Exploiting radiation-induced tumor vulnerabilities via targeted antibody-drug conjugates

Background : Antibody-drug conjugates (ADCs) demonstrate potent anti-cancer activity but may cause on-target off-tumor toxicity in normal tissues if the target antigen is not strictly tumor-specific. To address this, we leverage on radiation therapy (RT), a main pillar in gastric (GC) and colorectal cancer (CRC) treatment, to kill cancer cells while simultaneously triggering a cancer-specific response that relocates Heat Shock Protein A5 (HSPA5). HSPA5 is intrinsically localized to the endoplasmic reticulum (ER). The ability of RT to promote HSPA5 translocation from ER to the cell surface (cs) opens an opportunity to target cancer cells with anti-HSPA5 ADC. We seek to understand RT-induced csHSPA5 biology and target it via ADC. Methods : We mined the TCGA data for HSPA5 expression in normal and GC or CRC tissues and for its correlation with patient prognosis. Irradiated human (AGS and MKN45) and murine (YTN16) GC, human (HCT116 and HT29) CRC cells were subjected to flow cytometry (FC) and immunofluorescence (IF) for csHSPA5 levels. csHSPA5+ cells' tumorigenicity was examined by co-staining the cells with HSPA5 and Ki67 (proliferation), CD44 (stemness), N-cadherin (EMT) via FC. To examine the involvement of O-glycosylation in HSPA5 cell-surface presentation, we performed co-immunoprecipitation to detect potential O-glycosylation regulators and inhibited O-glycosylation in AGS (+/-RT). To assess HSPA5 antibody biodistribution , we injected GC/CRC nude mice with anti-HSPA5 antibody-Alexa Fluor 647 dye prior to imaging, with the tumors and organs harvested for FC and IF. Lastly, Annexin V/PI staining was performed with AGS and HUVEC cells treated with RT+anti-HSPA5 ADC. Results : Expression levels of HSPA5 are higher in GC and CRC against their normal counterparts and are correlated with worse patient prognosis. We detected RT-induced elevation of csHSPA5 in all GC/CRC cells, notably not in normal HUVEC or GES1. csHSPA5+ population is enriched for Ki67+, CD44+, and N-cadherin+ cells compared to csHSPA5- population, indicating the tumorigenic phenotypes of csHSPA5+ cells. Interestingly, our data revealed the cancer-specific csHSPA5 translocation mechanism, in which HSPA5 binds to N-acetylgalactosaminyltransferase 2 (GALNT2) and N-acetylgalactosamine (O-GalNAC) only in cancer cells but not normal cells. Inhibiting O-glycosylation blocks RT-induced HSPA5 cell-surface translocation. The in vivo models showed a significant accumulation of HSPA5 antibody in radiated tumors compared to non-irradiated tumors or healthy organs. Moreover, RT+ADC significantly decreased %live cells compared to RT or ADC alone. RT+ADC caused no decreased viability of the normal HUVEC cells, reflecting the low toxicity in healthy cells. Conclusion : The overexpression of HSPA5 in cancer, coupled with radiation-induced cell-surface expression, renders it a promising ADC target for achieving high anti-tumor specificity.