阅读说明：本技术 AP-2alpha抗体联合用药用于制备治疗***的药物中的用途 (Application of AP-2alpha antibody combined medicine in preparation of medicine for treating cervical cancer ) 是由 刘欢 朱小明 于 2020-07-02 设计创作，主要内容包括：本发明涉及AP-2alpha抗体与索拉非尼联合用药治疗宫颈癌的用途,其相比单独使用AP-2alpha抗体或索拉非尼,具有更好的治疗效果,两种药物产生了协调效应,有很好的临床使用前景。(The invention relates to an application of an AP-2alpha antibody and sorafenib combined drug in treating cervical cancer, which has a better treatment effect compared with the application of the AP-2alpha antibody or sorafenib alone, and the two drugs generate a coordination effect and have a good clinical application prospect.)

1. A combination, characterized by: consists of sorafenib and an AP-2alpha monoclonal antibody, wherein the AP-2alpha monoclonal antibody has a heavy chain variable region with an amino acid sequence of SEQ ID NO. 3 and a light chain variable region with an amino acid sequence of SEQ ID NO. 4.

2. The combination according to claim 1, wherein the amount of each drug in the pharmaceutical composition is 12mg/kg sorafenib and 5mg/kg of the AP-2alpha monoclonal antibody.

3. The application of the combination of sorafenib and AP-2alpha monoclonal antibody in preparing a medicament for treating cervical cancer; wherein the AP-2alpha monoclonal antibody has a heavy chain variable region with an amino acid sequence of SEQ ID NO. 3 and a light chain variable region with an amino acid sequence of SEQ ID NO. 4.

4. The use of claim 3, wherein the amount of each drug in the pharmaceutical composition is 12mg/kg of sorafenib and 5mg/kg of the AP-2alpha monoclonal antibody.

5. The use according to claim 4, wherein said anti-AP-2 alpha monoclonal antibody has a heavy chain type of IgG2 b; the light chain type is Kappa.

Technical Field

The invention relates to the field of antibodies, in particular to an AP-2alpha monoclonal antibody and application thereof in preparing a medicament for treating cervical cancer.

Background

Cervical cancer is an important disease threatening women. Statistically, there are about 50 million new cases and 24 million deaths worldwide each year. However, China is one of the high-incidence areas of cervical cancer in the world, and about 13.15 ten thousand new cervical cancer patients are newly added every year, and account for 28 percent of the total number of new cervical cancer cases in the world. With the widespread development of cervical cytology screening, the mortality rate of cervical cancer has decreased significantly in the united states for nearly 50 years. However, cervical cancer is a common cancer type in women because screening for cervical cancer in developing countries lags far behind in developed countries due to economic reasons.

The traditional treatment method for cervical cancer only has certain curative effect on early patients, has large treatment wound and cannot prevent HPV reinfection. Patients without HPV infection are best choice for vaccination with prophylactic vaccines, and early intervention and early treatment are particularly important for patients already infected with HPV. At present, no particularly effective medicine exists in the aspect of treating HPV infection, and serious side effects are often accompanied in the treatment process.

Over the past few decades, immunomodulatory antibodies targeting immune agonistic or inhibitory receptors have been able to enhance the anti-tumor immunity of the host, resulting in an effective therapeutic response. A general prerequisite for these therapies is that the cancer-bearing host has T cells that are specifically reactive to the tumor antigens present in its body, except that their function is inhibited by the tumor microenvironment. These immunomodulatory antibodies help to counteract this immunosuppression by increasing the expression and function of antigen presenting cells and T cells, ultimately leading to tumor regression.

In addition to the more mature therapeutic antibodies targeting the PD-1/PD-L1 pathway at present, many tumor necrosis factor receptor CTNFR) family members are also potential immunotherapeutic antibody targets, such as CD40, OX40 and 4-1BB, which are important co-stimulatory receptor molecules. It is well known that naive T cell activation requires strong T cell receptor-MHC antigen peptide interactions and the involvement of costimulatory molecules expressed by antigen presenting cells. In the absence of these costimulatory signals, activated naive T cells are either anergic or apoptotic; thus, costimulatory signals are essential for the response of effector T cells. Agonistic antibodies targeting CD40 and 4-1BB have received much attention due to their potential to stimulate activated T cells and enhance their clonal expansion and survival. Some studies have shown that therapeutic vaccines in combination with agonists of tumor necrosis factor receptor C TNFR family costimulatory molecules can induce effective and durable T cell responses, greatly facilitating the development of new combinatorial approaches to immune response.

By the effect of sorafenib and bevacizumab on mouse cervical cancer cell line U14, it is observed that the antibody drug combination can change the structure of transplanted tumor cells of cell line U14 and has the effect of inhibiting tumor growth. The S1 OOA 4-monoclonal antibody in combination with paclitaxel was found to have a considerable effect on the growth of Hela cells in vitro by tetramethylazotolonium (MTT) colorimetry and Flow Cytometry (FCM) detection.

AP-2 is an important transcription factor family, and research shows that AP-2 plays an important regulation role in cervical cancer. Among them, AP-2alpha plays an important role in cell growth and tumorigenesis (Cancer Res, 2004, 64: 631-638). The function of AP-2alpha is partly realized by binding with the promoter region of the gene downstream of AP-2alpha to promote the expression thereof. The oncogene ErbB2, which is widely overexpressed in cervical cancer, has been a target for therapeutic studies. Loss of function of ErbB2 in tumor cells will result in inhibition of cell growth and cause apoptosis. The recent research reports that the expression of the ErbB2 gene is down-regulated to promote the apoptosis of cervical cancer cells, thereby achieving the aim of treating the cervical cancer. Research shows that AP-2alpha is combined with the promoter of the oncogene ErbB2 gene to promote the transcription and protein expression of the ErbB2 gene, thereby showing the regulation and control function of AP-2alpha in the generation and development of cervical cancer.

CN 101705227A discloses that a biological database is used to obtain human AP-2alpha gene sequence, a group of siRNA capable of inducing RNA interference of human AP-2alpha is designed, a certain amount of siRNA is synthesized by a chemical method, thereby specifically reducing mRNA level and protein expression level of human AP-2alpha gene, and simultaneously the siRNA greatly reduces the activation of transcription level and protein level of transcription factor AP-2alpha to cancer gene ErbB2 in cervical cancer cell HeLa, thereby inhibiting the proliferation of tumor cell. However, monoclonal antibodies with high affinity specificity for AP-2alpha have been studied only rarely, and not much in combination.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an AP-2alpha immunogen fragment, the amino acid sequence of which is shown as SEQ ID NO:1, and the nucleotide sequence of which is shown as SEQ ID NO: 2, respectively.

The invention also provides an expression vector which comprises the nucleotide for coding the AP-2alpha immunogen fragment provided by the invention.

The invention also provides a host cell for transforming the expression vector.

The invention also provides a hybridoma cell strain for producing the anti-AP-2 alpha monoclonal antibody.

The invention also provides methods for producing the anti-AP-2 alpha monoclonal antibodies of the invention.

Furthermore, the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 3, and the light chain variable region is shown as SEQ ID NO. 4.

The preparation method of the anti-AP-2 alpha monoclonal antibody provided by the invention comprises the following steps:

step 1: after a mouse is immunized by the antigen provided by the invention, splenocytes of the mouse are obtained;

step 2: fusing the spleen cells and myeloma cells, screening hybridoma cell strains capable of being combined with AP-2alpha, and culturing in vitro to obtain the anti-AP-2 alpha monoclonal antibody.

The invention relates to a conjugate prepared by chemical labeling or biological labeling of the anti-AP-2 alpha monoclonal antibody.

The biomarker is a biotin, avidin, or enzyme label.

The invention relates to application of the anti-AP-2 alpha monoclonal antibody, the conjugate and/or the conjugate in preparing products for detecting AP-2alpha expression.

The invention also provides a kit comprising the anti-AP-2 alpha monoclonal antibody, a conjugate and/or a conjugate.

The invention relates to the application of the anti-AP-2 alpha monoclonal antibody in preparing the medicine for preventing and treating diseases; the disease is cervical cancer.

The invention also provides a medicament comprising the anti-AP-2 alpha monoclonal antibody.

A method for preventing and treating a disease, comprising administering the drug of the present invention; the disease is cervical cancer.

The invention further provides a pharmaceutical composition which consists of sorafenib and the AP-2alpha monoclonal antibody.

Furthermore, the dosage of each drug in the drug composition is 12mg/kg of sorafenib and 5mg/kg of AP-2alpha-3B6 monoclonal antibody.

Advantageous effects

The invention obtains the monoclonal AP-2alpha-3B6 with high affinity and specificity aiming at the AP-2alpha peptide segment by preparing the hybridoma cell, and has better effect of inhibiting the proliferation of the cervical cancer cell. Meanwhile, the combined administration finds that the sorafenib and AP-2alpha monoclonal antibody can have better effect of inhibiting cancer cell proliferation.

Drawings

FIG. 1 is a graph showing the results of purification of AP-2alpha protein, wherein lane 1 shows the results of purification by Ni column, and lane 2 shows the results of purification by blank vector control.

FIG. 2 is a graph showing the results of measurement of the titer of monoclonal antibodies

FIG. 3 is a graph showing the detection result of Western blot

FIG. 4 is a graph showing the effect of monoclonal antibodies on inhibiting cell proliferation

Detailed Description

The invention provides an anti-AP-2 alpha monoclonal antibody and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this 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 and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

An "antibody" refers to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are joined together and are responsible for binding to antigen, while the constant regions are responsible for the effector functions of the antibody.

"antibody" includes any isotype of antibody or immunoglobulin, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be labeled and detected, for example, by a radioisotope, an enzyme capable of producing a detectable substance, a fluorescent protein, biotin, or the like. The antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, and the like.