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TIM-3 single-domain antibody and application thereof

1. TIM-3, single-domain antibody technology, applied in the fields of antibodies, applications, antibody medical components, etc., can solve the problems of weakened affinity of single-domain antibodies, and achieve the effect of enhancing anti-tumor activity and improving tumor tissue permeability

Pending Publication Date: 2020-03-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the weakened affinity of single domain antibodies compared with traditional monoclonal antibodies
There are no reports of TIM-3 single domain antibodies that successfully block the binding of Galectin-9 and TIM-3

Method used

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  • TIM-3 single-domain antibody and application thereof
  • TIM-3 single-domain antibody and application thereof
  • TIM-3 single-domain antibody and application thereof

Examples

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specific Embodiment approach

[0066] The specific steps of the present invention are illustrated below through the examples, but are not limited to the scope of the examples. The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only for illustrating the present invention, rather than limiting the scope of the present invention in any way. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

[0067] The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto.

Embodiment 1

[0068] Example 1 Construction and screening of phage-displayed TIM-3 single domain antibody library

[0069] New Zealand white rabbits were immunized with TIM-3 recombinant protein (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.), the spleen was taken, total RNA was extracted, and the antibody variable region gene was amplified by reverse transcription, and the antibody gene was amplified The fragments were ligated with phage vectors, purified, and the ligated products were electrotransformed into TG1 competent cells to prepare a library of TIM-3 single domain antibodies with a capacity of 6×10 9 . At the same time, the synthetic human VH library (refer to Thomas Tiller, et al. A fully synthetic human Fabantibody library based on fixed VH / VL framework pairings with favorable biophysical properties. mAbs 5:3, 445–470; May / June 2013) was linked together with the phage vector, After purification, the ligated product was electrotransformed into TG1 competent cells. ...

Embodiment 2

[0070] Example 2 Prokaryotic expression and purification of single domain antibody

[0071] The single domain antibody gene obtained in Example 1 was subcloned into the prokaryotic expression vector pET28, and introduced into the expression strain BL21, and a single colony was picked from the overnight-grown ampicillin plate, induced by adding IPTG to the LB medium, and induced for 8 hours at 30°C . After the wall was broken by ultrasound, the supernatant was collected by centrifugation, and purified by His nickel column to obtain single domain antibodies TIM-3-CX101 (SEQ ID NO:1), TIM-3-CX106 (SEQ ID NO:2), TIM-3 - CX119 (SEQ ID NO:3), TIM-3-CX123 (SEQ ID NO:4), TIM-3-CX153, (SEQ ID NO:5).

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Abstract

The present invention provides a single-domain antibody or an antigen-binding fragment thereof capable of specifically binding to an immune checkpoint molecule TIM-3, a polynucleotide encoding the single-domain antibody or antigen-binding fragment, and uses based on the single-domain antibody or the antigen-binding fragment, and the polynucleotide. The provided single-domain antibody or the antigen-binding fragment thereof is composed of a frame region of an antibody heavy chain variable region (VH) and an antigen-binding complementarity determining region (CDR). The frame region and the CDR region can be combined according to different ways. The provided single-domain antibody or the antigen-binding fragment can block binding of Galectin-9 and TIM-3 and increase activity of T cells. The provided single-domain antibody or the antigen-binding fragment thereof can be used for diagnosis, treatment, and adjuvant treatment of diseases of tumors, immune diseases, infectious diseases, etc., and can also be used to detect presence of TIM-3 protein and content thereof in biological samples.

Description

technical field [0001] The invention belongs to the field of biotechnology. The present invention relates to a new anti-human TIM-3 protein single-domain antibody and its application. Background technique [0002] Tumor is one of the diseases that seriously threaten human health. In recent years, tumor immunotherapy technologies represented by chimeric antigen receptor T cells (CAR-T) and immune checkpoint inhibitor antibodies have brought revolutionary progress to the treatment of tumors. Immune checkpoints are a class of regulatory proteins expressed on the surface of immune cells (T cells, monocytes / macrophages). Highly expressed inhibitory ligands on the surface of tumor cells can inhibit the activity of immune cells after binding to immune checkpoint proteins. Help tumor cells escape the surveillance of the immune system. Monoclonal antibodies targeting immune checkpoints can block the inhibitory effect of tumor cells on immune cells and enhance the anti-tumor activi...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/46C07K19/00C12N15/13A61K39/395A61K45/06A61P35/00
CPCC07K16/2803C07K16/468A61K39/39566A61K45/06A61P35/00C07K2317/569C07K2319/00C07K2317/565A61K2039/505A61K2300/00
Inventor 丰明乾陈鑫左殿宝何慧侠杨柳
Owner HUAZHONG AGRI UNIV
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