Shih, T.-Y. | bioRxiv Science

bioRxiv2025

A Supramolecular Self-assembly Approach to Site-Specific Antibody Conjugates via a Coiled-coil Peptides Platform

Antibody conjugates play a central role across multiple healthcare sectors with a prime example being antibody-drug conjugates (ADCs). Although widely used lysine and hinge cysteine conjugation methods yield products, the lack of site-specificity and spatial control along with the highly heterogeneous composition are significant limitations. We describe a facile supramolecular assembly method based on heterodimer coiled-coil formation for site-specific antibody conjugation. The method affords uniform loading of diverse payloads including anti-cancer agents, polymers, enzymes, fluorophores, etc. under mild aqueous conditions. Further, the facile convergent approach capitalizes on the independent strengths and flexibility of protein expression and peptide chemistry culminating in a final self-assembly step. Coiled-coil conjugation perseveres both antibody antigen binding sites for target engagement and heavy chains constant domains for Fc binding and recycling. An ADC loaded with monomethyl auristatin E targeting HER2+ tumors significantly reduces tumor volume in a human ovarian cancer xenograft model outperforming the antibody alone with validated performance against a best-in-class therapeutic. Supramolecular assembly-driven bioconjugation expands the bioorthogonal chemistry toolbox for antibody modification and opens new avenues for advanced antibody conjugates with multiple payloads.

bioengineering↗

bioRxiv2023

Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA

Self-amplifying RNA (saRNA) will revolutionize vaccines and in situ therapeutics by enabling protein expression for longer duration at lower doses. However, a major barrier to saRNA efficacy is the potent early interferon response triggered upon cellular entry, resulting in saRNA degradation and translational inhibition. Substitution of mRNA with modified nucleotides (modNTPs), such as N1-methylpseudouridine (N1m{Psi}), reduce the interferon response and enhance expression levels. Multiple attempts to use modNTPs in saRNA have been unsuccessful, leading to the conclusion that modNTPs are incompatible with saRNA, thus hindering further development. Here, contrary to the common dogma in the field, we identify multiple modNTPs that when incorporated into saRNA at 100% substitution confer immune evasion and enhance expression potency. Transfection efficiency enhances by roughly an order of magnitude in difficult to transfect cell types compared to unmodified saRNA, and interferon production reduces by >8 fold compared to unmodified saRNA in human peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrate expression of viral antigens in vitro and observe significant protection against lethal challenge with a mouse-adapted SARS-CoV-2 strain in vivo. A modified saRNA vaccine, at 100-fold lower dose than a modified mRNA vaccine, results in a statistically improved performance to unmodified saRNA and statistically equivalent performance to modified mRNA. This discovery considerably broadens the potential scope of self-amplifying RNA, enabling entry into previously impossible cell types, as well as the potential to apply saRNA technology to non-vaccine modalities such as cell therapy and protein replacement.

bioengineering↗