Delayed neurotoxicity, enterocolitis, and BCMA-CART-associated immune-related adverse events (CirAE)  are caused by CD4+ CAR T-cells

Abstract Introduction. B-cell maturation antigen (BCMA)–directed CAR T-cell therapies (CART) are approved for second or third-line treatment of relapsed/refractory multiple myeloma (RRMM). Ongoing efforts to move BCMA-CART to earlier lines must be balanced against the risk of delayed toxicities, including cranial nerve palsies, Parkinsonism, and enterocolitis, collectively termed CART immune-related adverse events (CirAE). Methods & Results. We analyzed 198 patients (pts) treated with commercial BCMA-CART (idecabtagene-vicleucel: 73; ciltacabtagene-autoleucel: 125) between June 2021–Dec 2024, and report the incidence, spectrum, risk factors, and mechanisms of CirAE. CirAE occurred in 27 pts, significantly more often with cilta-cel (20%) than ide-cel (2.7%). The most common CirAE after cilta-cel were facial nerve palsy (7.2%), enterocolitis (5.6%), Parkinsonism (4.9%), and arthritis (1.6%). Less frequent events included delayed immune-effector cell-associated neurotoxicity syndrome (ICANS), optic neuritis, encephalopathy, and brachial plexopathy (0.8% each). Median onset was 28 days post-cilta-cel infusion. In contrast, both ide-cel-associated CirAE were pneumonitis and occurred ~1 year post-infusion. CirAE was associated with significantly higher nonrelapse mortality (1-year NRM: 17% vs 2.7%). All 7 CirAE-related deaths occurred in cilta-cel-treated patients (5.6% of that cohort). Among pts with NRM, 60% of CirAE pts died from infection vs. 14.3% in non-CirAE pts, likely due to 7-fold higher cumulative steroid exposure. Baseline MM burden and cytokine release syndrome did not predict CirAE. However, ICANS was associated with CirAE (OR: 3.8). Furthermore, pts who developed CirAE had significantly higher peak absolute lymphocyte count (ALC) within 14 days post-infusion (median ALCPeak: 3.3 vs 0.9×10³/μL), which correlated with greater CART expansion (median Day 14 CART count: 1.5 vs. 0.1×10³/μL). A day 14 ALCPeak≥2.4×103/μL (third quartile) was associated with 8-fold elevated CirAE odds. Flow cytometry of peripheral blood mononuclear cells (PBMC) prior to lymphodepletion showed that CirAE pts had T-cells enriched for CD4 central memory cells, while non-CirAE pts had more CD8 terminally differentiated cells. This CD4 skewing in the CirAE group was observed at apheresis (P<0.001), day 7 (P=0.045), and Months 1, 3, and 6 post-infusion. An apheresis CD4/8 ratio >1 conferred >3-fold higher CirAE risk. In multivariate analysis, product type was not an independent predictor of CirAE after adjusting for expansion and CD4 skewing, suggesting that the higher rate of CirAE with cilta-cel may reflect greater expansion. Analysis of enterocolitis biopsies (n=5) showed on-target off-tumor CAR–BCMA colocalization, with marked depletion of normal plasma cells in the lamina propria. Infiltrating CARTs were predominantly CD4+, with a median CD4/8 ratio of 9 (IQR: 7.5). Similarly, single-cell RNAseq of matched PBMC and CSF samples during neuro-CirAE (n=4) revealed CD4-predominance in the CSF (median CD4/8: 4; IQR: 4.5) and enrichment of resident memory-like, interferon-stimulated CD4+ CART (17% vs 0.06% in PB), marked by cytotoxic gene signatures and elevated expression of effector molecules (GNLY, GZMK, IFNG, PRF1, CD107a). Upon stimulation with MM cells, CSF-derived CD4⁺ CARTs produced more CD107a, GZMB, IL-17a, IL-2, and GM-CSF compared to CD8⁺ CARTs. Paired PBMC analysis showed a significant temporal decline in circulating CART CD4/8 ratio during CirAE episodes, consistent with preferential trafficking of CD4⁺ CAR T cells into affected tissues; supported by matched PBMC and CSF analysis demonstrating CD4⁺ CART enrichment at CirAE sites relative to PB. Proteomic analysis revealed enrichment of chemokines (CXCL10, CCL2, CCL3, IL-8) in the CSF and transwell assay using matched serum (top) and CSF (bottom) enriched for CD4⁺ CARTs in migrated cells. Finally, CD4+ CARTs had significantly higher BCMA-binding avidity compared to CD8+ CARTs. Conclusions. CirAEs are a heterogeneous group of toxicities with high NRM, unified by a shared immunologic profile. We corroborate the association of ICANS and high peak ALC with risk of delayed neurotoxicity after cilta-cel, extend these correlates to a broader set of clinically diverse CirAE, identify additional risk factors (apheresis CD4/8 >1), and demonstrate that CD4+ CARTs mediate on-target off-tumor CirAE, potentially via selective trafficking and enhanced tissue retention driven by higher BCMA-binding avidity.