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Activated anti-OX40 antibody, production method and application

A technology of antibody and heavy chain antibody, applied in the direction of biochemical equipment and methods, applications, antibodies, etc., can solve problems such as incomplete consistency, difficult to predict adverse reactions, etc., and achieve low heterology, high affinity, and clear amino acid sequence structure Effect

Pending Publication Date: 2021-07-20
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although there are several varieties of anti-OX40 activating antibodies in clinical research, due to the inconsistency between the early screening performance of anti-OX40 activating antibodies and the final clinical treatment effect, unpredictable adverse reactions, and effects for different indications For reasons such as these, it is still necessary to develop more activated anti-OX40 antibodies with high affinity and high biological activity, especially to better stimulate immune memory, so as to meet the urgent needs of clinical treatment

Method used

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  • Activated anti-OX40 antibody, production method and application
  • Activated anti-OX40 antibody, production method and application
  • Activated anti-OX40 antibody, production method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0170] Example 1: Preparation of anti-human OX40 antibody hybridoma cells

[0171] Immunization: Use human OX40 / mFc recombinant protein (sequence number: P43489-1, 29aa-216aa) to immunize Balb / c mice, and use 96-well microtiter plate coated with recombinant human OX40-his protein to detect serum titer by ELISA method ; The mice whose serum titer reached the fusion requirements were used for the next step of cell fusion.

[0172] Cell fusion and hybridoma preparation: On the 67th day after the initial immunization, select the mouse whose titer reached the requirement, aseptically take the spleen of the mouse, prepare B lymphocyte suspension, and mix it with FO myeloma cells at a ratio of 5:1. The two cells were fused under the action of PEG4000. After the fused cells were resuspended in HAT medium, they were divided into 96-well cell culture plates. Cultured in a 37°C, 5% CO2 incubator.

Embodiment 2

[0173] Example 2: Screening of anti-human OX40 antibody-positive hybridoma cell lines

[0174] Protocol 1. Positive Hybridoma ELISA Binding Screening

[0175] 10-14 days after fusion, coat the microtiter plate with human OX40-His recombinant protein (serial number: P43489-1, 29aa-216aa) (10ug / ml, pH9.6, 0.1M NaHCO3), 4°C, overnight; Block with 4% skimmed milk powder-PBS, 37°C, 2hr; wash three times with PBST (0.05% Tween20-PBS), add hybridoma clone culture supernatant, 37°C, 1hr. The following controls were set up: (1) positive control (PC): post-immunization mouse serum (diluted with PBS 1:1000); (2) negative control (NC): pre-immunized mouse serum (diluted with PBS 1:1000). Washed three times with PBST (0.05% Tween20-PBS), added HRP-goat anti-mouse IgG (Fcγ), diluted 1:20000, 37°C, 1hr; washed five times with PBST (0.05% Tween20-PBS), added OPD chromogenic solution, develop color in the dark for 10-15min, add 2M H 2 SO 4 Terminate the reaction; read the A492 value with a...

Embodiment 3

[0180] Example 3: Sequence determination of murine anti-human OX40 antibody

[0181] After expanding the hybridoma cells m25A7, m27G12, and m11F7 secreting anti-human OX40 antibodies, use MouseMonoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and MouseMonoclonal Antibody Light / Heavy Chain Test Card (Cat.: A12401, VicNovo ) according to the reagent operating procedures for subtype detection, and the subtype identification results showed that the m25A7, m27G12, and m11F7 heavy chains were all IgG1, and the light chains were all Kappa chains, which provided a basis for the cloning of antibody genes.

[0182] The total RNA of the m25A7, m27G12, m11F7 hybridoma cells was extracted according to the instructions of the TRIzol kit (Cat: 15596026, Invitrogen); the total RNA of the hybridoma cells was reversed using M-MuLV reverse transcriptase (Cat: M0253S, NEB) Recorded into cDNA; use degenerate primers and Phusion kit (Cat: E0553L, NEB) to amplify antibody light chai...

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Abstract

The present invention provides an antibody or fragment thereof that expresses an activated receptor (OX40) on the surface of activated CD4+T, CD8+T cells, and the use of the antibody or fragment thereof for the prevention or treatment of diseases. The antibody disclosed by the invention is high in affinity to OX40 and obvious in effect of activating an OX40 signal channel; the antibody has a relatively broad-spectrum immunopotentiation effect, and can enhance T cell response immune memory; and good tumor inhibiting and killing effects are achieved in animal model in-vivo experiments, and good clinical application prospects are achieved.

Description

technical field [0001] The invention belongs to the field of antibody engineering, and specifically relates to an anti-OX40 antibody, its production method and application, in particular to an anti-human OX40 humanized antibody, its recombinant expression method and its application in the treatment of solid tumors. Background technique [0002] OX40, also known as CD134, ACT45, TNFRSF4, is a member of the tumor necrosis factor receptor (TNFR) superfamily and is an activating receptor expressed on the surface of activated CD4+T and CD8+T cells. OX40 signaling can activate the downstream NF-κB, PI3K and PKB pathways, and the continuous activation of these pathways can ultimately prolong the survival time of T cells, expand T cell memory, and promote the cell killing ability of T cells; The differentiation and activity of regulatory T cells (Treg) improves the immunosuppressive effect in the tumor microenvironment and further enhances the function of effector T cells. [0003]...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12P21/02A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2878C07K2317/24C07K2317/33C07K2317/56C07K2317/565C07K2317/76C07K2317/92A61K39/395C07K16/00C07K16/28C12P21/02G01N33/574
Inventor 王双曾大地王荣娟焦莎莎张畅
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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