Work

233 Dual B7-H3 and CADM1 CAR T cells enhance antitumor activity and mitigate antigen escape in osteosarcoma

Year: 2026

Type: conference-abstract

Abstract: Background Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, and outcomes remain poor for patients with relapsed or metastatic disease. Although chimeric antigen receptor (CAR) T-cell therapy is promising, its efficacy in OS is limited by tumor heterogeneity and antigen escape. B7H3, an immune checkpoint molecule, is highly expressed in pediatric solid tumors and represents an attractive CAR target; however, heterogeneous expression may facilitate antigen-negative escape. To address this challenge, we identified and validated CADM1 as a highly expressed OS surface antigen through surfaceome profiling and functional studies using CADM1-directed antibody–drug conjugates. We therefore generated dual-targeting CAR T cells co-expressing B7H3 and CADM1 specific CARs and hypothesized that simultaneous targeting of both antigens would broaden tumor coverage, reduce antigen escape, and enhance antitumor activity in OS.Methods We generated second-generation dual-targeting CAR T cells co-expressing B7H3 and CADM1-specific CARs and evaluated them against osteosarcoma cell lines and PDXs with heterogeneous antigen expression. Antitumor activity was assessed by in vitro cytotoxicity, cytokine secretion, and serial tumor rechallenge assays. In vivo efficacy was evaluated in osteosarcoma xenograft models relative to single-target CAR T-cell controls.Results B7H3 and CADM1 dual-targeting CAR T cells demonstrated superior recognition and cytotoxicity against osteosarcoma cells expressing either or both target antigens compared with single-target CAR T cells. CAR expression was comparable between constructs following transduction, and engineered T cells expanded robustly to clinically relevant numbers (5.3e9 cells, ≥ 23 fold-expansion) with preservation of a memory-associated phenotype characterized by high dual expression of CD45RA and CD62L (>84%) alongside CD95 (82.43%) and CD28 (42.53%) T cells. Upon antigen-specific stimulation, dual-targeting CAR T cells exhibited enhanced in vitro function, including 1.5-fold higher IFN-γ and IL-2 production, superior cytotoxicity against osteosarcoma cell lines and PDXs, and sustained tumor control with reduced antigen escape during serial rechallenge assays compared with single-target CAR T cells. Ongoing in vivo studies in SJAS-1 osteosarcoma xenograft models are evaluating antitumor activity, CAR T-cell persistence, prevention of antigen escape, survival, and off-tumor toxicity. Preliminary findings suggest enhanced tumor control with dual-targeting CAR T cells compared with single-target CAR T-cell products.Conclusions By broadening tumor recognition and limiting the emergence of antigen-loss variants, B7H3 and CADM1 dual-targeting CAR T cells may overcome major barriers to successful CAR T-cell therapy in osteosarcoma. These findings support further translational development and clinical investigation approaches for patients with relapsed or refractory disease.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0233