A newly identified role for IL-5 in regulating CART-associated toxicity and efficacy

Abstract

Abstract Background: CD19-directed CART cell (CART19) therapy has transformed the treatment of B-cell malignancies but is limited by life-threatening toxicities, including cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), as well as low durable remission rates. Our understanding of the pathogenesis of CART-associated toxicities is limited, and although current treatments are effective, refractory cases remain challenging. Methods and Results: To investigate CRS/ICANS pathogenesis and identify new therapeutic targets, we analyzed plasma cytokine kinetics within the first month of CART19 cell therapy in 25 relapsed/refractory (r/r) large B-cell lymphoma (LBCL) and 14 r/r follicular lymphoma (FL) patients (NCT02030834). In this cohort, 22 (88%) patients developed any-grade CRS (73% LBCL, 27% FL) and 4 (16%) developed any-grade ICANS (50% LBCL, 50% FL). Across 30 analyzed cytokines, IL-5 emerged as the second most enriched cytokine based on the median peak-to-baseline (Day 0) ratio in patients who experienced CRS vs. those who did not. The IL-5 ratio was 6.6-fold higher (p<0.05) and 11.9-fold higher (p=0.093) in patients who experienced CRS or ICANS, respectively. Moreover, IL-5 was elevated in patients experiencing CRS on Days 1 and 2 (p<0.05). Prompted by our results, we evaluated the effect of IL-5 neutralization with a monoclonal antibody (mAb) on CART-associated toxicity in a patient-derived B-acute lymphoblastic leukemia (B-ALL) xenograft model. After engraftment (average of 110 human CD19+ cells/µL peripheral blood), NSG mice were randomized based on tumor burden and treated with 3.5×10⁶ CART19 plus either IL-5 mAb or IgG control (10 mg/kg, weekly), or no treatment. Mice receiving IL-5 mAb showed reduced weight loss (p<0.005, Days 13–16) and neuroscore (p<0.05, Day 15), as assessed by a daily, blinded 18-point assessment, vs. the IgG group. Additionally, while CRS-associated cytokines (GM-CSF, IFN-γ, MIP-1β) were significantly elevated in the IgG group (p<0.05) vs. the untreated group, they were not in the IL-5 mAb group. In this model, all mice treated with CART19 cells were able to clear the tumor burden. However, in a follow-up challenge model generated by engrafting NSG mice with 1x106 luciferase+ JeKo-1 cells, mice treated with a combination of 1x106CART19 cells + IL-5 mAb showed reduced weight loss (p<0.0001) and enhanced overall survival (p<0.01) vs. mice treated with CART19 + IgG. Next, we evaluated IL-5 neutralization in an immunocompetent toxicity model. BALB/c mice were treated with 10×10⁶ murine untransduced T cells, CART19 + IL-5 mAb, or CART19 + IgG (10 mg/kg, weekly). Mice receiving IL-5 mAb exhibited significantly reduced weight loss (p<0.05, Day 1) and lower neuroscore (p<0.05, Days 1, 6, and 8) vs. those receiving IgG control, with a consistent trend observed on other days, supporting a protective role for IL-5 blockade in mitigating CART-associated toxicities. To explore the direct effects of IL-5 on CART19 cells, we disrupted either IL-5 (IL-5KO-CART19) or its receptor (IL-5RαKO-CART19) using CRISPR-Cas9. In NSG mice engrafted with 1×10⁶ luciferase+NALM6 cells, mice receiving 1×10⁶ IL-5KO-CART19 vs. IL-5WT-CART19 showed a trend toward reduced weight loss (p=0.08), significantly increased antitumor activity by Day 20 (p<0.05), and improved overall survival (p<0.05), indicating that IL-5 production can significantly impair CART19 cell efficacy. We next evaluated changes in IL-5Rα gRNA representation in a published genome-wide CRISPR screen (PMID: 39266501). CART19 cells with IL-5Rα-targeting gRNAs were enriched following chronic stimulation, indicating a protective effect for IL-5 pathway interruption. In subsequent studies, IL-5RαKO-CART19 cells showed enhanced killing of JeKo-1 cells following 48 hours of co-culture (p<0.05) and reduced co-expression of multiple inhibitory markers (p<0.01) following chronic stimulation with JeKo-1 cells for one week vs. IL-5RαWT-CART19 cells, suggesting an improvement in CART19 cell activity and phenotype following disruption of IL-5Rα. Conclusion: Our data identify IL-5 as a previously unrecognized mediator of CART–associated toxicities and show significant improvement of CART19 activity with disruption of IL-5 signaling either indirectly with an IL-5 neutralizing antibody or directly with CRISPR gene editing. Thus, IL-5 could serve as a dual-purpose target to improve both safety and efficacy of CART cell therapy.