Anti-her2 antibody or antigen-binding fragment thereof, and chimeric antigen receptor comprising same

A technology that combines fragments and antibodies, applied in receptors/cell surface antigens/cell surface determinants, antibody medical components, antibody mimics/scaffolds, etc. Sexual decline, etc.

Active Publication Date: 2020-09-11
GREEN CROSS LAB CELL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although trastuzumab has been commercially successful, the use of trastuzumab for therapeutic purposes has been limited because of the existence of multiple cancer cell

Method used

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  • Anti-her2 antibody or antigen-binding fragment thereof, and chimeric antigen receptor comprising same
  • Anti-her2 antibody or antigen-binding fragment thereof, and chimeric antigen receptor comprising same
  • Anti-her2 antibody or antigen-binding fragment thereof, and chimeric antigen receptor comprising same

Examples

Experimental program
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Embodiment 1

[0188] Example 1: Development of anti-HER2 antibodies

[0189] To develop antibodies, the extracellular domain (ECD) of the HER2 protein was produced using animal cells. Using HindIII and BamHI restriction enzymes, the hinge region and Fc region of human IgG1 (CH 2 -CH 3 ) bound to the C-terminus of the ECD was cloned into pCEP4 (Invitrogen, catalog # V044-50). Then, the cloned vector was transiently transformed into FreeStyle 293F (Invitrogen, catalog number R790-07) cells using polyethyleneimine (Polyscience Inc., cat. No. 20078-028) to purify the cloned vector from cell culture. Purified protein was quantified using a protein assay dye (Bio-Rad, cat. no. 500-0006), and its concentration and purity were investigated by Coomassie brilliant blue staining after SDS-PAGE. 100 μg of protein antigen was mixed with Freund's adjuvant (Sigma, Cat. No. F5506), and injected intraperitoneally into BALB / c mice (Dae Han Bio). Two weeks later, 100 μg of antigen diluted in PBS was furt...

Embodiment 2

[0197] Example 2: Verification of the binding site of the developed HER2 protein antibody

[0198] The binding sites of the five selected antibodies (hz2G10, hz39D2, 24D3, 1G3, hz8G11) to the extracellular domain (ECD) of HER2 protein were verified by ELISA. For ELISA, extracellular domains (ECDs) of ERBB family proteins were produced using animal cells and used as antigens. Specifically, the hinge region and Fc region (CH 2 -CH 3 ) bound to the C-terminus of the ECD was cloned into pCEP4 (Invitrogen, catalog # V044-50). Then, the cloned vector was transiently transformed into FreeStyle 293F (Invitrogen, catalog number R790-07) cells using polyethyleneimine (Polyscience Inc., cat. 20078-028) Purification of HER2-ECD DI Fc, HER2-ECDDH Fc, HER2-ECD DIII Fc, HER2-ECD DIV Fc and HER2-ECD Fc fusion proteins from cell culture. Purified protein was quantified using a protein assay dye (Bio-Rad, cat. no. 500-0006), and its concentration and purity were investigated by Coomassie br...

Embodiment 3

[0203] Example 3: Comparison of the inhibitory effect of the developed antibodies on the growth of breast cancer cells

[0204] Cell viability was analyzed by treating HER2-overexpressing SKBR3 breast cancer cells or HER2-nonexpressing breast cancer cells with MCF-7 alone or in combination with trastuzumab. For combined administration, the developed antibody and trastuzumab were mixed in a 1:1 weight ratio. SKBR3 cells (Korean Cell Line Bank, Cat. No. 30030, 5000 cells / well) and MCF-7 cells (ATCC, Cat. No. HTB22, 5000 cells / well) were aliquoted onto 96-well plates and cultured for 24 hours. After treatment with the purified antibody at a final concentration of 20 μg / mL, the cells were further cultured for 4 days. To measure cell viability, CCK-8 (Dojindo, catalog number CK-04-13) was added to a final concentration of 10%, and absorbance was measured after treatment at 37°C for 3 hours. Relative cell viability was calculated relative to the absorbance of untreated wells as 10...

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Abstract

The present invention relates to a novel anti-HER2 antibody or antigen-binding fragment thereof used in the prevention or treatment of cancer, a chimeric antigen receptor comprising the same, and usesthereof. The antibody of the present invention is an antibody that specifically binds to HER2 which is highly expressed in cancer cells (particularly, breast cancer or gastric cancer cells), and binds to an epitope that is different from an epitope to which conventional trastuzumab binds. Compared to transtuzumab, the antibody of the present invention exhibits a better killing ability with respect to kill HER2-unexpressed cancer cells which have non reactivity (or resistance) to a trastuzumab antibody or have reduced sensitivity. In addition, when the anti-HER2 antibody of the present invention is administered in combination with trastuzumab, a synergistic killing ability with respect to a cancer cell line on which the trastuzumab antibody acts. Therefore, a composition of the present invention can be very usefully used for combined administration with a trastuzumab antibody for the treatment of cancer, and for the treatment of cancer not treated by trastuzumab.

Description

technical field [0001] This research was conducted with the support of the Korean Ministry of Trade, Industry and Energy under project number 1415118385. The R&D management agency of the project is the Korea Institute of Technology Advancement, the name of the R&D project is "Global Innovation Technology Alliance", and the research name is "Development of Global Antibody Drugs Based on New Epitope Screening Platform Technology". The study was conducted by AbClon Inc. from November 1, 2011 to October 31, 2014. [0002] This application claims priority from Korean Patent Application No. 10-2017-0151841 filed with the Korean Intellectual Property Office on November 14, 2017, the entire disclosure of which is incorporated herein by reference. [0003] The present disclosure relates to novel anti-HER2 antibodies or antigen-binding fragments thereof, including chimeric antigen receptors thereof, and uses thereof. Background technique [0004] The Her2 / neu (ErbB2) gene encodes a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32G01N33/574
CPCC07K16/32C07K2317/24C07K2317/33C07K2317/73C07K2317/92C07K2317/565G01N33/57415G01N2333/71G01N33/574G01N2333/70596A61P35/00A61K35/17A61K38/00A61K2039/5156A61K2039/5158C07K14/7051C07K14/70521C07K14/70578C07K2319/02C07K2319/03C07K2319/30C07K2319/33
Inventor 李钟瑞金奎兑李英河黄寅式高凤国
Owner GREEN CROSS LAB CELL CORP
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