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Humanized monoclonal antibody, preparation method thereof, and application thereof

A monoclonal antibody, humanized technology, applied in biochemical equipment and methods, chemical instruments and methods, and botanical equipment and methods, etc., can solve the problems of low production capacity of antibody drugs, ineffective treatment of patients or recurrence, etc.

Active Publication Date: 2020-01-10
SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, a large number of clinical data have shown that for low-grade lymphoma, The curative effect of a single drug is about 50%, the other 50% of patients are ineffective, and 60% of patients who are initially effective after treatment are ineffective; for patients with diffuse large B-cell lymphoma with high malignancy and chronic lymphocytic leukemia with low CD20 expression, Even when combined with CHOP regimen (cyclophosphamide + doxorubicin + vincristine + prednisone), half or more patients still fail or relapse
[0005] In addition, the current production capacity of antibody drugs in the world is far lower than the demand

Method used

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  • Humanized monoclonal antibody, preparation method thereof, and application thereof
  • Humanized monoclonal antibody, preparation method thereof, and application thereof
  • Humanized monoclonal antibody, preparation method thereof, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1 Optimization of Nucleotide Sequence and Evaluation of Transient Expression

[0056] The amino acid sequences of the light chain and heavy chain of the monoclonal antibody of the present invention are from WHO Drug Information, Vol.22, No.2, 2008. The amino acid sequence reported in the above literature was converted into a nucleotide sequence, and a series of parameters that may affect the expression of the antibody in mammalian cells were targeted: codon bias, GC content (that is, guanine G and ratio of cytosine C), CpG island (that is, the region with high density of CpG dinucleotides in the genome), secondary structure of mRNA, splicing site, pre-mature PolyA site, internal Chi site (in the genome A short DNA fragment, the probability of homologous recombination increases near this site) or ribosome binding site, RNA unstable sequence, inverted repeat sequence and restriction enzyme site that may interfere with cloning, etc. At the same time, related seq...

Embodiment 2

[0065] Example 2 Construction of CHO-DG44 Fut8- / - cell line

[0066] The host cell used is CHO-DG44Fut8- / - cell line, which is the cell line of CHO-DG44 with biallelic fucose knockout (Fut8- / -), provided by Nanjing KingScript Biotechnology Co., Ltd. Company development. The specific method is to transform the expression system through genetic engineering technology, namely CRISPR / Cas9 technology, and knock out the gene responsible for encoding fucose, namely the FUT8 gene, in the antibody expression host cell CHO-DG44. Specific technical routes such as figure 1 shown.

Embodiment 3

[0067] The construction of embodiment 3 expression vector

[0068] The pcDNA3.4-G418 and pcDNA3.4-DHFR vectors (purchased from invitrogen) were used as special vectors for expressing and transfecting CHO-DG44 cells. pcDNA3.4-G418 contains the promoter CMV Promoter used for the light chain, the eukaryotic screening marker G418 tag and the prokaryotic screening tag Ampicilline. pcDNA3.4-DHFR contains the heavy chain promoter CMVPromoter, the eukaryotic screening marker DHFR tag and the prokaryotic screening tag Ampicilline.

[0069]The nucleotide sequences of SH006-2 antibody expressing light chain and heavy chain were obtained by gene synthesis, and connected to pcDNA3.1(+) and pcDNA3.1-Hygro(+) vectors (synthesized by Nanjing GenScript) respectively. Using the pcDNA3.1(+) vector containing the SH006-2 light chain and the pcDNA3.1-Hygro(+) vector containing the SH006-2 heavy chain as templates, two restriction endonucleases HindIII and XhoI were used for double-enzyme Diges...

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Abstract

The invention discloses an anti-human-CD20 humanized monoclonal antibody, a nucleic acid molecule encoding the same, a recombinant vector containing the nucleic acid molecule, a recombinant cell containing the recombinant vector, a preparation method and medical application of the anti-human CD20 humanized monoclonal antibody. The nucleic acid molecule for encoding the antibody comprises a nucleotide sequence, shown in SEQ ID NO: 1, for encoding a light chain, and a nucleotide sequence, shown in SEQ ID NO: 2, for encoding a heavy chain; and a signal peptide and a termination codon are designedat the two sequences respectively. According to the invention, because of optimization of the codon, the expression quantity of the anti-human-CD20 humanized antibody expressed by the transgenic CHOcell is high. Moreover, with the disclosed fermentation method, the cell growth time is prolonged, the expression level is improved, the production cost is reduced, and a high-purity target protein isobtained especially after adding of a fed-batch culture medium.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and in particular relates to an anti-CD20 humanized monoclonal antibody SH006-2, a nucleic acid molecule encoding the antibody, a recombinant vector comprising the nucleic acid molecule, a recombinant cell comprising the recombinant vector, and the anti-CD20 The preparation method of CD20 humanized monoclonal antibody SH006-2 and its medical application. Background technique [0002] CD20 antigen is a hydrophobic transmembrane protein on pre-B cells and mature B lymphocytes, which plays a very important role in regulating the proliferation and differentiation of B lymphocytes. CD20 is expressed during early pre-B cell development and persists until plasma cell differentiation. Studies have found that more than 90% of non-Hodgkin's lymphoma patients, chronic lymphoma patients and large B-cell lymphoma patients highly express CD20 antigen on the surface of B cells. Anti-CD20 monoclonal antibo...

Claims

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Application Information

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IPC IPC(8): C12N15/13C12N5/10C07K16/28A61K39/395A61P35/00A61P37/02A61P29/00A61P35/02A61P19/02A61P25/00G01N33/68G01N33/574
CPCC07K16/2887A61K39/395A61P35/00A61P37/02A61P29/00A61P35/02A61P19/02A61P25/00G01N33/6893G01N33/57426C07K2317/24C07K2317/732C07K2317/92C12N2800/22A61K2039/505G01N2333/70596
Inventor 不公告发明人
Owner SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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