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Humanized modified anti-CD147 chimeric antibody hchab18 and its application

A chimeric antibody and humanized technology, applied in the biological field, can solve problems such as short half-life, lack of immunoglobulin, and blocking antibodies

Active Publication Date: 2016-06-08
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] However, numerous studies have shown that murine monoclonal antibodies have a relatively short half-life and, when used in humans, lack some of the basic functional properties of immunoglobulins, such as complement-dependent R cytotoxicity (ComplementDependentCytotoxicity, CDC) and antibody Dependent cell-mediated cytotoxicity (AntibodyDependentCellmediatedCytotoxicity, ADCC)
[0020] In addition, repeated injection of mouse-derived McAb into the human body will induce human antimouse antibody (human antimouse antibody, HAMA) reaction in patients, causing systemic allergic toxic reactions and blocking the efficacy of the antibody.

Method used

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  • Humanized modified anti-CD147 chimeric antibody hchab18 and its application
  • Humanized modified anti-CD147 chimeric antibody hchab18 and its application
  • Humanized modified anti-CD147 chimeric antibody hchab18 and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Cloning and sequencing of the light and heavy chain variable region genes of the mouse-derived anti-CD147 monoclonal antibody

[0046] According to the instructions of the TrizolReagent kit (Invitrogen Company), 2 × 10 6 Total RNA of hybridoma cells secreting anti-human CD147 monoclonal antibody HAb18 (patent number: 02114471.0). After identifying the quality of total RNA by 1% agarose electrophoresis, use PrimeScript TM RTreagentKit (TaKaRa) reverse transcription kit was used for reverse transcription to obtain cDNA of total RNA. According to the sequence information of the light and heavy chain variable region genes of HAb18 monoclonal antibody HAb18 published in the patent "anti-human liver cancer monoclonal antibody HAb18 light and heavy chain variable region genes and its application" (patent number: 02114471.0), the corresponding primers were designed and synthesized. Increase the light and heavy chain variable region genes of HAb18 monoclonal antibod...

Embodiment 2

[0049] Example 2: Optimization of VL and VH genes and construction of chimeric antibodies

[0050] After obtaining the VL and VH sequencing results. First, the light and heavy chain genes were modified using the codon bias of hamsters to solve the low protein expression caused by rare codons in CHO cells during translation. The principle is based on solving the problem of transporting amino acids during amino acid synthesis The tRNA bias, that is, the use of codons corresponding to more tRNA content, accelerates protein synthesis. According to the codon utilization table of the hamster, the codon bias in the VL and VH nucleotide sequences obtained in Example 1 is optimized to obtain the light, light, Amino acid sequence of heavy chain variable region and its nucleotide coding sequence.

[0051] Secondly, the oligonucleotide fragments of antibody light and heavy chain genes were artificially synthesized, and NheI and BamH1 restriction sites were synthesized at both ends of th...

Embodiment 3

[0053] Example 3: Transfection of CHO cells and screening of recombinant clones

[0054] The expression vector pQD-Hyg-GSeu-cHAb18 containing the humanized antibody gene constructed above was used to transform Escherichia coli DH-5α strain, and inoculated on 100ml LB medium for amplification.

[0055] The expression vector pQD-Hyg-GSeu-cHAb18 plasmid DNA was extracted according to the instructions of the kit using an ultrapure plasmid DNA purification kit (Qiagen).

[0056] The pQD-Hyg-GSeu-cHAb18 plasmid DNA was transfected into fucosyltransferase-deficient CHO cells (MAGE1.5 cells) by electroporation (patent: 200980145664.9).

[0057] The transfected MAGE1.5 cells were pressure-selected with a final concentration of 500ug / ml Hygromycin B (Invitrogen, HyB for short), and then L-Methionine Sulfoximine (L-MethionineSulfoximine, MSX) (Sigma) continued to carry out cell selection and culture; and ClonePixFL (Genentix, Inc) was used to perform multiple clone selections on cells t...

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Abstract

The invention provides a humanized modified anti-CD147 chimeric antibody HcHAb18 and its application. Specifically, on the basis of obtaining the hybridoma cell of anti-human CD147 monoclonal antibody and its antibody HAb18 gene, an anti-CD147 human-mouse chimeric antibody was prepared, and its light chain variable region has the expression shown in SEQ ID NO:1 The amino acid sequence of its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2. In specific host cells, fucose is not bound to N-acetylglucosamine in the reducing end of the sugar chain of the Fc segment of the antibody, sugar The type is mannose (MAN5), and the antibody has antibody-mediated cell-dependent cytotoxicity (ADCC). The present invention also includes the expression vector of the anti-CD147 chimeric antibody, the engineered cell line and its application as a cancer treatment drug.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a novel humanized modified anti-CD147 monoclonal chimeric antibody capable of inhibiting tumor growth and metastasis, and also relates to the amino acid sequence and nucleotide sequence encoding the antibody. The invention also includes the use of the antibody as a medicament for the treatment of cancer. Background of the invention [0002] CD147 molecule is a widely expressed cell transmembrane glycoprotein. In humans, CD147 consists of 269 amino acids, which can be divided into extracellular region, transmembrane region and intracellular region. The first 21 residues after N-terminal translation are signal peptides, 22-205 constitute the extracellular region, 206-229 are transmembrane regions with a typical leucine zipper structure, and C-terminal 230-269 is the intracellular region. [0003] CD147 is a member of the immunoglobulin superfamil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00
Inventor 陈志南张征张阳冯飞呼和牧仁杨向民
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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