Upregulation of mucin glycoprotein MUC1 in the progression to esophageal adenocarcinoma and therapeutic potential with a targeted photoactive antibody-drug conjugate

Abstract

Background: Mucin glycoprotein 1 (MUC1) is a glycosylated transmembrane protein on epithelial cells. We investigate MUC1 as a therapeutic target in Barrett's epithelium (BE) and esophageal adenocarcinoma (EA) and provide proof of concept for a light based therapy targeting MUC1.

Results: MUC1 was present in 21% and 30% of significantly enriched pathways comparing BE and EA to squamous epithelium respectively. MUC1 gene expression was x2.3 and x2.2 higher in BE (p=<0.001) and EA (p=0.03). MUC1 immunohistochemical expression increased during progression to EA and followed tumor invasion. HuHMFG1 based photosensitive antibody drug conjugates (ADC) showed cell internalization, MUC1 selective and light-dependent cytotoxicity (p=0.0006) and superior toxicity over photosensitizer alone (p=0.0022).

Methods: Gene set enrichment analysis (GSEA) evaluated pathways during BE and EA development and quantified MUC1 gene expression. Immunohistochemistry and flow cytometry evaluated the anti-MUC1 antibody HuHMFG1 in esophageal cells of varying pathological grade. Confocal microscopy examined HuHMFG1 internalization and HuHMFG1 ADCs were created to deliver a MUC1 targeted phototoxic payload.

Conclusions: MUC1 is a promising target in EA. Molecular and light based targeting of MUC1 with a photosensitive ADC is effective in vitro and after development may enable treatment of locoregional tumors endoscopically.

Keywords: Barrett’s esophagus; antibody-drug conjugate; esophageal adenocarcinoma; mucins; photodynamic therapy.

Figure 1. Gene set enrichment and microarray analysis of MUC1 in the progression to esophageal adenocarcinoma

Heat map A. and an example probability plot B. of the gene set enrichment analysis (GSEA) for non-dysplastic Barrett’s esophagus (NDBE) vs normal squamous esophageal epithelium (Sq). Heat map C. and an example probability plot D. of the GSEA for esophageal adenocarcinoma (EA) vs Sq. GSEA detail in supplementary (Supplementary Figure 1) and evaluated with Kolmogorov-Smirnoff test. Microarray analysis E.; raw expression values of MUC1 mRNA in Sq, NDBE and EA tissues, results show a 2.3 fold increase in MUC1 expression at the mRNA level in NDBE compared to Sq (Mann-Whitney; p < 0.001) and 2.2 fold increase in EA compared to Sq (Mann-Whitney; p = 0.03). Box plot presented as median and interquartile range.

Figure 2. Representation of MUC1 receptor structure in normal and tumor epithelium with binding sites for selected antibodies

Representation of MUC1 receptor glycosylation in normal and tumor epithelium. NCL-MUC1 binds a sialic acid on the glycosylated side chain, while NCL-MUC-1-CORE and HuHMFG1 bind the extracellular peptide backbone. The extracellular target antigens can be hidden in fully glycosylated normal tissue, but become increasingly exposed in cancer due to aberrant glycosylation. CT2 targets the intracellular cytoplasmic tail of MUC1.

Figure 3. Immunohistochemical staining patterns with anti-MUC1 antibodies in high grade dysplasia and HuHMFG1 staining in the squamous-metaplasia-dysplasia-carcinoma sequence

A. Immunohistochemical images of high-grade dysplasia in Barrett’s epithelium stained with four anti-MUC1 antibodies (brown), and hematoxylin (blue). B. HuHMFG1 staining in normal esophageal squamous epithelium (Sq), non-dysplastic Barrett’s esophagus (NDBE), low-grade dysplasia (LGD), high grade dysplasia (HGD) and invasive esophageal adenocarcinoma (EA). An increase in the intensity of staining is seen as pathological grades progress. Staining also follows the direction of epithelial maturation from basement membrane toward the lumen. In higher pathological grades, staining is seen throughout the epithelial layer.

Figure 4. Levels of expression of four MUC1 epitopes in the squamous-metaplasia-dysplasia-carcinoma sequence

A. The anti-MUC1 antibodies HuHMFG1, CT2, NCL-MUC-1 and NCL-MUC-1-CORE were evaluated by immunohistochemistry in esophageal tissue from incremental pathological grades including normal squamous epithelium (Sq), non-dysplastic Barrett’s esophagus (NDBE), low-grade dysplasia (LGD), high grade dysplasia (HGD) and invasive esophageal adenocarcinoma (EA). Positivity was defined by 2+/3+ intensity staining in ≥10% of the pathology examined. The proportion of positive samples for each tissue is shown with respectively colored polynomial lines of best fit. HuHMFG1 and CT2 staining increase at the metaplastic (NBDE) stage, whereas NCL antibodies increase staining after development of EA. B. All antibodies showed significant expression differences during progression to cancer (χ2 test; p < 0.00005), with all having a significant trend of increasing positivity (Pearson’s R; p < 0.001).

Figure 5. HuHMFG1 staining MUC1 in invasive esophageal adenocarcinoma and locoregional lymph node metastases

A. A transverse esophagectomy section from a patient with invasive esophageal adenocarcinoma (EA) stained with HuHMFG1 (brown) and hematoxylin (blue). HuHMFG1 stains surface mucin, invasive EA as it invades into muscularis externa and only the local tumor infiltrated lymph node. Normal mucosa and a benign local lymph node did not stain positively. A x20 magnification from a representative tumor region is inset. B. Analysis of 31 locoregional nodes resected from 11 patients highlight positive expression of MUC1 by HuHMFG1 in all malignant but no benign lymph nodes (Fishers exact; p <0.0001).

Figure 6. MUC-1 expression in esophageal cell lines of various pathological grades and internalization into esophageal adenocarcinoma

A. Binding of the antibody HuHMFG1 in vitro to esophageal cell lines originally isolated from tissue of various pathological grades was carried out using flow cytometry. Cells were incubated with varying concentrations of HuHMFG1 antibody which was detected using a FITC conjugated anti-human IgG secondary fluorescent antibody (shown in green) shown alongside nuclei staining with DAPI (shown in blue). An increase in fluorescence represents more HuHMFG1 bound to each cell and the saturation of the fluorescent signal indicates cell surface receptor saturation. HuHMFG1 did not bind the colonic line HT29 (negative control). It bound at a low level in normal squamous and non-dysplastic Barrett’s esophagus (NDBE) and a high level in high grade dysplasia (HGD) and esophageal adenocarcinoma (EA). B. Confocal microscopy images showing OE19 cells exposed to HuHMFG1 (i) or media alone (ii). OE19 internalize HuHMFG1 and the intracellular localization pattern is punctate.

Figure 7. Photophysical characterization and internalization of photoactive MUC1 targeting antibody drug conjugates

A. Absorbance spectra of HuHMFG1:PS1 antibody drug conjugate (ADC) highlights peak absorbance at 683nm (red spectral region) for laser excitation. Shown are any photophysical shifts away from the absorbent spectra of free antibody or free PS1 (spectra normalized to 280nm or 687nm respectively). B. SDS page gel showing proportion of covalently coupled antibody to free photosensitizer (PS1) in the ADC mixture; (i-iii) Coomassie stained protein gel; (i) molecular weight markers, (ii) HuHMFG1 and (iii) HuHMFG1:PS1 ADC. (iv-vi) Image of the same SDS gel before Coomassie staining for the PS1 dye via UV fluorescence; (iv) molecular weight markers, (v) HuHMFG1 and (vi) HuHMFG1:PS1 ADC. Covalently bound photosensitizer PS1 is that seen at the same height as antibody protein. C. Confocal microscopy images showing OE19 cells exposed to HuHMFG1 and PS1 in either a conjugated form or free un-conjugated form showing the covalently bound photosensitizer and antibody components remain co-localized after internalization. HuHMFG1 (in green) nuclei staining (in blue) PS1 (in red). D. HuHMFG1 was conjugated to a non-toxic dye Cy5.5 and OE19 cells exposed to the HuHMFG1:Cy5.5 conjugate with and without a marker of recycling endosomal localization. Cells were co-stained with the nuclear stain DAPI shown in blue. Cy5.5 fluorescence is shown in red, endosomal marker is shown in green and the partial co-localization of HuHMFG1 and endosomal marker is shown in yellow.

Figure 8. Light dependent cytotoxicity and superior efficacy over equivalent free photosensitizer of a MUC1 specific photoactive antibody drug conjugate

A. The cytotoxicity of HuHMFG1:PS1 ADC, was compared with equivalent free photosensitizer (PS1) concentrations in light and dark conditions in OE19 cells. Light activation was by laser at 0.33J/cm2 at 670nm over 10 seconds. Light activated ADC cytotoxicity was significantly more effective than light activated PS1 cytotoxicity (linear regression of dose response curves with F test for comparison; p=0.0002; F=26.09). B. The cytotoxic efficacy of HuHMFG1:PS1 ADC, HuHMFG1 antibody alone and vehicle control were compared in a MUC1 positive line (OE19) and a MUC1 negative line (HT29) with and without light activation. Light activation involved an energy exposure of 0.33J/cm2 at 670nm over 10 seconds. Light activated ADC cytotoxicity was significantly more effective in the MUC1 positive line compared to the MUC1 negative control (Students t-test; p=0.0006). No significant cytotoxic effect was seen in either line with the vehicle control, antibody alone or ADC without light activation.