Tumour accumulation of anti-carcinoembryonic antigen affibody-albumin-binding domain fusion depends on domains' order

Affibody affinity proteins are engineered scaffold proteins that can be selected by molecular display techniques for high-affinity, specific binding to various molecular targets. Conjugation of cytotoxic payloads (radionuclides or drugs) to affibodies has enabled successful treatment of human xenografts in mice. In this study, we evaluated a novel affibody-based system for in vivo targeting of tumours expressing the pancarcinoma antigen carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5). A CEACAM5-binding affibody head-to-tail dimer, C9-C9, was fused with the albumin-binding domain ABD₀₃₅ to extend its blood residence time. Two variants were designed and evaluated, one with ABD₀₃₅ placed at the N-terminus of the construct (ABD₀₃₅-C9-C9) and the other with ABD₀₃₅ at the C-terminus (C9-C9-ABD₀₃₅). The constructs were site-specifically labelled with ^99mTc, employing a C-terminal EYEC tetrapeptide extension to allow in vivo tracking. The radiolabelled affibody retained specific binding to CEACAM5-expressing cells in vitro. When compared in vivo to non-ABD₀₃₅-fused C9-C9, both constructs showed a 10-fold decrease in renal uptake, a 50-fold increase in blood concentration, and a 9-fold increase in tumour uptake 4 h after injection in mice bearing human CEACAM5-expressing xenografts. The tumour uptake was CEACAM5-specific and depended on the target's expression level. The biodistribution was dependent on the placement of ABD₀₃₅, and the construct with ABD₀₃₅ at the N-terminus, [^99mTc]Tc-ABD₀₃₅-C9-C9, had a longer half-life in blood and significantly higher uptake in tumours. In conclusion, a fusion of C9-C9 with ABD₀₃₅ and optimisation of the molecular architecture provides an affibody-based platform for targeting CEACAM5-expressing malignant tumours.