Poster abstracts
Poster number 88 submitted by Valerio Embrione
Optimization of human anti-NCL immunoagents for cancer therapy
Valerio Embrione (MCDB), Claudia Foray, Anna Tessari, Tyler J. Sheetz, Ashley E. Braddom, K. Parbhoo, Joseph J. Mills, Meghan E. Pawlikowski, Dario Palmieri, Vincenzo Coppola, Carlo M. Croce (Department of Cancer Biology and Genetics, Comprehensive Cancer Center The Ohio State University, Columbus, OH)
Abstract:
Nucleolin (NCL) is a nucleolar protein, associated to several RNA functions, such as ribosomal RNA (rRNA) processing, ribosomal assembly and transcript stabilization. It is also involved in the positive regulation of the biogenesis of several microRNAs related to cancer. NCL is often reported as upregulated in many different types of cancer. For reasons still not fully understood, in highly proliferative cancer cells, Nucleolin is also abundantly located on the cell surface. This property makes Nucleolin a potential target for cancer diagnosis and therapy. Several laboratories attempted to develop anti-neoplastic molecules targeting Nucleolin on the cell surface (aptamers, peptides), but their clinical relevance is still debated. We recently developed a human single chain fragment variable (ScFv) antibody, named 4LB5, which is internalized in the cytoplasm of those cells exposing Nucleolin on their surface. This occurrence interferes with the biogenesis of microRNAs regulated by Nucleolin itself, leading to decreased cell viability in vitro and reduced tumor volume in vivo. Our goal is improving the therapeutic potential of 4LB5, as well as optimizing the production and the purification process. To this aim, we cloned 4LB5 in pBAD-MycHis vector, a plasmid allowing a tight arabinose-modulated recombinant production of the protein of interest in E. Coli. The controlled expression, combined to the 6xHis and Myc tags carried by the plasmid at the C-terminus of the expression cassette, leads to increased protein solubility and enhanced purification efficiency. In order to improve the anti-neoplastic activity and the pharmacokinetics of the compound, we cloned 4LB5 also in a plasmid containing the Fragment crystallizable (Fc) region of human IgGs, making a functional compact form of the antibody. Finally, we analyzed the binding ability and the anti-neoplastic activity of both the compounds in an in vitro model of triple negative breast cancer.
References:
Pichiorri et al., J Exp Med (2013)
Frenzel et al., Front Immunol (2013)
Jager et al., BMC Biotechnol (2013)
Palmieri et al., PNAS (2015)
D’Avino et al., Oncotarget (2016)
Ohio State Innovation Foundation, Patent #WO 2017011411 A1 (2016)
Keywords: Nucleolin, Recombinant Proteins, Cancer Therapy