RP215 single chain fragment variable and single domain recombinant antibodies induce cell cycle arrest at G0/G1 phase in breast cancer.

Abstract

BACKGROUND:

Targeted therapy is an attractive approach to avoid the side effects of cancer treatment. Based on antibody-targeted superantigens, single chain variable fragment (scFv) and single domain (sdAb) antibodies, characterized by a low molecular weight, low immunogenicity and a high tumor penetration compared to monoclonal antibodies (mAb), have been increasingly used in gene-targeted therapy for cancer. In the present study, we aimed to develop the novel recombinant scFv-RP215 and sdAb-RP215 antibodies based on the variable regions of the RP215 monoclonal antibody (RP215-mAb) against CA215, a pan cancer marker expressed in various human tumor tissues, and to examine their biological activity in breast cancer cell lines.

METHODS:

The VH and VL genes were amplified from hybridoma cells secreting RP215-mAb by RT-PCR and joined with a linker using splicing by overlap extension PCR (SOE-PCR) to obtain the RP215-scFv gene, whereas the VH gene was used to generate the RP215-sdAb. Gene fragments of antibodies were subcloned into the pET32a(+) vector and expressed in Escherichia coli BL21. Western blot, indirect immunofluorescence (IF), ELISA and competitive ELISA were used to detect the immunoreactivity of scFv-RP215, sdAb-RP215, and RP215-mAb. The CCK-8 assay and cell cycle analysis were used to assess antibodies function.

RESULTS:

The novel recombinant scFv-RP215 and sdAb-RP215 antibodies were successfully developed based on the variable regions of the monoclonal antibody RP215 (RP215-mAb) against CA215. We verified that scFv-RP215 and sdAb-RP215 recognize CA215 on the surface of breast cancer cells (MB231, MCF7, MB468, SK-BR-3 and BT549) and characterized their activity and specificity. Our findings also indicate that scFv-RP215 and sdAb-RP215 induce cell cycle arrest at the G0/G1 phase in breast cancer cells.

CONCLUSION:

Our results showed that scFv-RP215 and sdAb-RP215 have excellent immunoreactivity and localize accurately to breast cancer cells in membrane-bound form, suggesting their potential as tumor targeting antibodies for breast cancer therapy.

• Authors: Fang Yu, Yanling Wang, Yun Xiao, Ying He, Cong Luo, Dong Duan, Chunhong Li, Shengsheng Xu, Tingxiu Xiang
• Journal: Molecular immunology (Mol Immunol) Vol. 59 Issue 1 Pg. 100-9 (May 2014) ISSN: 1872-9142 [Electronic] England
• PMID: 24534066 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Ltd. All rights reserved.

Chemical References

• Antibodies, Monoclonal
• Antigens, Neoplasm
• CA215 antigen, human
• Recombinant Proteins
• Single-Chain Antibodies
• Single-Domain Antibodies

Topics

• Amino Acid Sequence
• Antibodies, Monoclonal (immunology)
• Antibody Specificity (immunology)
• Antigens, Neoplasm (immunology, metabolism)
• Blotting, Western
• Breast Neoplasms (immunology, metabolism, pathology)
• Cell Cycle Checkpoints (immunology)
• Cell Line, Tumor
• Enzyme-Linked Immunosorbent Assay
• Escherichia coli (genetics)
• Female
• Flow Cytometry
• Fluorescent Antibody Technique, Indirect
• G1 Phase (immunology)
• Humans
• Hybridomas (immunology, metabolism)
• MCF-7 Cells
• Molecular Sequence Data
• Recombinant Proteins (immunology)
• Resting Phase, Cell Cycle (immunology)
• Single-Chain Antibodies (genetics, immunology)
• Single-Domain Antibodies (genetics, immunology)