Introduction: Chimeric antigen receptor (CAR) T-cell therapy is standard care for relapsed or refractory hematologic malignancies. As clinical use expands, rare but serious toxicities beyond cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are emerging. These include hyperleukocytosis and, specifically, following BCMA-directed CART (CART-BCMA) therapy, delayed neurotoxicity and enterocolitis. We named these post–CART-BCMA complications, distinct from CRS and ICANS, as CART immune-related adverse events (CirAE). CirAE are associated with elevated CART expansion in the first two weeks, suggesting that modulating CART-BCMA proliferation without affecting CART effector function could improve safety and expand therapeutic use.

Method: We retrospectively analyzed a cohort of 198 multiple myeloma (MM) patients treated with CART-BCMA (idecabtagene vicleucel [ide-cel] and ciltacabtagene autoleucel [cilta-cel]) at the University of Pennsylvania (June 2021-December 2024) to identify predictors of CirAE and to investigate the mechanisms underlying the early onset of CirAE. Serial serum samples were analyzed using 32-plex proteomics (Luminex). Immune cell subsets were characterized serially by multiparametric flow cytometry. T-cell proliferation was assessed using CellTrace Violet. Cytotoxic CART function was evaluated via luciferase assays.

Results: In this large cohort, both peak absolute lymphocyte count (ALC) ≥ 2.4 × 10³/μL and CD4:CD8 ratio >1 at apheresis independently predicted CirAE following CART-BCMA. To investigate mechanisms underlying elevated ALC, we analyzed a unique case (Cilta#1) marked by extreme, polyclonal CART expansion and three distinct post-infusion CirAE: facial palsy, delayed ICANS, and severe enterocolitis. Cilta#1 exhibited hyperleukocytosis (peak ALC: 197.5 × 10³/μL, Day 13) and profound CD4-skewed CART expansion (CD4:CD8 ratio: 12.6). Despite the magnitude, the CART population remained polyclonal, as confirmed by TCR Vβ sequencing and vector integration site analysis (>20,000 unique sites). Whole-exome sequencing of blood and marrow excluded clonal transformation or pathogenic mutations. Longitudinal flow cytometry from pre-lymphodepletion to month 15 showed persistent CD4 skewing, with CART displaying a highly activated, proliferative phenotype(HLA-DR+/Ki-67+). Serum proteomics at day 7, prior to peak expansion, revealed elevated lymphoproliferative cytokines (IL-2, IL-7, IL-15) and chemokines (CCL5, CXCL9, CXCL10). Given these findings, we assessed the dominant proliferative signal via cytokine-stimulated proliferation assays on Cilta#1 CART cells. IL-15 elicited the strongest proliferation and pSTAT5 activation. In vitro, IL-15 induced CCL5 secretion in both Cilta#1 and healthy donor CART (n=3); however, only Cilta#1 cells upregulated CCR5, a pattern absent in donor CART, where CCR5 was actually downregulated after cytokine exposure. These results suggest a potential IL-15–driven CCL5–CCR5 loop sustaining CART expansion in Cilta#1. We hypothesized that this axis contributes to the elevated ALC observed in patients with CirAE. To test this, we cultured healthy donor CART + 25% Cilta#1 day 7 serum, and observed significantly enhanced survival compared to serum from three other cilta-cel patients (p < 0.001). This effect was abrogated by 40 μM maraviroc,an FDA approved CCR5 antagonist, highlighting the key role of CCR5 signaling. Notably, maraviroc suppressed both IL-15–induced and antigen-dependent CART proliferation across multiple donors (n=4), suggesting broader applicability. Importantly, maraviroc did not impair CART viability or anti-myeloma cytotoxicity, as assessed by flow cytometry for CD107a, granzyme B, and MM.1S tumor killing. CCR5 blockade (20 µg/mL) inhibited IL-15–induced proliferation in Cilta#1 CART, further validating CCR5 as a key effector node. Finally, CCR5 knockout impaired antigen-driven proliferation and rendered CART insensitive to maraviroc, confirming on-target specificity.Conclusion: We identified the IL-15–CCL5–CCR5 circuit as a key driver of CART-BCMA proliferation in CirAE and demonstrated that CCR5 blockade safely restrains CART-BCMA expansion while preserving their anti-myeloma activity. These findings support CCR5-directed targeted strategies to selectively modulate CART expansion without compromising efficacy.

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2025
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