Humanization modified anti-CD147 chimeric antibody HcHAb18 and application thereof

A chimeric antibody, humanized technology, applied in the biological field, can solve the problems of blocking antibodies, short half-life, allergic and toxic reactions, etc.

Active Publication Date: 2014-10-08
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 lack some of the basic functional properties of immunoglobulins when used in humans, such as Complement Dependent Cytotoxicity (CDC) And antibody-dependent cell-mediated cytotoxicity (Antibody Dependent Cell mediated Cytotoxicity, ADCC)
[0020] In addition, repeated injections of mouse-derived McAbs into the human body will induce human anti mouse antibody (human anti mouse antibody, HAMA) reactions in patients, causing systemic allergic toxic reactions and blocking the efficacy of antibodies

Method used

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  • Humanization modified anti-CD147 chimeric antibody HcHAb18 and application thereof
  • Humanization modified anti-CD147 chimeric antibody HcHAb18 and application thereof
  • Humanization modified anti-CD147 chimeric antibody HcHAb18 and application thereof

Examples

Experimental program
Comparison scheme
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 Trizol Reagent 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 RT reagent Kit (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 ant...

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 was optimized to obtain the monoclonal as shown in SEQ ID NO:1 to SEQ ID NO:8 The amino acid sequence of the light and heavy chain variable regions of the antibody and its nucleotide coding sequence.

[0051] Secondly, the oligonucleotide fragments of antibody light and heavy chain genes were artificially synthesized, ...

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-Methionine Sulfoximine) with a final concentration of 25uM was used. , MSX) (Sigma) continued to perform cell selection and culture; and ClonePix FL (Genentix, Inc) was used ...

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Abstract

The invention provides a humanized modified anti-CD147 chimeric antibody HcHAb18 and an application thereof. The anti-CD147 human-rat chimeric antibody is prepared on the basis of obtaining hybridoma cells of an anti-human CD147 monoclonal antibody and antibody HcHAb18 genes of the hybridoma cells, wherein the variable region of light chains of the anti-CD147 human-rat chimeric antibody has the amino acid sequence shown in SEQ ID NO:1; the variable region of heavy chains of the anti-CD147 human-rat chimeric antibody has the amino acid sequence shown in SEQ ID NO:2; fucose are not combined in N-acetylglucosamine in a reducing end of a carbohydrate chain at a Fc segment of the anti-CD147 monoclonal antibody in a specific host cell; the glycoform of the anti-CD147 monoclonal antibody is of a mannose type (MAN 5); the anti-CD147 chimeric antibody has antibody dependent cell-mediated cytotoxicity (ADCC). The invention further provides an expression vector and an engineering cell strain of the anti-CD147 chimeric antibody and the application of the anti-CD147 chimeric antibody taken as a cancer treating medicine.

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 Applications(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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