Development of Programmable Nanobodies for Multi-functionalized Biologics

PROJECT SUMMARY Antibody Drug Conjugates (ADCs) have been developed to deliver cytotoxic payloads (CTPs) to patients, yet their efficacy is limited by serum instability, low tissue/tumor penetration, low cell internalization, and immunogenicity. Unstable linkers further impair the utility of ADCs leading to off-target toxicities, non-specific CTP conjugations and heterogeneity of Drug Antibody Ratios (DARs). Clinical trials have highlighted the diminished bioactivity of ADCs resulting from conjugations at natural residues critical to epitope binding or structural integrity, yielding only 11 FDA approved ADC therapeutics. Notably, two ADCs, Myolotarg (re-approved) and Blenrep were withdrawn from FDA approval due to safety concerns. Nanobodies (Nbs), derived from heavy chain camelid antibodies, have enhanced therapeutic properties due to their small size (~15 KDa vs. 150 KDa for Abs), and convex paratopes, which enable binding to unique epitopes inaccessible to Abs, and improved intra-cellular delivery of CTPs yielding more efficacious therapeutics. Nevertheless, the small size of Nbs is a double-edged sword limiting their utility with short half-lives (1-2 hours), necessitating frequent dosing and unfavorable peak-valley pharmacokinetics with dose-limiting toxicities. Current strategies to extend their half-life (e.g., fusion to albumin) create bulky molecules impairing tumor penetration. Anti- polyethylene glycol (PEG)-antibodies have similarly limited clinical efficacy of therapeutics with notable recent failures of pegilodecakin (PEG-IL-10), toxicity from Doxil (PEG-Doxurubicin) and reduced therapeutic efficacy of Oncaspar (PEG-asparginase). Pearl Bio’s innovative synthetic biology platform can overcome these challenges by combining Genomically Recoded Organisms and orthogonal translation systems to produce Nbs with 1-10 synthetic amino acids (sAAs) for precise tuning of therapeutic properties: (i) lipidation for half-life tunability, (ii) tunable fluorescence-tagging for tumor detection and (iii) tunable DAR-specific CTP conjugation with predictable homogenous products for enhanced tumor cytotoxicity. Our technology further enables multi-site encoding of 2+ distinct sAAs within one Nb, enabling multi-functionalization and combination of these properties without compromising tumor penetration or triggering immunogenicity. Building upon our prior work on anti-EGFR Nb (a target validated for primary tumors and metastases) and EgA1, we will demonstrate multi-site sAA incorporation, lipidation and CTP conjugation. Specifically, we will (1) demonstrate expression of engineered EgA1 Nb encoded with multiple sAAs, (2) show precise conjugation of EgA1 with fluorophores, lipidated moieties, and cytotoxic payloads at sites of sAA incorporation and (3) characterize tumor-cell targeted EgA1 nanobodies functionalized with lipids, fluorophores, and cytotoxic payloads for tumor cell-binding, detection, and cytotoxicity. Phase 2 animal studies would be used to validate these designs. This project is poised to establish Pearl’s platform as foundational technology for producing novel multi- functionalized Nb biologics across diverse disease indications.