Development of novel EGFR-targeting therapeutics for the treatment of pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains a highly challenging disease with minimal improvements in clinical outcomes since the 1970s. The efficacy of currently available treatments is strongly limited by systemic toxicities, PDAC inherent chemoresistance, and the immunosuppressive nature of PDAC tumour microenvironment (TME). These challenges underscore the urgent need for novel therapeutic strategies. Recent advancements have seen nanoparticles (NPs) loaded with cytotoxic drugs offering modest clinical efficacy in PDAC treatment. The therapeutic success of these treatments could hypothetically be improved by actively targeting NPs with ligands directed against tumour antigens, such as monoclonal antibodies (mAbs), with previous studies highlighting the importance of utilising site-specific conjugation strategies to afford NPs with conjugated targeting ligand paratopes possessing optimal orientation for cognate antigen engagement. In PDAC specifically, cetuximab (CTX), an anti-epidermal growth factor receptor (EGFR) antibody, has been identified as a promising ligand for active NP targeting. Another attractive therapeutic avenue for the treatment of pancreatic cancer is immunotherapy, which could provide an exciting solution to overcoming the stumbling block of PDAC immune evasion.

This thesis explores the development of two EGFR-targeting platforms, i.e., drug-loaded antibody-conjugated NPs (ACNPs) and a Fab – Fab bispecific antibody (bsAb), as alternatives to conventionally used therapeutics. The first body of work described in this thesis focused on developing “clickable” Fab fragments directed against EGFR and CD3 utilising click-enabled interchain disulfide bond rebridging linker molecules or dibromopyridazinediones (Br2PDs). Chemical modification of Fabs with Br2PDs allowed for quantitative and site-specific integration of biorthogonal "click" handles into the Fab structure, enabling selective coupling with desired ligands through strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC). The second part of this work discusses the development of CTX Fab-conjugated NPs loaded with SN-38, the active metabolite of irinotecan. These NPs, generated via SPIEDAC which was accessed through chemical modification of CTX Fab with a bicyclononyne (BCN)-bearing Br2PD, were assessed in terms of their anticancer activity alongside CTX mAb SN-38 NPs, generated using a less selective maleimide-thiol coupling approach. Both actively targeted NP systems displayed enhanced cellular internalization and cytotoxicity in EGFR-positive pancreatic cancer models compared to control non-targeted NP formulations, suggesting that endowing NPs with active targeting capabilities rather than relying only on passive targeting through the EPR effect may be preferable for expediting advancements in the field of PDAC care. The last part of this work details the development of FabCET – FabOKT3 bsAb, which was generated through SPIEDAC coupling of BCN-bearing CTX Fab and methyltetrazine (MTZ)-bearing OKT3 Fab. The bsAb was shown to retain binding to target antigens and was capable of successfully activating T cells upon CD3+ T cell/EGFR+ tumour cell crosslinking, promoting T cell-mediated cancer cell death. The activity FabCET – FabOKT3 bsAb was correlated with multiple factors, i.e., concentration of bsAb, levels of EGFR expression and effector to target (E:T) cell ratio. Furthermore, in all assays, bsAb-mediated T cell activation was dependent on the presence of EGFR-positive cancer cells, suggesting the activity of the construct may be preferentially restricted to the TME in vivo. Together, these results indicate the potential of fragment-based bsAb immunotherapy in PDAC therapeutic landscape.

Thesis is embargoed until 31st December 2029.

Date of Award	Dec 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	EU Horizon 2020 Marie Sklodowska-Curie ITN Programme
Supervisor	Christopher Scott (Supervisor), Karen McCloskey (Supervisor) & Vijay Chudasama (Supervisor)