Abstract 6118: Engineering p95HER2 CAR T cells for the treatment of HER2-positive tumors

Abstract Background: Clinical implementation of Chimeric Antigen Receptor (CAR) T cells in solid tumors has been more challenging than for hematological malignancies due in part to "on-target, off-tumor" toxicities. The tyrosine-kinase receptor HER2 is a potent oncogene amplified in almost 3% of all cancers. While most HER2-positive tumors diagnosed at early stages have already effective and available treatment, refractory and metastatic tumors still pose an unmet medical need. p95HER2, a truncated version of the HER2, is expressed in 30-40% of HER2-amplified tumors and, unlike HER2, its expression is restricted to tumor cells. Recent research in the CAR T field has explored novel logic gated CAR T cell designs using alternative signaling domains to CD3z and dual antigen targeting strategies to mitigate toxicity. Here we show the use of LINK CAR T cells to selectively target p95HER2 and HER2-positive tumors while sparing normal tissues expressing basal levels of HER2. Methods: We generated LINK CARs: a CAR incorporating LAT signaling domain and another with SLP-76 domain, targeting p95HER2 and HER2. Then, transduced both CAR constructs into T cells from healthy donors. In vitro cytotoxicity was assessed through co-cultures with cell lines derived from gastric, breast, ovarian, and lung tumors expressing endogenous p95HER2 and HER2. Safety was evaluated by co-culturing the CAR Ts with cell lines from healthy tissues expressing basal HER2 levels. In vivo efficacy was investigated using immunodeficient mice implanted with cell-derived xenografts. Results: We show the specific activation of these CAR T cells in the presence of both HER2 and p95HER2-expressing tumor cells. No cytotoxicity was observed in cells with only basal HER2 levels. Safety testing across a panel of healthy cell lines with varying HER2 expression confirmed the CAR T cells' specificity. In vivo studies demonstrated the p95HER2 LINK CAR's ability to eradicate tumors from cell lines expressing HER2 and p95HER2, while preserving MCF7 tumors representing tissues with basal HER2 expression. Patient-derived xenograft(PDX) models are currently being tested to evaluate the translational potential of this novel therapeutic. Conclusions: Our preliminary results show the feasibility of LINK CAR T cells as a targeted therapeutic approach for HER2-positive tumors, leveraging p95HER2 specificity. Experimentation in PDX models and expansion into different tumor types will further validate this findings. Citation Format: Ariadna Grinyo-Escuer, Marta Bort-Brusca, Pablo Ovejero, Julia Fernandez Torrente, Judit Gago, Marta Escorihuela, Macarena Roman, Joaquin Arribas. Engineering p95HER2 CAR T cells for the treatment of HER2-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6118.