# 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.

## Key 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.