MRNA (messenger Ribonucleic Acid) for coding HTRA1 protein antibody and application of mRNA

Abstract

The invention relates to the field of biological pharmacy, in particular to mRNA (messenger Ribonucleic Acid) for coding an HTRA1 protein antibody and application of the mRNA. The invention provides an mRNA (messenger ribonucleic acid) for coding an HTRA1 protein antibody. The mRNA comprises an mRNA for coding a heavy chain of the HTRA1 protein antibody and an mRNA for coding a light chain of the HTRA1 protein antibody, wherein the sequence of the mRNA is as shown in SEQ ID No.1, and the sequence of the mRNA is as shown in SEQ ID No.2. According to the specific embodiment of the invention, an in-vitro experiment is carried out by adopting mRNA for coding an HTRA1 protein antibody, and the mRNA is proved to be capable of inhibiting the expression of HTRA1 protein in pancreatic cells, limiting the proliferation, invasion, adhesion and migration of PANC-1 and SW1990 cells and promoting the apoptosis of the PANC-1 and SW1990 cells. Therefore, the mRNA for coding the HTRA1 protein antibody provided by the invention can be used for treating cancers.

Description

Technical Field

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

[0002] Pancreatic cancer (PC) is a malignant tumor with a very poor prognosis. Its 5-year survival rate is only 12%, so it is also called the king of cancer. It is estimated that by 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 stage, so it is easy to be ignored, which increases the difficulty of early diagnosis of pancreatic cancer. At the initial diagnosis, only 15%-20% of patients can undergo surgical resection. 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 effect of surgical treatment, prolonging patient survival, and improving the quality of life.

[0003] HTRA1 (High Temperature Requirement Factor A1) is a heat shock-induced, envelope-associated serine protease that has dual activities of binding to molecular chaperones and proteases, and can recognize and degrade misfolded proteins in the cytoplasm. As a secreted protein, HTRA1 is associated with the degradation of the extracellular matrix. In addition to being a tumor marker and / or prognostic factor, HTRA1 is also associated with tumorigenesis by regulating tumor cell proliferation, migration, apoptosis, and differentiation.

[0004] Currently, antibody-based drugs have made rapid progress in the field of biopharmaceuticals, but the fragile nature of monoclonal antibodies and the high production, storage, transportation and distribution costs limit their application worldwide. Nucleic acid-encoded monoclonal antibodies, especially mRNA-based monoclonal antibodies, have brought great hope for improving the efficacy of antibody therapy. mRNA (messenger RNA) therapy uses the natural process of protein synthesis in cells for treatment. By encoding immunostimulatory molecules or antigens, mRNA drugs can activate the immune system to target and destroy pathogens or cancer cells, thereby achieving the purpose of preventing and treating diseases. mRNA therapy is a new type of gene therapy. Synthetic mRNA provides a template for the synthesis of any given protein, protein fragment or peptide, and facilitates a wide range of drug applications, including different modes of cancer immunotherapy. mRNA therapy not only mediates better transfection efficiency and longer protein expression time, but also has greater advantages over DNA. There is currently no research on mRNA drugs targeting HTRA1 for the treatment of pancreatic cancer. Summary of the invention

[0005] The purpose of the present invention is 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 in SEQ ID No.1; the sequence of the mRNA encoding the light chain of the HTRA1 protein antibody is shown in SEQ ID No.2.

[0009] The present invention provides a pharmaceutical composition, which comprises the above 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 auxiliary materials 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 mRNA or the above 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 auxiliary materials include one or more of fillers, diluents, binders, disintegrants, emulsifiers and drug carriers with no toxic or side effects.

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

[0019] The present invention provides the 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 mRNA or the above pharmaceutical composition.

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

[0023] The pharmaceutically acceptable auxiliary materials 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] The specific embodiment of the present invention uses mRNA encoding HTRA1 protein antibody (Anti-HTRA1 mRNA) to conduct in vitro experiments, proving that Anti-HTRA1 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 Anti-HTRA1 mRNA provided by the present invention can be used for the treatment of cancer, especially pancreatic cancer. The present invention provides a basis for using Anti-HTRA1 mRNA as a new 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0028] Figure 1 The results of Western blot detection of HTRA1 protein expression in PANC-1 and SW1990 cells are shown in Figure 1. A is a Western blot detection diagram; B is a statistical diagram of HTRA1 protein expression, and the 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 The results of the Transwell experiment to detect the invasion ability of PANC-1 and SW1990 cells; A is the result of the Transwell experiment; B is the statistical diagram of the invasion rate in the Transwell experiment, the data are expressed as mean ± SEM, compared with Ctrl, ** represents P < 0.01;

[0031] Figure 4 The results of adhesion experiment for detecting the adhesion ability of PANC-1 and SW1990 cells; A is the result of adhesion experiment for detecting the adhesion ability of PANC-1 cells; B is the result of adhesion experiment for detecting the adhesion ability of SW1990 cells; data are expressed as mean ± SEM, compared with Ctrl, ** represents P < 0.01;

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

[0033] Figure 6 The results of flow cytometry detection of cell apoptosis are shown in Figure 1. A is a flow cytometric graph; B is a statistical graph of cell apoptosis percentage, and the data are expressed as mean ± SEM. Compared with Ctrl, * represents P < 0.05, and ** represents P < 0.01.

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

[0035] Figure 8 This is the secondary structure diagram of IVT-mRNA of 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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] Our previous research found that HTRA1 promotes the release of inflammatory factors and aggravates acute pancreatitis by inhibiting the anti-inflammatory response mediated by TGF-β. Further research found that HTRA1 can promote the development of pancreatic cancer and is a key factor in the progression of pancreatic cancer. Therefore, targeting HTRA1 for treatment may have a better effect on pancreatic cancer.

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

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

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

[0046]

[0047] Among them, the secondary structure of 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] JetMESSENGER was used for in vitro transfection. The specific steps of transfection were as follows: PANC-1 or SW1990 cells were cultured at 1×10 6 The cells were inoculated at a density of 100 / 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 were fused to 70%-80%, 5μg of IVT-mRNA (SEQ ID No.1) of the heavy chain of Anti-HTRA1 and 5μg of IVT-mRNA (SEQ ID No.2) of the light chain of Anti-HTRA1 were added to 1000μL mRNA buffer and mixed by pipetting; then 20μL of jetMESSENGER reagent was added, mixed by pipetting, and allowed to stand at room temperature for 10 minutes to obtain the Anti-HTRA1 mRNA complex. Among them, jetMESSENGER transfection reagent was added to the Ctrl group, and Anti-HTRA1 mRNA group added Anti-HTRA1 mRNA complex. 5mL of DMEM high-glucose medium was added to the cell culture dishes of the Ctrl group and Anti-HTRA1 mRNA group, mixed and incubated in an incubator for 24h. Subsequently, Western blot, CCK-8 cell proliferation assay, cell invasion assay, cell adhesion assay and flow cytometry were used to verify the efficacy of the Anti-HTRA1 mRNA drug.

[0052] 2. Experimental results:

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

[0054] The CCK-8 method was used to detect the effect of drugs 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 drugs inhibited the cell viability of PANC-1 and SW1990 ( Figure 2 ).

[0055] The results of Transwell, adhesion and scratch experiments also revealed this: when Anti-HTRA1 mRNA was transfected into PANC-1 and SW1990 cells, 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 proportions 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An mRNA encoding an HTRA1 protein antibody, characterized in that: The mRNA includes mRNA encoding the heavy chain of the HTRA1 protein antibody and mRNA encoding the light chain of the HTRA1 protein antibody; The sequence of the mRNA encoding the heavy chain of the HTRA1 protein antibody is shown in SEQ ID No.1; the sequence of the mRNA encoding the light chain of the HTRA1 protein antibody is shown in SEQ ID No.

2.

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the mRNA of claim 1 and a delivery vector.

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

4. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients; The pharmaceutically acceptable auxiliary materials include one or more of fillers, diluents, binders, disintegrants, emulsifiers and drug carriers with no toxic or side effects.

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

6. Use of the mRNA according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 5 in the preparation of a drug for treating pancreatic cancer.

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

8. A drug for treating pancreatic cancer, characterized in that: The drug comprises the mRNA of claim 1 or the pharmaceutical composition of claim 2.

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

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

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