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Humanized monoclonal antibody, its preparation method and its use

A monoclonal antibody, humanized technology, applied in biochemical equipment and methods, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of low production capacity of antibody drugs, ineffective treatment of patients or recurrence, etc., to make up for Drug resistance and ineffectiveness, improving the effect of tumor treatment, and enhancing the effect of affinity

Active Publication Date: 2022-07-12
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, its preparation method and its use
  • Humanized monoclonal antibody, its preparation method and its use
  • Humanized monoclonal antibody, its preparation method and its use

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0055] Example 1 Optimization of Nucleotide Sequences 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 sequences reported in the above literature were converted into nucleotide sequences and targeted for a series of parameters that may affect the expression of antibodies in mammalian cells: codon preference, GC content (i.e., guanine G and 4 bases in DNA). Cytosine C ratio), CpG islands (i.e., regions with high density of CpG dinucleotides in the genome), mRNA secondary structure, splice sites, pre-mature PolyA sites, internal Chi sites (in the genome) A short DNA fragment with an increased chance of homologous recombination near this site) or optimization of ribosome binding sites, RNA instability sequences, inverted repeats, and restriction sites that may interfere with cloning; At the same time,...

Embodiment 2

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

[0066] The host cell used is CHO-DG44Fut8- / - cell line, which is a fucose biallelic knockout (Fut8- / -) CHO-DG44 cell line, which was developed by Nanjing GenScript 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, that is, 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 expression and transfection of CHO-DG44 cells. pcDNA3.4-G418 contains the promoter CMV Promoter used for the light chain, the eukaryotic selection marker G418 tag and the prokaryotic selection tag Ampicilline. pcDNA3.4-DHFR contains the promoter CMVPromoter used for the heavy chain, the eukaryotic selection marker DHFR tag and the prokaryotic selection tag Ampicilline.

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

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Abstract

The present invention discloses an anti-human CD20 humanized monoclonal antibody, 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-human CD20 humanized monoclonal antibody The preparation method and medical use thereof. Wherein, the nucleic acid molecule encoding the antibody comprises the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 1 and the nucleotide sequence encoding the heavy chain as shown in SEQ ID NO: 2; The signal peptide and stop codon were designed on the two sequences. Due to the optimization of codons in the present invention, the transgenic CHO cells express a high expression level of anti-human CD20 humanized antibody; and the fermentation method provided by the present invention, especially after adding a fed batch medium, prolongs the cell growth time , improve the expression level, reduce the production cost, and obtain high-purity target protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly 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 antibody. Preparation method of CD20 humanized monoclonal antibody SH006-2 and medical use thereof. Background technique [0002] CD20 antigen is a hydrophobic transmembrane protein on pre-B cells and mature B lymphocytes, which has a very important regulatory effect on 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 lymphoma patients, chronic lymphoma patients and large B-cell lymphoma patients have high expression of CD20 antigen on the surface of B cells. Anti-CD20 monoclonal antibo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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