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Humanized anti-CD26 antibody and application thereof

A CD26, antibody technology, applied in the field of preparation, high-affinity fully human single-chain antibody, to achieve the effect of inhibiting invasion, enhancing the ability to inhibit the growth of tumor cells in vitro, and inhibiting proliferation

Active Publication Date: 2014-04-16
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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  • Humanized anti-CD26 antibody and application thereof
  • Humanized anti-CD26 antibody and application thereof
  • Humanized anti-CD26 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The preparation of embodiment 1 CD26 extracellular domain protein

[0064] 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.

[0065] 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).

[0066] 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. Accor...

Embodiment 2

[0068] Example 2 Isolation of Fully Human Anti-CD26 Single Chain Antibody

[0069] 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

[0074] Example 3 Soluble expression and separation and purification of anti-CD26 single chain antibody

[0075] 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. The ZHB-2dB3 single-chain antibody is soluble expressed in the periplasmic space of Escherichia coli, and the periplasmic protein...

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Abstract

The invention provides a novel high-affinity completely humanized antibody which can specifically bind with CD26 as well as a preparation method and application thereof and belongs to the technical field of genetic engineering antibodies. The CD26 is a ubiquitous multifunctional II type transmembrane glycoprotein, has various biological functions and can be interacted with various proteins such as ADA, CD45, FAP-alpha and the like. The invention provides the humanized antibody or a fragment thereof, wherein the antibody or the fragment thereof is capable of specifically binding with the human CD26, preferably the CD26 extracellular domain; the amino acid sequence of the antibody or the fragment thereof comprises an amino acid sequence containing a monoclonal antibody or a fragment thereof or a conjugate of the fragment in any one of 6 complementary determining regions in one of SED ID NO: 2, SED ID NO: 3, SED ID NO: 4, SED ID NO: 6, SED ID NO: 7, and SED ID NO: 8, or an amino acid sequence obtained through amino acid replacement or modification. The obtained anti-CD26 single-chain antibody provided by the invention can highly specifically bind with the CD26 and is simultaneously capable of obviously inhibiting the proliferation, the invasion and the metastasis of tumor cells.

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 single-chain 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 peptidas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21A61K39/395A61P35/00
Inventor 马永周雅琼高云霞黄梁敏徐春林陈晨王耀方
Owner ZONHON BIOPHARMA INST
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