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Human anti-CD20 monoclonal antibody and preparation method and application thereof

A fully human, antibody technology, applied in the biological field, can solve the problems of easy recurrence or drug resistance, reduced efficacy, and difficult to cure.

Active Publication Date: 2014-08-20
SHENZHEN SCIPROGEN BIO PHARMA
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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 radiotherapy is performed again, the curative effect is significantly reduced, so they are considered as difficult-to-cure malignant tumors

Method used

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  • Human anti-CD20 monoclonal antibody and preparation method and application thereof
  • Human anti-CD20 monoclonal antibody and preparation method and application thereof
  • Human anti-CD20 monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0020] Example Preparation of Antibody

[0021] (1) Cloning of human antibody light and heavy chain constant region genes

[0022] Lymphocyte separation fluid (product of Dingguo Biotechnology Development Co., Ltd.) was used to separate healthy human lymphocytes, and total RNA was extracted with Trizol reagent (product of Invitrogen Company). Research, 1982, 10: 4071-4079) respectively designed primers using RT-PCR reaction to amplify the antibody heavy chain and light chain constant region genes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector (promega company product). After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO: 1 and SEQ ID NO: 2 show the nucleotide sequence and amino acid sequence of the heavy chain constant region (CH), respectively. SEQ ID NO: 3 and SEQ ID NO: 4 show the nucleotide sequence and amino acid sequence of the light chain constant region (CL), resp...

experiment example 1

[0035] Experimental Example 1. CD20 Antibody Affinity Detection

[0036] The affinity of CD20 antibody was determined by radioimmunoassay【Cragg MS, Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW, French RR, Glennie MJ(2003) Complement-mediated lysis by anti-CD20mAb correlates with segregation intolipid rafts .Blood 101(3):1045-1052]. Each CD20 antibody (fully human antibody 3C5; chimeric antibody C4E5; humanized antibody H9D3; rituximab (C2B8, Roche); negative control antibody Trastuzumab Genentech) was labeled by iodobead method. The 125-iodine-labeled antibody was incubated with Daudi (ATCC CCL-213) cells for 2 hours at 37°C. Cell-bound and free iodine-labeled antibodies were separated by centrifugation, and the radioactivity of 125-iodine-labeled antibodies bound to cells was detected. The affinity constants of each CD20 antibody were obtained by Hill equation curve fitting titration binding curve (see Table 1). The KD value of the fully human antibody 3C5 was 1.9...

experiment example 2

[0040] Experimental example 2. Complement-dependent killing effect (CDC) experiment of anti-CD20 antibody 3C5 on target cells

[0041] Anti-CD20 antibody can induce the killing effect of CD20 positive cells Raii, Namalwa and Daudi cells through the complement-dependent killing effect. Raji (ATCC CCL-86), Namalwa (ATCC CRL-1432) and Daudi (ATCC CCL-213) cells in the logarithmic growth phase were washed twice with PBS, and Raji, For Namalwa and Daudi cells, adjust the cell density to 2×10 5 / mL spare. Fully human anti-CD20 monoclonal antibody 3C5, humanized antibody H9D3, chimeric antibody C4E5, Rituximab (Roche) and Transtuzumab (Genentech) were diluted to a concentration of 10 μg / ml with phenol red-free RPMI-1640 culture medium, and For doubling serial dilution, the diluted samples were added to 96-well cell culture plates, 100 μl / well, and duplicate wells were set. For Raji, Namalwa and Daudi cell suspensions with adjusted densities, 5 μl of fresh human serum was added to ...

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Abstract

The invention relates to the technical field of biology and particularly discloses a human anti-CD20 monoclonal antibody and a preparation method and application thereof. In the preparation method, a large-capacity natural human phage antibody library is constructed and the antibody library is screened to obtain a human anti-CD20 antibody 3C5, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8. In addition, the invention also discloses a preparation method of the antibody 3C5, a nucleotide sequence coding the antibody 3C5 and an expression vector and host cell containing the nucleotide sequence. Compared with the other anti-CD20 antibodies, the human monoclonal antibody 3C5 has higher antibody affinity and ADCC and CDC activities; and the antibody 3C5 can be used to prepare anticancer medicaments.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, the invention discloses a fully human monoclonal antibody, its preparation method and application. 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 radiotherapy is performed again, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00A61P35/02
Inventor 王淑蕙李川聂丽
Owner SHENZHEN SCIPROGEN BIO PHARMA
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