Antibody Modulation via Protein L-Linked Blockers for Precision Immunotherapy

Introduction: : Monoclonal antibodies (mAbs) are central to modern cancer therapy, yet their native active state often leads to systemic toxicity and off-target effects. Many modulation strategies require antibody re-engineering, which is costly, time-intensive, and inaccessible to most academic or clinical labs. To overcome these limitations, we developed an innovative, dual modality blocking construct to control mAb binding activity. Our approach uses a mutated protein L (PpL) construct covalently crosslinked to the antibody light chain and fused to steric blocking peptide via a cleavable linker. This dual modality blocking system enables site-specific antibody inactivation and tunable reactivation using either light or enzymatic triggers. Importantly, it functions across a range of commercial and therapeutic antibodies without requiring structural modification to the antibody itself. Herein, we applied this platform to cetuximab, an FDA-approved EGFR-blocking antibody, and demonstrated reversible control of both binding affinity and downstream signaling in vitro. Additionally, we developed a miniature, light-emitting implant (~4 mm LED, 365 nm) to facilitate localized activation for future in vivo studies. Altogether, this system enables spatial and temporal precision of antibody function with broad translational potential for modulating immune and signaling pathways in both preclinical and therapeutic contexts.

Materials and

Methods: : To enable controlled antibody inactivation, we used site-directed photo-crosslinking to attach a mutated protein L (PpL) domain to the light chain of cetuximab. The PpL was engineered with tethered inhibitory peptide that blocks the antigen-binding site via steric hindrance. The tether contains a cleavable linker sensitive to either 365 nm light or proteolytic enzymes, enabling reversible unmasking of antibody function. We optimized antibody-conjugate enrichment via size exclusion chromatography and validated purity via SDS-PAGE. An Enzyme-Linked Immunosorbent Assay (ELISA) was used to quantify binding affinity in the blocked and unblocked states. Epidermal Growth Factor Receptor (EGFR+) A431 cells were used to evaluate the ability of blocked Cetuximab to bind to target EGFR and measured by proliferation, motility, and immunofluorescence. To assess downstream signaling kinetics and functional modification of cetuximab, we employed an ERK-kinase translocation reporter cell line that render signaling of EGFR pathway activation. Concurrently, a 4 mm, implantable LED pellet was re-engineered and validated for localized light delivery through an array of embedded polydimethylsiloxane (PDMS) material to simulate tissue penetration for therapeutic applications in activating antibodies. All statistical analysis were performed using GraphPad Prism.

Results, Conclusions, and Discussions:: We demonstrated that our PpL-based blocking system significantly suppresses cetuximab function in both binding and downstream signaling assays. ELISA showed a 25-fold reduction in binding affinity for EGFR in the blocked state. Immunofluorescence microscopy confirmed decreased surface binding of blocked cetuximab to EGFR+ A431 cells, signal restoration upon linker cleavage. This reversible control was further validated using ERK-kinase translocation reporter assays, which showed suppressed pathway inhibition by blocked cetuximab and reactivation. These data confirm that our blocking system not only masks antibody binding but also tunes its influence on downstream cellular responses.

Importantly, our design does not require antibody sequence modification or re-expression, making it broad in utility. The modularity of the blocking domain allows for easy adaptation across different antibody kappa light chain isotypes and targets. We further demonstrated proof-of-concept for future translation by developing a miniaturized LED pellet emitting 365 nm light, capable of local antibody activation within tissue-mimicking polymer scaffolds, setting the stage for preclinical deployment.

Our approach addresses a critical unmet need in antibody therapy – spatial and temporal control over antibody function – without the limitations of genetic re-engineering. This technology enables context-dependent modulation of immune or oncogenic signaling in preclinical models, while offering potential for reduced side effects in therapeutic settings. By tuning when and where mAbs are active, this system opens potential avenues for safer activation of immunotherapies and signal-modulating biologics.

Acknowledgements and/or References (Optional):: This project was supported by funding from the Cancer Early Detection Advanced Research (CEDAR) center at the Oregon Health & Science University’s Knight Cancer Institute and the N.L. Tartar Trust Fellowship.