Abstract 1323: Anti-B7-H3 chimeric antigen receptor NK cells suppress the growth of atypical teratoid/rhabdoid tumor orthotopic xenografts

Abstract Background: Atypical teratoid/rhabdoid tumors (AT/RTs) are the most common malignant CNS tumor in infants. AT/RT patients have a 5-year overall survival rate of ~35% and high rates of relapse, emphasizing a dire need for new safe and effective therapies. These therapy-resistant tumors frequently overexpress the immune checkpoint cell surface molecule B7-H3 (CD276). CAR-T cell studies have an established safety profile in pediatric patients. We developed B7-H3 targeted chimeric antigen receptor (CAR) natural killer (NK) cells as a tumor-specific cell therapy for aggressive pediatric brain tumors. CAR-NK cells have several advantages over CAR-T cells. NK cells can be obtained from healthy donors and produced as an “off-the-shelf” product. NK cells also have a lower risk of inflammatory toxicity and graft-versus-host disease compared to T-cells when transferred across the HLA barrier from healthy donors to patients. Methods: We have designed a library of variable affinity B7-H3-targeted CARs, produced replication incompetent γ-retroviral vector, and used this for generation of B7-H3 CAR-NK cells. We verified B7-H3 expression in a panel of AT/RT cell lines, and further engineered firefly luciferase expressing AT/RT (CHLA06. ffLuc, BT12. ffLuc, BT37. ffLuc) as well as a CHLA06-derived B7-H3 knockout. We developed an orthotopic CHLA06. ffLuc xenograft model. We tested CAR-NK cell functionality using in vitro co-culture and cytotoxicity assays. In vivo study was performed in our xenograft with intratumoral delivery of CAR-NK cells. Results: B7-H3 targeted CAR-NK cells demonstrate target-specific cytotoxicity when compared to untransduced NK cells (36. 8±12. 5% vs. 72. 4±24. 2% at effector: target ratio of 1: 1, n=3). CRISPR/Cas9 mediated knockout of B7-H3 in target cells abolished the difference in CAR vs. no-CAR NK-mediated target killing. When delivered intracranially to CHLA-06 orthotopic xenograft bearing mice, B7-H3 CAR NK cells eliminate tumor cells and prolong survival, whereas unmodified NK cells did not (log-ranked median survival = 20 vs. 84 days, p0. 0001). We are currently testing intracranial delivery of our B7-H3 CAR NK against other AT/RT orthotopic xenografts. Conclusions: Targeting pediatric brain tumors with an anti-B7-H3 CAR-NK cell therapy may provide a safe and effective treatment for patients who have extremely limited therapeutic options. Direct intracranial injections of cell therapies into pediatric patients is currently being explored in clinical trials. As B7-H3 is a tumor associated antigen found to be overexpressed in a large variety of tumor types, this strategy may also be useful for treatment of other B7-H3 expressing cancers. Citation Format: Jun Choe, Sachiv Chakravarti, Natalie Holl, Ruyan Rahnama, Megan Zinsky, Danielle G. Jones, Stamatia Vorri, Arjun Modi, Eric H. Raabe, Challice L. Bonifant. Anti-B7-H3 chimeric antigen receptor NK cells suppress the growth of atypical teratoid/rhabdoid tumor orthotopic xenografts abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1323.