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The antibody-anticalin fusion protein (Mabcalin) targeting programmed cell death-ligand 1 (PD-L1) and T-cell costimulatory immunoreceptor CD137 (4-1BB) is designed to enhance T-cell reactivity while preventing T-cell inhibition by PD-L1/programmed cell death protein 1 (PD-1) checkpoint blockade. Using positron emission tomography (PET) imaging and ex vivo analysis, we investigated the factors influencing biodistribution, tumor uptake, and the influence of dose and target presence. Methods Murine or human PD-L1-reactive and human 4-1BB reactive Mabcalins (mPD-L1xh4-1BB and hPD-L1xh4-1BB) were generated, radiolabeled with ⁸⁹Zr, and administered to human 4-1BB knock-in (h4-1BB KI) or wild-type (WT) mice bearing PD-L1-positive mouse MC38 tumors. Mice underwent serial PET imaging on days 1, 2, and 4 or on days 2, 4, and 7 post intravenous injection, followed by ex vivo biodistribution. The intratumoral distribution of 2 protein doses of ⁸⁹Zr-radiolabeled mPD-L1xh4-1BB and hPD-L1xh4-1BB was examined using autoradiography on tumor tissue sections. These tumor sections were immunohistochemically stained for PD-L1, CD3, CD8, and 4-1BB to link uptake to target expression levels. Results ⁸⁹Zr-mPD-L1xh4-1BB, able to bind mPD-L1 and h4-1BB in h4-1BB KI mice, predominantly showed specific and rapid dose-dependent lymphoid tissue uptake. The tumor uptake of 200 µg ⁸⁹Zr-mPD-L1xh4-1BB in h4-1BB KI mice was also specific and increased over time. Tumor uptake in this group, where both targets PD-L1 and 4-1BB could be bound, was > 4-fold higher than in the groups that could bind only PD-L1 or 4-1BB. Dual PD-L1 and 4-1BB Mabcalin engagement at a therapeutic dose also resulted in elevated tumor protein expression levels for PD-L1, CD3, and CD8, which were lower when only PD-L1 or 4-1BB was engaged. The lowest expression was observed with the Mabcalin binding non-specifically (P _trend≤0.01 for PD-L1 and CD3, P _trend≤0.05 for CD8). Conclusion The biodistribution of mPD-L1xh4-1BB is specific, dose-dependent, and associated with the elevated target expression resulting from dual PD-L1 and 4-1BB engagement.