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Anti-hu-OX40 antigen nano antibody and application thereof

A technology of hu-ox40 and nano-antibody, which is applied in the direction of antibody, application, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. It can solve the problems of enhancing activation and proliferation, complex anti-tumor immune response, etc.

Pending Publication Date: 2022-06-24
ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the tumor microenvironment, immune activation can lead to the expression of OX40, which can enhance the activation and proliferation of effector T cells and suppress Tregs, resulting in a complex anti-tumor immune response

Method used

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  • Anti-hu-OX40 antigen nano antibody and application thereof
  • Anti-hu-OX40 antigen nano antibody and application thereof
  • Anti-hu-OX40 antigen nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of membrane-expressing human OX40 stably transfected cell line

[0041] Whole-gene synthesis of the full-length human OX40 gene, including signal peptide, extracellular segment, transmembrane region and intracellular segment, with appropriate restriction sites at both ends. Using the method of enzyme digestion, the full-length gene of OX40 was constructed into the eukaryotic expression vector GC-ID (modified from pMH3 plasmid, Hangzhou Anpu), and the GC-ID-OX40 plasmid was obtained and then transfected into CHO-DG44 cells to construct Membrane expression of human OX40 stably transfected cell line CHO-DG44-OX40 was used to detect the binding activity of OX40 mAb by flow cytometry; Jurkat-OX40-NFκB-Luc cells were prepared in the same process for the detection of OX40 antibody binding to OX40 at the cellular level Or OX40 antibody NFκB cell pathway activation detection.

Embodiment 2

[0042] Example 2: Preparation of rhOX40-Avi-His-bio antigenic protein

[0043] For the synthetic OX40 full-length gene, appropriate primers were designed to construct the immunoglobulin variable region (IgV, 29aa-214aa) domain of the extracellular segment into the vector pCD-Avi-His ( It was transformed from pCDNA3.1+, Invitrogen Company) to obtain the eukaryotic expression plasmid pCD-OX40-Avi-His ( figure 1 ). figure 1 Schematic diagram for the construction of the pCD-OX40-Avi-His plasmid, which can be transfected into eukaryotic cells to obtain the expressed rhOX40-Avi-His protein.

[0044] Biotin was covalently linked to the rhOX40-Avi-His protein containing the Avi-tagged peptide sequence using BirA biotinylase to complete biotinylase labeling. Both BiaA enzyme and Biotin solution were from GeneCopoeia, and the Hitrap Desalting column was from GE. , to obtain rhOX40-Avi-His-bio protein with higher concentration and better labeling efficiency, which is used for antibody ...

Embodiment 3

[0045] Example 3: OX40 mAb screening

[0046] Using the prepared rhOX40-Avi-His-bio antigenic protein, 3 rounds of liquid phase screening were performed on 3 fully synthetic human nanobody libraries built by our company, and picked from the bacterial culture plates in the second and third rounds A large number of monoclonal, soluble nanobodies were prepared.

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PUM

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-hu-OX40 antigen nano antibody which is a monoclonal nano antibody and comprises a heavy chain variable region; the heavy chain variable region comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region respectively comprise any one of amino acid sequences as shown in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39 or a homologous sequence with at least 80% sequence identity with any one of the amino acid sequences as shown in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39. According to the invention, 9 specific full-human anti-human OX40 nano antibodies A1, B7, C4, C7, C10, D1, D2, D7 and D12 are screened and optimized from a constructed large-capacity full-synthetic human phage antibody library, so that the immunostimulation of T effector cells can be enhanced, the secretion of cell factors can be promoted, and a remarkable tumor inhibition effect is shown in a mouse tumor model.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an antibody that specifically binds to human OX40, and its preparation and use, especially its use in the treatment of OX40-related diseases, such as cancer and the like. Background technique [0002] The emergence of antibody library technology provides a new way for the development of human antibodies. Winter et al. created the phage antibody library technology in 1994, which overcomes the disadvantage that the human body cannot be immunized at will, and does not require artificial immunization of animals and cell fusion technology. Humanized antibodies are prepared by genetic engineering technology. Among them, George P.Smith is the builder of phage display technology, and he creatively pioneered the platform method of this technology, and Gregory P.Winter used this technology to develop the first fully human antibody drug for the treatment of rheumatoid Arthritis, pso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2878A61P35/00C07K2317/569C07K2317/565A61K2039/505
Inventor 刘浩许文娟徐婷周伟崔智强叶洪涛鲍文英范清林宋礼华
Owner ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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