Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose: Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex including CUL4A, DDB1, and ROC-1. CC-220 is a novel immunomodulatory compound currently in development for the treatment of immune conditions. The effects of CC-220 on CRBN binding and ubiquitination, and on immune cell responses were profiled.
Methods: Binding studies to CRBN were conducted using endogenous CRBN from human U266 plasmacytoma cells and affinity beads in a competition assay by quantitative immunoblot determination. CRBN ubiquitination was measured in HEK293T cells transfected with a His-biotin-tagged CRBN construct, treated with the MG132 proteasome inhibitor (to arrest degradation of ubiquitinated proteins). Cells were lysed and processed to measure CRBN ubiquitination by SDS-PAGE and immunoblot analysis. T-cell costimulation was measured in purified primary human T cells stimulated using immobilized anti-CD3 antibody. Cytokine secretion was measured by ELISA. Immunoglobulin M and G (IgG and IgM) production was measured from normal donor peripheral blood mononuclear cells by culturing in the presence of the B cell differentiation factors IL-2, IL-6, IL-10, IL-15, CD40L, and TLR9 ligand. IgM and IgG were measured by ELISA. Cell proliferation studies were conducted in H929 plasmacytoma cells assessed by 7-aminoactinomycin D (7-AAD) staining. Toll-like Receptor 4 (TLR4) stimulation was performed in human PBMC from subjects with SLE and SSc using lipopolysaccharide (LPS).
Results: In the competitive CRBN binding studies, CC-220 had a 50% inhibitory concentration (IC50) of approximately 0.1 μM, compared with 3 μM for pomalidomide. CRBN ubiquitination studies in the transfected HEK293T cells resulted in an IC50 of 0.19 μM. CC-220 costimulated IL-2 production by T cells with an EC50 of approximately 0.29 nM, compared with 10 nM for pomalidomide. CC-220 inhibited IgM and IgG production with an IC50 of 0.35 and 2.1 nM, respectively, compared to 17 nM and 63 nM for pomalidomide. The IC50 value for inhibition of proliferation by CC-220 was 0.01 μM in the H929 plasmacytoma cell line. A 50% decrease in cell cycle (S-phase) was evident after 24 hours of treatment of H929 cells with CC-220. At 48 hours, CC-220 decreased expression of survivin and retinoblastoma protein (pRB) and increased expression of the cyclin-dependent kinase inhibitor p27. In LPS-stimulated PBMC from systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) patients, CC-220 significantly reduced production of pro-inflammatory cytokines including TNF-α, IL-1α, IL-1β, and IL-6, and increased production of IL-10.
Conclusion: The results indicate that CC-220 binds to CRBN and inhibits CRBN ubiquitination CC-220 enhances T cell IL-2 production, but inhibits B cell production of immunoglobulin and reduces plasma cell line proliferation. This compound also reduces the production of several key inflammatory mediators by SLE and SSc patients cells stimulated via the TLR4 pathway. In summary, CC-220 is a novel high affinity CRBN ligand with immunomodulatory effects and is currently in development for the treatment of immune and inflammatory diseases.
Disclosure:
P. Schafer,
Celgene,
3;
E. Rychak,
Celgene,
3;
D. Mendy,
Celgene,
3,
Celgene,
1;
S. Parton,
Celgene,
3;
L. Capone,
Celgene,
3;
A. Lopez-Girona,
Celgene,
3;
D. Cedzik,
Celgene,
3;
J. Kosek,
Celgene,
3;
L. H. Zhang,
Celgene,
3;
R. Chopra,
Celgene,
3.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-cereblon-with-the-high-affinity-immunomodulatory-compound-cc-220-a-novel-therapeutic-agent-for-autoimmunity/