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
Authors: Ioannou Nikolaos , Hagner Patrick R. , Towfic Fadi , Gandhi Anita K. , Stamatopoulos Kostas , Patten Piers E.M. , Vardi Anna , Ramsay Alan G. 
Published: October 2019

Abstract
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 naïve 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<.01, n=16), while combining avadomide with anti-PD-1 or anti-PD-L1 treatment significantly enhanced (P<.01) T cell killing function compared to these immunotherapies alone. Notably, anti-PD-1 monotherapy induced comparatively modest improvements of effector function.
We next performed RNA sequencing on highly purified T cells from treatment naïve 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<.01) T cell migration, and this response was significantly augmented by combination immunotherapy. Luminex protein analysis and cell migration assays confirmed the transcriptome data, highlighting the ability of avadomide and combination immunotherapy to significantly (P<.01) induce the release of CXCL10 (as well as IFN-γ, IL-2, GM-CSF and IL-8) from patient T cells, thereby attracting autologous T cells (CXCR3+). Avadomide as well as its pairing with anti-PD-L1 were also found to increase the proliferation of patient T cells (P<.01, n=6), particularly CD8+ 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<.01) that was associated with increased infiltration of PD-L1+ 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 'hot' 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 entity's 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.