Abstract 574: Detection of tumor cell surface calreticulin after doxorubicin treatment using a novel radiolabeled nanobody

Abstract Background: Targeted agents, such as nanobodies, can be covalently linked to radionuclide chelators for theranostics. Nanobodies are single-domain immunoglobulin variable regions with a short blood half-lives and high specificity. Calreticulin (CALR) is endoplasmic reticular (ER) protein that translocates to the cell surface upon induction with certain agents, namely doxorubicin. We hypothesized surface CALR on tumors could be targeted using a novel nanobody after induction with doxorubicin. Methods: Studies were conducted using in vitro and in vivo models of pancreatic ductal adenocarcinoma (PDAC). Surface CALR expression was determined by western blot on fractionated lysates, flow cytometry, or immunofluorescence (IF). Doxorubicin was administered by intraperitoneal injection to mouse models of PDAC. The anti-CALR nanobody was developed via the panning of a synthetic nanobody phage display library. The top CALR-binding nanobody was confirmed by ELISA, immunohistochemistry (IHC), and flow cytometry. For in vivo studies, the nanobody was conjugated with the chelator NOTA and radiolabeled with 64Cu. PET images were acquired 1-hour after injection. Significance was determined using an independent t-test and p values < 0.05 were considered statistically significant. Results: PDAC cells treated with doxorubicin showed peak surface CALR expression between 24 and 48 hours. Flow cytometry revealed that 61.7% of live PDAC cultured cells treated for 24 hours expressed surface CALR, compared to 2.2% in controls (p < 0.001). In a subcutaneous syngeneic PDAC mouse model, two doses of doxorubicin 48 hours apart significantly increased surface CALR, with 30.4% of live-cells expressing CALR in treated tumors versus 1.5% in controls (p < 0.001). Similarly, doxorubicin-treated tumors from a PDAC patient derived xenograft (PDX) model had a 6.7-fold increase in live-cells expressing surface CALR compared to controls (p < 0.01). IF confirmed a 1.4-fold increase in CALR expression in doxorubicin-treated tumors from the subcutaneous PDAC mouse model (p < 0.01) and a 1.7-fold increase in the PDAC PDX model (p < 0.01). Our anti-CALR nanobody has an apparent 37 nM affinity for recombinant CALR and successfully detected surface CALR by IHC and flow cytometry. In vivo PET imaging showed significant increased uptake of the 64Cu-NOTA-nanobody in tumors from doxorubicin-treated mice over controls, with no significant changes in heart, kidney, or liver accumulation by PET quantification and flow cytometry (p < 0.05). Conclusions: Systemic doxorubicin treatment induces CALR surface translocation in tumors that can be successfully targeted with a radiolabeled nanobody for PET imaging. Targeting chemotherapy-induced surface CALR using our radiolabeled nanobody could provide a theragnostic strategy for the detection and treatment of PDAC. Citation Format: Rachael Guenter, Yuvasri Golivi, Lucinda Hall, Amro Abdelrahman, Chloe La Prairie, Caleb Miller, Tejeshwar C. Rao, Danielle M. Carlson, Mark J. Truty, Benjamin Larimer, J. Bart Rose. Detection of tumor cell surface calreticulin after doxorubicin treatment using a novel radiolabeled nanobody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 574.