Work

220 Overcoming antigen heterogeneity in Ewing sarcoma using adaptable multi-targeted SNAP-CAR T cells

Year: 2026

Type: conference-abstract

Abstract: Background Targeted therapies for relapsed Ewing sarcoma remain severely limited by intratumoral heterogeneity. To address this barrier, we investigated the universal SNAP-chimeric antigen receptor (SNAP-CAR) T cell platform alongside antibody adaptors to target multiple surface antigens simultaneously. We tested the individual and combined targeting of interleukin-1 receptor accessory protein (IL1RAP) and B7 homolog 3 (B7H3), evaluating the hypothesis that simultaneous dual-targeting enhances antitumor response and restricts immune evasion compared to single-antigen approaches.Methods Flow cytometry was used to characterize surface expression profiles and co-expression of IL1RAP and B7H3 across six distinct Ewing sarcoma lines: A673, CHLA9, CHLA10, SK-N-MC, TC32, and TC71. A panel of nine benzylguanine-conjugated antibody adaptors was screened, including a novel bispecific against both target antigens. In vitro cytolytic performance was validated via co-culture assays against control A673 cells and knock-out (KO) variants (IL1RAP-/-, B7H3-/-, and dual-KO). Cytotoxicity evaluations and T cell activation profiling were conducted using flow cytometry at 5:1 and 1:1 effector-to-target (E:T) ratios, respectively. Unarmed SNAP-CAR T cells and mock T cells served as negative controls, with experiments replicated utilizing independent healthy donor T cells. Statistical significance (alpha = 0.01) was assessed using analysis of variance (ANOVA) with a post hoc Bonferroni correction.Results Screening assays confirmed robust yet variable co-expression of IL1RAP and B7H3 across the Ewing sarcoma cell line panel. The bispecific adaptor candidate, 7D12xm276, demonstrated potent efficacy, with SNAP-CAR cells from two separate donors achieving up to 87.3% specific lysis of control A673 cells. Notably, whereas single-antigen adaptors failed to eliminate their respective antigen-negative KO targets, the bispecific construct retained high cytolytic activity against both individual IL1RAP -/- (79.1% lysis) and B7H3-/- (70.7% lysis) lines. Lysis of the dual-KO line did not differ significantly between single-target and bispecific adaptors, validating the strict antigen-dependency of the platform.Conclusions These data indicate that the flexible SNAP-CAR system successfully mitigates Ewing sarcoma antigen heterogeneity in vitro. By sustaining high-level cytotoxicity in the event of single-antigen loss, the bispecific 7D12 x m276 adaptor strategy offers a compelling therapeutic avenue to counter immune escape and optimize clinical outcomes. Complementary in vivo investigations are underway.Acknowledgements KMB and JL share senior authorship of this work.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0220