Bispecific antibody capable of targeting CLDN18.2, and preparation method and applications of bispecific antibody

A bispecific antibody, antibody technology, applied in the field of biomedicine, to achieve the effect of low cost

Pending Publication Date: 2020-08-11
L&L BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In previous research, we developed an anti-CLDN18.2 antibody (CN201810610790.3) and its drug conjugate (CN201811295845.2), however, the CLDN18.2 target has not yet entered the clinical or pre-clinical stage Developed bispecific antibody

Method used

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  • Bispecific antibody capable of targeting CLDN18.2, and preparation method and applications of bispecific antibody
  • Bispecific antibody capable of targeting CLDN18.2, and preparation method and applications of bispecific antibody
  • Bispecific antibody capable of targeting CLDN18.2, and preparation method and applications of bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Cloning, expression and purification of embodiment 1 antigen and antibody

[0143] Human CLDN18.2, PD-1, PD-L1 extracellular region-human IgG1Fc fusion protein, -his tag protein, monoclonal antibody, designed bispecific antibody with different structures, etc. used in the present invention are all cloned and expressed by the present invention Purified. Parts were purchased from Beijing Baipusaisi Biological Technology Co., Ltd. and Beijing Yiqiao Shenzhou Technology Co., Ltd. (Sino biological).

[0144] Antigen sequences were retrieved from the NCBI database. Antibodies used in the present invention include recombinant antibodies and bispecific antibodies. Except for the human CLDN18.2 antibody sequence discovered in the present invention, other sequences can be obtained from published literature, including anti-PD-1 antibody Nivo, Pem, Ba08; anti-PD-L1 antibody Atezo (Atezolizumab / Tecentriq), Avel (Avelumab / Bavencio), Durv (Durvalumab / imfinzi); anti-CD47 antibody hu...

Embodiment 2

[0156] Example 2 Anti-CLDN18.2 Antibody Binding ELISA Experiment

[0157] The human CLDN18.2 high-expression cell line used in the present invention is completed through the company's stable cell line construction platform. Specific steps are as follows:

[0158] On the first day of the experiment, 293T cells (Cat#GNHu17, a cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were seeded in two 6cm culture dishes, and the number of cells in each culture dish reached 7.5×10 5indivual. On the second day, the plasmids (pGag-pol, pVSV-G, pBabe, etc. BioVector, Plasmid Vector Strain Cell Gene Collection Center) and cloned human CLDN18.1 gene (NCBI Reference Sequence: NP_057453.1), human CLDN18.2 gene (NCBIReference Sequence: NP_001002026.1), mouse CLDN18.1 (NP_062789.1) or mouse CLDN18.2 (NP_001181850.1) gene plasmid pBabe-CLDN18.2 each 4 μg was added to OPTI-MEM (Thermofisher ScientificCat#31985070), so that The final volume is 200 μl, prepare ...

Embodiment 3

[0160] Example 3 Antigen-antibody binding ELISA experiment

[0161] Human PD-1, PD-L1, CD47, LAG3, Tim3 expressed in Example 1, or TGFβ1 (Cat#100-21-10), TGFβ2 (Cat#100- 35B), TGFβ3 (Cat#100-36E). Antigens such as IL10 (product number SEKA10947) purchased from Sino biological were diluted to a concentration of 2 μg / ml according to different assays, and added to a 96-well microtiter plate (Corning, CLS3590-100EA) at a volume of 50 μl / well, and incubated in a 37°C incubator Leave for 2 hours. After discarding the liquid, add 5% skimmed milk diluted with PBS (Shanghai Sangon Bioengineering Co., Ltd., A600669-0250) blocking solution 200 μl / well, incubate at 37°C for 3 hours or place at 4°C overnight (16-18 hours ) to close. Discard the blocking solution, wash the plate 5 times with PBST buffer (pH7.4PBS containing 0.05% tweeen-20), add 50 μl / well of 5-fold serial dilution of the antibody to be tested with 1% BSA, incubate at 37°C for 1 hour, PBST The plate was washed 5 times, ...

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Abstract

The invention discloses a bispecific antibody. The bispecific antibody includes a first protein functional area and a second protein functional area; the first protein functional area is a protein functional area that targetedly binds to CLDN18.2 antigen, and includes a variable region of a light chain and/or a variable region of a heavy chain; the second protein functional area is a protein functional area that targetedly binds to non-CLDN18.2 antigen; and the sequences of the variable region of the light chain and the variable region of the heavy chain of the first protein functional area are shown in the description. Isolated nucleic acid encoding the bispecific antibody, a vector containing the isolated nucleic acid, a host cell containing the vector, and a preparation method and applications of the bispecific antibody are also disclosed. The bispecific antibody retains the binding activity and functional activity of double targets; and more convenient drug development options canbe brought for the combination of tumor immunotherapy and targeted therapy.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a bispecific antibody targeting CLDN18.2 and its preparation method and application. Background technique [0002] Cell junction claudin (Claudin or CLDN) is expressed in human, mouse and other species. It is an intercellular layer seal-associated protein and plays an important role in controlling ion flow between cell layers, maintaining cell polarity and intercellular signal transmission. As many as 29 kinds of CLDN family proteins have been discovered, and CLDN18 is one of them. CLDN18 has two homologous molecules called claudin 18.1 (CLDN18.1) and claudin 18.2 (CLDN18.2). Human claudin 18.1 (hCLDN18.1) and human claudin 18.2 (hCLDN18.2) are highly homologous, and the amino acid homology is as high as 92%. CLDN is expressed differently in different tissues, hCLDN18.1 is expressed in normal tissues, while hCLDN18.2 is expressed in tumor tissues and is related to tumor forma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61K47/68A61P35/00A61P35/02
CPCC07K16/28C07K16/2818C07K16/2827A61K47/6843A61P35/00A61P35/02C07K2317/31C07K2317/565C07K2317/622A61K2039/505
Inventor 刘佳建
Owner L&L BIOPHARMA CO LTD
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