Oligo-clonal nanobodies as an innovative targeting agent for cancer therapy: New biology and novel targeting systems

•Nanobodies are smallest intact antibody fragment with subnanomolar affinity against antigen.•Nanobodies are powerful tools as targeting agents for targeted delivery in cancer therapy.•Mutation is hallmark of cancerous cells, so antigen drift is probable in these cells.•Four different clones of nanobody, target different epitopes on HER2 receptor, were expressed and purified.•Using oligo-clonal nanobodies in targeting enhance affinity and avidity of targeting moiety in comparison to monoclonal one.

Variable heavy chain of HcAb (VHH), the smallest intact antibody fragment, possesses sub-nanomolar affinity to antigens. In spite of conventional antibodies, these fragments recognize concave and linear epitopes. VHHs are one the best weapon for targeted drug delivery in nanomedicine and biopharmaceutics. HER2 is overexpressed in 20-25% of breast and ovarian cancers. For many reasons, HER2 is a prominent target for drug delivery to breast tumor. In this study, we designed a robust prokaryotic expression system to express functional VHHs against HER2 receptor. This system showed high recombinant yields besides purified VHHs flow cytometry verified great capabilities of these molecules to pinpoint ecto-domain of HER2 receptor in MC4L2 HER2+ while insignificant non-specific binding to MC4L2 HER2-confirm nanobodies trivial cross-reaction. In the next step, we evaluated cooperative effect of four distinctive VHHs (oligoclonal VHHs) targeting different epitopes on HER2. As our result proved, using oligoclonal nanobodies as targeting moiety enhance targeting efficacy in comparison with monoclonal VHH.