Chimeric antigen receptor T (CAR-T) cell therapy is a revolutionary approach for treating relapsed or refractory B-cell lymphoproliferative disorders. However, significant toxicities, including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and macrophage activation syndrome (MAS), hinder its widespread application. CRS, often occurring within days of CAR-T cell infusion, is characterized by a systemic inflammatory response with high levels of cytokines, including IFNγ. ICANS, a neurological complication, may occur concurrently with or after CRS. The pathophysiology of both involves a systemic inflammatory cascade, with IFNγ playing a key role. While low-grade CRS can be managed symptomatically, severe CRS requires intervention with steroids or tocilizumab, an anti-IL-6 receptor antibody. Tocilizumab, while effective against CRS, has limited efficacy against ICANS, possibly due to poor blood-brain barrier (BBB) penetration. High IFNγ levels contribute to BBB permeability and subsequent brain damage. This study investigates the therapeutic potential of emapalumab, an FDA-approved anti-IFNγ monoclonal antibody, in mitigating CAR-T cell-associated toxicities.

The literature extensively documents the efficacy of CAR-T cell therapy in treating B-cell malignancies, but also highlights the substantial challenges posed by its associated toxicities. Studies show a wide range in the incidence and severity of CRS and ICANS, highlighting the need for improved safety measures. Existing management strategies, including corticosteroids and tocilizumab, have limitations in their efficacy and potential for compromising the anti-tumor activity of CAR-T cells. The role of IFNγ in the pathogenesis of CRS and neurotoxicity has been established, suggesting that targeting this cytokine could represent a valuable therapeutic strategy. The preclinical studies of anti-IFNγ therapies, and the clinical experience with emapalumab in primary hemophagocytic lymphohistiocytosis (HLH), provide a rationale for exploring its use in combination with CAR-T cell therapy.

This study employed both in vitro and in vivo models to evaluate the impact of IFNγ neutralization on CAR-T cell efficacy and toxicity. In vitro experiments used healthy donor-derived peripheral blood mononuclear cells (PBMCs) genetically modified with a 4-1BB-based CAR.CD19 construct. The cytotoxic activity of CAR.CD19 T cells against CD19+ lymphoma cell lines (Daudi and Raji) was assessed in the presence and absence of emapalumab at various concentrations (0–100 μg/mL). Cytotoxicity was evaluated using long-term co-culture assays, real-time live-cell imaging, and a luciferase-based assay. Gene expression analysis using Nanostring technology was performed to evaluate the impact of emapalumab on CAR-T cell activation. In vivo studies utilized two distinct murine models. The first model employed NOD-scid gamma (NSG) mice engrafted with low tumor burden and received CAR-T cells followed by emapalumab 7 days later. The second model, a humanized mouse model (hGM-CSF/hIL3 NOG mice engrafted with human CD34+ hematopoietic stem cells), was used to mimic the clinical situation of a high tumor burden with acute toxicity. Mice were monitored for tumor burden, survival, and brain damage. Cytokine levels and gene expression in brain tissue were analyzed to assess the inflammatory response and extent of brain injury. Statistical analysis using Student's t-test, Mann-Whitney test, and Kaplan-Meier survival curves were used to evaluate differences between groups. The study adhered to relevant ethical guidelines and research compliance standards, including approval from the Institutional Review Board and the Ethical Committee for Animal experimentation.

In vitro studies demonstrated that emapalumab, at clinically relevant concentrations, did not impair the anti-lymphoma activity of CAR.CD19 T cells against Daudi and Raji cells. Neither the kinetics of target cell killing nor the proliferation of CAR-T cells were affected by emapalumab. Gene expression analysis revealed minimal changes in the CAR-T cell gene signature following emapalumab exposure. In vivo experiments using a model with low tumor burden showed that emapalumab administration did not compromise lymphoma control. Critically, in the humanized mouse model with high tumor burden, emapalumab treatment significantly improved survival rates, reduced brain hemorrhages, and decreased levels of several IFNγ-driven inflammatory cytokines (CXCL9, CXCL10, and IL-16). Gene expression analysis of brain tissue from the humanized model indicated that emapalumab significantly downregulated genes associated with oxidative stress, IFN signaling, and inflammation, consistent with its protective effects. Histopathological analysis showed reduced inflammation in the bone marrow of emapalumab-treated mice. In summary, the results show that emapalumab mitigates the severe toxicities associated with CAR-T cell therapy, specifically CRS and brain damage, without compromising its anti-leukemic efficacy.

This study provides compelling preclinical evidence supporting the clinical development of combination therapy using emapalumab and CAR-T cells. The findings directly address the significant clinical need for safer and more effective CAR-T cell therapies. The use of a humanized mouse model capable of recapitulating the severe toxicities observed in human patients, including brain damage, strengthens the translational relevance of these findings. The observation that IFNγ neutralization does not impair the anti-tumor activity of CAR-T cells is crucial, demonstrating that emapalumab could significantly improve the therapeutic index of this treatment modality. The reduction in several pro-inflammatory cytokines and the improvement in survival rates in the humanized model highlight the therapeutic efficacy of emapalumab in preventing severe toxicity. The results warrant further investigation in clinical trials to confirm the safety and efficacy of this combination approach in human patients.

This study demonstrates that IFNγ neutralization with emapalumab significantly reduces the severe toxicities associated with CAR-T cell therapy, including CRS and neurotoxicity, without affecting its anti-leukemic activity. The preclinical data strongly support the clinical evaluation of emapalumab in combination with CAR-T cell therapy to improve the safety and efficacy of this promising treatment approach. Future research should focus on conducting clinical trials to assess the clinical translation of these findings and investigate the potential of similar strategies for other types of adoptive cell therapies.

The study is limited by its preclinical nature. While the humanized mouse model provides a more clinically relevant setting than traditional murine models, it does not fully capture the complexities of the human immune system. Further, the study focused on CD19-targeted CAR-T cells, and the findings may not be directly generalizable to other CAR targets. Long-term effects of emapalumab on CAR-T cell persistence and the potential for emergence of resistance remain to be investigated.