CD20-resistant monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody and nucleic acid molecule technology, applied in the field of biomedicine, can solve the problems of high cost of drug treatment and difficult for patients to receive treatment, and achieve the effect of strong pharmacological effect and safe use.

Inactive Publication Date: 2013-01-09
张 爱晖
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the high cost of these drugs makes it difficult for many patients in our country to receive treatment

Method used

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  • CD20-resistant monoclonal antibody as well as preparation method and application thereof
  • CD20-resistant monoclonal antibody as well as preparation method and application thereof
  • CD20-resistant monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Preparation and Screening of Mouse Monoclonal Antibody

[0063] (1) Preparation of antigen The human B lymphocyte-specific membrane antigen CD20 gene was amplified from the human gene transcriptome by PCR. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The sequence is as follows:

[0064] GSP1-H, 5'-ACC TGG GTAGGT GCG GAC ACC ACT-3';

[0065] GSP2-H, 5'-CAC ACT TTC ACG TCAAGG ACA-3';

[0066] GSP3-H,5'-CTT GTC CAG GGA TCC TAG AGT-3';

[0067] GSP1-L,5'-TTG CAG TCC TGA TCA GGC CAA CT-3';

[0068] GSP2-L,5'-TGT CGT GCA CTG GCAACA ATC TT-3':

[0069] GSP3-L,5'-TTG TTC ATG AAG CTC AAG GCT GA-3'.

[0070] Using BamHI and Xho I double enzyme digestion, the gene fragment was connected into the prokaryotic expression vector pGEM-T (promega company product of the United States), PCR and double enzyme digestion identified positive clones and then sequenced. After the sequence was determined correctly, the plasmid was extracted. Prokaryotic...

Embodiment 2

[0088] Example 2 Humanization and Expression Purification of Mouse Monoclonal Antibody

[0089] (1) Humanization of mouse monoclonal antibody The amino acid sequence of the mouse antibody was compared with the amino acid sequence of the human antibody, and the structure of the consensus sequence of the human subclass with the highest homologous sequence was obtained, that is, the human light chain VLκ subgroup II ( VLκII) and heavy chain VH subgroup I (VHI), as humanized frameworks. According to the definition of kabat et al., CDR1:24-34, CDR2:50-56, CDR3:89-97 of the L chain; CDR1:26-35, CDR2:50-65, CDR3:95-102 of the H chain (sequences of proteins of immunological Interest, (5th), Public Health Service, National Institutes of Health, Bethesda, MD (1991). Utilize computer to construct the three-dimensional model of mouse monoclonal antibody VL-VH region (Carter etc., Proc.Natl.Acad. Sci. USA 89: 4285-4289 (1992) Eigenbrot. J. Mol. Biol.) 229: 969-995 (1993)), determining whi...

Embodiment 3

[0091] Example 3 Affinity Analysis of Humanized Monoclonal Antibody CD20

[0092] The affinity of monoclonal antibody 31-7, BD31-7 and Rituximab was determined by Scatchard method. Table 1 shows the results of the assay, and the experimental results show that the two monoclonal antibodies have strong binding affinity to VEGF.

[0093] Table 1 Monoclonal antibody affinity results

[0094]

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Abstract

The invention discloses a humanized CD20-resistant monoclonal antibody. A heavy chain variable region of the humanized CD20-resistant monoclonal antibody has an amino acid sequence as follows: VH: SEQ ID:NO.6; and a light chain variable region has an amino acid sequence as follows: VL:SEQ ID:NO.8. The invention further comprises an application of the monoclonal antibody to the aspects of researches as well as diagnosis and treatment. The invention provides the humanized CD20-resistant monoclonal antibody with high specificity. Compared with a positive medicament, the monoclonal antibody has the advantages of better medicinal effect, simple and convenient preparation process, lower cost, wide application range, and capability of meeting the social requirements, and supplies an accurate, easy, convenient and effective monitoring means for the prevention, treatment, early-stage screening and diagnosis work of cancers and eye diseases. Above all, the CD20-resistant monoclonal antibody positively adapts to the working requirements and the requirement of user-friendly service in the fields of modern biology, prevention and health care, clinical diagnosis and the like; the humanized CD20-resistant monoclonal antibody is researched and developed to provide a treatment medicament with higher cost performance for the medicament market in China; and the CD20-resistant monoclonal antibody has a very wide application prospect.

Description

technical field [0001] The invention belongs to the technical fields of biomedicine, monoclonal antibody technology, antibody humanization technology, etc., specifically relates to a monoclonal antibody and its preparation method and application, and more specifically relates to a monoclonal antibody for anti-CD20 Cloned antibody and its preparation method and use. Background technique [0002] The mortality rate of tumors ranks first in the mortality rate of various diseases in the world, and malignant tumors have become a killer that seriously affects human health and life. Non-Hodgkin's Lymphoma (NHL) is one of the common hematologic malignancies, and is the most common lymphoid system malignancy in clinical practice. It is caused by the malignant growth of B lymphocytes (B cells account for about 85%). Occurs in young adults. In recent years, the morbidity and mortality of NHL have been increasing year by year, seriously endangering human health. The treatment and dia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00A61P29/00A61P19/02A61P7/06A61P37/02C12P21/08
Inventor 张爱晖
Owner 张 爱晖
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