Co-Treatment with Milatuzumab (Anti-CD74 mAb) and Rituximab (Anti-CD20 mAb) Results in the Induction of Mantle Cell Lymphoma Cell Death That Is Dependent On Actin Polymerization and Inhibition of NF-Kb.

Abstract 1694

Poster Board I-720

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of rituximab in treatment of B-cell lymphoma, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and is implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since rituximab and milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that 6 different MCL cell lines (Mino, JeKo, SP53, Rec-1, Hbl2, Granta-519) and MCL patient primary tumor cells, express variable levels of CD74, with Mino cells showing highest level and Rec-1 the lowest. Incubation of the 6 MCL cell lines and primary cells (7 patients) with immobilized milatuzumab (5 μg/ml) and rituximab (10 μg/ml) resulted in mitochondrial depolarization and in statistically significant enhanced induction of apoptosis determined by Annexin V/PI and flow cytometry. The combination of both agents resulted in additive induction of apoptosis that was caspase independent in 5 MCL cell lines (synergistic in JeKo cells) and in primary cells, at 8, 24 and 48 hours. Importantly, while sensitivity to milatuzumab depends on the level of CD74 expression, the combination of milatuzumab and rituximab was able to induce enhanced cell death in all MCL cell lines and MCL primary cells, regardless of antigen density.

We demonstrated that the combination of milatuzumab and rituximab induced enhanced apoptosis in a caspase-independent fashion with no apparent involvement of apoptotic key regulatory proteins such as Bax, Bcl-2, Bcl-Xl and Mcl-1. However, changes in the nuclear level of p65 were observed with either drug alone and with the combination, starting as early as 4 hours after treatment. The association of CD74 with MHC class II led us to explore pro-death mechanisms that become operable during HLA-DR-specific mAb treatment of lymphoma cells (Ivanov A et al., J Clin Invest 2009). We therefore investigated the role of actin polymerization by addition of cytochalasin D and latrunculin B, inhibitors of actin polimerization, prior to treatment with milatuzumab and/or rituximab. These studies showed that milatuzumab-induced MCL cell (Jeko and Mino) death was dependent on actin polymerization.

To examine the in vivo activity of rituximab and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, i.v. injection of 40×10⁶ JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal trastuzumab mAb control (300 μg qod), milatuzumab (300 μg qod), rituximab (300 μg qod), or a combination of milatuzumab and rituximab. The mean survival for the combination-treated group was 44.5 days (95%CI:39,51), compared to 28 days for trastuzumab-treated mice (95% CI:24,30), 33.5 days for the milatuzumab-treated mice (95% CI:28,36), and 38 days for the rituximab-treated mice (95%CI:36,42). The combination treatment prolonged survival of this group compared to trastuzumab control (P<0.0001), milatuzumab (P<0.0001) or rituximab (P=0.03). No overt toxicity from milatuzumab or the combination regimen was noted. These preliminary results provide justification for further evaluation of milatuzumab and rituximab in combination in MCL.