RT Journal Article SR Electronic T1 Preclinical optimization of a GPC2-targeting CAR T-cell therapy for neuroblastoma JF Journal for ImmunoTherapy of Cancer JO J Immunother Cancer FD BMJ Publishing Group Ltd SP e005881 DO 10.1136/jitc-2022-005881 VO 11 IS 1 A1 Ming Sun A1 Yingying Cao A1 Reona Okada A1 Jeyshka M Reyes-González A1 Hannah G Stack A1 Haiying Qin A1 Nan Li A1 Charlie Seibert A1 Michael C Kelly A1 Eytan Ruppin A1 Mitchell Ho A1 Carol J Thiele A1 Rosa Nguyen YR 2023 UL http://jitc.bmj.com/content/11/1/e005881.abstract AB Background Although most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their cancer. Therapeutic strategies to target MRD may benefit these children. We developed a new chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and conducted iterative preclinical engineering of the CAR structure to maximize its anti-tumor efficacy before clinical translation.Methods We evaluated different GPC2-CAR constructs by measuring the CAR activity in vitro. NOD-SCID mice engrafted orthotopically with human NB cell lines or patient-derived xenografts and treated with human CAR T cells served as in vivo models. Mechanistic studies were performed using single-cell RNA-sequencing.Results Applying stringent in vitro assays and orthotopic in vivo NB models, we demonstrated that our single-chain variable fragment, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H.BBζ) elicits the best preclinical anti-NB activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. Finally, we also showed that the CT3.28H.BBζ CAR we developed was more potent than a recently clinically tested GD2-targeted CAR to control NB growth in vivo.Conclusion Given the robust preclinical activity of CT3.28H.BBζ, these results form a promising basis for further clinical testing in children with NB.Data are available in a public, open access repository. Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information.