Fc-Engineering Improves PET Imaging of Anti-Mesothelin VH-Fc across Multiple Tumor Mouse Models and Reveals Sex-Specific Renal Clearance

Mesothelin (MSLN) is overexpressed in various malignancies, making it a promising target for molecular imaging and therapeutic strategies. Anti-MSLN VH-Fc fusion proteins show high tumor uptake as compared with monoclonal antibodies; however, elevated accumulation in Fc-rich organs (liver, spleen) can compromise tumor-to-background ratios and limit clinical applicability. To overcome this, we developed Fc mutant anti-MSLN VH-Fc fusion proteins incorporating G236R/L328R (GRLR) and L234A/L235A/P329G (LALAPG) mutations to eliminate FcγRs interactions. Engineered mutants exhibited high purity (>95%), retained strong MSLN binding (KD 2.2-3.7 nM), and effectively silenced FcγR binding by <i>ex vivo</i> and <i>in vivo</i> analyses. Following zirconium-89 radiolabeling, PET imaging was conducted across multiple xenograft models with varying MSLN expression. In HCT116 xenografts, [⁸⁹Zr]Zr-2A10-VH-Fc_LALAPG demonstrated substantially higher uptake (13.0 ± 0.1%ID/g at 120 h p.i.) than [⁸⁹Zr]Zr-2A10-VH-Fc_WT (4.2 ± 0.6%ID/g), while substantially reducing liver (LALAPG: 4.3 ± 0.6%ID/g vs WT: 19.8 ± 2.8%ID/g) and spleen (LALAPG: 9.3 ± 0.1%ID/g vs WT: 95.0 ± 39.3%ID/g) uptake. Biodistribution studies in additional xenograft models confirmed a high specific uptake for [⁸⁹Zr]Zr-2A10-VH-Fc_LALAPG in tumors with moderate to high MSLN expression. Notably for the mutants, females exhibited higher renal retention than males, indicating sex-dependent pharmacokinetics. These findings highlight Fc-engineered VH-Fc fusion proteins, particularly the LALAPG, as promising agents with enhanced tumor specificity, improved pharmacokinetics, and significantly reduced off-target uptake, supporting their use in PET imaging-guided therapeutic applications.