The human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in a variety of cancers and therapies targeting HER2 are routinely used in the clinic. Recently, small engineered scaffold proteins, such as affibody molecules, have shown promise as carriers of cytotoxic drugs, and these drug conjugates may become complements or alternatives to the current HER2-targeting therapies. Here, we investigated if a monovalent HER2-binding affibody molecule, Z_HER2:2891, fused with a plasma half-life extending albumin binding domain (ABD), may be used as carrier of the cytotoxic maytansine derivate mcDM1. We found that the resulting drug conjugate, Z_HER2:2891-ABD-E₃-mcDM1, had strong affinity for its cognate molecular targets: HER2 and serum albumin. Z_HER2:2891-ABD-E₃-mcDM1 displayed potent cytotoxic activity towards cells with high HER2 expression, with IC₅₀ values ranging from 0.6 to 33 nM. In vivo, an unspecific increase in uptake in the liver, imparted by the hydrophobic mcDM1, was counteracted by incorporation of hydrophilic and negatively charged glutamate residues near the site of mcDM1 conjugation. A dose-escalation experiment showed that increasing doses up to 15.1 mg/kg gave a proportional increase in uptake in xenografted HER2-overexpressing SKOV3 tumors, after which the tumors became saturated. Experimental therapy with four once-weekly injection of 10.3 or 15.1 mg/kg led to efficient regression of tumors in all animals and complete regression in some. Weight loss was detected for some animals in the group receiving the highest dose, suggesting that it was close to the maximum tolerated dose. In conclusion, the monovalent HER2-targeting affibody drug conjugate presented herein have potent anti-tumor activity in vivo.

Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderate- to high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (Z_HER2:2891)₂–ABD–MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate–spacer–, (Z_HER2:2891)₂–ABD–E₃–MC-DM1, or a hexaglutamate–spacer–, (Z_HER2:2891)₂–ABD–E₆–MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (Z_HER2:2891)₂–ABD–MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

Affibody molecules hold great promise as carriers of cytotoxic drugs for cancer therapy due to their typically high affinity, easy production, and inherent control of the drug molecules’ loading and spatial arrangement. Here, the impact of increasing the drug load from one to three on the properties of an affibody drug conjugate targeting the human epidermal growth factor receptor 2 (HER2) was investigated. The affibody carrier was recombinantly expressed as a fusion to an albumin-binding domain (ABD) for plasma half-life extension. One or three cysteine amino acids were placed at the C-terminus to which cytotoxic mcDM1 molecules were conjugated. The resulting drug conjugates, Z_HER2–ABD–mcDM1 and Z_HER2–ABD–mcDM1₃, were characterized in vitro, and their biodistribution in mice carrying HER2-overexpressing SKOV3 xenografts was determined. Increasing the drug load from one to three led to a decrease in affinity for HER2, but a significantly more potent cytotoxic effect on SKOV3 cells with high HER2 expression. The difference in cytotoxic effect on other cell lines with high HER2 expression was not significant. In vivo, an increase in drug load led to a 1.45-fold higher amount of cytotoxic mcDM1 delivered to the tumors. The increase in drug load also led to more rapid hepatic clearance, warranting further optimization of the molecular design.

Treatment with antibody drug conjugates targeting receptors over-expressed on cancer cells is well established for clinical use in several types of cancer, however, resistance often occurs motivating the development of novel drugs. We have recently investigated a drug conjugate consisting of an affibody molecule targeting the human epidermal growth factor receptor 2 (HER2), fused to an albumin-binding domain (ABD) for half-life extension, loaded with the cytotoxic maytansine derivative DM1. In this study, we investigated the impact of the cytotoxic payload on binding properties, cytotoxicity and biodistribution by comparing DM1 with the auristatins MMAE and MMAF, as part of the drug conjugate. All constructs had specific and high affinity binding to HER2, human and mouse albumins with values in the low- to sub-nM range. Z_HER2-ABD-mcMMAF demonstrated the most potent cytotoxic effect on several HER2-over-expressing cell lines. In an experimental therapy study, the MMAF-based conjugate provided complete tumor regression in 50% of BALB/c nu/nu mice bearing HER2-over-expressing SKOV3 tumors at a 2.9 mg/kg dose, while the same dose of Z_HER2-ABD-mcDM1 provided only a moderate anti-tumor effect. A comparison with the non-targeting Z_Taq-ABD-mcMMAF control demonstrated HER2-targeting specificity. In conclusion, a combination of potent cytotoxicity in vitro, with minimal uptake in normal organs in vivo, and efficient delivery to tumors provided a superior anti-tumor effect of Z_HER2-ABD-mcMMAF, while maintaining a favorable toxicity profile with no observed adverse effects.

The epithelial cell adhesion molecule (EpCAM) is intensively overexpressed in 40–60% of prostate cancer (PCa) cases and can be used as a target for the delivery of drugs and toxins. The designed ankyrin repeat protein (DARPin) Ec1 has a high affinity to EpCAM (68 pM) and a small size (18 kDa). Radiolabeled Ec1 might be used as a companion diagnostic for the selection of PCa patients for therapy. The study aimed to investigate the influence of radiolabel position (N- or C-terminal) and composition on the targeting and imaging properties of Ec1. Two variants, having an N- or C-terminal cysteine, were produced, site-specifically conjugated to a DOTA chelator and labeled with cobalt-57, gallium-68 or indium-111. Site-specific radioiodination was performed using ((4-hydroxyphenyl)-ethyl)maleimide (HPEM). Biodistribution of eight radiolabeled Ec1-probes was measured in nude mice bearing PCa DU145 xenografts. In all cases, positioning of a label at the C-terminus provided the best tumor-to-organ ratios. The non-residualizing [¹²⁵I]I-HPEM label provided the highest tumor-to-muscle and tumor-to-bone ratios and is more suitable for EpCAM imaging in early-stage PCa. Among the radiometals, indium-111 provided the highest tumor-to-blood, tumor-to-lung and tumor-to-liver ratios and could be used at late-stage PCa. In conclusion, label position and composition are important for the DARPin Ec1.