Iberdomide (CC-220) is a potent cereblon E3 ligase modulator with antitumor and immunostimulatory activities in lenalidomide- and pomalidomide-resistant multiple myeloma cells with dysregulated CRBN

Immunomodulatory compounds, IMiDs® lenalidomide and pomalidomide mediate their anti-myeloma activities via cereblon, a component of the Cul4A E3 ligase complex [1, 2]. Here we describe preclinical experiments with a next generation cereblon targeting agent, iberdomide (CC-220) and explore its activity in lenalidomideand pomalidomidesensitive and -resistant multiple myeloma (MM) cell lines, and its pharmacodynamic effects in the bone marrow from relapsed/refractory MM (RRMM) patients. We observed order of antiproliferative activity of iberdomide > pomalidomide > lenalidomide in matched lenalidomide-sensitive (H929) and an acquired lenalidomide-resistant (H929/LR) cell line (Fig. S1A). In a panel of MM cell lines across a range of concentrations, iberdomide had pronounced antiproliferative effects (Fig. S1B) compared to lenalidomide and pomalidomide measured by a ‘sensitivity shift’ of the relative percentage of AUC reduction (Supplementary Methods) (Fig. S1C). Analysis of the substrates Aiolos/Ikaros show degradation by both pomalidomide and iberdomide in the H929/LR cells (not shown), consistent with previous observations [3]. Further, treatment of H929 cells, with either pomalidomide or iberdomide resulted in timedependent increases in G0/G1 and sub-G1 cell cycle fractions (Fig. S1D). Consistently, iberdomide induced greater apoptosis than pomalidomide in all MM cell lines tested, at a tenfold lower concentration, estimated to be in the range of clinical activity [4] (Fig. S1E). Pomalidomide and lenalidomide bind cereblon with similar affinity (~3 μM) [5]. We previously reported that faster rate of degradation of targeted substrates, Ikaros and Aiolos, and the down regulation of c-Myc/IRF4 expression were associated with greater antitumor effects of pomalidomide [6]. Treatment with 0.1 μM iberdomide led to a faster decrease in the relative abundance of these proteins than with pomalidomide (1 μM) (Fig. S1F). Cereblonbinding affinity IC50 of iberdomide is ~150 nM [5]. Thus the faster degradation of the substrates may be due to increased cereblon-binding affinity and/or improved processivity of the iberdomide-bound E3 ligase. Current clinical application of IMiDs compounds include doublet and triplet combinations with dexamethasone, bortezomib, and/or daratumumab. We initially compared the antiproliferative and pro-apoptotic activity of iberdomide to pomalidomide in combination with bortezomib in MM1.S cells. Due to the potent cytotoxic effects of bortezomib, pomalidomide, and iberdomide, and the narrow window of observable combinatorial effects, we titrated either pomalidomide (0.001–10 μM) or iberdomide (0.0001–1 μM) against bortezomib (0.0625–1 nM) (Figs. S2A, S3A, left). Using these concentrations, inhibition of proliferation induced by the combinations of iberdomide/bortezomib and pomalidomide/bortezomib were both synergistic [7] (Fig. S2B). In MM1.S cells, while single agent bortezomib, pomalidomide, or iberdomide induced * Anjan Thakurta athakurta@celgene.com

quantitative measure of the degree of drug interaction in terms of additive effect (CI=1), synergism (CI<1), or antagonism (CI>1) for a given endpoint of the effective measurement.

Apoptosis and Cell Cycle Analysis by Flow Cytometry
Apoptosis was measured by To-Pro-3 and Annexin V incorporation.(1) Cell cycle analysis was measured by propidium iodide staining. (4) Immunoblotting Cells for immunoblotting were prepared(1) and probed with respective primary antibodies overnight, followed by incubation with IR-dye secondary antibodies (LI-COR Biotechnology, Lincoln, NE, USA). Protein bands were visualized (1) followed by densitometric quantification and analysis using Image Studio Software (LI-COR). Protein density was normalized to β-actin loading controls and parental controls as indicated, arbitrarly set at 100%. The percentage of

Peripheral Blood Mononuclear (PBMC) Co-culture with MM Cells
Immune-stimulated PBMC co-culture killing assay was performed as previously described. (5) Peripheral blood mononuclear cells (PBMCs) were obtained from healthy untreated donors by Ficoll gradient. Anti-CD3 (OKT3; eBiosciences, Springfield, NJ, USA) stimulated PBMCs were cultured with iberdomide (0.0001-1 μM) for 72 hours and culture supernatants for IL-2 were assessed using ELISA. PBMCs were washed to remove residual compound, andsubsequently cocultured with carboxyfluorescein succinimidyl ester (CFSE; ThermoFisher, Waltham, MA, USA)-labelled MM cells at a 3:1 ratio for 4 hrs. The culture supernatants were collected and assayed for Granzyme B secretion by ELISA. Co-cultured cells were washed and stained with Annexin-V-PE and To-Pro3-APC for apoptosis measurement on CFSE-labelled MM cells. (6)

CDC Assays
For complement-dependent cytotoxicity (CDC) assay, target cells were treated with the indicated drug (iberdomide or daratumumab) with and without the presence of 15% human serum (Sigma-Aldrich; St, Louis, MO, USA) containing complement. Heat inactivated serum was also utilized as a control. Cells were plated in triplicate or quadruplicate wells in 96-well plates with the indicated drug for 72 hrs, followed by 3 H-thymidine addition to the culture medium for the final 6 hours. The 3 H-thymidine incorporation was determined in a liquid scintillation counter.(1) All data was normalized to either vehicle or isotype control.

ADCC Assay
Isolated, CD3-stimulated PBMCs (effector cells), H929 MM cells (target), or both were treated with either vehicle (DMSO), daratumumab (dara (0.1 μg/mL)), iberdomide (0.008 μM) (iber), or both drugs ( Figure 2C), and measured ADCC on the target H929 cells. Following washout of drug (iberdomide or daratumumab at the indicated concentrations) on either target cells or PBMCs, target cells were labeled with CSFE and then combined with PBMCs at a 3:1 ratio for 4 hrs. Co-cultured cells were washed and stained with Annexin-V-PE and To-Pro3-APC and target cells were then gated by CFSE and measured for apoptosis.

IL-2 and Granzyme B ELISA
Supernatants from co-culture experiments were analyzed for IL-2 production and Granzyme B release using ELISA kits and according to following manufacturer's protocol (R&D and Biolegend, respectively).

Quantitative RT-PCR (qRT-PCR)
Total RNA was isolated from harvested cell pellets using RLT Buffer (Qiagen; Germantown, MD, USA) per manufacturer's directions. cDNA was synthesized using a cDNA Reverse Transcription kit (Applied Biosystems; Foster City, CA, USA) according to the manufacturer's protocol. qRT-PCR was performed using the TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assays as triplicate/quadruplicate samples on a Viaa7 PCR System (Applied Biosystems). Relative quantification was assessed by comparative CT method after normalization to internal GAPDH control;all samples were then normalized to either parental or vehicle controls (arbitrarily set to 1).

NGS of CRBN in Pomalidomide -Resistant MM Cell Lines
We generated a set of pomalidomide-resistant cell lines (n=9).