Eliciting Anti-Tumor T Cell Immunity in Chronic Lymphocytic Leukemia (CLL) with PD-L1/PD-1 Blockade Is Enhanced By Avadomide Immunotherapy through the Triggering of Immunogenic Interferon Signaling

Immune checkpoint blockade has demonstrated potential to reactivate anti-tumor immunity and regress tumors. However, response rates in B-cell non-Hodgkin lymphoma patients have been lower compared to Hodgkin lymphoma, with no activity reported in a recent trial of anti-PD-1 immunotherapy in relapsed CLL. This suggests that certain lymphoma subtypes may harbor non-immunogenic tumor microenvironments (TME). Our preliminary studies revealed that avadomide (CC-122), a cereblon modulator, can enhance T cell immune synapse signaling with autologous CLL cells, resulting in a concomitant increase in PD-1/PD-L1 expression at repaired synapses, indicating that avadomide may represent a complementary treatment partner for checkpoint inhibition. Here, we have extended our pre-clinical studies to investigate how these immunotherapy drugs alter the function and gene signatures of previously exhausted T cells from treatment naive CLL patients (representing disease heterogeneity). Cytotoxicity assays revealed that treating primary T cells and autologous CLL cells with avadomide (1 μM, 48h) activated anti-tumor T cell killing function ( P P We next performed RNA sequencing on highly purified T cells from treatment naive CLL patients, representing extremes of prognosis (n=6 good and n=6 poor including disease harboring TP53 abnormalities), following 18 h treatment with avadomide (100 nM) or anti-PD-1 (nivolumab) or PD-L1 (durvalumab) alone (10 μg/ml) or in combinations. Differential expression pathway analysis revealed that the top functional gene categories common for all the avadomide and combination treated samples (independent of anti-PD-1 or anti-PD-L1 monotherapy) were related to the response to type I and II IFN signaling, as well as inflammatory/stimulatory cytokine TNF-α, proliferative IL-6/JAK/STAT3, and IL-2/STAT5 responses. Type I IFN drives expression of chemokines CXCL10 and CXCL9, which are linked to enhanced tumor-infiltrated lymphocyte recruitment, and these chemoattractant genes were significantly upregulated in the avadomide and combination treated patient samples. In addition to their immunostimulatory roles, type II IFN (IFN-γ) and chronic type I IFN signaling have been linked to T cell resistance/exhaustion. We detected upregulated PD-L1 transcript in avadomide and combination treated T cells - supporting blockade of this inhibitory ligand. To investigate the ability of avadomide to modulate T cell migration, we performed comparative quantitative time-lapse microscopy analysis of pretreated patient T cells (n=12). These assays revealed that avadomide, as well as anti-PD-1 or anti-PD-L1 monotherapies, significantly enhanced ( P P + ). Avadomide as well as its pairing with anti-PD-L1 were also found to increase the proliferation of patient T cells ( P + T cells. Patient-derived xenograft models demonstrate that the therapeutic treatment of established tumors (3 weeks post-engraftment) with avadomide treatment (0.5 mg/kg) can activate anti-tumor T cells and significantly reduce disease volume ( P + CD8 + T cells. Pairing avadomide with anti-PD-L1 durvalumab led to maximal activation of anti-tumor T cell immunity. In conclusion, our findings support the concept that PD-L1/PD-1 blockade in CLL could be enhanced when combined with avadomide through the promotion of immunogenic IFN signaling that enhances T cell infiltration and function. We believe these data support the rationale for combination immunotherapy that could convert a non-immunogenic TME into a 9hot9 T cell inflamed TME which would be sensitive to checkpoint blockade. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership. Towfic: Celgene Corporation: Employment, Equity Ownership. Gandhi: Celgene Corporation: Employment, Equity Ownership. Stamatopoulos: Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Patten: L Hoffman La Roche: Honoraria, Research Funding; Gilead Sciences: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: travel, Research Funding; AbbVie Inc: Honoraria, Other: travel; Janssen: Honoraria, Other: travel. Vardi: Gilead: Research Funding; Janssen: Honoraria. Ramsay: MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding.