Introduction Most patients with oesophageal adenocarcinoma (OA) are unfit for curative treatment but are fit for photodynamic therapy (PDT). PDT has been limited by poor tumour selectivity, poor tissue penetration of light and protracted side effects. These restrictions may be overcome with targeted PDT. We have previously demonstrated aberrantly glycosylated MUC1 (AG-MUC1), bound by humanised anti-human milk-fat globule-1 (Hu-HMFG1) antibody is significantly expressed in Barrett's epithelium (BE) with high grade dysplasia and OA. In this study, we characterise photoimmunoconjugates (PIC) of Hu-HMFG1 with the photosensitisers (PS) pyropheophorbide-α (PPa), and PS1, a water-soluble derivative of PPa activated in the near infra-red.
Methods PPa succinimidyl ester (PPA-SE) and PS1 were first synthesised (PhotoBiotics) from PPa. Both PS were then conjugated with Hu-HMFG1. The resulting PICs were dialysed, centrifuged and the supernatant and resultant pellet were resuspended in PBS and retained for spectroscopic and SDS-Page gel analysis. Fluorescence of PICs in the gels were photographed and the gels stained with Coomasie to confirm accurate loading. AIDA image analyser software was used for densitometry measurement.
Results HuHMFG1:PPa PICs, although well conjugated by fluorescent photography of SDS-page gels, were very insoluble. Further characterisation was limited to PS1 PICs. UV/Vis analysis of the soluble HuHMFG1:PS1 PIC supernatant, suitable for subsequent in vitro testing, confirmed maximum absorption at 683 nm (Abstract PWE-012 figure 1). SDS page analysis indicated up to 52% of PS1 photosensitiser was conjugated in the supernatant mixture post-dialysis. AIDA image analysis confirmed that in those antibodies conjugated, a loading ratio of PS1:HuHMFG1 of up to 7:1 was achieved.
Conclusion This pilot study is the first to successfully conjugate HuHMFG1 with the photosensitisers PPa and PS1. These PICs could potentially selectively target photosensitisers to tissues of interest preserving the surrounding architecture during PDT. HuHMFG1:PS1 PICs have shown enhanced absorption in the red spectral region which will translate clinically into deeper tissue penetration than currently licenced photosensitisers. Further experiments are needed to both optimise the conjugation protocol and purify the product to GLP standards prior to clinical studies.
Competing interests None declared.
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