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Ant CD20 chimeric antibody

A chimeric antibody and sequence listing technology, applied in the field of medical bioengineering, can solve problems such as clearance, achieve long half-life, improve clinical treatment effects, and reduce incidence.

Inactive Publication Date: 2006-12-20
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is very likely that the murine antibody is neutralized before binding to the target cells, and then cleared

Method used

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  • Ant CD20 chimeric antibody
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  • Ant CD20 chimeric antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Synthesis of antibody light and heavy chain variable regions

[0036] The sequences of the light and heavy chain variable regions were designed and synthesized as follows. This sequence is not only different from Rituximab in the CDR3 region, but also uses human gene sequences in the FR4 region, so the degree of humanization is higher than that of Rituximab. Portions different from Rituximab are underlined.

[0037] Light chain variable region coding sequence:

[0038] ATGGATTTTCAGGTGCAGATTATCAGCTTCCTGCTAATCAGTGCTTCAGTCATAATGTCCAGAGGAC

[0039] AAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTG

[0040] CAGGGCCAGCTCAAGTGTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACC

[0041] CTGGATTTATGCCACATCCAACCTGGCTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGG

[0042] ACTTCTTACTCTCTCACCATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGT

[0043] GG AGTTTT AACCCACCCACGTTCGGAGGGGGGACCAAG GTG GAAATCAAA

[0044] Heavy chain variable region coding sequenc...

Embodiment 2

[0070] Example 2. Transfer of light and heavy chain constant region genes and connection of variable region and constant region genes

[0071] According to the antibody κ and IgG1 gene sequences retrieved in GENEBANK, the primers L up, L down, H up and H down were designed and retrieved. Isolate healthy human lymphocytes according to conventional methods, extract RNA, and obtain light and heavy chain constant region sequences through RT-PCR amplification. After the above sequences are sequenced correctly, the variable region and the constant region are connected by overlap extension PCR to obtain the complete antibody gene.

[0072] Call primer: L: 5'-TTCGGAGGGGGGACCAAGGTG-3'

[0073] Lower L: 5'-TCAACACTTCTCCCCTGTTGAAG-3'

[0074] Upper H: 5'GGCCAAGGGACCACGGTCAC-3'

[0075] Lower H: 5'-TCATTTAGCCGGAGACAGGGAG-3'

[0076] RT-PCR reaction system: 2×reaction mix 25μl; RNA 1μl; 5’ primer 2μl; 3’ primer 1μl; RT / Taq enzyme 1μl; Mg 2+ (50mM) 1.8μl; ...

Embodiment 3

[0078] Example 3. Linking genes to cloning vectors

[0079] 1) Double digestion of light and heavy chain variable regions and PUC19 vector, reaction system: HindIII Xba I 2 μl each; 10×Buffer 5 μl; 0.1% BSA 5 μl; DNA 5 μl; water to 50 μl. 37°C water bath for 2h.

[0080] According to the instructions of the kit, the digested product was recovered using the Promega Wizard DNA clean-μp kit.

[0081] 2) Linking the variable region gene and pUC19 vector: T4 ligase 1 μl; 10×B μffer 2.5 μl; gene 5 μl; pUC19 2 μl; water to 25 μl. 16°C water bath, overnight.

[0082] 3) Ligate the constant region gene and pGEM-T Easy vector: 5 μl of 2×Rapid ligation Buffer; 1 μl of T Easy vector; 3 μl of gene fragment; 1 μl of T4DNA ligase; overnight at 4°C.

[0083] 4) Transformation: Transform the ligation product into Escherichia coli, perform blue-white screening, and identify recombinants.

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Abstract

A chimeric antibody for CD20 has human IgGl or K constant region and murine variable region. The amino acid sequence of light chain is shown by SEQ ID No.1. The amino acid sequence of heavy chain is shown by SEQ ID No.2. It can be used to prepare the medicines for treating lymphocyte cancer B. The CHO cell strain (CGMCC 1400) can express it.

Description

technical field [0001] The invention relates to an anti-CD20 chimeric antibody, its coding gene and a CHO cell line expressing the antibody, belonging to the field of medical bioengineering. Background technique [0002] Non-Hodgkin's lymphoma (NHL) is a group of malignant tumors of the lymphatic system with different histomorphology and biological characteristics. It is the most common hematological malignancy and the fastest growing malignant tumor One of the tumors, about 30,000 to 40,000 people die of the disease in my country every year. According to the prognosis classification, NHLs are divided into two groups, indolent lymphoma (indolent lymphoma) and aggressive lymphoma (aggressive lymphoma). Indolent lymphoma accounts for 25%-40% of NHLs, most of which are nodular or follicular in histology, with a good prognosis and a median survival period of up to 10 years. There is no complete cure. Aggressive lymphoma accounts for 60%-75% of NHLs, mainly including mantle cel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K19/00A61K39/395A61P35/00
Inventor 胡显文师明磊陈惠鹏高丽华李世崇冯立
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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