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.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%–75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with ¹²⁵I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [¹²⁵I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [¹²⁵I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [¹²⁵I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [¹²⁵I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [¹²⁵I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [¹²⁵I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

ABY-027 is a scaffold-protein-based cancer-targeting agent. ABY-027 includes the second-generation Affibody molecule Z_HER2:2891, which binds to human epidermal growth factor receptor type 2 (HER2). An engineered albumin-binding domain is fused to Z_HER2:2891 to reduce renal uptake and increase bioavailability. The agent can be site-specifically labeled with a beta-emitting radionuclide ¹⁷⁷Lu using a DOTA chelator. The goals of this study were to test the hypotheses that a targeted radionuclide therapy using [¹⁷⁷Lu]Lu-ABY-027 could extend the survival of mice with HER2-expressing human xenografts and that co-treatment with [¹⁷⁷Lu]Lu-ABY-027 and the HER2-targeting antibody trastuzumab could enhance this effect. Balb/C nu/nu mice bearing HER2-expressing SKOV-3 xenografts were used as in vivo models. A pre-injection of trastuzumab did not reduce the uptake of [¹⁷⁷Lu]Lu-ABY-027 in tumors. Mice were treated with [¹⁷⁷Lu]Lu-ABY-027 or trastuzumab as monotherapies and a combination of these therapies. Mice treated with vehicle or unlabeled ABY-027 were used as controls. Targeted monotherapy using [¹⁷⁷Lu]Lu-ABY-027 improved the survival of mice and was more efficient than trastuzumab monotherapy. A combination of therapies utilizing [¹⁷⁷Lu]Lu-ABY-027 and trastuzumab improved the treatment outcome in comparison with monotherapies using these agents. In conclusion, [¹⁷⁷Lu]Lu-ABY-027 alone or in combination with trastuzumab could be a new potential agent for the treatment of HER2-expressing tumors.

Radionuclide molecular imaging could provide an accurate assessment of the expression of molecular targets in disseminated cancers enabling stratification of patients for specific therapies. B7-H3 (CD276) is a transmembrane protein belonging to the B7 superfamily. This protein is overexpressed in different types of human malignancies and such upregulation is generally associated with a poor clinical prognosis. In this study, targeting properties of an Affibody-based probe, AC12, containing a -GGGC amino acid sequence as a chelator (designated as AC12-GGGC) labelled with technetium-99m (^99mTc) were evaluated for imaging of B7-H3-expressing tumours. AC12-GGGC was efficiently labelled with ^99mTc. [^99mTc]Tc-AC12-GGGC bound specifically to B7-H3 expressing cells in vitro with affinities in nanomolar range. In mice bearing B7-H3-expressing xenografts, [^99mTc]Tc-AC12-GGGC showed tumour uptake of 2.1 ± 0.5 %ID/g at 2 h after injection. Its clearance from blood, normal organs and tissues was very rapid. This new targeting agent, [^99mTc]Tc-AC12-GGGC, provided high tumour-to-blood ratio already at 2 h (8.2 ± 1.9), which increased to 11.0 ± 0.5 at 4 h after injection. Significantly (p < 0.05) higher tumour-to-liver and higher tumour-to-bone ratios at 2 h in comparison with 4 h after injection were observed. Thus, [^99mTc]Tc-AC12-GGGC could be a promising candidate for further development.

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 of patients with HER2-expressing tumors using the monoclonal antibody trastuzumab increases survival. The affibody-based peptide nucleic acid (PNA)-mediated pretargeted radionuclide therapy has demonstrated efficacy against HER2-expressing xenografts in mice. Structural studies suggest that affibody molecules and trastuzumab bind to different epitopes on HER2. The aim of this study was to test the hypothesis that a combination of PNA-mediated pretargeted radionuclide therapy and trastuzumab treatment of HER2-expressing xenografts can extend the survival compared to monotherapies. Methods: Mutual interference of the primary pretargeting probe ZHER2:342-SR-HP1 and trastuzumab in binding to HER2-expressing cell lines was investigated in vitro. Experimental therapy evaluated the survival of mice bearing HER2-expressing SKOV-3 xenografts after treatment with a) vehicle; b) trastuzumab only; c) pretargeting using affibody-PNA chimera ZHER2:342-SR-HP1 and complementary probe ¹⁷⁷Lu-HP2; and d) combination of trastuzumab and pretargeting. The ethical permit limited the study to 90 days. The animals’ weights were monitored during the study. After the study termination, samples of liver and kidneys were evaluated by a veterinary pathologist for toxicity signs. Results: The presence of a large molar excess of trastuzumab had no influence on the affinity of ZHER2:342-SR-HP1 binding to HER2-expressing cells in vitro. The affinity of trastuzumab was not affected by a large excess of ZHER2:342-SR-HP1. The median survival of mice treated with trastuzumab (75.5 d) was significantly longer than the survival of mice treated with a vehicle (59.5 d). Median survival of mice treated with pretargeting was not reached by the day 90. Six mice of ten in this group survived, and two had complete remission. All mice in the combination treatment group survived, and tumors in seven mice had disappeared at the study termination. There was no significant difference between animal weights in the different treatment groups. No significant pathologic alterations were detected in livers and kidneys of treated animals. Conclusion: Treatment of mice bearing HER2-expressing xenografts with the combination of trastuzumab and affibody-mediated PNA-based radionuclide pretargeting significantly increased the survival compared to monotherapies. Co-treatment was not toxic for normal tissues.