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Chimeric anti-ROR1 antibody Fab molecule as well as preparation method and application thereof

An antibody and molecular technology, applied in the field of genetic engineering technology and immune targeting diagnosis and treatment, can solve the problems of poor tissue specificity, low average expression level and expression rate of target molecules, and unclear application prospects of clinical treatment, so as to achieve large expression and reduce curative effect , reduce the effect of rat-derived ingredients

Active Publication Date: 2018-03-23
张慧林 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the field of ovarian cancer, most monoclonal antibody-mediated targeted therapy is still in the preclinical stage. Although monoclonal antibodies against tumor angiogenesis (Bevacizumab, etc.) have been used in the treatment of ovarian cancer, due to poor tissue specificity, It will bind to normal tissues to varying degrees, and the average expression level and expression rate of the target molecules it targets in ovarian cancer tissues are low, so the clinical application prospect is unclear

Method used

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  • Chimeric anti-ROR1 antibody Fab molecule as well as preparation method and application thereof
  • Chimeric anti-ROR1 antibody Fab molecule as well as preparation method and application thereof
  • Chimeric anti-ROR1 antibody Fab molecule as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0026] Example 1. Preparation method of anti-ROR1 antibody Fab molecule:

[0027] 1) Amplification and verification of antibody variable region gene fragments:

[0028] Immunize three 8-week-old female BALB / c mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.) with recombinant human ROR1 protein (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.), once every 2 weeks, a total of 3 times . After the third immunization, the mouse serum was taken to measure the titer, and the mouse with a higher titer was taken for sprint immunization, and the spleen of the mouse was taken 3 days later to perform cell fusion with myeloma cells. After three times of subcloning, the murine hybridoma cells highly expressing the anti-ROR1 antibody were screened out. The mouse-derived anti-ROR1 antibody hybridoma cells were cultured to the logarithmic growth phase, the total RNA of the cells was extracted with an RNA extraction kit, and single-stranded cDNA was obtained by re...

Embodiment 2

[0045] Example 2 Activity identification of chimeric Fab antibody fragments:

[0046] IELISA

[0047] Dilute the recombinant human ROR1 protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number 13968-H08H) with coating solution (0.1M carbonate buffer, pH9.6) to 0.5 μg / mL to coat ELISA 96 wells Add 100 μL to each well, overnight at 4°C; block with PBST (PBS containing 0.5% Tween20) 5% skimmed milk-washing buffer, incubate at 37°C for 2 hours; after washing with PBST for 3 times, add 100 μL of anti-ROR1 antibody Fab to each well Molecule (200 μg / mL initial concentration, 11 concentration gradient dilutions) overnight at 4 °C; 100 μL / well of goat anti-human Fab secondary antibody diluted 1:5000 was added to the well, incubated at 37 °C for 1 h; peroxidase substrate Chromogenic solution 100 μL / well, stop the reaction with 2 mol sulfuric acid after 10 minutes at room temperature, and read the OD450 value with a microplate reader. The result is as F...

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Abstract

The invention discloses a chimeric anti-ROR1 antibody Fab molecule as well as a preparation method and application thereof. The chimeric anti-ROR1 antibody Fab molecule includes a recombinant light chain L and a recombinant heavy chain Fd which are linked by disulfide bonds between the chains, the amino acid sequence of the recombinant light chain L is shown as SEQ ID NO.1, and the amino acid sequence of the recombinant heavy chain Fd is shown as SEQ ID NO.2. The chimeric anti-ROR1 antibody Fab molecule provided by the invention greatly reduces mouse-derived components of an anti-ROR1 antibodyand still preserves the characteristics of a human-mouse cross reaction, thereby facilitating further experiments in vivo; the mimic antigen characteristics of a mouse-derived antibody are retained,and the chimeric anti-ROR1 antibody Fab molecule can potentially be used for targeting therapy of ROR1 high-expression tumors; a prokaryotic expression vector is easy to construct, can be expressed inlarge quantities, and is produced rapidly and convenient for purification; and the preparation of a chimeric whole-molecule antibody or humanization modification of the antibody are facilitated, andthe mouse-derived components of the anti-ROR1 antibody are further reduced for developing targeting therapy research of the ROR1 high-expression tumors.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering technology and immune targeting diagnosis and treatment, and specifically relates to a chimeric anti-ROR1 antibody Fab molecule and its preparation method and application. Background technique: [0002] Since the US FDA first approved the monoclonal antibody drug Muromumab (Muromomab-CD3) for clinical treatment in 1986, dozens of targeted agents have been used in tumor treatment, all of which have shown good tumor specificity and low Toxicity has become a highlight of tumor biotherapy. Targeted therapy has good selectivity. Through the recognition of target molecules by antibodies, it can specifically bind to tumor cell membrane receptors, block the signal transmission process, and then inhibit the growth and spread of tumor cells. In recent years, antibody-mediated tumor targeting therapy technology has made remarkable achievements in the clinical treatment of colorectal cancer, breast cancer...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/70A61K39/395A61P35/00G01N33/574
CPCA61K2039/505C07K16/2803C07K2317/24C12N15/70
Inventor 张慧林殷郑娜童华朱进
Owner 张慧林
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