Cancer Therapy: Preclinical Preclinical Assessment of CD171-Directed CAR T-cell Adoptive Therapy for Childhood Neuroblastoma: CE7 Epitope Target Safety and Product Manufacturing Feasibility Annette K€ unkele 1 , Agne Taraseviciute 1,2,3 , Laura S. Finn 4 , Adam J. Johnson 1 , Carolina Berger 2,5 , Olivia Finney 1 , Cindy A. Chang 1 , Lisa S. Rolczynski 1 , Christopher Brown 1 , Stephanie Mgebroff 1 , Michael Berger 2 , Julie R. Park 3 , and Michael C. Jensen 1,3,6 Abstract Purpose: The identification and vetting of cell surface tumor- restricted epitopes for chimeric antigen receptor (CAR)–redir- ected T-cell immunotherapy is the subject of intensive inves- tigation. We have focused on CD171 (L1-CAM), an abundant cell surface molecule on neuroblastomas and, specifically, on the glycosylation-dependent tumor-specific epitope recognized by the CE7 monoclonal antibody. Experimental Design: CD171 expression was assessed by IHC using CE7 mAb in tumor microarrays of primary, meta- static, and recurrent neuroblastoma, as well as human and rhesus macaque tissue arrays. The safety of targeting the CE7 epitope of CD171 with CE7-CAR T cells was evaluated in a preclinical rhesus macaque trial on the basis of CD171 homol- ogy and CE7 cross reactivity. The feasibility of generating bioactive CAR T cells from heavily pretreated pediatric patients with recurrent/refractory disease was assessed. Results: CD171 is uniformly and abundantly expressed by neuroblastoma tumor specimens obtained at diagnoses and relapse independent of patient clinical risk group. CD171 expression in normal tissues is similar in humans and rhesus macaques. Infusion of up to 1  10 8 /kg CE7-CAR þ CTLs in rhesus macaques revealed no signs of specific on- target off-tumor toxicity. Manufacturing of lentivirally trans- duced CD4 þ and CD8 þ CE7-CAR T-cell products under GMP was successful in 4 out of 5 consecutively enrolled neuroblastoma patients in a phase I study. All four CE7-CAR T-cell products demonstrated in vitro and in vivo antitumor activity. Conclusions: Our preclinical assessment of the CE7 epitope on CD171 supports its utility and safety as a CAR T-cell tar- get for neuroblastoma immunotherapy. Clin Cancer Res; 23(2); 466–77. Ó2016 AACR. Introduction Neuroblastoma is the most common extracranial solid tumor of childhood with a heterogeneous clinical course (1). While neuroblastomas with favorable biology spontaneously regress or differentiate without therapeutic intervention, neuroblasto- mas with unfavorable biology often fatally progress despite intensive multimodal therapy (1–3). Maximally tolerated front- line intensive chemotherapy, radiation, consolidative auto- logous hematopoietic stem cell transplantation followed by retinoids and anti-GD2 antibody may cure up to 50% of high- risk patients. Accordingly, the development of new therapeutic modalities, which are tolerable in this patient population, is needed. Immunotherapy is an attractive approach because it invokes immunologic effector mechanisms to which chemotherapy/ radiation-resistant tumor cells are susceptible (4). In neuro- blastoma, anti-disialoganglioside (GD2) antibodies have been most extensively utilized for antigen-specific immunotherapy; however, GD2 is not tumor specific and their antitumor effects are limited by passive biodistribution and the short half-life of the antibody (5–7). T cells expressing a chimeric antigen receptor (CAR) engage tumor cells independent of expression of HLA molecules and are activated via coordinated costimula- tion and CD3zeta signaling. A phase I study comparing EBV- cytotoxic T cells and peripheral blood T cells, both expressing first-generation GD2-specific CARs showed safety and transient responses (8). Further studies investigating the use of first- generation GD2 CAR expressing donor derived virus-specific cytotoxic T cells after allogeneic stem cell transplantation 1 Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, Washington. 2 Fred Hutchinson Cancer Research Center, Seat- tle, Washington. 3 Seattle Children's Hospital, Department of Pediatrics, Univer- sity of Washington, Seattle, Washington. 4 Seattle Children's Hospital, Depart- ment of Pathology, University of Washington, Seattle, Washington. 5 Depart- ment of Medicine, University of Washington, Seattle, Washington. 6 University of Washington, Department of Bioengineering, Seattle, Washington. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Current address for A. K€ unkele: Department of pediatric hematology and oncology, Charit e, Berlin, Germany. Corresponding Author: Michael C. Jensen, Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, 1100 Olive Way, Suite 100, Seattle, WA 98101. Phone: 206-987-1241; Fax: 206-884-4100; E-mail: michael.jensen@seattlechildrens.org doi: 10.1158/1078-0432.CCR-16-0354 Ó2016 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 23(2) January 15, 2017 466 on June 13, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst July 7, 2016; DOI: 10.1158/1078-0432.CCR-16-0354