Aims: We investigated whether radiation-induced nontargeted effects are involved in the cytotoxic effects of anticell surface monoclonal antibodies labeled with Auger electron emitters, such as iodine 125 (monoclonal antibodies labeled with 125I [125I-mAbs]). Results: We showed that the cytotoxicity of 125I-mAbs targeting the cell membrane of p53+/+ HCT116 colon cancer cells is mainly due to nontargeted effects. Targeted and nontargeted cytotoxicities were inhibited in vitro following lipid raft disruption with Methyl-β-cyclodextrin (MBCD) or filipin or use of radical oxygen species scavengers. 125I-mAb efficacy was associated with acid sphingomyelinase activation and modulated through activation of the AKT, extracellular signal-related kinase ½ (ERK1/2), p38 kinase, c-Jun N-terminal kinase (JNK) signaling pathways, and also of phospholipase C-γ (PLC-γ), proline-rich tyrosine kinase 2 (PYK-2), and paxillin, involved in Ca2+ fluxes. Moreover, the nontargeted response induced by directing 5-[(125)I]iodo-2′-deoxyuridine to the nucleus was comparable to that of 125I-mAb against cell surface receptors. In vivo, we found that the statistical significance of tumor growth delay induced by 125I-mAb was removed after MBCD treatment and observed oxidative DNA damage beyond the expected Auger electron range. These results suggest the involvement of nontargeted effects in vivo also. Innovation: Low-energy Auger electrons, such as those emitted by 125I, have a short tissue range and are usually targeted to the nucleus to maximize their cytotoxicity. In this study, we show that targeting the cancer cell surface with 125I-mAbs produces a lipid raft-mediated nontargeted response that compensates for the inferior efficacy of non-nuclear targeting. Conclusion: Our findings describe the mechanisms involved in the efficacy of 125I-mAbs targeting the cancer cell surface. Antioxid. Redox Signal. 25, 467–484.

BackgroundTriple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC.MethodsCath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned in the human IgG1 λ format (F1 and E2). Anti-cath-D antibody biodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumor xenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs).ResultsHigh CTSD mRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D was detected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodies accumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival without apparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in the MDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15 upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγ cytokine. The F1 antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGFβ decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy.ConclusionCath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients with TNBC.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0498-z) contains supplementary material, which is available to authorized users.