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

Abstract

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 (^99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [^99mTc]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 [^99mTc]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 ^99mTc) 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.