Abstract 2159: B7-H3 as a strategic target for CAR-CIK immunotherapy to eliminate chemoresistant cancer stem cells across diverse tumor histotypes

Abstract Purpose. The objective of our study is the translational exploration of a cellular immunotherapy strategy with CAR-engineered lymphocytes to target solid tumors resistant to conventional treatments. Specifically, we focus on attacking the tumor stem-like component, considered responsible for relapses and resistance to therapies. We have chosen B7-H3 as the target molecule due to its broad expression across multiple tumor types and its association with stemness and tumor aggressiveness. As effectors we exploited Cytokine-induced Killer lymphocytes(CIK) that, given their T-NK function and intrinsic HLA-independent antitumor activity, could be advantageous against tumors with heterogeneous CAR target expression. Experimental Design. Our preclinical platform is based on tumor cell lines from multiple histotypes (lung and ovarian cancer, osteosarcoma, soft tissue sarcomas) and patient-derived CIK engineered with a second-generation anti-B7-H3 CAR, including CD28 as costimulatory molecule. To visualize putative cancer stem cells (CSCs) within tumor cultures, we employed a strategy previously developed and published by our group. This involves transducing tumor cultures with a lentiviral vector (CSC-detector) encoding GFP under the control of the OCT4 stemness gene promoter, enabling the identification and tracking of CSCs as “GFP-positive”. Results. Across all the various tumors studied, we confirmed homogeneous and intense expression of B7-H3 (80-100%), as well as key NKG2D ligands recognized by CIK (MIC A/B and ULBPs, > 70%). The rate of CSCs (GFP+) varied among tumor types (mean 24%±4). As expected, CSCs were more quiescent, with reduced proliferative potential in vitro compared to their differentiated counterparts, showing an extended G2 phase (2.5fold) in the cell cycle suggestive of a potential enhanced apoptotic resistance. Furthermore, GFP+CSC subsets across diverse histotypes were relatively resistant in vitro to reference chemotherapeutics (doxorubicin, cisplatin, PARPi). Functionally, anti-B7-H3 CAR-CIK exhibited potent killing activity against all tumor types, even at low effector-to-target (E:T) ratios (70-85% at E:T 1/1: >60% E:T 1/2). Unlike observed with chemotherapeutic drugs, anti-B7-H3 CAR-CIK effectively eliminated the CSC tumor component, with enhanced activity (2.5 fold) against this subset compared to surrounding tumor cells. Conclusions. B7-H3 is a valuable and relevant target across diverse solid tumors, allowing to eliminate cell subset with stemness features, resistant to conventional treatments and potentially capable to promote disease relapse. CIK are a suitable platform for CAR-based approaches and our data provide rationale to promote clinical trials with anti-B7-H3 CAR-CIK for relapsed or refractory tumors. Citation Format: Letizia Vitali, Alessia Proment, Annamaria Massa, Chiara Donini, Alessandra Merlini, Federica Galvagno, Andrea Maria Lombardi, Elisa Vigna, Lucilla Minori, Elisa Landoni, Gianpietro Dotti, Claudia Chiodoni, Patrizia Gasparini, Silvia Novello, Valeria Leuci, Sandro Pasquali, Dario Sangiolo. B7-H3 as a strategic target for CAR-CIK immunotherapy to eliminate chemoresistant cancer stem cells across diverse tumor histotypes. [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 2159.