Abstract
Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, constituting 30-40% of all new cases. Avadomide, a small molecule cereblon modulator currently being developed in DLBCL, binds to cereblon in the CRL4CRBN E3 ligase, leading to ubiquitination and subsequent proteasomal degradation of transcription factors Aiolos and Ikaros. This results in decreased proliferation and increased apoptosis of DLBCL cells, independent of cell-of-origin, and immunostimulatory effects in T and NK cells, as measured by increased cytokine production, cell surface activation markers, and enhanced antibody-dependent cellular cytotoxicity. A novel gene expression-based classifier, which detects DLBCL patients with T cell and macrophage infiltration within the tumor microenvironment, has been shown to enrich for responders to avadomide. Avadomide, as a single agent and in combination with rituximab, is currently being investigated in relapsed/refractory DLBCL (NCT01421524 and NCT02031419).
Methods: Eighty-one DLBCL patients were enrolled in the expansion phase of the CC-122-ST-001 study (NCT01421524). Peripheral blood T cell subsets were enumerated at screening (baseline), cycle 1 day 15 (C1D15) and cycle 2 day 15 (C2D15) by flow cytometric immunophenotyping. Ex vivo production of IL-2 and IFNγ, as a measure of T cell activation, was determined using the α-CD3 TruCulture Assay. Changes from baseline were evaluated using the t-test with P<0.05 considered significant. T cell receptor (TCR) repertoire analysis through TCRB CDR3 region sequencing was done to derive metrics of population diversity and composition. RNAseq was performed on screening and on-treatment (C1D10/15) biopsies; gene expression deconvolution analyses were used to identify immune cell populations within the tumor microenvironment.
Results: Avadomide treatment results in decreased peripheral CD4+ and CD8+ naïve (CD45RA+/CD45RO-) T cells and increased memory (CD45RA-/CD45RO+) and activated (HLA-DR+) T cells, without significantly affecting the absolute numbers of total CD3+, CD4+ or CD8+ populations (Table). High-dimensional single-cell mass cytometry of longitudinally collected peripheral blood samples confirmed the significant increase in CD8+ memory T cells and identified an increase in Treg populations and decreases in CD16+ monocytes and dendritic cells (adj. P<0.02). A single dose of avadomide on C1D1 significantly activated T cells, as indicated by a 300% increase in IL-2 (P=0.018) and 185% increase in IFNγ (P=0.003) secretion. Assessment of TCR B clonotypes revealed that avadomide increases the TCRB repertoire breadth, while reducing its clonality. To understand the influence of avadomide treatment on the tumor microenvironment, we performed RNA sequencing on tumor biopsies collected at screening and two weeks after initiating avadomide treatment (n=18 patients). Deconvolution analyses identified an increase in the expression of genes indicative of various T cell populations, dendritic cells and macrophages, while B cell associated gene expression decreased in on-treatment biopsies compared to screening biopsies. Gene set enrichment analysis (GSEA) revealed significantly increased expression of genes associated with "HALLMARK Interferon Alpha Response" (adj. P=0.04), indicative of an increase in Type I/II interferon production by cells such as T and NK cells. Buttressing the in vitro observations of avadomide-mediated inhibition of DLBCL cell proliferation, GSEA identified a decrease in "E2F targets" (adj. P=0.007) consistent with decreased proliferation of malignant B cells.
Conclusion: Avadomide is a potent immunomodulating agent with multiple immune activating properties, including positive effects on T cell activation, as well as a broad expansion of T cell populations as defined by an increase in the richness of the T cell repertoire in blood. In addition, our data demonstrate decreased proliferation of malignant B cells in the tumor, with concomitant increased trafficking of immune cells, such as dendritic cells and macrophages, to the tumor microenvironment. These data further delineate the immune enhancing activity of avadomide in DLBCL patients beyond T-cell activation and provide rational combination strategies.
Hagner:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership.
