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Human Epidermal Growth Factor Receptor 2 (HER2) is over-expressed in several cancers, including breast, gastric, ovarian, and lung cancers. Although HER2-targeted monoclonal antibodies have improved clinical outcomes, resistance remains a major challenge. Their large molecular size also limits tumor penetration and leads to prolonged circulation, increasing off-target toxicity. Affibody molecules offer an attractive alternative due to their small size (~7 kDa), high stability, and strong, specific HER2 binding. Their rapid tumor penetration and fast blood clearance make them suitable for imaging and Targeted Radionuclide Therapy (TRT). Therapeutic radionuclides like lutetium-177 and rhenium-188 can be site specifically labeled with Affibody molecules via conjugated chelators, enabling selective delivery of cytotoxic radiation to HER2-expressing tumors. A major limitation of Affibody-based TRT is high renal uptake. Strategies such as non-residualizing labels and fusion with albumin-binding domains (ABD) aim to improve biodistribution. Non-residualiz-ing labels utilize the slow internalization of HER2-bound Affibody molecules in tumors while allowing rapid renal clearance, resulting in higher tumor-to-kidney ratios. ABD fusion prolongs circulation by binding to serum albumin, reducing renal filtration and enhancing tumor accumulation. These approaches form the basis of the work summarized in this thesis.

Paper I showed that the non-residualizing label, [¹⁸⁸Re]Re-Z_HER2:41071 pro-vided favorable tumor-to-kidney ratios and improved survival in mice without organ toxicity. Paper II evaluated chelator positioning in ABD-fused constructs and demonstrated that placing DOTA to helix 1 of ABD did not reduce renal uptake. Paper III showed that chemo-enzymatic peptide synthesis enables production of ABD-fused Affibody molecules with preserved structure, HER2 affinity, albumin binding, and in vivo targeting. Paper IV demonstrated that [¹⁷⁷Lu]Lu-ABY-027, alone or combined with trastuzumab, significantly prolonged survival in xenografted mice, with combination therapy providing better outcome. Paper V introduced a variant with deimmunized ABD, PEP49989, which improved biodistribution profile and provided potent therapeutic efficacy, further enhanced by trastuzumab, with minimal renal and hepatic toxicity.

In conclusion, strategies such as non-residualizing label and ABD fusion enable effective tumor targeting and therapeutic effects. These advances validate Affibody-based TRT as a promising complement to existing HER2-directed therapies and provide important design principles for next-generation radio-pharmaceuticals with improved safety, efficacy, and translational potential.