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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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      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.BB\u03b6) elicits the best preclinical anti-NB activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8\n                      <jats:sup>+<\/jats:sup>\n                      effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as\n                      <jats:italic>GNLY<\/jats:italic>\n                      ,\n                      <jats:italic>GZMB<\/jats:italic>\n                      ,\n                      <jats:italic>ZNF683<\/jats:italic>\n                      , and\n                      <jats:italic>HMGN2<\/jats:italic>\n                      . Finally, we also showed that the CT3.28H.BB\u03b6 CAR we developed was more potent than a recently clinically tested GD2-targeted CAR to control NB growth in vivo.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>Given the robust preclinical activity of CT3.28H.BB\u03b6, these results form a promising basis for further clinical testing in children with NB.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/jitc-2022-005881","type":"journal-article","created":{"date-parts":[[2023,1,11]],"date-time":"2023-01-11T10:52:36Z","timestamp":1673434356000},"page":"e005881","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":36,"title":["Preclinical optimization of a GPC2-targeting CAR T-cell therapy for neuroblastoma"],"prefix":"10.1136","volume":"11","author":[{"given":"Ming","family":"Sun","sequence":"first","affiliation":[{"name":"Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingying","family":"Cao","sequence":"additional","affiliation":[{"name":"Cancer Data Science Laboratory, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reona","family":"Okada","sequence":"additional","affiliation":[{"name":"Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeyshka M","family":"Reyes-Gonz\u00e1lez","sequence":"additional","affiliation":[{"name":"Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah G","family":"Stack","sequence":"additional","affiliation":[{"name":"Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiying","family":"Qin","sequence":"additional","affiliation":[{"name":"Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nan","family":"Li","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charlie","family":"Seibert","sequence":"additional","affiliation":[{"name":"Center for Cancer Research Single Cell Analysis Facility, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael C","family":"Kelly","sequence":"additional","affiliation":[{"name":"Single Cell Analysis Facility, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4299-7657","authenticated-orcid":false,"given":"Eytan","family":"Ruppin","sequence":"additional","affiliation":[{"name":"Cancer Data Science Laboratory, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitchell","family":"Ho","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carol J","family":"Thiele","sequence":"additional","affiliation":[{"name":"Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5143-2055","authenticated-orcid":false,"given":"Rosa","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Pediatric Oncology Branch, NCI, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"239","published-online":{"date-parts":[[2023,1,11]]},"reference":[{"key":"2025100313562375000_11.1.e005881.1","unstructured":"Ries LAG SM , Gurney JG , Linet M , et al . 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