Functional Tuning of CARs Reveals Signaling Threshold above Which CD8+ CTL Antitumor Potency Is Attenuated due to Cell Fas–FasL-Dependent AICDChimeric antigen receptor (CAR) development is biased toward selecting constructs that elicit the highest magnitude of T-cell functional outputs. Here, we show that components of CAR extracellular spacer and cytoplasmic signaling domain modulate, in a cooperative manner, the magnitude of CD8(+)CTL activation for tumor-cell cytolysis and cytokine secretion. Unexpectedly, CAR constructs that generate the highest in vitro activity, either by extracellular spacer length tuning or by the addition of cytoplasmic signaling modules, exhibit attenuated antitumor potency in vivo, whereas CARs tuned for moderate signaling outputs mediate tumor eradication. Recursive CAR triggering renders CTLs expressing hyperactive CARs highly susceptible to activation-induced cell death (AICD) as a result of augmented FasL expression. CAR tuning using combinations of extracellular spacers and cytoplasmic signaling modules, which limit AICD of CD8(+)CTLs, may be a critical parameter for achieving clinical activity against solid tumors.
Preclinical Assessment of CD171-Directed CAR T-cell Adoptive Therapy for Childhood Neuroblastoma: CE7 Epitope Target Safety and Product Manufacturing FeasibilityAbstract Purpose: The identification and vetting of cell surface tumor-restricted epitopes for chimeric antigen receptor (CAR)–redirected T-cell immunotherapy is the subject of intensive investigation. 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, metastatic, 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 homology 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 × 108/kg CE7-CAR+ CTLs in rhesus macaques revealed no signs of specific on-target off-tumor toxicity. Manufacturing of lentivirally transduced CD4+ and CD8+ 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 target for neuroblastoma immunotherapy. Clin Cancer Res; 23(2); 466–77. ©2016 AACR.
EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma// Ali C. Ravanpay 1 , 2 , * , Juliane Gust 3 , 4 , * , Adam J. Johnson 1 , Lisa S. Rolczynski 1 , Michelle Cecchini 2 , Cindy A. Chang 1 , Virginia J. Hoglund 1 , Rithun Mukherjee 1 , Nicholas A. Vitanza 5 , 6 , Rimas J. Orentas 1 , 6 and Michael C. Jensen 1 , 6 , 7 1 Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, WA, U.S.A 2 University of Washington, Department of Neurological Surgery, Seattle, WA, U.S.A 3 University of Washington, Department of Neurology, Seattle, WA, U.S.A 4 Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, U.S.A 5 Center for Clinical and Translational Research, Seattle Children’s Research Institute, Seattle, WA, U.S.A 6 University of Washington, Department of Pediatrics, Seattle, WA, U.S.A 7 University of Washington, Department of Bioengineering, Seattle, WA, U.S.A * These authors contributed equally to this work Correspondence to: Juliane Gust, email: juliane.gust@seattlechildrens.org Keywords: glioblastoma; CAR T cell; EGFR; immunotherapy; mAb806 Received: August 14, 2019     Accepted: December 02, 2019     Published: December 17, 2019 ABSTRACT Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.