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Humanized anti-Periostin monoclonal antibody as well as preparation method and application of humanized anti-Periostin monoclonal antibody

A monoclonal antibody and humanized technology, applied in the field of medicine, can solve the problems that hinder the survival and continuous benefit of colorectal cancer patients, achieve the effects of inhibiting growth and recurrence, easy operation, and simple preparation method

Inactive Publication Date: 2019-12-10
杨澜
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Targeted therapy resistance is the biggest barrier to sustained survival benefits in colorectal cancer patients

Method used

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  • Humanized anti-Periostin monoclonal antibody as well as preparation method and application of humanized anti-Periostin monoclonal antibody
  • Humanized anti-Periostin monoclonal antibody as well as preparation method and application of humanized anti-Periostin monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1: Preparation method of humanized anti-Periostin monoclonal antibody

[0116] The preparation method of the humanized anti-Periostin monoclonal antibody of the present embodiment comprises the following steps:

[0117] Step 1: Extract total RNA from mouse hybridoma cell LC-5A6

[0118] The mouse hybridoma cell LC-5A6 was purchased from the China General Microorganism Culture Collection and Management Center, the preservation number is CGMCCNo.10898, and it was stored in liquid nitrogen. Extract the total RNA of mouse hybridoma cell LC-5A6, operate according to the instructions of Trizol (Gibco Company), specifically: collect 1 × 10 7 Mouse monoclonal antibody hybridoma cells were centrifuged at 10,000 rpm for 1 min, and the supernatant was discarded. Add 1mL Trizol to fully dissolve the cells, let stand at room temperature for 3-5min, then add 0.2mL chloroform, mix upside down; centrifuge at 12,000rpm at 4°C for 10min, transfer the supernatant to a clean 1.5m...

Embodiment 2

[0147] Example 2: Construction of humanized antibody gene and expression vector of anti-Periostin mouse monoclonal antibody

[0148] Using the gene synthesis method, the framework region of the variable region gene of the anti-Periostin mouse monoclonal antibody was replaced with the framework region of the human antibody, and the humanized antibody variable region gene fragment of the anti-Periostin mouse monoclonal antibody was synthesized by the total synthesis method, and recombined Into the vector containing the regulatory sequence and the human antibody constant region gene, the complete gene of the anti-Periostin humanized antibody and the eukaryotic expression vector containing the gene were constructed.

[0149] Synthesis of the humanized antibody variable region gene of the anti-Periostin mouse monoclonal antibody: using the humanized 4D5 monoclonal antibody variable region as a template, the CDR region was replaced by the corresponding 3B8 monoclonal antibody CDR reg...

Embodiment 3

[0163] Example 3: Induced Expression in Cells (CHO Cells) of Humanized Anti-Periostin Antibody Expression Vector

[0164] Will contain 10% FBS, 0.03mmol / L hypoxanthine (H), 0.003mmol / L thymidine (T), 0.1mmol / L proline (Pro), 0.1mmol / L glycine (Gly), DMEM medium with 100U / mL penicillin and streptomycin and 2mmol / L glutamine was placed in 5% CO 2 , in a 37°C environment, recover and culture CHO-dhfr - cell. According to the ratio of 1:10, subculture once every 3-4 days. Using the gene transfection method, use Lipofect AMINE reagent (Gibco Company) for transfection, co-transfect the light and heavy chain expression vectors of the anti-Periostin humanized antibody into the cells, and use medium without H, T, and Gly Screening was carried out, and after the clones were formed, selection medium containing 200 μg / mL G418 (Gibco Company) was used for screening. As a result, 4 μg of light and heavy chain antibody gene expression vectors were transfected into CHO-dhfr- cells, and th...

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Abstract

The invention discloses a humanized anti-Periostin monoclonal antibody as well as a preparation method and application of the humanized anti-Periostin monoclonal antibody, and belongs to the technicalfield of medicines. The humanized anti-Periostin monoclonal antibody comprises a light chain complementary determination zone and a heavy chain complementary determination zone, wherein an amino acidsequence of the light chain complementary determination zone is shown in SEQ ID NO.1-3, and an amino acid sequence of the heavy chain complementary determination zone is shown in SEQ ID NO.4-6. The invention also discloses the preparation method and the application of the humanized anti-Periostin monoclonal antibody. Besides the specificity being combined with Periostin proteins and Periostin positive tumor cells, the humanized anti-Periostin monoclonal antibody provided by the invention also has the humanization property and also can reduce toxic side effects of human application.

Description

technical field [0001] The invention relates to a humanized anti-Periostin monoclonal antibody, its preparation method and application, and belongs to the technical field of medicine. Background technique [0002] Since the 1980s, tumors have gradually become the most malignant disease that threatens human health and life worldwide. In the past two decades, with the development of China's economy and society and the improvement of people's living standards, the incidence of malignant tumors in China has continued to rise. Chemotherapy, neoadjuvant chemotherapy and surgery are still the main strategies in the clinical treatment of most solid tumors. Since 2001, tumor molecular targeted therapy has opened up a new horizon for the treatment of solid tumors. Molecularly targeted cancer therapy usually uses monoclonal antibodies or small molecular substances to block specific signal transduction pathways or molecules in the pathogenesis of tumors, reducing side effects caused b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/85A61K39/395A61P35/00A61P15/14
CPCA61K2039/505A61P15/14A61P35/00A61K2039/812C07K16/18C07K2317/24C07K2317/565C07K2317/76C12N15/85C12N2800/107
Inventor 杨澜舒雄
Owner 杨澜
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