PT  - JOURNAL ARTICLE
AU  - Ming Sun
AU  - Yingying Cao
AU  - Reona Okada
AU  - Jeyshka M Reyes-González
AU  - Hannah G Stack
AU  - Haiying Qin
AU  - Nan Li
AU  - Charlie Seibert
AU  - Michael C Kelly
AU  - Eytan Ruppin
AU  - Mitchell Ho
AU  - Carol J Thiele
AU  - Rosa Nguyen
TI  - Preclinical optimization of a GPC2-targeting CAR T-cell therapy for neuroblastoma
AID  - 10.1136/jitc-2022-005881
DP  - 2023 Jan 01
TA  - Journal for ImmunoTherapy of Cancer
PG  - e005881
VI  - 11
IP  - 1
4099  - http://jitc.bmj.com/content/11/1/e005881.short
4100  - http://jitc.bmj.com/content/11/1/e005881.full
SO  - J Immunother Cancer2023 Jan 01; 11
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.