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Group of 4-1BB monoclonal antibodies and medical application thereof

A monoclonal antibody and antigen technology, applied in the field of tumor immunotherapy and molecular immunology, which can solve the problem of weak anti-tumor effect

Active Publication Date: 2021-03-23
DONGDA BIOSCIENCE INC (SUZHOU)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with Urelumab, Utomilumab has a good safety profile, but its antitumor effect is relatively weak

Method used

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  • Group of 4-1BB monoclonal antibodies and medical application thereof
  • Group of 4-1BB monoclonal antibodies and medical application thereof
  • Group of 4-1BB monoclonal antibodies and medical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation of 4-1BB hybridoma antibody

[0032] Use the fusion protein (4-1BB-mFc) of the extracellular region of human 4-1BB and mouse Fc as antigen, fully emulsify with an equal volume of complete Freund's adjuvant (Sigma, Cat No: F5581), and subcutaneously immunize 6- Eight-week-old Balb / c mice (purchased from Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.), with an antigen immunization dose of 50 μg / mouse. Subsequently, mice were subcutaneously immunized three times with the same dose of antigen fully emulsified with incomplete Freund's adjuvant (Sigma, Cat No: F5506) every 2 weeks. After the three immunizations, the serum titer of the mice was determined, and a booster immunization was performed intraperitoneally 3 days before the fusion. Using PEG Hybri-Max (Sigma, Cat No: 7181) as a fusion agent, mouse spleen cells and SP2 / 0 cells were mixed at a ratio of 4:1. The fused cells were added to a 96-well plate (1×10 5 cells / well), and each well contained 0.1 ...

Embodiment 2

[0049] Effect of 4-1BB Hybridoma Antibody on Cytokines Secreted by T Lymphocyte Line Jurkat-4-1BB

[0050] In a 96-well plate, add 50 μL of 2×10 per well 6 / ml Jurka-4-1BB cells, 50 μl 4×10 5 / ml 293T-OKT3 cells and 50 μl 4×10 5 / ml 293T-CD32a or 293T-CD32b cells and 50 μL of different concentrations of 4-1BB antibody (initial concentration is 20 μg / mL, 10-fold serial dilution), put the 96-well cell culture plate at 37 ° C, 5% CO 2 Incubate in the incubator for 48 hours, collect the supernatant, and use the IL-2ELISA kit (R&D Systems, Cat No: DY202) to detect the concentration of cytokines, such as Figure 6 , 7, the selected 4-1BB antibodies could significantly activate the expression of IL-2.

Embodiment 3

[0052] Effects of 4-1BB Hybridoma Antibody on Cytokines Secreted by Human CD4+ T Cells and PBMC Cells

[0053] Lymphocyte separation solution Histopaque (Sigma, Cat No: 1077-1) was added to a 50 mL sterile centrifuge tube, and then an equal volume of fresh blood was added, and centrifuged at 1500 rpm for 30 min at room temperature. The sample is divided into four layers in the centrifuge tube, which are plasma layer, white blood cell layer, lymphocyte separation fluid and red blood cell layer from top to bottom. Collect the white blood cell layer in the middle into a new centrifuge tube, add 5 times the volume of washing buffer (PBS+3%FBS) to mix and wash, centrifuge at 1500rpm for 10min, repeat the washing for 3 times, and resuspend the cells in washing buffer And count. CD4+ T cells were isolated from PBMCs using the CD4+ T cell isolation reagent according to the instructions. One day in advance, coat Corning 96 wells with 2 μg / mL Goat anti-mouse Fc (Jackson, Cat: 115-005-...

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Abstract

The invention belongs to the field of tumor treatment and molecular immunology. The invention particularly relates to a group of anti-4-1BB monoclonal antibodies and medical application thereof. According to the invention, a group of anti-4-1BB monoclonal antibodies with excellent effects in the aspect of activating T cells are obtained through a hybridoma technology, and humanized modification issuccessfully carried out on the anti-4-1BB monoclonal antibodies. The antibody has a huge application prospect in the preparation of related drugs for activating and regulating the action and level of 4-1BB and remarkably enhancing the immunity of the organism, especially drugs related to cancer treatment.

Description

technical field [0001] The invention belongs to the fields of tumor immunotherapy and molecular immunology, and relates to 4-1BB antibody and its application. Specifically, the present invention relates to various monoclonal antibodies to 4-1BB. [0002] technical background [0003] 4-1BB, also known as CD137, belongs to the tumor necrosis factor receptor superfamily (TNFRSF9), and is a receptor with synergistic activation (Vinay, et al., (2012), Mol. Cancer Ther. 11:1062– 70). Initially, 4-1BB was shown to be expressed in activated T lymphocytes, but not in quiescent T lymphocytes (Kwon, et al., (1989), Proc. Natl. Acad. Sci. USA 86:1963–1967); later It is also found to be expressed on some non-T lymphocytes, such as monocytes, neutrophils, B cells, NK cells and NKT cells (Pollok, et al., (1993), J.Immunol.150:771-781; Vinay, et al., (1998), Sem. Immunol.10:481-9; Vinay et al., (2012), PLoS One 7:e50272; Melero, et al., (1998), Cell Immunol 190:167 –72). The ligand of ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61K47/68A61P35/00
CPCC07K16/2878A61K47/68A61P35/00C07K2317/24C07K2317/56C07K2317/565C07K2317/92
Inventor 孙锴邱均专孙自勇王振生周漫陈均勇孙键区日山
Owner DONGDA BIOSCIENCE INC (SUZHOU)
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