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

TL;DR

Scientists improved a protein used for imaging tumors by modifying its Fc region, which reduced unwanted accumulation in organs like the liver and spleen and enhanced tumor targeting.

Contribution

The novel Fc mutations (LALAPG) significantly improved tumor uptake and reduced off-target accumulation in Fc-rich organs.

Findings

Fc-engineered mutants showed high tumor uptake (13.0%ID/g) and reduced liver/spleen accumulation compared to wild-type.

Biodistribution studies confirmed high tumor specificity in multiple xenograft models with moderate to high MSLN expression.

Female mice showed higher renal retention of the engineered protein, indicating sex-specific pharmacokinetics.

Abstract

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 ex vivo and in vivo analyses.
Following zirconium-89 radiolabeling, PET imaging was conducted across
multiple xenograft models with varying MSLN expression. In HCT116
xenografts, [89Zr]­Zr-2A10-VH-FcLALAPG demonstrated
substantially 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 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-FcLALAPG 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.

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