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A kind of anti-tigit monoclonal antibody and application thereof

A monoclonal antibody and variant technology, applied in the direction of antibodies, applications, anti-inflammatory agents, etc., to achieve a wide range of application prospects and the effect of relieving immunosuppression

Active Publication Date: 2022-06-14
EXCELMAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] With the deepening of research on TIGIT-mediated immune response, researchers have designed an optimal combination strategy of TIGIT blockers for cancer patients, and more than 10 A humanized anti-TIGIT antibody of the IgG1 isotype has entered clinical trials, but no related drugs have been approved

Method used

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  • A kind of anti-tigit monoclonal antibody and application thereof
  • A kind of anti-tigit monoclonal antibody and application thereof
  • A kind of anti-tigit monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] The preparation of embodiment 1 murine anti-TIGIT antibody

[0147] (1) Preparation of TIGIT antigen

[0148] In this example, first prepare the TIGIT antigen (NCBI accession number: NP_776160.2, amino acids 21-141 in the extracellular region): clone the gene of the TIGIT antigen into a eukaryotic expression vector, and transfect the recombinant eukaryotic expression plasmid into For 293F host cells, the cell supernatant was collected after culture, subjected to protein A affinity purification to obtain TIGIT-Fc fusion protein, then digested with TEV protease, and purified by affinity chromatography to obtain recombinant human TIGIT protein antigen.

[0149] (2) Preparation and screening of single-chain antibody phage library

[0150] After the recombinant human TIGIT protein antigen was emulsified with complete Freund's adjuvant, the BALB / c mice aged 6-8 weeks were injected and immunized; every two weeks, the recombinant human TIGIT protein antigen emulsified with inc...

Embodiment 2

[0153] Example 2 Functional verification of mouse-derived anti-TIGIT single-chain antibody

[0154] (1) Expression and purification of single-chain antibody

[0155] In this example, the mouse-derived anti-TIGIT single-chain antibody was expressed using the 293F transient transfection system:

[0156] The mouse-derived anti-TIGIT single-chain antibody mT251 gene was cloned into the eukaryotic expression vector pcDNA3.1-Fc, and the mT251 scFv-Fc recombinant eukaryotic expression plasmid was constructed; the plasmid was amplified and extracted, then transiently transformed into 293F cells, cultured and collected on the cells The supernatant was purified by protein A affinity chromatography, and the purified product was mouse-derived anti-TIGIT single-chain antibody-Fc recombinant protein.

[0157] (2) ELISA detection of binding activity of anti-TIGIT single-chain antibody to human TIGIT antigen

[0158] Add TIGIT antigen to the microtiter plate and incubate overnight at 4°C; b...

Embodiment 3

[0166] Example 3 Murine Anti-TIGIT Antibody Blocks the Binding of TIGIT and Ligand CD155

[0167] (1) Competitive ELISA to detect the binding activity of anti-TIGIT antibody blocking TIGIT and CD155

[0168] Add TIGIT antigen to the microtiter plate and incubate overnight, wash with PBST and block at room temperature; after washing, add serially diluted anti-TIGIT antibody and incubate at room temperature; after washing, add biotin-labeled CD155 antigen, incubate at room temperature and then wash. Add Straptavidin-HRP to incubate at room temperature, add TMB chromogenic solution after washing again. Use a microplate reader to detect the absorbance at 450 nm, and draw a curve with log (antibody concentration) as the abscissa and OD450 as the ordinate.

[0169] The result is as Figure 4 As shown, mT251 antibody can effectively block the binding of TIGIT and CD155.

[0170] (2) FACS detection of anti-TIGIT antibody blocking the binding activity of TIGIT and CD155 at the cellula...

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Abstract

The present invention provides an anti-TIGIT monoclonal antibody and application thereof, the anti-TIGIT monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises SEQ ID NO:1 The heavy chain CDR1, the heavy chain CDR2 shown in SEQ ID NO:2 and the heavy chain CDR3 shown in SEQ ID NO:3; the light chain variable region includes the light chain CDR1 shown in SEQ ID NO:4, SEQ ID NO:4 Light chain CDR2 shown in ID NO:5 and light chain CDR3 shown in SEQ ID NO:6. The anti-TIGIT monoclonal antibody of the invention has strong specific recognition and binding ability to the antigen TIGIT, can effectively block the combination of TIGIT and its ligand CD155, and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of antibody engineering, and relates to an anti-TIGIT monoclonal antibody and its application. Background technique [0002] T cell immunoglobulin and ITIM domain (TIGIT) is a member of the poliovirus receptor (PVR) / Nectin family, composed of extracellular immunoglobulin variable region (IgV) domain, type I transmembrane domain, Composed of the intracellular domain of the classical immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoglobulin tyrosine tail (ITT) motif, it is a co-inhibitory receptor expressed in lymphocytes and plays a role in effector and regulation It is highly expressed in sex CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells and natural killer (NK) cells, and has become an emerging target for cancer immunotherapy. [0003] Currently, several mechanisms for TIGIT-mediated suppression of effector T cells and NK cells have been proposed. TIGIT may act as a ligan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C07K16/46C12N15/13A61K39/395A61P35/00A61P35/02A61P31/12A61P31/16A61P31/18A61P31/22A61P37/02A61P19/02A61P29/00A61P25/00A61P19/08A61P21/04A61P7/06A61P1/16
CPCC07K16/2803C07K16/2818A61P35/00A61P35/02A61P31/12A61P31/16A61P31/18A61P31/22A61P37/02A61P19/02A61P29/00A61P25/00A61P19/08A61P21/04A61P7/06A61P1/16C07K2317/31C07K2317/24C07K2317/56C07K2317/565C07K2317/622C07K2317/76C07K2317/92A61K2039/505
Inventor 张文军李峰刘敏谭芳慧华亚南方春华
Owner EXCELMAB INC
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