Anti-ox40 antibody and use thereof

An antibody and ligand technology, applied in the direction of antibodies, applications, anti-tumor drugs, etc., can solve the problem of losing the activation effect of OX40

Active Publication Date: 2020-03-31
KYINNO BIOTECHNOLOGY (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current data show that most of these agonists compete with OX40L for OX40 binding, so when such agonists activate OX40, they also lose the activation effect of natural ligands on OX40
For example, most of the existing OX40 antibodies in the world compete with OX40L for binding to OX40, that is, the binding site between the antibody and OX40 partially overlaps with the binding site between OX40L and OX40, so when the antibody acts as an agonist to activate OX40 and its downstream signals pathway, but loses the function of OX40L as a natural ligand and activates OX40 at the same time

Method used

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  • Anti-ox40 antibody and use thereof
  • Anti-ox40 antibody and use thereof
  • Anti-ox40 antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Preparation of hybridoma cells of the present invention

[0086] Using the fusion protein comprising the extracellular region (SEQ ID NO: 49) of OX40 protein (from Genbank accession number NM_003327.3) and mouse IgG2a-FC (from Genbank accession number AAH31470.1) as immunogen to immunize mice, 5 One mouse was immunized subcutaneously, 5 mice were immunized intramuscularly, and the adjuvant was quick antibody 5W water-soluble adjuvant. The titer was measured 2 weeks after the booster immunization, and two mice with high titer were selected for immune shock, and then carried out 3 days later. Cell fusion as described below.

[0087] Take two mice to be fused, take serum, and then dissect, take spleen, separate spleen cells, fuse with cultured myeloma cells, lay 96-well plates, add selective medium for screening, and change the medium after 7 days 10 days later, ELISA detection was performed, and the OD value was selected to be more than 10 times greater than ...

Embodiment 2

[0089] Example 2 ELISA detection of the combination of the culture supernatant of the hybridoma cells of the present invention and OX40

[0090] Dilute the extracellular region (SEQ ID NO: 49) containing OX40 protein (from Genbank accession number NM_003327.3) and human IgG1-FC (from Genbank accession number CAC20454.1) to 1-2 μg / ml with coating solution , and then add 50-100 μl / well into the wells of the microtiter plate, and place at 4°C for overnight or 37°C for 2 hours for adsorption. Discard the liquid in the well, wash with washing solution 3 times at the same time, each time for 3-5 minutes, and pat dry. Add 200 μl of blocking solution to each well to block overnight at 4°C or 2 hours at 37°C. Wash 3 times with washing solution. At this time, the coated plate can be stored at -20°C or 4°C for future use.

[0091] Add 50-100 μl of the culture supernatant of hybridoma cells to be tested to each well, and set up a positive control (adding the serum of the fusion mouse)...

Embodiment 3

[0094] Example 3 FACS detection of the combination of the culture supernatant of the hybridoma cells of the present invention and OX40

[0095] The extracellular region (SEQ ID NO: 49) of the OX40 protein (from Genbank accession number NM_003327.3) was constructed into the PLVX virus packaging vector (clontech, virus package mix, product number 631275), and the virus packaged by transfecting 293T cells was used. Infect HEK293 cells, add drug puromycin (puromycin) to screen out the drug-resistant cell line, which is HEK293 stably transfected with OX40. HEK293-OX40 cells were then prepared in PBS containing 2% FBS at a cell concentration of 10 7 cells / ml of cell suspension.

[0096] Put 50 μl of cell suspension into each flow tube (sample tube), then add 50 μl culture supernatant of hybridoma cells to be tested, and incubate at 4° C. for 60 minutes. Add 1ml of flow buffer to each flow tube, centrifuge at 1200rpm for 5 minutes, discard the supernatant, and repeat the washing ...

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Abstract

The invention provides an anti-OX40 antibody. In addition, the present invention provides nucleic acid sequences encoding the antibodies, vectors comprising the nucleic acid sequences, and host cells transformed or transfected with the vectors. Furthermore, the present invention provides the pharmaceutical use of the antibody.

Description

technical field [0001] The present invention relates to the field of antibody medicine, in particular, the present invention relates to an anti-OX40 antibody and its use. Background technique [0002] OX40 is a type 1 transmembrane glycoprotein that was reported nearly 30 years ago as a cell surface antigen expressed by activated T cells. Since its discovery, it has been shown to be a co-stimulatory molecule for T cells and members of the tumor necrosis factor receptor family, also known as "tumor necrosis factor receptor superfamily member 4" or CD134, and the name of the gene is TNFRSF4. [0003] OX40 has a molecular weight of 50 kD and has a cytoplasmic tail, a transmembrane domain and an extracellular region. Currently, OX40 has only one known ligand, namely OX40L (CD252), which is expressed on activated APC cells. The combination of OX40 and its ligand will co-stimulate T cell proliferation and cytokine production. The stimulation of T cells by OX40 is achieved throug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00A61P35/02G01N33/574
CPCA61P35/00A61K2039/505C07K16/2875C12N15/85G01N33/68G01N33/57415G01N33/57407C07K2317/565G01N2333/70575A61K39/395C07K16/28A61P35/02G01N33/574
Inventor 宁金鹰郝锋贺锋宁明萱
Owner KYINNO BIOTECHNOLOGY (BEIJING) CO LTD
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