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Fully humanized anti-CD20 recombinant antibody

A recombinant antibody, fully human technology, applied in recombinant DNA technology, antibody mimic/scaffold, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of lack of Fc segment function

Active Publication Date: 2019-10-29
武汉雁达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After scFv and Fab antibodies combine with CD20 antigen, they trigger apoptosis to protect the body, but they do not have the function of Fc segment

Method used

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  • Fully humanized anti-CD20 recombinant antibody
  • Fully humanized anti-CD20 recombinant antibody
  • Fully humanized anti-CD20 recombinant antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Expression and Identification of CD20 Antigen

[0032] The CD20 eukaryotic expression vector was constructed to highly express CD20 protein in HEK293T cells.

[0033] Methods: Using the total RNA of human peripheral blood mononuclear cells (PBMC) as a template, the CD20 gene was amplified by reverse transcription PCR and inserted into the eukaryotic expression vector pCMV3-C-His. After preliminary verification by PCR, it was verified by sequencing. The recombinant plasmid was transiently transfected into HEK293T cells for protein expression, and the expression of CD20 protein was detected by ELISA and Western blot, and the expression conditions were optimized.

[0034] The specific experimental steps of this example have been disclosed in this document: "Shi Mei, Nian Siji, Gao Yan, Wu Yuchuan, Song Zhangyong, Wang Mingxue, Yuan Qing. High expression of soluble CD20 in HEK293T cells[J]. Cellular and Molecular Immunology Journal of Science, 2018, 34(10):870-8...

Embodiment 2

[0037] Example 2: Anti-CD20 single-chain antibody screening

[0038] 1. Biotinylation of CD20 protein

[0039] Take about 40 μl of 50 μg / ml CD20 protein (purchased from China Sino Biological Company) dissolved in 1×PBS for use; weigh 1 mg biotin biotin, add 160 μl ddH 2 O was dissolved, and 1 μl was added to the aforementioned 40 μl CD20 protein solution, and incubated on ice for 2 hours or at room temperature at 25°C for 1 hour. The biotinylated protein was added to the desalting column, and the biotinylated protein was collected by centrifugation at 1000g for 2 minutes and stored at -80°C.

[0040] 2. Phage library screening

[0041] Our laboratory has previously constructed a natural fully human phage antibody library with a library capacity of 2.5×10 8 . Take 5×10 11 TU scFv antibody library phages were blocked with blocking solution (6%BSA / 1×PBST) for 1h at room temperature; biotinylated CD20 protein was added and incubated at 37°C for 2h to form CD20 protein-specif...

Embodiment 3

[0048] Example 3. Construction and expression of anti-CD20 scFv966-Fc recombinant expression vector

[0049] 1. Construction of sp-scFv966-Fc / pcDNA3.1 recombinant expression vector

[0050] 1. PCR amplification of scFv fragments

[0051] The scFv966 fragment was obtained by PCR amplification with high-fidelity Pfu DNA polymerase using scFv966-F (containing ClaⅠ restriction site) as the upstream primer and scFv966-R as the downstream primer (containing XhoⅠ restriction site). The sequence of the upstream primer scFv966-F is: 5'-CCCATCGATATGGGCCCAGGTGCAGCT-3' (SEQ ID No: 4), and the sequence of the downstream primer scFv966-R is: 5'-CCGCTCGAGACCTAGGACGGTCAGCTTGG-3' (SEQ ID No: 5). The 20μl PCR reaction system is: 10×Pfu PCRBuffer 2μl, 2.5mM dNTPs 1.6μl (final concentration 200μM), scFv966-F (10μM) 1μl, scFv966-R (10μM) 1μl, 2.5U ClonedPfu DNApolymerase 0.4μl, scFv966 DNA template 1μl (50ng / μl), add ddH 2 0 to 20 μl. PCR amplification conditions: 94°C for 2min; 98°C for 30sec...

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Abstract

The invention discloses an anti-CD20 single chain antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID No:2 and a light chain variable region amino acid sequence as shown in SEQ ID No:3. The invention further discloses a fully humanized anti-CD20 recombinant antibody, which comprises the single chain antibody and a human antibody constant region Fc fragment, the amino acid sequence of the recombinant antibody is as shown in SEQ ID No:17. The invention further discloses an eukaryotic expression vector. The eukaryotic expression vector is obtained by inserting thesequence of the anti-CD20 singlechain antibody into a universal expression vector sp-Fc / pcDNA3.1, and the nucleic acid sequence of the universal expression vector is as shown in SEQ ID No:18. The invention further discloses a recombinant expression vector.The eukaryotic expression vector is used as a template, an amplification primer is used for amplifyinga fragment sp-scFv966-Fc, and the obtained amplified fragment is inserted into a pMH3 vector.

Description

technical field [0001] The invention relates to the technical field of antibody engineering, in particular to a fully human anti-CD20 recombinant antibody. Background technique [0002] In recent years, biomedicine has revolutionized the pharmaceutical industry, bringing new hope to patients who have failed traditional treatments or previously had no treatment options. The development of monoclonal antibody drugs has gone through four stages: murine monoclonal antibody, chimeric monoclonal antibody, humanized monoclonal antibody and fully human monoclonal antibody. The traditional production of monoclonal antibodies relies on the immunization of laboratory animals and subsequent screening of specific hybridoma cells. This method is time-consuming, laborious, and costly to obtain antibodies, and its effectiveness depends on the ability of the immune system to produce humoral immunity to potential antigens. Ability is limited by many conditions. The emergence of recombinant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/85C12N15/66
CPCC07K16/2887C07K2317/622C07K2317/734C07K2319/02C07K2319/30C12N15/66C12N15/85
Inventor 袁青年四季石梅叶迎春于红徐文峰
Owner 武汉雁达生物技术有限公司
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