Therapeutic affinity proteins offer a targeted mode of action due to their typically high affinity and specificity for disease-associated molecules. In cancer therapy, such target molecules are often overexpressed receptors on tumor cells. However, their presence in healthy tissues can lead to on-target, off-tumor toxicity, necessitating strategies to enhance tumor selectivity. Here, we present an affibody-based prodrug concept that exploits tumor-associated proteases for selective activation. As proof of concept, we designed, produced, and characterized HER2-specific prodrug candidates, each incorporating a distinct protease substrate for selective activation by tumor-associated proteases. Their activation by corresponding proteases and subsequent HER2 binding were assessed. The most promising prodrug candidate was conjugated to the cytotoxic agent DM1 and evaluated for cytotoxicity in HER2-positive cancer cells. The results demonstrated potent, HER2-dependent cell killing, with markedly reduced cytotoxicity in the absence of prodrug activation. These findings support the feasibility of affibody-based prodrugs as a strategy to enhance tumor selectivity and minimize off-tumor toxicity in targeted cancer therapy.

Safety and efficacy of cancer-targeting treatments can be improved by conditional activation enabled by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumourigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease -dependent activation has the potential to increase tumour-selective targeting while decreasing exposure to healthy tissues, thus improving the safety profile for patients. Higher selectivity could also allow for adminis-tration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). We could show that binding to endogenous EGFR on cancer cells in vitro was restored following proteolytic removal of ZB05. In this study we evaluate a novel affibody-based pro -drug design, which incorporates a protease substrate sequence recognized by cancer-associated proteases and demonstrate the potential of this approach for selective tumour-targeting and shielded uptake in healthy tissues in vivo using tumour-bearing mice. This may widen the therapeutic index of cytotoxic EGFR-targeted thera-peutics by decreasing side effects, improving selectivity of drug delivery, and enabling the use of more potent cytotoxic drugs.

Cancer-directed therapies targeting membrane-bound receptors are often limited by systemic toxicity. The epidermal growth factor receptor (EGFR) is a receptor commonly overexpressed in cancer but its abundance in healthy tissues, particularly in skin, often results treatment-induced toxicities. Efficacy and safety of EGFR-targeting drugs could be dramatically improved by increasing the selectivity for tumours. The tumour microenvironment differs significantly from normal tissues which can be exploited to achieve local conditional drug activation and selective tumour-targeting. Proteases are often upregulated in cancer to promote tumour growth, invasion, and metastasis. We have developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). Binding to EGFR is restored following removal of ZB05 by cancer-associated proteases. We show that cleavage by proteases is necessary for EGFR-mediated targeting of cancer cells and delivery of cytotoxic drugs in vitro. These results warrant further evaluation of the potential pharmacokinetic advantages of an EGFR-targeting affibody prodrug in vivo compared to non-masked EGFR-targeting variants.