Inhibition of B Cell Differentiation to the Plasmablast and Plasma Cell Lineage by CC-220, a Potent Modulator of the CRBN E3 Ubiquitin Ligase Complex

CC-220 is an immunomodulatory compound which binds to the CUL4 family E3 ubiquitin ligase complex protein cereblon (CRBN) with high affinity and modulates the ubiquitination of substrate proteins. To explore the effects of CRBN targeting on the differentiation of B cells to the plasmablast and plasma cell lineages, an in vitro model of primary human B cell differentiation was developed based on the methods of Jourdan et al. (Jourdan M, et al. Blood. 2009).

CD19+ peripheral blood human B cells from normal donors, or total PBMC for patients with systemic lupus erythematosus (SLE), were cultured in the presence of interleukin (IL)-2, IL-10, IL-15, TLR9 agonist, and CD40L for 4 days, followed by IL-2, IL-6, IL-10, and IL-15 for another 3 days. Cells were counted, viability assessed, and expression of CD20, CD38, CD44, and CD83 were measured by flow cytometry. Plasmablast lineage factors IRF-4, BLIMP-1, XBP-1, and IgJ, and germinal center markers PAX-5 and BCL-6 were measured by qRT-PCR. Intracellular protein expression was measured by laser scanning cytometry. Secreted immunoglobulins IgG and IgM were measured by ELISA.

In normal B cell cultures, CC-220 reduced the percentage of total viable cells in these cultures after 4 days to 69.6% (P≤0.05) or 35.8% of control (P≤0.001) at 20 nM or 200 nM CC-220, respectively. CC-220 decreased the percentage of viable CD20-CD38+ plasmablasts on day 7 from 30.4% in control cultures in a dose-dependent manner to 27.3%, 2.1%, and 0.4% at 2 nM, 20 nM, and 200 nM CC-220, respectively. On Day 7, qRT-PCR analysis showed that CC-220 (20 nM) reduced expression of the plasmablast lineage factors IRF-4, BLIMP-1, XBP-1, and IgJ gene expression to 20.5%, 14.3%, 15.1%, and 31.5% of control, respectively (P ≤0.001). By intracellular flow cytometry, CC-220 (20 nM) significantly decreased IRF-4 (P<0.5), BLIMP-1 (P< 0.05), and XBP-1 (P<0.05) protein expression at Day 4, but significantly increased BCL-6 (P<0.05) protein expression on Day 7. By laser scanning cytometry on Day 7, CC-220 (20 nM) reduced CD38+ cell intracellular protein expression of IRF-4 (P≤0.001), and BLIMP-1 (P≤0.001), and increased BCL-6 expression (P≤0.05) (n=3). CC-220 inhibited secreted IgG production with an IC₅₀=1.8 nM (n=3).

In PBMC from SLE patients, CC-220 (20 nM) had similar effects as in normal B cells, reducing BLIMP-1, XBP-1, and IgJ gene expression to 52.8%, 49.2%, and 13.6% of control, respectively (P≤0.001, n=3). CC-220 (20 nM) significantly reduced CD38+ plasmablast intracellular protein expression of BLIMP-1 (P≤0.01) and IRF-4 (P ≤0.001), and increased BCL-6 (P≤0.05) (n=3). CC-220 inhibited secreted IgM and IgG production by SLE patient PBMC with IC₅₀s of 0.9 nM and 3.2 nM, respectively (n=3).

These results demonstrate that targeting of the E3 ubiquitin ligase complex substrate co-receptor CRBN with the small molecule immunomodulator compound CC-220 results in potent inhibition of B cell differentiation to the plasmablast lineage, as shown by a reduction in the percentage of viable CD38+ cells, a decrease in BLIMP-1, XBP-1, IRF4, and IgJ gene and protein expression, and inhibition of secreted immunoglobulin production. These data implicate the CUL4-CRBN complex in the differentiation of B cells to the plasma cell lineage. CC-220 is currently entering clinical development for the treatment of immune diseases associated with autoantibody production.

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