Abstract P4-07-18: FerOX, an anthracyclines nanoformulation to lead toward immuno-mediated cancer remission, preserving T-cell immune competence
Authors: Mazzucchelli Serena , Mainini Francesco , Andreata Francesco , Truffi Marta , Bonizzi Arianna , Piccotti Francesca , Sevieri Marta , Corsi Fabio 
Published: March 2023

Abstract
Background Chemotherapy with anthracyclines, such as doxorubicin (DOX), still remains a mainstay in cancer treatment. DOX kills tumor cells by direct interference with DNA replication, thus it is very effective against highly proliferating cells. Since DOX is not cell-specific drug, it displays severe side effects that limit its clinical use. Besides its direct cytotoxicity, it enhances also tumor immunogenicity, since DOX-induced immunogenic cancer cell death facilitates tumor antigens presentation. However, the generation of an adaptive immune response is one of the most highly- proliferative processes, thus it may be affected by DOX cytotoxicity, paradoxically preventing the establishment of a proper adaptive antitumor immune response. Therefore, the effect of DOX on dividing T-cells urges investigation. Methods We explored the phenotypic and functional effects of DOX on lymphocytes from healthy donors and from breast cancer (BC) patients. Purified peripheral blood mononuclear cells (PBMC) were subjected to an uptake assay and to a CFSE-based proliferation assay in vitro. Characterization of lymphocytes was made by flow cytometry (FC) and confocal microscopy. Innovative DOX nanoformulation with proved efficacy in cancer cell line, BC Patient-derived Organoids (PDO) and murine model of BC, has been assessed in parallel to liposomal DOX, evaluating both DOX uptake and their capability to preserve proliferation potential in different PBMC subpopulation. Results PBMC incubated in vitro with DOX displayed a dose-dependent DOX uptake that results in a dramatic proliferative impairment. The same behavior has been observed in BC patient-derived PBMC, isolated immediately after the end of first cycle of chemotherapy, which show a prompt DOX internalization coupled with a dramatic impairment of proliferative potential in comparison to the match-paired PMBC isolated before the treatment. Interestingly, FC characterization showed that DOX internalization is higher in CD8+ than CD4+ T-cells and mainly affects central memory, effector memory and terminal differentiated effector. Since pivotal to every adaptive immune response is the activation and massive proliferation of T cells from their resting state, we have exploited a DOX formulation in ferritin nanocages (FerOX), that displays a TfR1-mediated tumor specific homing in order to circumvent drug internalization in those immune cells. FerOX efficacy has been proved in BC-PDO, evidencing an inverse correlation between TfR1 expression and HFn-DOX IC50. HFn nanoformulation protects immune cells from DOX toxic effects, reducing DOX uptake and proliferative impairment, mainly preserving Naïve T-cells. The capability of FerOX to preserve T-cells competence to generate an immune response has been corroborated by an in vitro experiment aimed to mimic what could happens to patient’s PBMCs if they could be treated with FerOX. Conclusions As a whole, our data indicate that preventing T- cells chemotherapy-related toxicity through FerOX could allow the host adaptive immune system to fully benefit from the DOX-induced immunogenic cell death to vaccinate against cancer. This strategy could be promising for complete tumor eradication and might generate a long- term immunosurveillance of recurrences and metastases.
Citation Format: Serena Mazzucchelli, Francesco Mainini, Francesco Andreata, Marta Truffi, Arianna Bonizzi, Francesca Piccotti, Marta Sevieri, Fabio Corsi. FerOX, an anthracyclines nanoformulation to lead toward immuno-mediated cancer remission, preserving T-cell immune competence [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-18.