Abstract
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 malignancies, 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 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 the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). 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, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment 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 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.
Gene Methylation and Silencing of WIF1 Is a Frequent Genetic Abnormality in Mantle Cell Lymphoma
