High-affinity CD20-resistance monoclonal antibody

A monoclonal antibody, affinity technology, applied in the field of genetic engineering products, can solve the problems of reduced curative effect, easy recurrence or drug resistance, difficult to cure, etc., and achieve obvious anti-tumor effect.

Active Publication Date: 2011-04-27
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

They are sensitive to the first chemotherapy, but they are prone to relapse or drug resistance. When chemotherapy or radiotherap

Method used

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  • High-affinity CD20-resistance monoclonal antibody
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  • High-affinity CD20-resistance monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Cloning of human antibody light and heavy chain constant region genes

[0040]Healthy human lymphocytes were isolated with lymphocyte separation solution (product of Dingguo Biotechnology Development Co., Ltd.), and total RNA was extracted with Trizol reagent (product of Invitrogen company). According to literature (Cloned human and mouse kappaimmunoglobulin constant and J region genes conserve homology in functional segments) . Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980 Nov; 22(1 Pt 1): 197-207.) and literature (The nucleotide sequence of a human immunoglobulin C gamma1 gene. Ellison JW, Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10; 10(13): 4071-9.) designed primers respectively to amplify antibody heavy chain and light chain constant region genes by RT-PCR reaction. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into pGEM-T vector (promega company). After sequencing and verification, it was c...

Embodiment 2

[0041] Example 2. Construction of expression vector of anti-CD20 chimeric antibody C2B8

[0042] Referring to the anti-CD20 monoclonal antibody data and sequence disclosed in US Patent 6,399,061, Shanghai Sangon Bioengineering Co., Ltd. was entrusted to fully synthesize the anti-CD20 monoclonal antibody C2B8 heavy chain variable region gene (C2B8VH) and light chain variable region gene (C2B8VL) . figure 1 The nucleotide and amino acid sequences of the C2B8 heavy and light chain variable regions are shown.

[0043] The chimeric antibody heavy chain gene was synthesized by overlapping PCR using the C2B8VH gene and pGEM-T / CH vector as templates. The reaction conditions were: 95°C for 15 minutes; 94°C for 50 seconds, 58°C for 50 seconds, and 72°C for 50 seconds, 30 cycles ; 72°C for 10 minutes. The 5' end of the chimeric antibody heavy chain gene contains the restriction enzyme site HindIII and the signal peptide gene sequence, and the 3' end contains the translation stop codon ...

Embodiment 3

[0045] Example 3. Stable expression and purification of chimeric antibodies

[0046] Inoculate 3×10 in a 3.5cm tissue culture dish 5 CHO-K1 cells (ATCC CRL-9618), transfection when the cells are cultured to 90%-95% confluence: take 10 μg of plasmid (plasmid pcDNA3.1(+)(C2B8VHCH) 4 μg, plasmid pcDNA3.1(C2B8VLCL) 6 μg) and 20 μl Lipofectamine2000 Reagent (product of Invitrogen) were dissolved in 500 μl serum-free DMEM medium, left at room temperature for 5 minutes, the above two liquids were mixed, and incubated at room temperature for 20 minutes to form DNA-liposome complexes. Serum-free DMEM medium replaces the serum-containing medium in the dish, then the formed DNA-liposome complexes are added to the plate, CO 2 After 4 hours in the incubator, add 2 ml of DMEM complete medium containing 10% serum, and place in CO. 2 continue to grow in the incubator. After 24 hours of transfection, the cells were changed to selection medium containing 600 μg / ml G418 to select resistant cl...

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Abstract

The invention discloses a high-affinity CD20-resistance monoclonal antibody and application thereof in preparing antineoplastic drugs. The antibody comprises amino acid substitutions on one or a plurality of amino acid sites of a C2B8 variable region of a heavy chain and / or a variable region of a light chain. Compared with Rituximab, the mutation antibodies have higher affinity activity, the antibody specificity is strengthened, and the lethal effect on the tumor is improved obviously.

Description

technical field [0001] The invention relates to the field of genetic engineering products, more specifically, the invention discloses a high-affinity anti-CD20 monoclonal antibody and its use in the preparation of anti-tumor drugs. 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 chemotherapy or...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61P35/00A61P35/02
Inventor 郭亚军李博华王皓侯盛赵磊
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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