Impact of Radiometal Chelates on In Vivo Visualization of Immune Checkpoint Protein Using Radiolabeled Affibody Molecules

The immune checkpoint protein B7–H3 (CD276) is overexpressed in various cancers and is an attractive target for the treatment of malignant tumors. Radionuclide molecular imaging of B7–H3 expression using engineered scaffold proteins such as Affibody molecules is a promising strategy for the selection of potential responders to B7–H3-targeted therapy. Feasibility of B7–H3 imaging was demonstrated using two ^99mTc-labeled probes, AC12 and an affinity-matured SYNT179 using a [^99mTc]Tc-GGGC label. This study aimed to evaluate whether the use of a residualizing ¹¹¹In-based label provides better imaging contrast compared with a nonresidualizing label. To do that, SYNT179 and AC12-GGGC Affibody molecules were labeled with ¹¹¹In using (4,10-bis-carboxymethyl-7-{[2-(2,5-dioxo-3-thioxo-pyrrolidin-1-yl)-ethylcarbamoyl]-methyl}-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid (maleimide-DOTA) chelator, site-specifically coupled to the C-terminus of Affibody molecules. The binding affinities of the ¹¹¹In-labeled conjugates to B7–H3-expressing living cells were higher compared with the affinities of the ^99mTc-labeled variants. In mice with B7–H3-expressing xenografts, the tumor uptake of ¹¹¹In-labeled proteins (3.6 ± 0.3 and 1.8 ± 0.5%ID/g for [¹¹¹In]In-SYNT179-DOTA and [¹¹¹In]In-AC12-DOTA, respectively) was significantly (p < 0.05, ANOVA) higher than those for ^99mTc-labeled counterparts (1.6 ± 0.2%ID/g and 0.8 ± 0.2%ID/g for [^99mTc]Tc-SYNT179 and [^99mTc]Tc-AC12-GGGC, respectively). The best variant, [¹¹¹In]In-SYNT179-DOTA, provided a tumor-to-blood ratio of 31.1 ± 2.9, which was twice higher than that for [^99mTc]Tc-SYNT179 and 7-fold higher than that for [^99mTc]Tc-AC12-GGGC. Both ¹¹¹In-labeled Affibody molecules had higher renal retention compared with ^99mTc-labeled ones, but the hepatobiliary excretion of ¹¹¹In-labeled proteins was appreciably lower, potentially improving the imaging of abdominal metastases. Overall, [¹¹¹In]In-SYNT179-DOTA is the most promising tracer for visualization of B7–H3 expression.