Fc-EngineeringImproves PET Imaging of Anti-MesothelinVH-Fc across Multiple Tumor Mouse Models and Reveals Sex-SpecificRenal Clearance

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Mesothelin (MSLN) is overexpressed in various malignancies,makingit a promising target for molecular imaging and therapeutic strategies.Anti-MSLN VH-Fc fusion proteins show high tumor uptake as comparedwith monoclonal antibodies; however, elevated accumulation in Fc-richorgans (liver, spleen) can compromise tumor-to-background ratios andlimit clinical applicability. To overcome this, we developed Fc mutantanti-MSLN VH-Fc fusion proteins incorporating G236R/L328R (GRLR) andL234A/L235A/P329G (LALAPG) mutations to eliminate FcγRs interactions.Engineered mutants exhibited high purity (>95%), retained strongMSLNbinding (KD 2.2–3.7 nM), and effectively silenced FcγRbinding by ex vivo and in vivo analyses.Following zirconium-89 radiolabeling, PET imaging was conducted acrossmultiple xenograft models with varying MSLN expression. In HCT116xenografts, [89Zr]­Zr-2A10-VH-FcLALAPG demonstratedsubstantially higher uptake (13.0 ± 0.1%ID/g at 120 h p.i.) than[89Zr]­Zr-2A10-VH-FcWT (4.2 ± 0.6%ID/g),while substantially reducing liver (LALAPG: 4.3 ± 0.6%ID/g vsWT: 19.8 ± 2.8%ID/g) and spleen (LALAPG: 9.3 ± 0.1%ID/gvs WT: 95.0 ± 39.3%ID/g) uptake. Biodistribution studies in additionalxenograft models confirmed a high specific uptake for [89Zr]­Zr-2A10-VH-FcLALAPG in tumors with moderate to highMSLN expression. Notably for the mutants, females exhibited higherrenal retention than males, indicating sex-dependent pharmacokinetics.These findings highlight Fc-engineered VH-Fc fusion proteins, particularlythe LALAPG, as promising agents with enhanced tumor specificity, improvedpharmacokinetics, and significantly reduced off-target uptake, supportingtheir use in PET imaging-guided therapeutic applications.