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Anti-OX40 fully human antibody and preparation method and application thereof

An antibody and whole-human technology, applied in botany equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of high immunogenicity reduction, toxicity, and limited clinical application

Active Publication Date: 2019-11-19
CURON MACAO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As an agonist against co-stimulatory receptors, toxicity may be the most concern, such as cytokine storm, which limits clinical application
In addition, anti-OX40 antibodies currently tested in clinical trials are human-mouse chimeric or humanized antibodies, which reduce efficacy due to high immunogenicity due to protein sequences of mouse origin

Method used

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  • Anti-OX40 fully human antibody and preparation method and application thereof
  • Anti-OX40 fully human antibody and preparation method and application thereof
  • Anti-OX40 fully human antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0428] material preparation

[0429] 1.1 Production of immunogen

[0430] A cDNA encoding the extracellular domain (ECD) of the OX40 protein was synthesized by Sangon Biotech (GenBank refCAB96543.1) and inserted into a modified expression vector pcDNA3.3 (ThermoFisher). Plasmid DNA is prepared on a large scale and the inserted DNA sequence is verified by sequencing. The fusion protein OX40ECD coupled with human Fc or His tag was obtained by transfecting human OX40ECD gene into Freestyle 293F (ThermoFisher) or Expi-293F cells (ThermoFisher). After 5 days, supernatants were harvested from transiently transfected cell cultures. Fusion proteins are purified and quantified for immunization and screening.

[0431] 1.2 Production of reference antibody

[0432] Four reference antibodies, BMK1, BMK5, BMK7 and BMK10, were used as positive controls in the examples. BMK1 was synthesized according to the cloning of 11D4 of US Patent No. US8236930B2 (Pfizer). BMK5 was synthesized acco...

Embodiment 2

[0436] Production of Antibody Hybridomas

[0437] 2.1 Immunization and cell fusion

[0438] OMT rats were used to generate fully human monoclonal antibodies against OX40 comprising chimeric polynucleotides useful for optimal production of functional immunoglobulins with a human idiotype. This rat strain carries human heavy and light chain transgenes as described in PCT Publication WO2014 / 093908. In order to produce a fully human monoclonal antibody against OX40, use aluminum phosphate (Alum-Phos) as an adjuvant to mix 20 μg human OX40 ECD protein to immunize OMT rats aged 6-8 weeks at the footpad, and mix TiterMax with 20 μg human OX40 ECD protein was injected subcutaneously for the first booster and repeated every two weeks with Alum-Phos and TiterMax mixed human OX40ECD protein. Serum antibody titers were measured every 1 or 2 weeks by enzyme-linked immunosorbent assay (ELISA). When the serum antibody titer was high enough, rats were given a final booster immunization wit...

Embodiment 3

[0445] Construction and purification of fully human antibody molecules

[0446] 3.1 Hybridoma sequencing

[0447] RNA was extracted from monoclonal hybridoma cells using RNeasy Plus Mini Kit (Qiagen) and Trizol reagent. The heavy chain variable region (VH) and light chain variable region (VL) of the OX40 chimeric antibody were amplified as follows. Briefly, RNA is first reverse transcribed into cDNA using reverse transcriptase, as described below.

[0448] Table 1. cDNA amplification reaction (20μL)

[0449]

[0450] Table 2 cDNA amplification reaction conditions

[0451] step 1 step 2 step 3 step 4 temperature 25 50 85 4 time 10min 50min 5min ∞

[0452] The resulting cDNA is used as a template for subsequent PCR amplification using primers specific for the gene of interest. The PCR reaction was performed as follows.

[0453] Table 3. PCR reaction system (50μL)

[0454] components Dosage cDNA 2.0 μL P...

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Abstract

The application provides a fully human monoclonal antibody directed against member 4 of tumor necrosis factor receptor superfamily (TNFRSF4) (also known as OX40 and CD134). The application also provides a method for producing hybridomas using humanized transgenic rats, a nucleic acid molecule encoding the anti-OX40 antibody, an expression vector and a host cell for expressing the anti-OX40 antibody. The invention also provides a method for verifying antibody function in vitro and antibody efficacy in vivo. The antibody of the invention provides a very effective agent for treating a variety ofcancers by modulating human immune function.

Description

[0001] priority information [0002] This application claims the priority of PCT Application No. PCT / CN2018 / 086574 filed on May 11, 2018 and Chinese Application No. 201810529840.5 filed on May 29, 2018. [0003] sequence listing [0004] This application contains a Sequence Listing and is hereby incorporated by reference in its entirety. technical field [0005] This application relates generally to antibodies. More specifically, the present application relates to a fully human monoclonal antibody against OX40, its preparation method and its use. [0006] Background of the invention [0007] Accumulating evidence from preclinical and clinical results indicates that targeting immune checkpoints is emerging as the most promising approach to treat cancer patients. Tumor necrosis factor receptor superfamily member 4 (TNFRSF4, also known as OX40, CD134, and ACT35) is one of the immune checkpoint proteins that plays a role in T cell function by enhancing T cell receptor signalin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00A61P37/02A61P29/00A61P31/00
CPCC07K16/2878C07K2317/565C07K2317/56A61P29/00A61P31/00A61P35/00A61P37/02A61P37/00C07K2317/21C07K2317/92C07K2317/33C07K2317/732C07K2317/734A61K2039/505C07K2317/34C12N5/163A01K2227/105A01K2267/0331C07K2317/75C07K16/2875C07K2317/76A61P35/04C07K2317/52
Inventor 郑勇杨宝田李竞
Owner CURON MACAO LTD
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