Interferon γ Enhances the Anti-Myeloma Activity of the Fully Human Anti-HLA-DR Monoclonal Antibody 1D09C3.
Authors: Carlo-Stella Carmelo , Guidetti Anna , Di Nicola Massimo , Lavazza Cristiana , Cleris Loredana , Longoni Paolo , Milanesi Marco , Magni Michele , Nagy Zoltan , Corradini Paolo , Carbone Antonino , Formelli Franca , Gianni Alessandro M. 
Published: April 2020

Abstract
The fully human anti-HLA-DR antibody 1D09C3 exerts a potent anti-lymphoma activity both in vitro and in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. 1D09C3 is currently tested in phase I clinical trials in patients with relapsed/refractory B-cell malignancies. To investigate whether 1D09C3 might represent a treatment modality to target malignant plasma cells, we re-evaluated HLA-DR expression on CD138+ plasma cells. Additionally, we investigated the capacity of interferon γ (IFN-γ ) to upregulate HLA-DR expression on myeloma cell lines, and tested in vitro and in vivo the anti-myeloma activity of 1D09C3 alone or in combination with IFN-γ . Bone marrow CD138+ cells were enriched using an immunomagnetic method from 60 multiple myeloma (MM) patients. Three-color flow cytometry revealed a highly heterogeneous HLA-DR expression on plasma cells. CD138+HLA-DR+ cells were detected in 31/60 patients (52%), with 15/60 patients (25%) having ≥ 20% CD138+HLA-DR+ cells (median, 50%; mean, 54%; range, 23 - 100), and 3 patients (5%) displaying 100% CD138+HLA-DR+ cells. Two thirds of HLA-DR+ patients expressed CD45 on CD138+ cells, suggesting that 1D09C3 might target self-renewing plasma cells. HLA-DR expression was not associated with distinct cytogenetic abnormalities. Since primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines (n = 6) with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin-V/propidium iodide double staining showed that 1D09C3-induced cell death strongly correlated with constitutive HLA-DR expression. Interestingly, induction of HLA-DR by IFN-γ restored the sensitivity of HLA-DR dim cell lines (i.e., RPMI-8226, KMS-11) to the cytotoxic activity of 1D09C3. As compared to controls, exposure of RPMI-8226 and KMS-11 cell lines to IFN-γ and 1D09C3 significantly increased cell death to 45% (P ≤ 0.0001) and 40% (P ≤ 0.0001), respectively. The in vivo activity of 1D09C3 was analyzed by xenografting NOD/SCID mice with the KMS-11 cell line. A significant increase of median survival over controls was detected in mice treated with 1D09C3 alone at either 3 mg/mouse (92 vs 48 days, P ≤ 0.0001) or 6 mg/mouse (89 vs 48 days, P ≤ 0.0001). The combined treatment with IFN-γ plus 1D09C3 (3 mg/mouse) resulted in a significant increase of median survival as compared to controls (147 vs 48 days, P ≤ 0.0001) or mice receiving 1D09C3 alone (147 vs 92 days, P ≤ 0.03). The better therapeutic activity of the combined IFN-γ /1D09C3 treatment over 1D09C3 alone was further demonstrated by a 2-fold increase of mice being disease-free at 150 days after xenograft (47% vs 25%). No mice experienced any apparent treatment-related toxicity. In conclusion, our data demonstrate that:
one third of patients with MM express significant levels of HLA-DR on CD138+ cells; IFN-γ-induced upregulation of HLA-DR results in a potent enhancement of the in vivo anti-myeloma activity of 1D09C3.