include one or more of fillers, diluents, binders, disintegrants, emulsifiers and drug carriers with no toxic or side effects.

[0018] More preferably, the dosage form of the drug includes an injection preparation.

[0019] The present invention provides use of the above mRNA or the above pharmaceutical composition in preparing a drug for treating pancreatic cancer.

[0020] Preferably, the drug achieves the effect of treating pancreatic cancer by inhibiting the expression of HTRA1 protein.

[0021] The present invention provides a medicine for treating pancreatic cancer, which comprises the above-mentioned mRNA or the above-mentioned pharmaceutical composition.

[0022] Preferably, the drug further comprises pharmaceutically acceptable excipients;

[0023] The pharmaceutically acceptable excipients include one or more of fillers, diluents, binders, disintegrants, emulsifiers and drug carriers with no toxic or side effects.

[0024] Preferably, the dosage form of the drug includes injection.

[0025] The present invention discloses the following technical effects:

[0026] Specific examples of the present invention used mRNA encoding an antibody against the HTRA1 protein (Anti-HTRA1 mRNA) to conduct in vitro experiments, demonstrating that Anti-HTRA1 mRNA can inhibit HTRA1 protein expression in pancreatic cells, while also limiting the proliferation, invasion, adhesion, and migration of PANC-1 and SW1990 cells and promoting apoptosis of these cells. This demonstrates that the Anti-HTRA1 mRNA provided by the present invention can be used to treat cancer, particularly pancreatic cancer. This invention provides a basis for utilizing Anti-HTRA1 mRNA as a novel drug for treating pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Figure 2 is the result of Western blot detection of HTRA1 protein expression in PANC-1 and SW1990 cells; A is the Western blot detection image; B is the statistical graph of HTRA1 protein expression, data are expressed as mean ± SEM, compared with Ctrl, ** represents P < 0.01;

[0029] Figure 2The results of CCK8 detection of PANC-1 and SW1990 cell viability are shown in the figure; the data are expressed as mean ± SEM, compared with Ctrl, * represents P < 0.05, ** represents P < 0.01;

[0030] Figure 3 Figure 3 is the result of Transwell assay to detect the invasion ability of PANC-1 and SW1990 cells; A is the result of Transwell assay; B is the statistical graph of invasion rate in Transwell assay. Data are expressed as mean ± SEM. Compared with Ctrl, ** indicates P < 0.01.

[0031] Figure 4 Figure 3 is the result of adhesion assay for PANC-1 and SW1990 cells; A is the result of adhesion assay for PANC-1 cells; B is the result of adhesion assay for SW1990 cells; data are expressed as mean ± SEM, ** indicates P < 0.01 compared with Ctrl;

[0032] Figure 5 The migration ability of PANC-1 and SW1990 cells was detected by scratch wound assay; A is the result of scratch wound assay; B is the statistical graph of cell migration rate in scratch wound assay. Data are expressed as mean ± SEM. Compared with Ctrl, * represents P < 0.05, ** represents P < 0.01.

[0033] Figure 6 Figure 2 is the result of flow cytometry detection of cell apoptosis; A is the flow cytometry graph; B is the statistical graph of cell apoptosis percentage, data are expressed as mean ± SEM, compared with Ctrl, * represents P < 0.05, ** represents P < 0.01;

[0034] Figure 7 is the secondary structure diagram of IVT-mRNA of Anti-HTRA1 heavy chain;

[0035] Figure 8 This is the secondary structure diagram of the IVT-mRNA of the Anti-HTRA1 light chain. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Unless otherwise specified, the reagents used in the present invention can be purchased by those skilled in the art, and the methods used in the present invention are all well known to those skilled in the art.

[0042] Previous research by our research group has found that HTRA1 promotes the release of inflammatory factors, exacerbating acute pancreatitis, by inhibiting TGF-β-mediated anti-inflammatory responses. Further research has revealed that HTRA1 can promote the development of pancreatic cancer and is a key factor in its progression. Therefore, targeting HTRA1 for treatment may have a promising therapeutic effect in pancreatic cancer.

[0043] Example 1 Design and Synthesis of Anti-HTRA1 mRNA

[0044] (1) Obtain the CDS region of the Anti-HTRA1 mRNA sequence;

[0045] (2) Use AI algorithm to optimize the CDS region of Anti-HTRA1 mRNA sequence;

[0046]

[0047] Among them, the secondary structure of the IVT-mRNA of Anti-HTRA1 heavy chain is as follows Figure 7 As shown, the secondary structure of the IVT-mRNA of Anti-HTRA1 light chain is as follows Figure 8 shown.

[0048] Example 2 In vitro experiment

[0049] 1. Experimental methods:

[0050] In vitro transfection was performed using jetMESSENGER. The specific steps of transfection were as follows: PANC-1 or SW1990 cells were plated at 1×10 6 Cells were seeded at a density of 100 cells / mL in a 100 mm cell culture dish and cultured overnight to reach 70%-80% confluence.

[0051] The cell experiment was divided into two groups: a control group (Ctrl) and an experimental group (Anti-HTRA1 mRNA). After the cells reached 70%-80% confluency, 5μg of IVT-mRNA for the heavy chain of Anti-HTRA1 (SEQ ID No. 1) and 5μg of IVT-mRNA for the light chain of Anti-HTRA1 (SEQ ID No. 2) were added to 1000μL of mRNA buffer and mixed thoroughly by pipetting. Then, 20μL of jetMESSENGER reagent was added and mixed thoroughly by pipetting. The cells were allowed to stand at room temperature for 10 minutes to obtain the Anti-HTRA1 mRNA complex. JetMESSENGER transfection reagent was added to the Ctrl group, while the Anti-HTRA1 mRNA group was added to the Anti-HTRA1 mRNA complex. 5mL of DMEM high-glucose medium was added to the cell culture dishes in the Ctrl and Anti-HTRA1 mRNA groups, mixed thoroughly, and incubated in an incubator for 24 hours. Subsequently, the efficacy of the Anti-HTRA1 mRNA drug was verified by Western blot, CCK-8 cell proliferation assay, cell invasion assay, cell adhesion assay, and flow cytometry.

[0052] 2. Experimental results:

[0053] Western blot results are as follows Figure 1 After transfection of Anti-HTRA1 mRNA (IVT-mRNA for Anti-HTRA1 heavy chain and IVT-mRNA for Anti-HTRA1 light chain), the HTRA1 protein level in PANC-1 and SW1990 cells was significantly downregulated.

[0054] The CCK-8 assay was used to detect the effect of the drug on cell proliferation. The results showed that the cell viability of PANC-1 and SW1990 cells decreased 24h and 48h after transfection with Anti-HTRA1 mRNA, indicating that Anti-HTRA1 mRNA inhibited the cell viability of PANC-1 and SW1990 cells. Figure 2 ).

[0055] The results of Transwell, adhesion and scratch experiments also revealed this: when PANC-1 and SW1990 cells were transfected with Anti-HTRA1 mRNA, the number of invasive cells, cell adhesion activity and cell migration ability were significantly reduced ( Figure 3-Figure 5 ).

[0056] In addition, flow cytometry showed that the total cell ratios of Q2 and Q4 in Anti-HTRA1 mRNA treatment were significantly higher than those in the Ctrl group ( Figure 6 These results confirmed that Anti-HTRA1 mRNA could inhibit the proliferation, migration, invasion and adhesion of PANC-1 and SW1990 cells and promote cell apoptosis.

[0057] Example 3

[0058] Preparation of the drug containing Anti-HTRA1 mRNA: IVT-mRNA of Anti-HTRA1 heavy chain and IVT-mRNA of Anti-HTRA1 light chain are mixed with lipid (SNP) to obtain lipid nanoparticles (SNP-encapsulated lipid nanoparticles), namely, the drug containing Anti-HTRA1 mRNA.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition includes mRNA encoding HTRA1 protein antibody and a delivery vector; the mRNA includes mRNA encoding HTRA1 protein antibody heavy chain and mRNA encoding HTRA1 protein antibody light chain; The sequence of the mRNA encoding the heavy chain of the HTRA1 protein antibody is shown as SEQ ID No. 1; the sequence of the mRNA encoding the light chain of the HTRA1 protein antibody is shown as SEQ ID No.

2.

2. The pharmaceutical composition according to claim 1, characterized in that The delivery vehicle comprises lipid nanoparticles.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises pharmaceutically acceptable excipients; The pharmaceutically acceptable excipients include one or more of fillers, diluents, binders, disintegrants, emulsifiers and drug carriers with no toxic or side effects.

4. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the pharmaceutical composition includes an injection preparation.

5. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for treating pancreatic cancer.

6. The use according to claim 5, characterized in that The drug achieves the effect of treating pancreatic cancer by inhibiting the expression of HTRA1 protein.

7. A drug for treating pancreatic cancer, characterized in that: The medicine comprises the pharmaceutical composition according to claim 1 or claim 2.

8. The drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients; The pharmaceutically acceptable excipients include one or more of fillers, diluents, binders, disintegrants, emulsifiers and drug carriers with no toxic or side effects.

9. The drug according to claim 8, characterized in that The dosage form of the drug includes injection.

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