Cell-Based Models of ‘Cytokine Release Syndrome’ Endorse CD40L and Granulocyte–Macrophage Colony-Stimulating Factor Knockout in Chimeric Antigen Receptor T Cells as Mitigation Strategy

Abstract

While chimeric antigen receptor (CAR) T cell therapy has shown promising outcomes among patients with hematologic malignancies, it has also been associated with undesirable side-effects such as cytokine release syndrome (CRS). CRS is triggered by CAR T-cell-based activation of monocytes, which are stimulated via the CD40L–CD40R axis or via uptake of GM-CSF to secrete proinflammatory cytokines. Mouse models have been used to model CRS, but working with them is labor-intensive and they are not amenable to screening approaches. To overcome this challenge, we established two simple cell-based CRS in vitro models that entail the co-culturing of leukemic B cells with CD19-targeting CAR T cells and primary monocytes from the same donor. Upon antigen encounter, CAR T cells upregulated CD40L and released GM-CSF which in turn stimulated the monocytes to secrete IL-6. To endorse these models, we demonstrated that neutralizing antibodies or genetic disruption of the CD40L and/or CSF2 loci in CAR T cells using CRISPR-Cas technology significantly reduced IL-6 secretion by bystander monocytes without affecting the cytolytic activity of the engineered lymphocytes in vitro. Overall, our cell-based models were able to recapitulate CRS in vitro, allowing us to validate mitigation strategies based on antibodies or genome editing.

Mathematical modeling unveils the timeline of CAR-T cell therapy and macrophage-mediated cytokine release syndrome

“…Simulating a reduction of the different activation mechanism showed that targeting CD40 leads to the most substantial reduction in IL-6 peak height, causing an average reduction of 42% in the case of constant blocking and 30% reduction when blocking from day 2 after infusion to 1 day after CAR-T cell peak. This aligns with a recent cell-based in vitro model for CRS that showed that IL-6 supernatant levels decreased 20-30% when blocking cytokine release by bystander macrophages with neutralizing antibodies for GM-CSF and CD40, and decreased 30-40% due to the genetic disruption of the CD40L and/or CSF2 knock-out CAR-T cells [32].…”

“…Currently, the primary drug used to address CRS is Tocilizumab, a monoclonal antibody that acts as an IL-6 antagonist [12]. However, targeting macrophage activation with GS-CSF and CD40 antibodies has also been explored in cell-based studies [32].…”

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“…The granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a vital role in the clinical manifestations of CRS and ICANS [111]. The knockout of genes interplaying in the transcription of GM-CSF reduced the inflammation mediated by cytokine release in patients who received CAR-T immunotherapies [112][113][114][115][116]. Guercio et al conducted a study in vivo and administered the inducible caspase 9 (iC9) gene in a model of CAR-T cell lines.…”