Characterization of Epidermal Growth Factor (EGF) receptor targeting Nanobodies and ABIN for the treatment of EGF receptor-dependent tumours

At this point, use of the generated EGFR-specific Nanobodies as therapeutic agents, even in bivalent form and fused to an adaptor that can increase residence time, is considered no to be expedient. The tumour harming effect is not comparable to cetuximab. It would be interesting however to investigate whether the induced change in morphology reflects a change in sensitivity to radio- or chemotherapy. If so, combining Nanobody treatment with conventional therapy could improve the therapeutic outcome. Especially because EGFR-specific Nanobodies, even in trivalent format, are able to readily reach all areas of the tumour mass as opposed to cetuximab. Screening for Nanobodies with subnanomolar affinity for binding EGFR, could yield a Nanobody with increased therapeutic value. On the other hand further insight into the EGFR activation mechanism, could pinpoint new regions of clinical relevance and could motivate the generation of a new set of Nanobodies with hopefully enhanced therapeutic efficacy. The versatile nature of Nanobodies also allows fusion of adaptor molecules which facilitate tumour uptake or which can increase the overall cytotoxic effect. The in vivo biodistribution data indicate that the EGFR-specific Nanobodies are perfectly suited for in vivo molecular imaging studies that aim the detection of EGFR-overexpressing tumours. For this, the 7C12 Nanobody performed outstandingly well. From the literature it is clear that in various cancers the NF-kappaB pathway is heavily exploited to resist conventional therapy and to enchance proliferation. EGFR was found to be one of the upstream activators of NF-kappaB. We demonstrated that ABINs can effectively inhibit the EGFR-induced NF-kappaB activation. In addition proliferation of A431 and DU145 tumour cells could be halted by ectopic expression of ABIN-1. The excellent tumour targeting capability of EGFR-specific Nanobodies offers the opportunity to specifically deliver anti-proliferative or tumor cell killing agents to cancer cells that overexpress EGFR. In this context, it would allow the intracellularly delivery of NK-kappaB inhibitory proteins such as ABINs. In this case, the translocation domain (TLD) of exotocin A from Pseudomonas aeruginosa can be fused between the homing Nanobody and the NF-kappaB inhibitory protein. After uptake in the endosomes TLDs are cleaved by the endosomal protease furine, resulting in cytoplasmic translocation of the ABIN protein fused to the Nanobody. In order to minimize immunogenicity and to preserve optimal tumour penerating capabilities, the fused protein should preferably be as small as possible. ABIN-1 MAD, a 20 kDa fragment derived from ABIN-1 for which we showed that it could convincingly inhibit both EGF-induced and basal NF-kappaB activity, might be an ideal candidate for Nanobody conjugation. Similarly, other NK-kappaB inhibitory proteins such as the well established 37 kDA IkappaBalpha superrepressor, as well as pro-apoptotic peptides like the mitochondrial membrane destabilizing peptide(KLAKKLAK)2 are also valuable candidates that may enhance the therapeutic effect of EGFR-specific Nanobody-based therapy.