Engineering affibody-based prodrugs for enhanced tissue selectivity in targeted cancer therapies

QC 2024-11-14

Abstract

Cancer remains one of the leading causes of death worldwide, with approximately 40% of the population expected to receive a cancer diagnosis during their lifetime. Conventional treatments such as surgery, chemotherapy, and radiotherapy have been essential in improving patient outcomes. However, these approaches often lack specificity, partly due to the inherent heterogeneity of tumors both between and within patients. Precision medicine has emerged to address these challenges by developing therapies tailored to the specific molecular and genetic profiles of tumors. Targeted therapies, particularly monoclonal antibodies, have shown great promise in this field, yet these therapies face limitations such as toxicity, poor tissue penetration, and high production costs.  This thesis focuses on the development of innovative prodrug strategies, including affibody-based prodrugs and antibody prodrugs with affibody masking domains, aimed at enhancing tissue selectivity and reducing systemic toxicity in cancer therapy. Additionally, substrate engineering for tumor-associated proteases is explored to optimize prodrug activation. Through five research papers, these strategies are investigated for their potential to improve next-generation cancer therapeutics.

In Paper I, a masking domain was identified for an epidermal growth factor receptor (EGFR)-targeting affibody using Staphylococcus carnosus display. This study screened an affibody library to isolate a domain capable of efficiently masking EGFR-binding activity. A proof-of-concept prodrug demonstrated that the masking domain could inhibit EGFR binding, with restored activity upon proteolytic cleavage. In Paper II, the initial affibody-based prodrug was further optimized to improve its biodistribution in vivo. Key modifications included the introduction of a suitable tumor protease substrate and a high-affinity albumin-binding domain to extend blood circulation time. The optimized prodrug exhibited favorable biodistribution in tumor xenografted mice, with strikingly reduced uptake in healthy tissues, demonstrating a significant improvement in tumor selectivity in vivo.            

In Paper III, affibodies were explored as masking domains for the anti-EGFR monoclonal antibody cetuximab. Using Escherichia coli display, affibodies were selected to specifically bind and mask cetuximab's paratope. A cetuximab prodrug was engineered with an affibody masking domain, and in vitro studies revealed a 400-fold reduction in cetuximab’s growth inhibitory effects until proteolytic activation. This study validated the use of affibody masking domains in antibody-based prodrugs. Paper IV aimed to demonstrate the versatility of the E. coli display platform by isolating affibodies capable of masking nivolumab, an anti-PD-1 monoclonal antibody. The screening identified non-conventional affibody molecules that appear to mimic PD-1 and block nivolumab’s binding capacity. Structural modeling and bio-layer interferometry confirmed effective masking and restoration of PD-1 binding upon cleavage, suggesting potential for improved immune checkpoint inhibition with reduced systemic side effects.

With the aim of enhancing prodrug activation, in Paper V, a high-throughput E. coli display platform was engineered to identify optimized substrates for matriptase, a tumor-associated protease. A large substrate library was designed and screened for cleavage efficiency, leading to the discovery of several substrate candidates with enhanced cleavage kinetics. These optimized substrates were incorporated into prodrug designs, demonstrating significantly improved activation in vitro compared to previously reported reference substrates.

In conclusion, this thesis demonstrates the potential of affibody molecules as both masking and targeting domains in prodrugs, offering a promising strategy for improving the selectivity and efficacy of cancer therapeutics. These findings provide a strong foundation for future advancements in the design of precision cancer treatments.

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