P854: HEXABODY-CD38 INDUCES TROGOCYTOSIS AND EFFECTIVELY INDUCES COMPLEMENT-MEDIATED TUMOR CELL LYSIS AFTER TREATMENT WITH DARATUMUMAB OR ISATUXIMAB IN VITRO (2022)

Background: HexaBody-CD38 (GEN3014) is a next-generation CD38-specific IgG1 antibody with a hexamerization-enhancing mutation. HexaBody-CD38 is designed to induce strong anti-tumor activity in patients with CD38-expressing hematological malignancies through potent complement-dependent cytotoxicity (CDC) and other Fc-mediated effector functions. The safety and preliminary efficacy of HexaBody-CD38 are currently being evaluated in a first-in-human trial in relapsed/refractory multiple myeloma (MM) patients (NCT04824794). Trogocytosis has been suggested as effector mechanism of daratumumab: reduction of CD38 expression on tumor and immune cells is thought to reduce local immunosuppression and contribute to improved adaptive immune responses against MM cells (Krejcik, 2018, Oncotarget 9, 33621). Aims: The present study aimed to increase our understanding of the MoA of HexaBody-CD38, by studying its capacity to induce trogocytosis as well as its capacity to induce CDC in CD38+ tumor cells in the presence of daratumumab or isatuximab in vitro. Methods: Trogocytosis was evaluated as the amount of membrane transfer in absence of transfer of cytoplasm in a flow cytometry-based assay using Wien-133 cells as target cells and healthy donor monocytes as effector cells. Binding of HexaBody-CD38 to CD38+ cell lines SU-DHL-4, NCI-H929, and Wien-133 that were pre-treated with daratumumab or isatuximab was allowed for 15 min, 1 h, 4 h or 24 h, while daratumumab or isatuximab remained present at saturating concentrations. HexaBody-CD38 mediated CDC of daratumumab or isatuximab-opsonized Wien-133 cells, which were insensitive to CDC induction by daratumumab or isatuximab, was assessed by flow cytometry at 45 min, 4 h, or 24 h after adding human complement in the presence of daratumumab or isatuximab. Results: HexaBody-CD38 induced dose-dependent transfer of plasma membrane from CD38+ tumor cells to human monocytes (n=7). The mean EC50 for trogocytosis activity of HexaBody-CD38 was 8.79 ± 2.49 ng/mL (0.018 ± 0.014 nM). This was in the same range as EC50s of daratumumab and the HexaBody-CD38 parental antibody without E430G mutation, suggesting that the hexamerization mutation has limited impact on trogocytosis induction. HexaBody-CD38 was found to compete with daratumumab and isatuximab for binding to CD38. Binding of HexaBody-CD38 to CD38+ cells increased in time and with increasing concentration, generally faster and more extensive in the presence of isatuximab compared to daratumumab. At equimolar concentrations, near-complete displacement of isatuximab and daratumumab was observed after incubations ≥1 h and ≥4 h, respectively. Accordingly, after 24 h HexaBody-CD38 induced comparable maximum CDC of CD38+ cells in presence or absence of daratumumab. Comparable CDC activity in the presence or absence of isatuximab was already observed after 4 h. Summary/Conclusion: HexaBody-CD38 was shown to induce efficient monocyte-mediated trogocytosis of CD38+ tumor cells in vitro, suggesting HexaBody-CD38 may reduce CD38-associated immunosuppression in the tumor microenvironment. In addition, it has previously been reported that the main differentiating effector mechanism activity of HexaBody-CD38 compared to daratumumab and isatuximab is its increased capacity to induce CDC. Here we confirmed the CDC potency of HexaBody-CD38 even in the presence of saturating concentrations of daratumumab and isatuximab. This suggests that HexaBody-CD38 is capable of inducing CDC of CD38+ myeloma cells in patients who have received prior anti-CD38 mAb treatment with residual daratumumab or isatuximab present in their circulation.