Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Abstract

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.

Programmable polyproteams built using twin peptide superglues

“…These polyproteams were used to probe the spatial integration between different signaling pathways. Cancer cells are frequently sensitized to cell death induced by Death Receptor signaling, but clinical results have been disappointing using bivalent IgG, which is not potent at cell death induction (35,36). We set out to explore higher multivalency and how Death Receptor signaling interacts with signaling by growth factor receptors frequently overexpressed in cancer.…”

RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis

“…In addition to full-length antibodies, different scaffolds such as atrimers (31) or multivalent monobodies (32,33) are under evaluation to target DRs. However, the lack of clinical efficacy observed with untargeted DR5 agonistic antibodies illustrates that the activity of these molecules in vitro may not necessarily translate into clinical efficacy.…”

Unexpected hepatotoxicity in a phase I study of TAS266, a novel tetravalent agonistic Nanobody® targeting the DR5 receptor

“…In human tumor cell death assays, TAS266 was ≥1000-fold more potent when compared with a crosslinked DR5 antibody LBY135 or TRAIL [17]. In vivo, TAS266 elicited single-dose tumor regressions in multiple human tumor xenograft models.…”

“…1) [17]. TAS266, a novel agonistic 60 kDa Nanobody ® that targets the DR5 receptor, is tetravalent, consisting of four identical humanized VHH antibody fragments connected through three linkers of 35 amino acids each.…”