Abstract Our aim is to raise nanobodies, antigen-binding fragments derived from heavy chain-only antibodies of the Camelidae family, against the N-myc oncoprotein and demonstrate that these can directly and potently cripple the oncogenic activity of N-myc in vitro and in vivo. If successful, this will support the rationale for their use as N-myc-specific inhibitors for the treatment of neuroblastoma, which accounts for 15% of all pediatric cancer-related deaths. Nanobodies, a novel class of therapeutic proteins, have unique and advantageous properties over conventional antibodies. They are small, only ~15 kDa, and have excellent solubility making them ideal for use as an intracellular antibody or ‘intrabody’. Nanobodies have specificities and affinities comparable to that of conventional antibodies and are amenable to high-throughput engineering to modify aspects of their function. MYCN, the gene encoding N-myc, is amplified in ~20% of neuroblastomas and significantly contributes to tumorigenesis.MYCN amplification strongly correlates with poor prognosis. Its inhibition in vitro abrogates oncogenic activity and inhibition in mouse models has resulted in tumor shrinkage and increased survival, making N-myc a rational therapeutic target. However, translating these results into humans has had limited success due to problems such as physically targeting an intracellular transcription factor. We propose that an N-myc-specific nanobody will provide a new, specific way to target N-myc. Established protocols will be used to obtain N-myc-specific nanobodies. To design the immunogen, we have used secondary structure prediction and Clustal alignment software and selected 3 suitable N-myc fragments. These have been successfully expressed as glutathione S-transferase (GST)-fusion proteins, and we are currently optimizing large-scale purification and testing folding. A llama will be immunized with these immunogens, and high-affinity N-myc-specific nanobodies will be selected by a series of methods including phage display technology and their binding and physical properties characterized. Two approaches will be investigated to express nanobodies in N-myc-expressing cancer cells: viral transduction and a novel protein transduction technology known as small molecule carriers (SMoCs). SMoCs are biphenyl mimics of α-helical protein transduction domains which can be coupled to any cargo via a free cysteine residue. They have been shown to enhance cellular uptake of proteins which retain functionality inside the cell. We have demonstrated that a nanobody in development for Parkinson's Disease (NbPD) can be coupled to a SMoC, and demonstrated that this dramatically increases its cellular uptake, providing validation of this proof-of-principle. We will utilize this technology for transporting N-myc-specific nanobodies. We are currently preparing to test SMoCs and SMoC-NbPD in mice. In conclusion, this work provides a basis for the development of N-myc-specific nanobodies for the treatment of MYCN-amplified neuroblastomas and other N-myc-driven cancers. Citation Format: Lisa Kent, Christopher Dobson, Erwin de-Genst, Heike Laman. Targeting the N-myc oncoprotein using nanobody technology. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B35.
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Jan 1, 2017
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