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Optimized monoclonal antibody

A technology of antibody and species, applied in the biological field, can solve the problems of high culture cost, high cost of antibody drug expression, slow growth rate, etc.

Active Publication Date: 2009-06-03
SINO CELL TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the slow growth rate, high culture cost and low protein expression level of mammalian cells, the cost of antibody drug expression is very high.

Method used

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  • Optimized monoclonal antibody
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  • Optimized monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Optimization of light chain and heavy chain variable region genes of anti-CD20 monoclonal antibody

[0045] The original sequence of the anti-CD20 monoclonal antibody comes from IDEC's patent US.5,736,137, and its expressed gene is collected by our company. The variable region sequence of the antibody can also be obtained through oligonucleotide chain connection.

[0046] Host cells expressing glycosylated anti-CD20 monoclonal antibodies can be derived from various tissues, but vertebrate cells are preferred. Available cell lines include SV40-transformed monkey kidney CV1 cell line (ATCC CRL 1651), human embryonic kidney cells (293 or 293 in suspension culture) [Graham et al., J. Gen Virol. 1977 36:59], infant Hamster kidney cells (BHK-21 ATCC CCL 10), Chinese hamster ovary cells (CHO / dhfr - ATCCCRL 9096), monkey kidney cells (CV1 ATCC CRL90), African green monkey kidney cells (VERO-76 ATCCCRL 1587), human uterine cancer cells (HELA ATCC CCL2), canine kidney...

Embodiment 2

[0057] Example 2, Preparation of anti-CD20 monoclonal antibody gene

[0058] (1) Preparation of human κ light chain constant region and human IgG1 heavy chain constant region

[0059] Collect 5ml of fresh human whole blood and centrifuge at 300g for 5min to pellet red blood cells and white blood cells. White blood cell RNA was extracted according to the operation protocol of the classical total RNA isolation kit manual of BBI Company. The leukocyte mRNA was reverse-transcribed into cDNA according to the operation protocol of the MMLV first strand cDNA synthesis kit manual of BBI Company. The reaction system was 11 μl RNA (2.7 μg), 4 μl 5×reaction buffer, 1 μl RNase inhibitor (20U / μl), 2 μl dNTP mix (10 mmol / L), 1 μl M-MuLV reverse transcriptase, and the total reaction volume was 20 μl.

[0060] PCR primers were designed based on known human κ and IgG1 sequences. Using the reverse-transcribed cDNA as a template, the target gene was amplified by PCR.

[0061] Synthetic prime...

Embodiment 3

[0074] Example 3. Construction of Anti-CD20 Monoclonal Antibody Expression Vector

[0075] The expression of anti-CD20 monoclonal antibody gene in eukaryotic cells needs the help of an efficient eukaryotic expression vector to mediate the antibody gene into the host cell. These vectors need to contain signal sequence, origin of replication, one or more marker genes, enhancer sequence, promoter and transcription termination sequence. The present invention adopts the commercialized pIRESneo3 and pIREShyg (Clontech) as the basis, utilizes the principle of polycistronics that can be formed by IRES to optimize the expression vector, and weakens multiple marker genes, thereby screening anti-CD20 monoclonal antibody highly expressed Cell Cloning.

[0076] Using conventional molecular biology techniques well known to those skilled in the art, insert the CD20 monoclonal antibody heavy chain into the multiple cloning site region of the mammalian expression vector pIRESneo3d (obtained b...

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Abstract

The invention discloses an optimized method of nucleotide codon of eukaryotic expressive protein (including antibody), which also provides a variable area DNA molecule of the anti-CD20 monoclonal antibody of the optimized nucleotide codon. The invention also relates to an optimized mammal expressive carrier, which provides a more effective expressing method of anti-CD20 monoclonal antibody in the CHO cell.

Description

field of invention [0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to a vector highly expressed in mammalian cells, a technology for improving the expression of antibodies in mammalian cells through codon optimization, and a codon-optimized anti-CD20 monoclonal antibody Sequence, and high-efficiency expression of anti-CD20 monoclonal antibody in mammalian cells using an optimized expression system. Background of the invention [0002] Analysis of the coding codons of the expressed protein genes found that the coding codons of amino acids are not evenly used [Ikemura T.JMol Biol 1981, 151(3): 389; Grantham R et al., Nucleic Acids Res1981, 9(1) :43]. Codon usage can be divided into three levels, preferred codons, more preferred codons and non-preferred codons [Hooper SD et al., Nucleic Acids Res 2000, 28(18):3517]. In addition, lower organisms and higher organisms have different preferences for coding codons. It...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/85C12N15/13C12N15/65C12P21/08
Inventor 谢良志
Owner SINO CELL TECH INC