Evaluation of Otlertuzumab (TRU-016), an Anti-CD37 ADAPTIR^TM Therapeutic in Preclinical Combination Studies with Kinase Inhibitors and a Next Generation Anti-CD20 Mab in Vitro and in Animal Models of Non-Hodgkin’s Lymphoma

Background: CD37 is a 50-55 kDa heavily glycosylated member of the tetraspanin superfamily of molecules. This cell surface protein is expressed on normal and transformed B-cells, and has been implicated in diverse processes including cellular activation and proliferation, cell motility, and cell-cell adhesion. Otlertuzumab is a novel humanized anti-CD37 therapeutic, built on the ADAPTIR^TM (modular protein technology) platform that has been shown to mediate caspase-independent direct killing of normal and malignant B-cells, a mechanism of action that appears to be distinct from CD20 therapies. In addition, otlertuzumab results in killing through antibody directed cellular cytotoxicity (ADCC), mediated in part by NK cells. The therapeutic potential of otlertuzumab in the treatment of chronic lymphocytic leukemia (CLL) is currently being investigated in Phase 2 clinical studies in combination with bendamustine or rituximab. Preclinical vitro and in vivocombination studies for NHL to evaluate otlertuzumab in combination with other emerging drugs including kinase inhibitors (PI3K and BTK) and the next generation anti-CD20 mAb obinituzumab are reported here.

Methods: The ability of otlertuzumab to interact and increase malignant B cell killing with kinase inhibitors was investigated, including a pan PI3K inhibitor (LY294002), a PI3K delta inhibitor (CAL101(GS-1101, idelalisib)), a PI3K delta/gamma inhibitor (IPI-145, (INK1197)) and an inhibitor of BTK (PCI-32765). Combination studies were assayed in vitro using the Minos (mantle cell lymphoma), DoHH-2 (follicular lymphoma) and Ramos (Burkitt’s B cell lymphoma) cell lines. In addition, studies were performed in vitro to test the combination of otlertuzumab and obinituzumab. Individual drugs were tested alone or in combination with otlertuzumab. Combination index analyses were performed for drug combinations over the 20-90% effect levels. To determine whether in vitro synergy could be repeated in vivo, the tumor line with the best in vitro combination characteristics was utilized in xenograft tumor models and treated with otlertuzumab ± LY294002 or otlertuzumab ± PCI-32765 or otlertuzumab ± obinituzumab.

Results: Combination index analyses determined that the killing effects of otlertuzumab were synergistic with the pan PI3K inhibitor LY294002, demonstrating comparable results in all three cell lines tested in vitro. The combination of otlertuzumab and the BTK inhibitor PCI-32765 demonstrated synergy in vitro with the Ramos and Minos cell lines. The PI3K delta inhibitor (idelalisib) also demonstrated synergistic activity with all three cell lines when tested in combination with otlertuzumab in vitro. Finally, combination index analyses determined that the killing effects of otlertuzumab were synergistic with the obinituzumab, demonstrating comparable results in all three cell lines tested in vitro.

In vivo, the combination of otlertuzumab plus the pan PI3K inhibitor (LY294002) or otlertuzumab plus obinituzumab resulted in greater efficacy relative to each agent alone in the DoHH-2 xenograft tumor models. The combination of otlertuzumab with PCI-32765 resulted in significant delay of tumor outgrowth compared to PCI-32765 alone in the MINO xenograft model of NHL. In vivo results indicated that the in vitro synergy results were applicable to a more complex in vivodisease model.

Conclusions: Otlertuzumab tested in combination with multiple kinase inhibitors or next generation anti-CD20 had increased cell killing of NHL cell lines in vitro over that observed for each agent alone. Furthermore, the combinations of otlertuzumab with either obinituzumab, LY294002 or PCI-32765 displayed greater anti-tumor activity in vivo than each of the agents alone. These results provide preclinical rationale for the potential combinations of otlertuzumab with several emerging therapeutics for the treatment of NHL and related B-cell malignancies, including CLL.