Counteracting PD-L1/PD-L2 Immune Suppression in Multiple Myeloma through Kinase Inhibition

Introduction: Multiple myeloma (MM) is clonal plasma cell malignancy that remains incurable. Checkpoint inhibitors represent a revolutionary form of cancer therapy that empowers the immune system to defeat cancer. Programmed death-1 (PD-1) is an inhibitory receptor expressed on immune cells, particularly cytotoxic T cells, that interacts with two ligands, PD-ligand 1 (PD-L1) and PD-L2 expressed on tumor cells. PD-L1 and PD-L2 engage PD-1 on the T cell surface to negatively modulate the magnitude of T-cell-mediated responses. Such negative feedback is critically important in maintaining homeostasis of the immune response to prevent autoimmunity during infection or inflammation in normal tissue. However, in cancers, it presents a major problem in blocking cellular antitumor responses. PD-L1/PD-L2 ligation with PD-1 provides a mechanism of immune escape for tumor cells by turning off the cytotoxic T cells. Currently available monoclonal antibodies (mAbs) that disrupt the PD-1/PD-L1 interaction have exhibited remarkable responses in selected patients and tumor types. However, mAbs demonstrate many drawbacks that include a lack of tumor cell specificity, low response rates in unselected patient populations and induction of de novoautoimmune disease that excludes many patients from therapy. We hypothesize that small molecule checkpoint inhibitors can provide greater specificity, shortened half-lives to diminish autoimmune or other adverse events, increased oral bioavailability, enhanced bio-efficacy, and higher stability at ambient temperature facilitating purification during production. Here, we identified novel, small molecule PI3K inhibitors that reduce PD-L1/PD-L2 levels on MM cells and enhance the antimyeloma activity of autologous T cells.

Methods: To determine the effect of proteasome inhibitors on PD-L1/PD-L2 surface expression, RPMI8226 cells were incubated with bortezomib (BTZ), carfilzomib (CFZ) or ixazomib (IXZ) (1nM) for 36 h. Cells were then incubated in PBS/10% normal goat serum followed by antibodies (1/100) for 30 min at 4ºC and then treated with Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2. RPMI8226 cells were also incubated with the small molecule PI3K inhibitor DT97 (500nM) alone or combined with BTZ, CFZ, or IXZ. Cells were similarly stained using Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2 and a BD LSRFortessa™ cell analyzer was used for multicolor flow cytometry to acquire >10,000 events/sample. To determine the effect of DT97 on T cell-mediated cytolysis, bone marrow biopsy was performed on an MM patient and CD138⁺ cells were isolated by Miltenyi positive selection and CD4⁺ T cells by MojoSort™ CD4⁺ Human T Cell isolation. CD138⁺ cells were incubated with CD4⁺ T cells, proteasome inhibitors, or DT97 as indicated for 16 h at 37ºC. CD138⁺cells were then affinity-isolated, incubated with a FITC-conjugated anti-Annexin-V antibody and quantitated using a BD LSRFortessa™ cell analyzer.

Results: Treatment of MM cells with BTZ, CFZ, or IXZ significantly increased the surface expression of PD-L1 and PD-L2 (Fig. 1). In contrast, treatment with the PI3K inhibitor DT97 suppressed the expression of PD-L1 and PD-L2 on MM cells (Fig. 2). Importantly, DT97 co-treatment with either BTZ, CFZ, or IXZ suppressed the induction of PD-L1 and PD-L2 seen after treatment with proteasome inhibitors alone. Treatment with the proteasome inhibitors alone or combined with T cells did not promote significant killing of MM cells (Fig. 3). However, DT97 treatment significantly enhanced autologous T cell-mediated MM death and the effect was further enhanced by addition of proteasome inhibitors.

Conclusions: The results presented here establish the proof-of-principle that small molecule PI3K inhibitors reduce PD-L1/PD-L2 levels on tumor cells and enhance the anti-myeloma effect of autologous T cells. Small molecules checkpoint inhibitors represent a safe therapeutic alternative that can avoid the problems associated with antibodies, while retaining their functionality. Taken together, our studies indicate the promise of small molecules checkpoint inhibitors and support further translational and clinical development as a transformative form of cancer immunotherapy.