Oncolytic Reovirus Sensitizes Multiple Myeloma Cells to Anti-PD-L1 Therapy

Adaptive resistance mediated by inhibitory ligands such as programmed death 1 ligand (PD-L1) has emerged as an important mechanism of malignant cell survival. This has spurred the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. Analysis of patient specimens from clinical trials of novel immune checkpoint inhibitors indicates that high basal expression of PD-L1 on tumor cells may predict sensitivity to and be necessary to elicit significant clinical benefit from this drug class. Notably, many multiple myeloma (MM) cell lines and primary CD138+ cells from MM patients do not overexpress PD-L1 compared to normal plasma cells and this may preclude patients with MM from optimally benefitting from immune checkpoint inhibitor therapy. These data suggest that strategies that transiently increase PD-L1 levels could potentially sensitize malignant cells with low PD-L1 expression to anti-PD-1/PD-L1 blockade. The oncolytic reovirus-based anticancer agent Reolysin is known to have significant immunomodulatory effects and has demonstrated promising preclinical efficacy in MM models and favorable safety and tolerability in early MM clinical trials. We demonstrated that Reolysin selectively replicates in MM cells and possesses significant activity in preclinical in vitro and in vivo MM models. These findings established the framework for an ongoing investigator-initiated phase 1b clinical study of Reolysin in combination with bortezomib and dexamethasone in patients with relapsed/refractory MM. Recent gene ontology analyses of RPMI-8226 and U266 MM cells treated with Reolysin revealed that reovirus exposure triggers a highly significant transient increase in CD274 (PD-L1) in MM cell lines. Reolysin-mediated PD-L1 upregulation was confirmed by immunoblotting, qRT-PCR, and flow cytometric analyses in MM cell lines and primary patient specimens treated with Reolysin. Increased PD-L1 expression was also detected by immunohistochemistry in MM tumor samples collected from mice treated with Reolysin. A comparison of the anti-MM effects achieved by live reovirus versus UV-inactivated reovirus demonstrated that live reovirus is required to decrease MM cell viability and upregulate PD-L1 expression. Our data demonstrate proof of concept that reovirus infection and replication in MM cells can efficiently and selectively upregulate PD-L1 levels in malignant cells with low target expression.

We hypothesized that Reolysin treatment could be used as a precision priming strategy to potentiate the anti-MM efficacy of PD-1/PD-L1 targeted therapy by promoting myeloma immune recognition and PD-L1 upregulation. To investigate this therapeutic approach, 5TGM1-luc murine MM cells were injected IV into immunocompetent mice to generate MM bone disease. After disease was established, mice were randomized into groups and treated with vehicle, Reolysin (5 x 10⁸ TCID50, Q7D), murine anti-PD-L1 antibody (200 mg/mouse, Q2D) or the combination for 5 weeks. Mice treated with the combination demonstrated decreased disease burden as measured by bioluminescent imaging and also showed reduced IgG2bk levels (specific IgG secreted by 5TGM1 cells) by ELISA. Importantly, the combination also led to increased overall animal survival compared to vehicle control and either single agent treatment (P<0.01). Analysis of bone marrow specimens from mice in all experimental groups showed that Reolysin stimulated elevated PD-L1 levels in vivo in a manner that was directly linked to the enhanced efficacy of the combination. Our findings demonstrate that Reolysin has dramatic PD-L1-related immune priming effects in clinically relevant models of MM and support its use as a precision agent to sensitize MM cells to immune checkpoint inhibitor therapy. Based on these promising data, we are currently planning a clinical study of Reolysin in combination with bortezomib and a PD-1 inhibitor in patients with relapsed/refractory MM.