Evaluation of a novel 177Lu-labelled therapeutic Affibody molecule with a deimmunized ABD domain and improved biodistribution profile.

PURPOSE: Fusion of Affibody molecules with an albumin-binding domain (ABD) provides targeting agents, which are suitable for radionuclide therapy. To facilitate clinical translation, the low immunogenic potential of such constructs with targeting properties conserved is required. METHODS: The HER2-targeting Affibody molecule ZHER2:2891 was fused with a deimmunized ABD variant and DOTA was conjugated to a unique C-terminal cysteine. The novel construct, PEP49989, was labelled with 177Lu. Affinity, specificity, and in vivo targeting properties of [177Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated. RESULTS: The maximum molar activity of 52 GBq/µmol [177Lu]Lu-PEP49989 was obtained. [177Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [177Lu]Lu-PEP49989 was similar to the affinity of [177Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [177Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [177Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [177Lu]Lu-PEP49989 (> 90 days) was significantly longer than the survival of mice treated with vehicle (38 days) or trastuzumab (45 days). Treatment using a combination of [177Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild. CONCLUSION: In preclinical studies, [177Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

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 ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=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.