King's College London

Therapeutic monoclonal antibodies (mAbs) can inhibit the function of tumour antigens (TA) and additionally those of IgG1 subclass can recruit natural killer (NK) cells to kill tumours via antibodydependent cellular cytotoxicity (ADCC). One such example is cetuximab, which is specific for the epidermal growth factor receptor (EGFR) that is overexpressed in more than 70% of colorectal cancer cases. Importantly the efficacy of cetuximab is limited in patients who have the low affinity FCγRIIIa (CD16) polymorphism, thus a glyco-engineered (GE) anti-EGFR mAb was developed called GA201GE (imgatuzumab), that reportedly mediates high affinity CD16 binding and enhanced ADCC, irrespective of CD16 genotype. In a similar vein, the anti-CD20 GE-mAb obinutuzumab (GA101GE) was developed to improve upon the unmodified anti-CD20 rituximab currently used in the treatment of B-cell malignancies. It is important to evaluate whether ADCC facilitated by therapeutic mAbs has the potential to stimulate CD8+ T-cell mediated immunity, which is essential for long-term tumour immune-surveillance. The complex cross talk between NK cells and dendritic cells (DC) is a precursor to the generation of TA-specific T cells. DC can ingest and process exogenous antigen released from target cells lysed by NK cells, and subsequently cross present TA epitopes to TA-specific CD8+ T cells. Cetuximab-activated NK cells are known to induce DC maturation but whether GE mAb-activated NK cells can induce DC maturation is currently unknown. This study therefore aimed to assess the ability of GE-mAbs to induce DC maturation and other functions necessary for the stimulation of adaptive immunity. An in vitro coculture model was established to assess interactions between tumour cells, NK cells, DC and TAspecific CD8+ T cells. In keeping with a precedent set by previous studies of GE-mAbs, comparisons were made with wild type counterpart mAbs or clinically utilised unmodified mAbs. Principally, GE-mAbs were shown to induce NK-cell activation in terms of both activation marker up-regulation and cytokine production. Imgatuzumab-mediated NK-cell activation was achieved across various tumour-cell EGFR expression levels, indicating an ability to provoke responses to heterogeneous tumours and GE-mAb-activated NK cells were shown to induce DC maturation. Antigen uptake by DC and cross presentation of TA to TA-specific T cells were also examined in the presence of GE-mAbs. In a secondary manner, when compared at equivalent concentrations, GE-mAbs induced multiple measures of NK-cell function and DC maturation to a significantly greater extent than unmodified mAbs. These findings suggest that GE-mAbs may have greater potential to promote presentation of TA to CD8+ T cells than unmodified mAbs.