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Anti-CD 20 embedded antibody mutant gene and its use

A mutated gene and antibody technology, applied in the anti-CD20 chimeric antibody mutant gene and its application field, can solve the problems of expensive price, high cost, unacceptable to patients, etc. Effect

Inactive Publication Date: 2004-07-07
CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 3) CDs 20 Relatively exposed and not masked by other surface molecules and thus easily accessible
[0010] As therapeutic and diagnostic agents, CD 20 The production of antibodies is low. On the one hand, it cannot meet the needs of a large number of patients;

Method used

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  • Anti-CD 20 embedded antibody mutant gene and its use
  • Anti-CD 20 embedded antibody mutant gene and its use
  • Anti-CD 20 embedded antibody mutant gene and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Site-directed mutation of the gene of the light chain of the anti-CD20 antibody

[0021] anti-CD 20 The Fab expression vector pAYZcd20 (preserved in the laboratory of the inventor) was used as a template, and the following primers ① ② and primers ③ ④ were used to amplify the anti-CD 20 The genes before and after the light chain, and then use the Overlap method to amplify the two PCR fragments to obtain a complete anti-CD 20 For the light chain gene fragment, a mutated gene is obtained.

Embodiment 2

[0022] Embodiment 2, anti-CD 20 Construction of Fab antibody expression vector

[0023] Amplify anti-CD with the following primers⑤⑥ 20 heavy chain gene fragment. PCR amplified anti-CD 20 The light and heavy chain genes of the antibody were digested with MluI, NheI and NheI, ApaI respectively, and the pAYZ expression vector was digested with MluI and ApaI. Connect and transform Escherichia coli 16C9, and pick a single colony for identification.

[0024] Primers:

[0025] ①5′-GCTACAAACGCGTACGCTGACATCGAGCTC-3’

[0026] ②5'-ACTTGAGTTGGCCCTGCAAGT-3'

[0027] ③5'-ACTTGCAGGGCCAACTCAAGA-3'

[0028] ④5'-AACTTGCGTGCTAGCGCCAC-3'

[0029] ⑤5′-GCTACAAACGCGTACGCTCAGGTGAAGCTG-3′

[0030] ⑥5′-GACCGATGGGCCCTTGGTGGAGGCTGAGGAGACGGT-3′

Embodiment 3

[0031] Example 3, Expression and Purification of Anti-CD20 Fab Fragments

[0032] 3.1 Anti-CD 20 Expression and purification of antibody fragments

[0033] a. Pick the correct single colony, inoculate in 5ml 2×YT medium (containing 50ug / ml ampicillin), culture at 37°C with shaking at 200r / min until A 600 =0.7; the cells were collected by centrifugation, and the cells were resuspended in 20ml AP 5 Culture medium (containing 50ug / ml ampicillin), shake culture at 30°C for 24 hours; collect the cells by centrifugation, freeze the cells at -20°C for 1 hour, after thawing, add 1ml periosteum extract to break up the cells , shake gently at 4°C for 1 hour, centrifuge at 22,000g at 4°C for 20 minutes, and collect the supernatant.

[0034] b. The collected supernatant was separated and purified by Protein G affinity column.

[0035] c. 12% SDS-PAGE reduction electrophoresis analysis: the molecular weight of the expression product is about 25KD (see Figure 1).

[0036]It can be seen...

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Abstract

A light chain variable region gene resisting the CD 20 monoclonal antibody HI47 mutation, the polypeptide coded by said gene, and the application of the fusion protein containing said gene and polypeptide in preparing the medicines for diagnosing and treating tumor are disclosed. The purified expression product of said mutant has the activity to combine with Raji cells expressing CD20 antigen. Its expression product and activity are increased.

Description

Technical field: [0001] The present invention relates to anti-CD 20 The variable region gene of the light chain mutation of the monoclonal antibody, the polypeptide encoded by the gene, and the application of the gene and polypeptide containing the gene and polypeptide in the preparation of medicaments for the diagnosis and treatment of tumors. Specifically, the light chain variable region gene involved in the present invention is derived from anti-CD 20 Antibody HI 47 A mutant subtype of . Background technique: [0002] Malignant tumors are one of the key diseases in my country. B-lymphocytoma is a common hematological disease with an incidence rate of about 2 / 10,000. According to this estimate, there are more than 20,000 cases in my country every year. At present, the treatment methods for B lymphocytoma mainly include chemotherapy, radiotherapy and biological adjuvant therapy. Biological therapy as an adjuvant therapy is mainly used for chemotherapy replacement, combi...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P35/00C07H19/00C07H21/04C12N15/11C12N15/62G01N33/68
Inventor 杨纯正熊冬生刘银星许元富范冬梅彭晖邵晓枫许元生赖增祖朱祯平杨铭齐静王金宏纪庆王彩云
Owner CHINESE ACAD OF MEDICAL SCI
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