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Antibody with double mic binding activity and application thereof

A technology that combines activity and antibodies, applied in the field of proteins, can solve problems such as instability and weak binding force, and achieve the effect of good affinity.

Active Publication Date: 2022-03-25
GUANGZHOU KONCEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the method used in this document, the affinity connection between protein A and antibody IgG is mainly used, and the antibody adsorbed on the surface of the filler is then adsorbed to MICA. This binding force is weak and unstable.

Method used

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  • Antibody with double mic binding activity and application thereof
  • Antibody with double mic binding activity and application thereof
  • Antibody with double mic binding activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Preparation of membrane-expressed MIC protein and animal immunization

[0066] The full-length cDNAs of MICA*002, MICA*008, MICA*009, MICA*010 and MICB*005 will be expressed (see http: / / hla.alleles.org / alleles / text_index.html for the full-length sequence of MICA Contains signal peptide, amino acid sequence 1-366; extracellular region sequence contains signal peptide, amino acid sequence 1-284. MICB full-length sequence contains signal peptide, amino acid sequence 1-360; extracellular region sequence contains signal peptide, amino acid sequence 1- 286) was connected to the lentiviral vector pCDH-CMV-MCS-EF1-Puro, and then transferred into 293T cells together with the helper plasmid. The 293T cells were used to produce lentivirus, and the virus supernatant was collected to infect CHO cells. Take 5*10 6 3 transfected cells (5 types of cells with equal content) were mixed with complete Freund's adjuvant, shaken and mixed, and injected subcutaneously in the abdome...

Embodiment 2

[0067] Example 2 Preparation of secreted MIC protein

[0068] Use the cDNA of the extracellular region of MICA*002, MICA*008, MICA*009, MICA*010 and MICB*005 (the C-terminal his tag is designed) to connect with the lentiviral vector pCDH-CMV-MCS-EF1-Puro, and the auxiliary The plasmids were transferred together into 293T cells to produce virus, and the virus supernatant was collected to infect CHO cells. The CHO cell culture fluid was collected and purified with metal ion chelating filler to obtain the target protein. The molecular weight of MICA protein is about 55KD, and the molecular weight of MICB protein is about 45KD.

Embodiment 3

[0069] Example 3 Screening of monoclonal antibody cell lines

[0070] A total of 30 monoclonal cell lines were screened, and the monoclonal cells were picked up under a microscope, inoculated into 96-well plates, and placed in 37°C, 5% CO 2 After culturing in an incubator for 3 days, the supernatant was collected. The ELISA plate was coated with natural MICA*008, 100ng / well, incubated overnight at 4°C, and the primary antibody was 100µL / well of serially diluted cell culture supernatant (respectively diluted 2 times, 4 times, 8 times, 16 times, 32 times with PBS) , 64 times, 128 times), incubate at 37°C for two hours, secondary antibody: goat anti-mouse antibody (HRP labeled) diluted 1:5000, 100µL / well, incubate at 37°C for 1h, use TMB chromogenic solution for 10min, 450nm Detect absorbance. The five cell lines screened with higher binding ability to MICA*008 are 1E7, 3F5, 8B6, 9C9, and 10A1 cell lines, and the results are shown in figure 1 .

[0071] The anti-MIC antibodie...

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Abstract

The present invention discloses an antibody with double MIC binding activity and application thereof. The antibody comprises at least one heavy chain variable region comprising three CDRs and at least one light chain variable region comprising three CDRs, the light chain variable region and the heavy chain variable region thereof The CDR sequence of the chain variable region is described in SEQ ID NO.1-30. The anti-MIC antibody of the present invention has a good affinity effect on various MICA and MICB proteins, especially has binding activity on soluble MICA*008, MICA*010, MICA*002, MICA*009 and MICB*005, through Adsorption of NKG2D ligands in human blood or plasma, including soluble MICA, MICB molecules and immune complexes composed of MICA / B and anti-MIC monoclonal antibody drugs, can relieve the tumor immunosuppressive effect caused by the abnormal increase of MIC protein.

Description

technical field [0001] The invention relates to the field of proteins, in particular to an antibody with dual MIC binding activity and its application, and in particular to the treatment of tumors related to the abnormal expression of soluble MICA and MICB molecules. By expressing anti-MICA and MICB antibodies, coupling them to a solid-phase carrier, and removing free soluble MIC family proteins in the serum of tumor patients by immunoadsorption, the immune monitoring function of NK cells is enhanced. Background technique [0002] The prevention and treatment of cancer is a major problem facing human health and longevity in this century. In recent years, immunotherapy has provided a safe and effective tumor treatment plan. Natural killer cells (NK cells) are important members of the innate immune system, mainly fighting pathogens and cancer. NKG2D is expressed in NK cells and CD8 + T cells, also expressed on some CD4 + T cells, invariant natural killer T cells (iNKT) and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13G01N33/574G01N33/577A61K39/395A61P35/00B01J20/26
CPCC07K16/2833G01N33/57488G01N33/577G01N33/57407G01N33/5743G01N33/57423G01N33/57426G01N33/57449G01N33/57446G01N33/57434G01N33/57438G01N33/57419G01N33/57442G01N33/57411G01N33/57415A61P35/00B01J20/26C07K2317/565C07K2317/56C07K2317/92C07K2317/622G01N2333/70539A61K2039/505
Inventor 张海珍杨正根杨睿祯李家萍陈校园
Owner GUANGZHOU KONCEN BIOSCI
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