Engineering a targeted delivery platform using Centyrins

Abstract

Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload. Overall, 26 of the 94 positions were identified as ideal for cysteine modification, conjugation and drug delivery. Conjugation-tolerant positions were mapped onto a crystal structure of the Centyrin, providing a structural context for interpretation of the mutagenesis experiment and providing a foundation for a Centyrin-targeted delivery platform.

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…This Tenascin-FN3 derived monobody is now under clinical development as 'Tn3 by Viela Bio (spun out from AstraZeneca's Medimmune) [31,32]. Finally, in order to design more robust monobodies with substantially higher initial stabilities, the consensus bioinformatic technique [33] has been applied to generate Aro Therapeutic's Centyrins from a consensus sequence of 14 FN3 domains (spun out from Janssen) [34,35] and the hyper-stable "Consensus FN3" FN3Con domain [36] from a consensus of 2123 FN3 sequences. As a result of this differentiation, these synthetic domains share the parent FN3 fold but feature varying sequence similarity ( Figure 1D,E).…”

“…The absence of cysteines in FN3 domains allows for the introduction of a single conjugation-compatible cysteine for site-specific loading with drugs, thus avoiding potential disulphide-mediated aggregation [60,61]. This feature is critical as cytotoxic drugs can be disruptive to protein structure due to their hydrophobic nature, and small protein scaffolds require well-controlled conjugation to ensure resistance to aggregation [35,57]. In practice, these advantages have been demonstrated with a glypican-3 specific Adnectin monobody domain loaded with a cytotoxic derivative of tubulysin [58].…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…Although these monobody derivatives maintain a similar type 3 fibronectin structure, they vary widely in their amino-acid sequences, optimisation for expression and thermal stability [2,4,5,26,27,31,[33][34][35]. The sequences of 6 major variants have been summarised in Figure 1D,E.…”

“…The absence of cysteines in FN3 domains allows for the introduction of a single conjugation-compatible cysteine for site-specific loading with drugs, thus avoiding potential disulphide-mediated aggregation [59,60]. This feature is critical as cytotoxic drugs can be disruptive to protein structure due to their hydrophobic nature and a well-controlled conjugation in a small protein scaffold is required to ensure resistance to aggregation [35,56]. In practice, these advantages have been demonstrated with a glypican-3 specific Adnectin monobody domain loaded with a cytotoxic derivative of tubulysin [57].…”

Adnectin–drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors

“…Conjugates and genetic fusions between small, engineered, non-antibody domains that bind tumor-associated targets and cytotoxic payloads have been described previously ( Martin-Killias et al , 2011 ; Simon et al , 2013 ; Cox et al , 2016 ; Currier et al , 2016 ; Goldberg et al , 2016 ; Sochaj-Gregorczyk et al , 2016 , 2017 ; Serwotka-Suszczak et al , 2017 ; Sokolova et al , 2017 ). In contrast to the conjugates described here, the majority of the previously reported scaffold–drug conjugates that have been tested in animal models had been modified to extend their naturally short half-life to approximate the pharmacokinetics of antibodies, with the goal of maximizing exposure of tumors to the cytotoxic payloads and thus maximizing efficacy.…”