Background/Purpose: CC-220 is an immunomodulatory compound that binds to cereblon (CRBN), part of the CUL4^CRBN E3 ubiquitin ligase complex, which has been shown to ubiquitinate the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). Polymorphisms at the IKZF1 and IKZF3 loci have been associated with risk of systemic lupus erythematosus (SLE). We explored CRBN, IKZF1, and IKZF3 gene expression in peripheral blood mononuclear cells (PBMC) from SLE patients, the effect of CC-220 on Ikaros and Aiolos protein levels and SLE autoantibody production in vitro, and the impact of CC-220 on immunological parameters in a phase 1, double-blinded, placebo-controlled, single ascending dose, healthy volunteer study.

Methods: CRBN, IKZF1, and IKZF3 gene expression was measured by qRT-PCR. Ikaros and Aiolos protein levels were measured by western blot and flow cytometry. Anti-dsDNA and anti-phospholipid autoantibodies were measured from SLE PBMC cultures treated for 7 days with CC-220. In the phase 1 healthy volunteer study, 56 subjects were randomized and enrolled in 7 cohorts, with 6 subjects per cohort receiving a single oral dose of CC-220 (0.03 to 6 mg) and 2 subjects per cohort receiving placebo. CD19+ B cells, CD3+ T cells, and intracellular Aiolos were measured by flow cytometry. IL-2 and IL-1β production were stimulated with anti-CD3 or lipopolysaccharide, respectively, in the TruCulture ex vivo whole blood assay system.

Results: Compared to normal PBMC, SLE PBMC expressed significantly higher levels of CRBN (1.5-fold), IKZF1 (2.1-fold), and IKZF3 (4.1-fold). CC-220 treatment of whole blood significantly reduced Ikaros and Aiolos protein levels in B cells, T cells, and monocytes, but not in granulocytes. In cultures of SLE PBMC, CC-220 inhibited anti-dsDNA and anti-phospholipid autoantibody production with an IC₅₀ of ≈10 nM. Following administration of single doses of CC-220 to healthy volunteers, there was a treatment-related decrease in intracellular Aiolos, with minimum mean percent of baseline values of ≈12% to 28% in B cells and ≈0% to 33% in T cells for 0.3 to 6 mg. There was also a treatment-related decrease in absolute CD19+ B cells and CD3+ T cells, with minimum mean percent of baseline values of ≈41% to 67% for B cells and ≈66% to 73% for T cells for 2 to 6 mg. CC-220 administration also resulted in increased IL-2 (maximum mean percent of baseline values ranging from 247% to 1,896% for 0.1 to 6 mg), and a decrease in IL-1β (minimum mean percent of baseline values of ≈11% for 6 mg).

Conclusion: These results demonstrate that CRBN, IKZF1, and IKZF3 mRNA are overexpressed in PBMC from SLE patients. Targeting the CUL4^CRBN E3 ubiquitin ligase with CC-220 resulted in a potent reduction in Ikaros and Aiolos protein levels in B cells, T cells, and monocytes, and inhibited autoantibody production. Administration of single doses of CC-220 (0.3 to 6 mg) reduced intracellular Aiolos protein expression in B cells and T cells, reduced absolute B cell and T cell counts in the peripheral blood, increased T cell-derived IL-2 production, and decreased LPS-induced IL-1β production in whole blood ex vivo. These findings support the further development of CC-220 for the treatment of SLE and other autoimmune diseases.