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Preparation method and application of bispecific antibody targeting CD24 (cluster of differentiation 24) and activating NK cells (natural killer cells)

A bispecific antibody, CD24 technology, applied in the direction of antibodies, applications, specific peptides, etc., can solve problems such as shedding, and achieve the effect of inhibiting growth

Inactive Publication Date: 2019-04-26
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that during the growth of tumors, MICA molecules will be shed by proteolytic enzymes, resulting in tumor tissues that are still MICA-positive, but can avoid immune surveillance and immune escape

Method used

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  • Preparation method and application of bispecific antibody targeting CD24 (cluster of differentiation 24) and activating NK cells (natural killer cells)
  • Preparation method and application of bispecific antibody targeting CD24 (cluster of differentiation 24) and activating NK cells (natural killer cells)
  • Preparation method and application of bispecific antibody targeting CD24 (cluster of differentiation 24) and activating NK cells (natural killer cells)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of bispecific antibody cG7-MICA targeting CD24.

[0034] First, use the rG7s-MICA independently developed by the laboratory as a template to design primers to transfer the heavy and light chain variable region genes, and use the lab-protected IgG1 constant region gene as a template to design primers to transfer the heavy and light chain constant region genes. The variable region and the constant region of the light chain were respectively connected by overlapping PCR technology to construct the heavy chain (H-chain) and light chain (L-chain) genes of cG7. At the same time, continue to use rG7s-MICA as a template to design primers to transfer the gene of extracellular region 1-3 of human MICA, and design primers to transfer the heavy chain gene of cG7, and use the overlap PCR technology to pass the flexible peptide (Gly 4 Ser) connects these two genes to form the heavy chain (H'-chain) of cG7-MICA, and cG7-MICA is consistent with the light chain of...

Embodiment 2

[0035] Example 2 Expression, purification and identification of bispecific antibody cG7-MICA targeting CD24.

[0036]First, the four recombinant plasmids H'-pMH3, H'-pCApuro, L-pMH3 and L-pCApuro were introduced into CHO-s cells by electric shock method (in a 0.4cm electric shock cup, 160V, 15ms) in equal proportions, and two After rounds of pressurized screening (Dot Blot semi-quantitative screening), cell lines with stable and high expression of cG7-MICA were obtained. The screened high-expression cell lines were expanded step by step, the cell culture fluid was collected, and the sample was filtered through a 0.22 μm filter membrane, followed by protein A affinity chromatography purification, and finally a large amount of the target antibody was obtained. 10% SDS-PAGE protein electrophoresis to identify the molecular weight and purity, and Western blot to initially verify the target protein to see if it is assembled correctly.

Embodiment 3

[0037] Example 3 SPR experiment of bispecific antibody cG7-MICA targeting CD24.

[0038] In this experiment, the interaction of cG7-MICA with the antigen CD24 and the MICA receptor NKG2D was measured using BiacoreX100 as an SPR-dependent biosensor. Use the Human Antibody capture kit to couple 25 μg / mL anti-human IgG (Fc) antibody to the surface of the activated CM5 chip (coupling is limited to channel 2). Adjust the concentration of cG7 and cG7-MICA to 10 μg / mL, and use the anti-human IgG (Fc) antibody on the CM5 chip to specifically capture cG7 and cG7-MICA. Afterwards, the naked CD24 peptide was diluted (from 100 nM to 0.39 nM) in a buffer ratio. At 25°C and a flow rate of 30 μL / min, samples were injected to detect the binding / dissociation ability between cG7 and cG7-MICA and CD24. Meanwhile, channel 1 acts as a reference channel without coupling or capturing any protein. Therefore, the signal detected by channel 1 will be used as reference data. After the natural dissoc...

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Abstract

The invention belongs to the technical field of genetic engineering antibodies, and particularly relates to a preparation and application of a bispecific antibody targeting a CD24 (cluster of differentiation 24) and activating NK cells (natural killer cells). According to the preparation and application of the bispecific antibody, an anti-CD24 humanized chimeric antibody cG7 and an MICA molecule capable of recruiting and activating the activity of the NK cells are connected through flexible peptide (Gly4Ser) and are stably transferred to a CHO-s eukaryotic expression system, and a stable high-expression cell strain is selected for propagation and expression of cG7-MICA; the bispecific antibody cG7-MICA utilizes the function of remodeling the MICA by utilizing the targeting effect of the cG7 to selectively display the MICA on the surface of a CD24+ tumor cell, the immune surveillance effect of the NK cells is induced, in addition, the NK cells are further activated through the effect ofan Fc part, and the antibody-dependent cell-mediated cytotoxicity (ADCC) is achieved; and through experimental verification, the immunocompetence of killing the CD24+ hepatocellular carcinoma cells is achieved in vitro and vivo.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a new activating receptor NKG2D (NK cell receptor NKG2D) that can simultaneously bind to the leukocyte differentiation antigen CD24 (cluster of differentiation 24) and natural killer cells (natural killer cells; NK cells) on the surface. group 2, member D) The specific binding anti-CD24 humanized chimeric antibody is fused with the bispecific antibody cG7-MICA of human MICA molecule, which can reshape the function of MICA by using the targeting effect of cG7 to selectively select MICA Displayed on the surface of liver cancer cells, NK cells are recruited and activated to exert antibody-dependent cell-mediated cytotoxicity (Antibody-Dependent Cell-mediated Cytotoxicity; ADCC). in CD24 + In the human liver cancer cell Huh-7 subcutaneous tumor-bearing mouse model, it can effectively inhibit the growth of tumor tissue. The design of cG7-MICA can avoid tumor immune evasion ca...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00A61P1/16
CPCA61K2039/505A61P1/16A61P35/00C07K16/2851C07K16/2896C07K2317/24C07K2317/31C07K2317/732
Inventor 张娟王旻韩月孙福谋王阳王斐蔡佳玲
Owner CHINA PHARM UNIV
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