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Anti-TIGIT antibodies and uses thereof

An antibody and antigen technology, applied in the direction of antibodies, applications, anti-infective drugs, etc., can solve the problems of no drugs, listing, etc.

Inactive Publication Date: 2020-05-26
SICHUAN KELUN BIOTECH BIOPHARMACEUTICAL CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no related drugs on the market

Method used

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  • Anti-TIGIT antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0637] Embodiment 1, preparation of anti-human TIGIT mouse monoclonal antibody

[0638] Human TIGIT ectodomain antigen (Uniport entry: Q495A1, produced by Anyuan Pharmaceutical Technology (Shanghai) Co., Ltd.) 50 μg was fully emulsified with complete Freund's adjuvant, and then immunized male Balb / C mice by multipoint immunization. The immunization cycle was Once every three weeks. On the 10th day after the third immunization, blood was collected from the tail vein, and the plasma anti-human TIGIT antibody titer was tested by ELISA to monitor the degree of immune response of the mice. Mice were boosted once. After 3 days, the mice were sacrificed and the spleens of the mice were taken out for fusion with the mouse myeloma Sp 2 / 0 cell line. Mix 2×10 8 Sp 2 / 0 cells with 2×10 8 Splenocytes were fused in 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) solution. Adjust the number of spleen cells with HAT selection medium (DMEM medium containing...

Embodiment 2

[0639] Example 2, ELISA method to measure the binding ability of murine antibody and human TIGIT antigen

[0640] Coat the plate with human TIGIT and leave overnight at room temperature. The coating solution was discarded, the wells were blocked with skim milk dissolved in phosphate-buffered saline (PBS) for 1 hour, and the wells were washed with PBS containing 0.05% Tween-20. Then add 50 μL of purified anti-human TIGIT murine antibodies Mab21 and Mab7 to each well, use mouse IgG (prepared by purifying mouse serum with ProteinG) as a negative control, and PBS as a blank control, incubate at room temperature for 1 hour, and use 0.05% The wells were washed with Tween-20 PBS, and then 50 μL of HRP-labeled goat anti-mouse IgG polyclonal antibody (purchased from Jackson Laboratory, Cat. No.: 115-035-071) was added to each well as a detection antibody.

[0641] The result is as figure 1 As shown, the murine antibodies Mab21 and Mab7 have high affinity with human TIGIT, both EC 5...

Embodiment 3

[0642] Example 3, Subtype Identification and Variable Region Amplification of Anti-TIGIT Mouse Monoclonal Antibody

[0643] Antibody subtype identification: take hybridoma cell culture supernatant and use IsoStrip TM Mouse monoclonal antibody subtype identification kit (Santa Cruz Biotechnology, Cat. No. sc-24958) was used to identify antibody subtypes. The subtype of mouse monoclonal antibody Mab21 was identified as IgG2b (Kappa) type. The mouse monoclonal antibody Mab7 subtype was identified as IgG1 (Kappa) type.

[0644] Antibody variable region amplification: Candidate hybridoma cells #21 and #7 were cultured to a total of 10 7 Cells were collected by centrifugation at 1000 rpm for 10 min, and total RNA was extracted with an mRNA extraction kit (purchased from NEB Company, catalog number: S1550S), and the first Strand cDNA, using the first-strand cDNA as a subsequent template to amplify the DNA sequence of the antibody variable region corresponding to the hybridoma cell...

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Abstract

The present invention relates to the field of disease treatment and immunology, and in particular, to anti-TIGIT antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the anti-TIGIT antibody and the antigen-binding fragment, and a preparation method thereof. The anti-TIGIT antibody or the antigen binding fragment thereof provided by the invention has high specificity andhigh affinity to TIGIT, can effectively block the binding of TIGIT and a ligand thereof, and inhibits and / or blocks intracellular signal transduction mediated by the binding of TIGIT and the ligand thereof. Therefore, the invention further relates to a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, and to the use thereof in the preparation of a medicine for enhancing immune cell activity, enhancing immune response, or for preventing and / or treating tumors, infections or infectious diseases.

Description

technical field [0001] The present invention belongs to the field of therapeutic monoclonal antibodies, and more particularly, the present invention relates to an antibody against T cell immune receptor (TIGIT) having Ig and ITIM domains; limited to use in cancer, infection, etc.). Background technique [0002] TIGIT (T cell immune receptor with Ig and ITIM domains) belongs to the immunoglobulin superfamily member, also known as Wucam, Vstm3 or Vsig9, with an extracellular immunoglobulin domain, type I transmembrane domain and two Immunoreceptor Tyrosine Inhibition Motif (ITIM). TIGIT is mainly distributed in regulatory T cells (Tregs), activated T cells, and natural killer cells (NK). It is a co-inhibitory receptor protein that can bind to the positive protein CD226 (Dnam-1) and APC on T cells. The expressed ligands CD155 (Pvr or Necl-5) and CD112 (Pvrl-2 or Nectin2) constitute a co-stimulatory network. Among them, TIGIT competes with CD226 for binding to CD155 and CD112...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N5/20C12N5/0783A61K39/395A61P37/04A61P35/00A61P35/02A61P31/00A61P33/00G01N33/68
CPCA61K2039/505A61P31/00A61P33/00A61P35/00A61P35/02A61P37/04C07K16/2809C07K2317/24C07K2317/33C07K2317/565C07K2317/732C07K2317/734C07K2317/92C12N5/0636C12N5/0638C12N5/0646C12N2501/515A61K39/395C07K16/28G01N33/00G01N33/68
Inventor 肖亮周利刘登念孙见宇胡江江刘雪梅喻海旻郑云程李强薛彤彤
Owner SICHUAN KELUN BIOTECH BIOPHARMACEUTICAL CO LTD
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