Suppression of Myeloid Cell–Derived Proinflammatory Cytokines with Celmod Agents: Implications for CRS with T-Cell Engagers (TCEs)

Background: Alnuctamab (ALNUC; BMS-986349; CC-93269) is an asymmetric 2-arm, humanized IgG TCE that binds to B-cell maturation antigen (BCMA) and CD3ε in a 2+1 format. The ALNUC-mediated interaction between T cells and BCMA-expressing myeloma cells induces T-cell receptor/CD3 crosslinking that promotes T-cell activation, release of proinflammatory cytokines and cytolytic enzymes, and myeloma cell death. CRS is a serious adverse event frequently observed with several T cell-based immune therapies and is characterized by rapid release of proinflammatory cytokines from immune cells, including T cells and myeloid cells. Key cytokines implicated in CRS include interleukin 6 (IL-6), IL-1β, and TNFα. Cereblon E3 ligase modulator (CELMoD^®) agents, including immunomodulatory (IMiD^®) agents, have been shown to reduce secretion of proinflammatory cytokines while also demonstrating immune-stimulatory effects and antimyeloma activity. We hypothesized that pre-treatment of myeloid cells with CELMoD agents would prime them and mitigate the secretion of proinflammatory cytokines that are associated with TCE-related CRS upon ALNUC exposure, while preserving the distinct immune activation and anti-myeloma properties of ALNUC and CELMoD agents. To test this hypothesis, we developed an in vitro myeloid proinflammatory cytokines mitigation model/assay.

Methods: The inhibitory effects of CELMoD agents on stimulated cytokine release was investigated in healthy donor monocytes, macrophages, and peripheral blood mononuclear cells (PBMCs). Cells were pretreated overnight (~17 h) with clinically relevant concentrations of lenalidomide (LEN), pomalidomide (POM), iberdomide (IBER), mezigdomide (MEZI; BMS-986348; CC-92480), or dimethyl sulfoxide (DMSO), then stimulated with lipopolysaccharide (LPS) or anti-CD3/-CD28 beads to trigger cytokine release. Components of the in vitro model included: (a) PBMCs as a source of T and myeloid cells, (b) target human myelogenous leukemia cells (K562) engineered to overexpress BCMA or multiple myeloma (MM) cells with high endogenous BCMA expression (H929); BCMA-null K562 cells served as a control, and (c) ALNUC. The in vitro assay was performed by pretreating PBMCs overnight with CELMoD agents before the addition of target cells at a 1:5 target:effector ratio and ALNUC. Secreted cytokines were measured using the MSD^® V-Plex^® proinflammatory panel at various timepoints.

Results: Pretreatment of monocytes and macrophages with CELMoD agents inhibited LPS‑induced IL-6 secretion, with the greatest inhibition (up to 3-fold) reported with MEZI. Pretreated PBMCs also had reductions in LPS-induced IL-6, IL-1β, and TNFα secretion of up to 2-, 5- and 6-fold, respectively. In T-cells stimulated with anti-CD3/-CD28 beads, CELMoD agent pretreatment resulted in enhanced secretion of T-cell derived cytokines. Thus, CELMoD agents had differential impact on cytokines secreted by T cells vs myeloid cells.

In the in vitro assay, non-pretreated PBMCs in the presence of K562-BCMA cells and ALNUC secreted ≥ 800 pg/mL of IL-6 within 6 h of stimulation, demonstrating the robustness of the assay. In PBMCs pretreated with MEZI prior to co-culture with K562-BCMA cells, ALNUC-induced IL-6 secretion was suppressed by up to 50% and IL-1β secretion by up to 95% vs PBMCs pretreated with DMSO. Secretion of IL-2, TNFα, and IFNγ, critical cytokines related to T-cell lytic activity, was not suppressed in our model following MEZI pretreatment.

Conclusions: CELMoD agents can suppress secretion of the proinflammatory cytokines IL-6 and IL-1β by monocytes/macrophages and PBMCs in vitro, with the strongest suppression observed with MEZI. In an ex vivo co-culture system of PBMCs with BCMA-expressing target cells, pretreatment with MEZI suppressed ALNUC-induced secretion of IL-6 and IL-1β, but not secretion of T cell-mediated cytokines. Our data demonstrate strong reductions in proinflammatory myeloid cell-derived cytokines by CELMoD agents while maintaining these agents’ pro-immune properties, suggesting that potent CELMoD agents such as MEZI have the potential to mitigate CRS in patients with MM receiving TCEs.