Human anti-CD26 antibody and applications thereof

A technology of CD26 and antibody, applied in the field of high-affinity fully human antibody preparation, to achieve the effect of inhibiting proliferation, invasion and metastasis

Active Publication Date: 2014-04-09
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the remaining few sequences of murine antibodies may still potentially trigger HAMA

Method used

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  • Human anti-CD26 antibody and applications thereof
  • Human anti-CD26 antibody and applications thereof
  • Human anti-CD26 antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of CD26 extracellular region protein

[0062] The purpose of this study is to transfect eukaryotic cells with recombinant plasmids, G418 resistance screening to obtain stable transfected cell lines, secrete and express CD26 extracellular domain protein, and separate and purify CD26 extracellular domain protein by affinity chromatography.

[0063]The CD26 extracellular region gene is a synthetic gene selected from the "Extracellular" region of amino acid 29-766 in the Uniprot: P27487 sequence. The CD26 extracellular region gene is connected into the plasmid p3XFLAG-CMV9, and the restriction sites are Hind III, Xba I, Construction of recombinant plasmids (J. Sambrook. Molecular Cloning Experiment Guide. Third Edition. Science Press. 2002.P68).

[0064] Lipofectamine LTX Reagent (purchased from Invitrogen) was used to transfer the recombinant plasmid containing CD26 extracellular region gene into HLF cells according to the instructions. According to ...

Embodiment 2

[0066] Example 2 Isolation of fully human anti-CD26 single chain antibody

[0067] The single-chain antibody is obtained from a human single-chain antibody phage display library by solid-phase affinity screening of the CD26 extracellular region protein. The human single-chain antibody phage display library was constructed by Jiangsu Zhonghong Bioengineering Pharmaceutical Research Institute Co., Ltd. The phage display library containing the heavy chain and light chain variable regions of antibodies produced by human cells was constructed from human peripheral blood lymphocytes. The first round of amplification was carried out by using primers specific to the antibody variable region genes, and the second round of amplification was repeated. The variable regions of the light and chain chains were connected with a flexible linker peptide gene to form a single-chain antibody gene, and the single-chain antibody gene was cloned into the plasmid pHEN2 to transform Escherichia coli TG...

Embodiment 3

[0072] Example 3 Soluble expression and isolation and purification of anti-CD26 single chain antibody

[0073] pHEN2 is a bifunctional phagemid vector with an amber stop codon (Amber) TAG between the expression detection tag (c-my tag) and the coat protein gene. If the phage is infected with an amber mutation (SupE) suppressor strain, such as TG1, the TAG codon is translated into glutamic acid, the sequence can be read through and translated, and the antibody fragment is fused to the coat protein p3 and expressed on the surface of the phage; when the phage is infected with a non-amber mutation suppressor For strains, such as HB2151, translation is terminated at TAG, and soluble antibody fragments can be obtained. The C-terminus of the antibody fragment has 6×His tag and c-myc tag, which is convenient for purification, detection and identification. ZHB-2cF9 single-chain antibody is soluble expressed in the periplasmic space of Escherichia coli, purified by high-osmotic method ...

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Abstract

The invention provides a high-affinity human antibody capable of being combined with the specificity of CD26, and a preparation method and applications thereof, belonging to the technical field of a genetically engineered antibody. CD26 is of a ubiquitously multifunctional II type transmembrane glycoprotein, has multiple biological functions, can act with various proteins such as ADA, CD45, FAP-alpha and the like. The invention provides an antibody or a segment thereof from a human, the antibody or segment thereof specifically combines the human CD26, preferably, specifically combines the CD26 extracellular region, the amino acid sequence of the antibody or the segment thereof comprises an amino acid sequence obtained by performing amino acid replacement or modification to the monoclonal antibody or segments thereof or conjugates of the segments in any one of six complementary decision regions containing a group of sequences: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 AND SEQ ID NO:8. The anti-CD26 single-chain antibody has high specificity combination with the CD26, and the proliferation and invasion and metastasis of tumor cells can be obviously inhibited.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering antibodies, and in particular relates to a new high-affinity fully human antibody capable of specifically binding to CD26, a preparation method and application thereof. Background technique [0002] CD26 is a ubiquitous multifunctional type II transmembrane glycoprotein with multiple biological functions and can also exist in plasma in a dissolved form. CD26 often exists in the form of homodimers, and its monomer contains 766 amino acids with a relative molecular mass of about 110kDa. Amino acid residues are divided into 5 parts from inside to outside: intracellular region (1-6), transmembrane region (7-28), highly glycosylated region (29-323), cysteine-rich region (324 ~551) and the C-terminal catalytic domain (552~766), the three-dimensional structure of CD26 molecule is closely related to its function. The C-terminal catalytic domain of CD26 exerts dipeptidyl peptidase IV (Dipepti...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21A61K39/395A61P35/00
CPCA61P35/00C07K16/2896C07K16/40C07K2317/21
Inventor 马永周雅琼高云霞黄粱敏徐春林陈晨王耀方
Owner ZONHON BIOPHARMA INST
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