Immunosuppressant-anti-CD25 mosaic monoclonal antibody

A monoclonal antibody, CDR2 technology, applied in the fields of genetic engineering and immunology, can solve problems such as rejection, loss of target antigen affinity and stability, and expensive treatment

Inactive Publication Date: 2008-07-02
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical trials have shown that monoclonal antibody drugs are more effective than chemical drugs, but due to the high production cost of monoclonal antibody drugs, the treatment cost is quite expensive
[0007] Moreover, there are some major problems in the existing monoclonal antibody products: mouse-derived monoclonal antibodies are heterologous proteins, and entering the human body will cause rejection in the body, and antibodies against mouse-derived monoclonal antibodies that require multiple repeated treatments can be It is rapidly cleared from the body, which is the biggest obstacle limiting the therapeutic clinical application of murine monoclonal antibodies
However, during the process of antibody engineering, humanized monoclonal antibodies usually lose their affinity and stability to the target antigen. At the same time, there are difficulties such as the difficulty in establishing production cell lines, and there has been a lack of effective solutions.
[0008] In addition, a monoclonal antibody usually targets a specific epitope, and in many cases a monoclonal antibody cannot completely inhibit the activity of the corresponding antigen

Method used

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  • Immunosuppressant-anti-CD25 mosaic monoclonal antibody
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  • Immunosuppressant-anti-CD25 mosaic monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Expression vector construction and transformation

[0084] 1. Acquisition of heavy chain variable region VH and light chain variable region VL of anti-CD25 monoclonal antibody

[0085] The steps for obtaining the heavy chain variable region VH and the light chain variable region VL of an anti-CD25 monoclonal antibody are as follows: figure 1 shown.

[0086] Human lymphocytes activated by phytohemagglutinin (PHA) were used to immunize LOU / C rats (purchased from the Experimental Animal Center of Chinese Academy of Medical Sciences), and then the splenocytes and non-secreting bone marrow cell line IR983F were used (see Bazin H (1982) Production of rat monoclonal antibodies with the LOU ratnon-secreting IR983F myeloma cell line. Protein Biol Fluids 29: 615-618) were fused to obtain hybridoma cells with anti-CD25 monoclonal antibody.

[0087] Total RNA (Gibco BRL. Gaithersburg. USA) was isolated from anti-CD25 monoclonal antibody-producing hybridomas using Trizol reagent a...

Embodiment 2

[0129] Selection of the best medium

[0130] One serum-free cell line recovered from the seed cell bank was inoculated into a T25 square bottle for cultivation. After 48 hours, the cells were inoculated into three T25 square bottles at a ratio of 1:3, and then the cells could be inoculated at a ratio of 1:3. Carry out expansion culture.

[0131] Cells were inoculated into different media at the same density, and the number and expression of cells were investigated after 48 hours. The media tested are listed in Table 2.

[0132] Table 2

[0133]

[0134] In Table 2, BD CHO medium was purchased from BD Company of the United States, Excell 620 medium was purchased from JRH Company, and Pramatone and DOMA were purchased from Life Technologies Company.

[0135] The experimental data showed that the cell growth rate and the expression level of the product reached the highest in the B+E+P medium, and the expression level of the antibody exceeded 100 mg / L. Therefore, B+E+P medi...

Embodiment 3

[0137] Study on High Density Serum-free Suspension Culture of Engineering Cells

[0138] The present inventor investigated the adaptation process of engineered cells producing anti-CD25 chimeric monoclonal antibody in BD CHO, BD CHO+excel 620+Pramatone, Excell 620+DOMA+Pramatone and Excell 620 medium, and the results are shown in Table 3 .

[0139] table 3

[0140] culture medium

[0141] To sum up, it is preferable to use BD CHO+Excel 620+Pramatone medium as the serum-free medium for cell expansion, expand and culture the cells in a 250ml spinner, obtain a certain amount of culture supernatant, and provide purification for the purification process Research, and obtain a certain amount of pure product to provide quality control for structural confirmation.

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Abstract

The invention discloses a monoclonal antibody resisting CD25 with special variable region of the light chain and variable region of heavy chain structure aiming at special CD25 epitope (IL-2 acceptor). The invention also discloses the coded sequence of the monoclonal antibody, the expression vector and host cell containing the coded sequence and the preparation method thereof. The monoclonal antibody of the invention has excellent immunosuppressive action and low immunogenicity and high specificity.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and immunology, more specifically, the present invention relates to an anti-CD25 chimeric monoclonal antibody and its coding sequence, an expression vector and a host cell comprising the coding sequence of the antibody, and the production of the antibody Methods. Background technique [0002] Organ transplantation is the most effective treatment for end-stage organ disease. Taking kidney transplantation as an example, allogeneic kidney transplantation is currently the most effective method for the treatment of end-stage renal disease. Kidney transplantation is the earliest large organ transplantation in China with the largest number of cases and the most mature technology. It has become a routine operation for the treatment of end-stage renal disease. In recent years, the number of organ transplant cases has been increasing. Take kidney transplantation as an example: By the end of 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P37/06
Inventor 黄阳滨张翊孙九如任军游磊顾小伟徐晓燕
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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