High-mannose N-glycans in breast cancer: Implications for targeted therapies

Abstract

Breast cancer (BC) is the leading cause of cancer-related deaths in women, responsible for ~685,000 global deaths in 2020. The global incidence of BC continues to rise annually, despite advancements in targeted therapies. These treatments are not universally effective, can be accompanied by drug toxicity, and nearly all patients eventually develop therapeutic resistance. Additionally, therapeutic options for patients with the most aggressive forms of BC, namely triple-negative breast cancer (TNBC) and metastatic disease, are limited with the fewest number and most toxic targeted therapies available. Thus, there is a clear unmet clinical need for new treatment options. Aberrant glycosylation is considered one of the established hallmarks of cancer. Cancer-associated glycans are recognised for their involvement in various aspects of oncogenesis, including epithelial-mesenchymal transition, invasion, metastasis, and immunosuppression. This research has assisted in identifying glycan serum cancer biomarkers and novel molecular targets which have led to the approval of glycan based-therapeutics in the clinic. While high-mannose glycans have been associated with many cancers, including BC, previous studies have yielded conflicting results, highlighting the heterogeneity of BC glycosylation. The aim of this thesis was to investigate the potential significance of high-mannose glycans in BC progression and determine whether therapeutically targeting high-mannose antigens could enhance current BC therapeutics. High-mannose glycans were identified on the cell surface of BC cell lines and within clinical tissues. Cell surface levels of mannose on BC cells were variable, however, levels of high-mannose glycans were higher in non-metastatic BC cell lines compared to metastatic ones. Moreover, we observed that mannose processing enzymes were aberrantly regulated in BC clinical datasets, emphasising the high heterogeneity of expression across samples. Despite being prevalent in BC, we demonstrated that the induction of high-mannose reduced BC cell migration and invasion in vitro and impacted tumour growth in vivo. High-mannose glycans were induced by methods of mannosidase inhibition using the inhibitor, Kifunensine, and CRISPR-cas9 targeting of MGAT1. These findings suggest that high-mannose glycans may be critical in modulating cellular behaviours linked to BC progression possibly through complex interactions between high-mannose and highly branched glycoforms. This suggests a potential mechanism for the transition from non-metastatic to metastatic phenotypes. We also developed three novel bi-specific antibody constructs targeting both HER2 and high-mannose glycans and demonstrated their therapeutic potential. One of these antibodies, 2G12-Hn 2, provided advantages over the standard anti-HER2 therapy, Trastuzumab. To address difficulties in production and increased aggregation faced with larger multitargeting antibodies, such as 2G12-Hn 2, we investigated a novel alternative delivery strategy utilising Replicon RNA. For this, we used Trastuzumab as it is a well-characterised BC mAb of standard format. We established that replicon-produced Trastuzumab retained similar target binding to HER2 and mediated comparable effector functions as traditional recombinant-made Trastuzumab. This paves the way for future experiments to determine the potential of utilising Replicons for the delivery of larger multi-targeting antibodies such as 2G12-Hn 2.

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Identifiers

ORCID for Charlie Birts: orcid.org/0000-0002-0368-8766

ORCID for Max Crispin: orcid.org/0000-0002-1072-2694

ORCID for Paul Skipp: orcid.org/0000-0002-2995-2959

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

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