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ROR1 (receptor tyrosine kinase-like orphan receptor) monoclonal antibody, preparation method and application of ROR1 monoclonal antibody

A monoclonal antibody, heavy chain technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem that the ROR1 monoclonal antibody has not been discovered yet.

Inactive Publication Date: 2019-12-20
艾美斐生物医药科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No ROR1 monoclonal antibodies targeting other domains of ROR1 have been found

Method used

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  • ROR1 (receptor tyrosine kinase-like orphan receptor) monoclonal antibody, preparation method and application of ROR1 monoclonal antibody
  • ROR1 (receptor tyrosine kinase-like orphan receptor) monoclonal antibody, preparation method and application of ROR1 monoclonal antibody
  • ROR1 (receptor tyrosine kinase-like orphan receptor) monoclonal antibody, preparation method and application of ROR1 monoclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0041] Preparation method of anti-ROR1 antibody

[0042] The VH and VL sequences of the ROR1 antibody in the present invention come from a natural phage display library (ProMabBiotechnologies, Inc).

[0043] The sequences of the heavy chain and light chain of the antibody are obtained by gene synthesis (ie, SEQ ID NO.9 and 10, and the corresponding secretion signal peptides SEQID NO.11 and 15 are added), and are respectively constructed on the pcDNA3.4 vector. After Expi-CHO-S cells were expanded to a certain density, the plasmid DNA was transfected into Expi- In CHO-S, feeding was carried out on the 1st day and 5th day after transfection according to the operation protocol of the transfection kit, and purification was carried out on the 7th day after transfection.

[0044] After the expression, the protein A (GE mabselect Sure) was used to purify to obtain the purified antibody. Finally, SDS-PAGE and SEC-HPLC were used to analyze the quality of the antibody.

[0045] The m...

Embodiment 2

[0047] Activity identification of ROR1 antibody

[0048] I enzyme-linked immunosorbent (ELISA) method

[0049] Coating solution (3.56g Na 2 CO 3 and 8.4g NaHCO 3 , pH 9.5) to dilute recombinant human ROR1 protein (purchased from Beijing Baipusaisi Biotechnology Co., Ltd., product number: RO1-H522y) to 1 μg / mL coated ELISA 96-well plate, add 100 μL to each well, and incubate at 4 °C Overnight; PBS containing 2% BSA buffer to block the 96-well plate, incubate at room temperature for 1 h; after washing with PBST (PBS containing 0.05% Tween 20) for 3 times, add 100 μL of ROR1 antibody molecule G3 (starting from 0.6 μg / ml) to each well The initial concentration was diluted by 5 times into 8 concentration gradients) and incubated at room temperature for 1 h; horseradish peroxidase-coupled goat anti-human IgG Fc secondary antibody (purchased from Abcam, product number: ab97225) was diluted 1:10000, Add 100 μL / well into the wells, incubate at room temperature for 1 h; prepare hors...

Embodiment 3

[0059] Antigen-binding epitope identification of ROR1 antibody

[0060] The recombinant ROR1 protein contains three extracellular domains: Ig domain, Frizzled domain, and Klinger domain (purchased from Beijing Baipu Saisi Biotechnology Co., Ltd., product numbers are: RO1-H5221, RO1-H5222, RO1-H5223). Dilute the extracellular domains of the three ROR1 proteins to 1 μg / mL with coating solution (3.56g Na2CO3 and 8.4g NaHCO3, pH9.5), coat the ELISA 96-well plate, add 100 μL to each well, and incubate overnight at 4 °C; The 96-well plate was blocked with buffer solution containing 2% BSA in PBS and incubated at room temperature for 1 h; after washing 3 times with PBST (PBS containing 0.05% Tween 20), 100 μL of ROR1 antibody molecules G3 and C3 (starting at 20 μg / ml) were added to each well. Concentration, according to 5-fold dilution into 8 concentration gradients) incubate at room temperature for 1 h; after washing 3 times with PBST, dilute horseradish peroxidase-coupled goat ant...

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Abstract

The invention provides an ROR1 (receptor tyrosine kinase-like orphan receptor) monoclonal antibody. The ROR1 monoclonal antibody includes a heavy chain structure and a light chain structure, wherein aheavy chain variable region of the heavy chain structure and a light chain variable region of the light chain structure are provided with three complementary determinant regions CDRs separately, andsecretion-optimized signal peptides are added, so that the ROR1 monoclonal antibody is expressed efficiently; and specific binding to a Frizzled structural domain of an ROR1 is achieved, transmissionof related pathways is inhibited, cancer is treated effectively, and significant application effects are achieved especially in treatment of non-small-cell lung adenocarcinoma.

Description

technical field [0001] The invention belongs to the field of biotechnology and the field of antibodies, and specifically relates to a ROR1 monoclonal antibody, a preparation method and an application thereof. Background technique [0002] Receptor tyrosine kinase-like orphan receptor-1 (receptor tyrosine kinase-like orphan receptor, ROR1) is a transmembrane protein with a molecular weight of about 104kDa and a modified glycosylation size of about 130kDa. The extracellular domain of the ROR1 receptor consists of three distinct domains: an Ig-like domain distal to the membrane; a Frizzled domain in the middle; and a Kringle domain proximal to the membrane. [0003] Studies have shown that ROR1 is expressed at a high level during embryonic development and plays an important role in the regulation of embryonic, muscle, bone, lung and nervous system development. After maturation, the expression of ROR1 protein is restricted, and it is only expressed in a small amount in some org...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62A61K39/395A61P35/00
CPCC07K16/2803A61P35/00C07K2317/565C07K2317/92C07K2317/76C07K2319/02
Inventor 范国煌丁成立
Owner 艾美斐生物医药科技(上海)有限公司
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