Expression-Dependent Tumor Pretargeting via Engineered Avidity

Abbigael Harthorn; Tse-Han Kuo; Sarah W Torres; Roy R Lobb; Benjamin J Hackel

doi:10.1021/acs.molpharmaceut.4c01177

Expression-Dependent Tumor Pretargeting via Engineered Avidity

Mol Pharm. 2024 Dec 20. doi: 10.1021/acs.molpharmaceut.4c01177. Online ahead of print.

Authors

Abbigael Harthorn¹, Tse-Han Kuo², Sarah W Torres¹, Roy R Lobb³, Benjamin J Hackel^{1

2

3}

Affiliations

¹ Department of Biomedical Engineering, University of Minnesota-Twin Cities, Minneapolis, Minnesota 55455, United States.
² Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota 55455, United States.
³ MRB Biotherapeutics, Newton Center, Massachusetts 02459, United States.

PMID: 39704255
DOI: 10.1021/acs.molpharmaceut.4c01177

Abstract

Selective delivery of therapeutic modalities to tumor cells via binding of tumor-selective cell-surface biomarkers has empowered substantial advances in cancer treatment. Yet, tumor cells generally lack a truly specific biomarker that is present in high density on tumor tissue while being completely absent from healthy tissue. Rather, low but nonzero expression in healthy tissues results in on-target, off-tumor activity with detrimental side effects that constrain the therapeutic window or prevent use altogether. Advanced technologies to enhance the selectivity for tumor targeting are sorely needed. We have engineered a binding platform that is quantitatively dependent upon expression levels, via avidity-driven specificity, rather than binarily reliant on the presence or absence of a biomarker. We systematically varied monomeric binding affinity by engineering affibodies to target carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and folate receptor 1 (FolR1). Two identical affibody ligands were tethered, with varying polypeptide linker lengths, to a nanobody that binds Alfa peptide to create a bispecific, trivalent protein for use in pretargeted radioligand therapy. Expression-dependent targeting was achieved in both systems: with 110 nM monomeric affinity to CEACAM5 with a two-amino-acid linker or with 250 nM monomeric affinity for FolR1 and a 10 amino acid linker. The latter bispecific, trivalent achieved over 25-fold differentiation between FolR1^high and FolR1^low cells in a mixed culture. Similar selectivity was achieved in a size-efficient bivalent molecule lacking a central nanobody. Moreover, the avid bivalent affibody molecule exhibited minimal inhibition by soluble antigen, whereas high-affinity bivalent antibody was inhibited by 97 ± 2%, which is indicative of serum inhibition of shed antigen. This work advances design principles for achieving expression-dependent tumor targeting via low-affinity, high-avidity ligands.

Keywords: avidity; carcinoembryonic antigen; folate receptor; ligand; multivalency; targeting.