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Anti CD20 tetravalent antibody, preparation method and uses thereof

A technology of antibodies and preparations, which is applied in the field of preparation of anti-B cell lymphoma drugs, and can solve the problems of short half-life and large molecular weight of dimer antibodies

Inactive Publication Date: 2008-06-25
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many defects in the way of producing dimers by chemical cross-linking, mainly because of the large molecular weight (300kDa) and short half-life of dimer antibodies [Wolff EA, Schreiber GJ, Cosand WL, Raff HV. Monoclonal antibody homodimers: enhanced Antitumor activity in nude mice. Cancer Res. 1993; 53: 2560-2565. Ghetie MA, Podar EM, Ilgen A, Gordon BE, Uhr JW, Vitetta ES. Homodimerization of tumor-reactive monoclonal antibodies markedly increases their ability to induce growth arrest orapo of tumor cells. Proc Natl Acad Sci USA. 1997; 94: 7509-7514.]

Method used

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  • Anti CD20 tetravalent antibody, preparation method and uses thereof
  • Anti CD20 tetravalent antibody, preparation method and uses thereof
  • Anti CD20 tetravalent antibody, preparation method and uses thereof

Examples

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Effect test

Embodiment 1

[0047] Example 1. Gene Construction of Anti-CD20 Single Chain Antibody

[0048] Referring to the anti-human CD20 monoclonal antibody data and sequence disclosed in US Patent No. 6,399,061, Shanghai Sangon Bioengineering Co., Ltd. was commissioned to synthesize the heavy chain variable region (V H ) and the light chain variable region (V L )Gene. SEQ ID NO: 1 and SEQ ID NO: 2 respectively show the nucleotide sequence and amino acid sequence of the heavy chain variable region of C2B8 mAb. SEQ ID NO: 3 and SEQ ID NO: 4 respectively show the nucleotide sequence and amino acid sequence of the light chain variable region of 2B8 mAb. The 3'-end of the 2B8 heavy chain variable region was passed through the Overlapping PCR method (Gly 4 Ser) 3 The linker was fused to the 5'-end of its light chain variable region, constituting the single-chain antibody gene C2B8 (ScFvHL). SEQ ID NO: 5 and SEQ ID NO: 6 show respectively (Gly 4 Ser) 3 Nucleotide and amino acid sequences of linkers....

Embodiment 2

[0050] Example 2. Cloning of Human Antibody Heavy Chain Constant Region Gene

[0051] Lymphocytes were isolated from healthy human lymphocytes with lymphocyte separation medium (product of Dingguo Biotechnology Development Co., Ltd.), and total RNA was extracted with Trizol reagent (product of Invitrogen Company). Design primer CH sense: GCT TCCACC AAG GGC CCA TC and primer CH antisense TTT ACC GGG AGA CAG PCR reaction to amplify the antibody heavy chain constant region gene. The PCR reaction adopts hot start, and the reaction conditions are: 94°C for 5 minutes; 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 1 minute and 10 seconds, 30 cycles; 72°C for 10 minutes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector. After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO: 11 and SEQ ID NO: 12 show the heavy chain constant region (C H ) nucleotide sequence and amino acid sequenc...

Embodiment 4

[0052] Example 4. Construction of anti-CD20 tetravalent antibody

[0053] pGEM-T / C H As a template, design primers Fc sense GAA TTC GCC GCT GCA GAG CCC AAATCT CCC GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA and Fc antisense TCT AGA TTTACC GGG AGA CAG GGA Amplify the Fc gene of human antibody IgG1 by PCR reaction , so that its 5' contains the restriction enzyme site EcoRI, the 3' end contains the restriction enzyme site XbaI, and the Hinge region in the Fc gene is changed to HingeM. SEQ ID NO: 13 and SEQ ID NO: 14 show the nucleotide sequence and amino acid sequence of Fc, respectively. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector, which was designated as pGEM-T(Fc). Positive clones were screened and sequenced. The correctly sequenced Fc gene was excised from the pGEMT vector with EcoRI and XbaI double enzyme digestion, cloned into pcDNA3.1(+) (product of Invitrogen Company), and constructed into pcDNA3.1(Fc) vector. ...

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Abstract

The invention discloses an anti- CD20 tetravalent antibody and a preparation method as well as application thereof, in particular the invention disclsoes an anti- CD20 tetravalent antibody C2B8(ScFvHL)4-Fc and 2F2(ScFvHL)4-Fc, and the preparation method as well as the use thereof in the preparation of drugs for inhibiting B-cell lymphoma.

Description

technical field [0001] The invention relates to the field of antibodies, and more specifically, the invention discloses an anti-quaternary antibody, its preparation method and its use in the preparation method of anti-B cell lymphoma medicine. Background technique [0002] Malignant tumor is a disease that seriously endangers human health in the world today, ranking second among the deaths caused by various diseases. Non-Hodgkin's lymphoma (NHL) is the most common malignant tumor of the lymphatic system in clinical practice, of which about 85% are derived from B cells, and it is more common in young adults, and its morbidity and mortality are increasing year by year. According to the characteristics of the disease, NHL can be divided into low, medium and high degrees. Among them, low-grade and some moderate-grade NHL progress slowly, collectively referred to as indolent NHL. They are sensitive to the first chemotherapy, but are prone to relapse or drug resistance. When che...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/46A61K39/395A61P35/00
Inventor 郭亚军李博华张大鹏钱卫珠侯盛王皓
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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