MPA Case Study
Membrane Proteome Array™ confirms specificity of novel antibody VH 3C9 for ADC discovery
The Need: Potent, Specific Biotherapeutics to Target Mesothelin
Mesothelin is an ideal target for antibody-based therapeutics due to its high expression in cancer cells, coupled with low expression in healthy adult tissues. Several mesothelin-targeting therapeutics have shown promising antitumor capabilities in clinical trials. Yet a need remains for more potent, yet safe, treatments that can better penetrate solid tumors.
Researchers at the University of Pittsburgh and Abound Bio* identified a novel antibody heavy chain variable (VH) domain, VH 3C9, with excellent targeting specificity and binding affinity to mesothelin. The team fused VH 3C9 to a human immunoglobulin (Ig)G1 Fc protein scaffold to form the bivalent VH-Fc 3C9 for further testing as a therapeutic candidate.
For improved efficacy, the team sought to develop their candidate as an antibody-drug conjugate (ADC). ADCs stand out as highly potent cancer killing agents—yet because they are so potent, any off-target binding in healthy tissues raises serious toxicity concerns. Thus, before proceeding, the team needed to evaluate their molecule’s specificity for mesothelin.
* Abound Bio was acquired by Galápagos in 2022.
The Solution: Membrane Proteome Array Confirms Target Specificity
Integral Molecular’s cell-based Membrane Proteome Array (MPA) assesses off-target binding by screening antibody therapeutics against a library of 6,000 human membrane-associated proteins, the largest and most comprehensive library of its kind.
Screening on the MPA revealed no off-target binding for VH-Fc 3C9, confirming its specificity for mesothelin (MSLN). While minor Fc gamma receptor (FCGR) interactions are expected with the Fc scaffold region, they do not compromise the molecule’s targeting abilities.
These findings indicate that VH-Fc 3C9 is a highly specific candidate suitable for development as an ADC against mesothelin-expressing cancers (data from Sun et al., 2023, Fig. 1G).
The Outcome: Paving the Way for Improved ADC Therapeutics
VH antibody domains have gained attention for their improved solid tumor penetration and reduced immunogenicity. And VH-Fc 3C9 is a promising candidate for safer and more precise therapeutics against mesothelin-expressing tumors.
Screening on Integral Molecular’s MPA successfully confirmed the antibody’s high specificity, detecting no off-target cross-reactivity across the membrane proteome.
When coupled with the drug monomethyl auristatin E, VH-Fc 3C9 exhibited remarkable targeting and tumor-killing capabilities, forming a powerful ADC. This work will allow for the optimization of VH 3C9 in constructing antibody therapeutics, improving outcomes for mesothelin-expressing cancers.
"Non-specific interactions can lead to off-target binding, which results in toxicity or fast antibody clearance in vivo. 3C9 reported here did not bind the 6,000 human membrane-associated proteins in the MPA assay, indicating low potential for off-target toxicity.”
Data featured in Molecular Therapy Oncology, Sun et. al, 2023