Immunoliposomes Incorporated with Humanized Monoclonal Antibody, Milatuzumab, Induce Cell Death in CLL by Retention of the CD74 Receptor On the Surface of B Cells.
Authors: Hertlein Erin K , Triantafillou Georgia , Hessler Joshua , Zhang Xiaoli , Jarjoura David , Lucas David M. , Lee Robert J , Muthusamy Natarajan , Goldenberg David M , Byrd John C. 
Published: October 2019

Abstract
Abstract 721
Chronic lymphocytic leukemia (CLL) is a progressive disease for which most patients require treatment. Therapy with monoclonal antibodies (mAbs) such as rituximab (anti-CD20) and alemtuzumab (anti-CD52) have improved the outcomes of patients with CLL; however, patients display varied levels of response to these agents and many develop resistance. We describe here a humanized mAb, milatuzumab (Immunomedics, Inc.), against CD74. The CD74 antigen has several ligands, including macrophage inhibitory factor, MIF, whose binding promotes internalization and nuclear localization with concomitant activation of NF-κB. Milatuzumab is currently in clinical trials in multiple myeloma and CLL; however, the mechanism by which this antibody induces apoptosis is still unknown. We demonstrate surface expression of CD74 on CLL and circulating normal CD19+ B cells, but no expression on T cells. Milatuzumab-induced cytotoxicity occurs very rapidly, with 70% cell survival at 4 h and 45% at 24 h, which is superior to that observed with the CD20 antibody rituximab. No toxicity was detected in NK and T cells treated with milatuzumab. The mode of cell death induced by milatuzumab was found to be independent of caspase activation, with no detectable caspase-3 processing, PARP cleavage, or inhibition of death with the pan-caspase inhibitor, BocD-fmk. In vitro, the addition of a-Fc crosslinking antibody is necessary for milatuzumab-induced cell death. However, previous reports noted milatuzumab does not mediate antibody-dependent cellular cytotoxicity (ADCC) in lymphoma cell lines and this was verified in CLL using peripheral blood mononuclear cells, NK cells, and granulocytes as effector cells. Thus, milatuzumab-mediated death appears to be mediated directly through interaction with CD74 and effects on signal transduction. We further investigated the role of anti-Fc crosslinking antibody in mediating milatuzumab-induced cell death, and found that upon milatuzumab treatment in the presence of anti-Fc, CLL cells aggregated in culture. Furthermore, the mean fluorescent intensity (MFI) of CD74 on the cell surface increased, which did not occur following treatment with anti-Fc alone, milatuzumab alone, or anti-Fc along with other anti-B-cell mAbs such as rituximab. This suggests that milatuzumab, when crosslinked, promotes the maintenance of CD74 on the cell surface and thereby prevents receptor internalization and subsequent signaling. However, association with Fc receptors on other cells in the microenvironment may not be sufficient to mediate this effect, as indicated by the lack of ADCC. Furthermore, in vitro co-culture of NK cells in the presence of milatuzumab did not induce the same cellular aggregation and elevated CD74 surface expression as seen with the anti-Fc antibody. These results indicate that milatuzumab treatment in vivo may not be as efficient as suggested by the in vitro studies without a sufficient method to sequester CD74 on the cell surface. We tested this hypothesis by incorporating milatuzumab into liposomes (Mila-ILP). Our results show that this conjugate mediates the same receptor aggregation on the cell surface as milatuzumab combined with anti-Fc crosslinking antibody. This effect was not evident with incorporation of irrelevant immunoglobulin into liposomes (IgG-ILP). Furthermore, Mila-ILP induced the same degree of cell death as observed previously with in vitro antibody crosslinking. Therefore, the incorporation of milatuzumab into liposomes induces cell death independent of other cell types in the microenvironment. Together, these data describe a mechanism of milatuzumab-induced cytotoxicity in CLL, and encourage the clinical application of milatuzumab-immunoliposome-based therapy in CLL and other CD74-positive lymphoid malignancies.

Disclosures:
Goldenberg: Immunomedics: Employment, Equity Ownership.