Comparison of approaches for increasing affinity of affibody molecules for imaging of B7-H3: dimerization and affinity maturation

Background Radionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast. Results Two dimeric variants of anti-B7-H3 Affibody molecules were produced (designated Z AC12* -Z AC12* -GGGC and Z AC12* -Z Taq_3 -GGGC). Both variants were labelled with Tc-99m ( 99m Tc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [ 99m Tc]Tc-Z AC12* -Z AC12* -GGGC showed subnanomolar affinity (K D1 =0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [ 99m Tc]Tc-Z AC12* -Z Taq_3 -GGGC (K D =2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with 99m Tc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule. Conclusion The improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes.