Antibody engineering for improving breast cancer therapeutics.

Abstract

Breast cancer is the most commonly diagnosed cancer worldwide, with an estimated 2.3 million new cases reported in 2020. Breast cancers expressing human epidermal growth factor receptor -2 (HER2) represent approximately 20% of total breast cancer diagnoses, and are associated with poor prognosis and low rates of progression free survival. Patients with HER2+ breast cancer may be treated with trastuzumab either as a monotherapy or in combination with chemotherapy, which has been shown to significantly increase overall survival. Trastuzumab is an IgG1 humanised anti-HER2 monoclonal antibody (mAb) which binds to the extracellular region of HER2. The therapeutic efficacy of trastuzumab has been attributed to direct mechanisms, including blockade of HER2 shedding from the tumour cell surface and inhibition of pro-tumoral signalling pathways, as well as immune-mediated Fcγ receptor (FcγR) mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Despite the significant increase in survival, 40-50% of patients do not respond to trastuzumab therapy, and most will develop resistance within a year of treatment. Many reasons for poor in vivo responses to trastuzumab have been postulated, including low expression of HER2 on the tumour cell surface and high concentrations of endogenous IgG in human serum which may compete with trastuzumab for FcγR engagement on effector cells. Tumour cell removal by immune effectors depends upon the interaction with FcγRs which includes FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB. The only receptor with high affinity for Fc is FcγRI, which is cable of binding monomeric IgG. The remaining FcγRs are low affinity, and depend on multiple Fc-FcγR interactions to initiate downstream signalling and effector function. Low affinity FcγRs therefore depend upon avidity exhibited by antibody opsonised targets or immune complexes to enable cross-linking with Fc to occur. Engineering strategies which may overcome competition from endogenous IgG include the introduction of mutations into the mAb Fc domain which enhance affinity to the low affinity FcγR, FcγRIIIA. Margetuximab is an anti-HER2, chimeric mAb which features such affinity -enhancing Fc mutations and was found to demonstrate superior progression free survival compared to trastuzumab in the SOPHIA trial. A less explored strategy includes mAbs featuring multiple Fc domains which exhibit higher avidity for low affinity FcγRs. mAbs with multiple Fc domains arranged in tandem (tandem Fcs) have previously been reported to stimulate more potent levels of ADCC or ADCP against tumour targets. However, tandem Fcs are yet to enter the clinic. The focus of this project was to characterise a panel of trastuzumab-based tandem Fc constructs and determine their clinical potential. Poor yield and the presence of high mannose glycans at the Fc glycan site were observed in a wild-type tandem Fc construct. Moreover, extensive misfolding and aberrant quaternity structures associated with the tandem Fc architecture were revealed by negative-electron microscopy, which were found to limit effector function. Notably, the addition of knob-in-hole (KH) pair mutations to the lower Fc domain was associated with an improvement in yield and a reduction the abundance of oligomannose type glycans at the Fc glycan site. An enhancement in avidity to the low affinity FcγRs was also observed when tandem Fcs exhibiting KH pairs were analysed via surface plasmon resonance, which translated into increased levels of phagocytosis compared to their single Fc counterparts as demonstrated in a series of in vitro assay. Significantly, these tandem Fcs incorporating KH pair Fc mutations were found to maintain high levels of phagocytosis in the presence of competing IgG, and induce potent phagocytotic responses against tumour targets with low HER2 expression. Data from in vivo studies also supported the notion that these tandem Fc constructs induced stronger anti-cancer immune responses than their single Fc counterparts. Tandem Fc mAbs were then benchmarked against margetuximab in a series of in vitro ADCC and ADCP assays. Although margetuximab was found to induce more potent ADCC responses, significantly higher levels of ADCP were observed following opsonisation with the tandem Fc mAb format. This indicates that affinity-enhancing Fc mutations may be the more effective approach for increasing ADCC. In contrast, increasing FcγR avidity via Fc multimerization may be more a superior strategy for increasing ADCP. Ultimately, the data outlined in this thesis demonstrates the utility of KH pair mutations in the development of tandem Fcs. Moreover, these studies have indicated that tandem Fcs may be an effective mAb format for overcoming competition from endogenous IgG and enhancing innate immune responses against tumoral targets with low antigen expression. Finally, the significant improvement in ADCP induction compared to margetuximab underscores the clinical potential of tandem Fc mAbs.

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Identifiers

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

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

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