Tianqi Xu: Scaffold Protein-based Theranostics of HER2-overexpressing Ovarian Cancer: Imaging-guided Therapy

Abstract

This thesis is based on five original articles aiming to develop HER2-targeting affibody-albumin-binding-domain (ABD)-drug conjugates (AffiDCs) for the treatment of ovarian cancer. The research focused on several aspects of molecular design, including the number of HER2-binding domains (ZHER2), the number and position of functional domains targeting HER2 or albumin, the number of conjugated drug molecules, the composition of linkers between domains, and the drug composition. The cytotoxic payloads DM1, MMAE or MMAF were conjugated via a maleimidocaproyl (mc) linker. All affibody-based constructs were radiolabeled with technetium-99m to quantitatively assess their properties in vitro and in vivo. The selected conjugates were evaluated in therapeutic studies using the HER2-overexpressing SKOV3 ovarian cancer xenograft model in BALB/c nu/nu mice. 

In Paper I, the influence of HER2-binding valency on targeting properties, internalization, cytotoxicity, and drug delivery was evaluated. The ZHER2-ABD-E3-DM1 conjugate showed highly potent anti-tumor activity in vivo. Imaging using SPECT/CT visualized the HER2 expression during the treatment. In Paper II, the influence of the number and position of functional domains on cancer cell proliferation was investigated. While dimeric anti-HER2 affibody molecules stimulated the proliferation of cancer cells in vitro and promoted tumor growth in vivo, the additional stimulation of proliferation did not improve the therapeutic effect of DM1. ZHER2-ABD was selected as the most optimal format for targeted delivery of DM1. In Paper III, the influence of the number of conjugated drug molecules on biodistribution and tumor-targeted drug delivery was investigated. Increasing the DM1 number from one to three increased the amount of drug delivered to tumors; however, it also raised the risk of normal organ toxicity. In Paper IV, the influence of linker composition between the affibody domain and ABD on biodistribution was evaluated. Introducing additional serine in the inter-domain linker decreased the hepatic uptake. However, the linker length should be optimal to preserve cytotoxicity and high-affinity binding to the target. In Paper V, the impact of the drug composition on binding properties, cytotoxicity, and biodistribution was examined. ZHER2-ABD-MMAF showed higher efficacy than ZHER2-ABD-DM1 for HER2-targeted therapy in vivo. 

In conclusion, modification of molecular design impacts functional properties, biodistribution, and tumor targeting of AffiDCs. Careful optimization of molecular design allowed for selecting several candidates with high anti-tumor efficacy and favorable toxicity profiles in mice.