Abstract A09: Glypican-2 targeted CAR T cells designed to effectively eradicate endogenous site density solid tumors in the absence of toxicity
Authors: Heitzeneder Sabine , Bosse Kristopher R. , Zhu Zhongyu , Majzner Robbie G. , Theruvath Johanna , Xu Peng , Dhingra Shaurya , Anbunathan Hima , Alag Anya , Dimitrov Dimiter S. , Maris John M. , Mackall Crystal L. 
Published: August 2020

Abstract
Background: Glypican-2 (GPC2) is a promising immunotherapeutic target, due to very low/absent expression on normal tissues (Bosse et al, Cancer Cell 2017). High cell surface GPC2 is found in numerous pediatric and adult malignancies, such as neuroblastoma, medulloblastoma, retinoblastoma, SCLC, and GBM. Here, we engineered GPC2-targeted CAR T-cells and optimized numerous modules (orientation of the variable heavy and light chain, hinge/transmembrane and co-stimulatory domains) to render CAR T cells highly efficacious against tumors expressing endogenous GPC2 site density in neuroblastoma xenograft models.
Methods: GPC2 scFvs (GPC2.19 and GPC2.27) were isolated from a human Fab phage library and screened alongside with previously identified scFvs GPC2.D3 and GPC2.D4 in two orientations (N-terminal variable heavy or light chain) in second-generation retroviral vectors, possessing CD8a hinge/transmembrane (H/TM) and 41BB co-stimulatory domains. Potency was assessed in vitro for antigen-dependent cytokine production and killing and in vivo in neuroblastoma orthotopic subrenal capsule and/or flank PDX models.
Results: While all constructs showed potent in vitro efficacy against isogenic target cells engineered to express GPC2 at supraphysiologic levels (GPC2Hi_Kelly-GPC2), efficacy against cell lines possessing endogenous site density (GPC2E_NBSD, SMS-SAN, KCNR) was modest. Based on cytokine production (IFNy, IL-2), killing capacity, and cross-reactivity against murine GPC2, GPC2.19VLVH was prioritized for in vivo studies, recapitulating in vitro findings. While CARs failed against GPC2E-models, significant antitumor effects were achieved against GPC2Hi, however, failing to cure. Target antigen density is an emerging determinant of CAR potency and our lab has recently demonstrated that incorporating CD28-H/TM and co-stimulatory domains exhibit advantages when targeting low site-density tumors (Majzner et al., ASH 2018). Strikingly, replacing H/TM domains derived from CD8a with those derived from CD28 in either 41BBζ or CD28ζ constructs resulted in a dramatic increase in potency and eradicated established GPC2E-tumors in orthotopic (NBSD) or PDX (COG-N-421x) neuroblastoma models, without signs of toxicity. While the majority of animals remained cured (&gt;75 days), tumors relapsed in 30% of mice after a prolonged tumor-free interval. Principal component analysis (RNA-seq) of control-treated tumors and late relapses suggested strong clustering of transcriptional profiles based on these phenotypes and relapses were associated with ultralow protein and/or gene expression of GPC2 and other NB tumor antigens.
Conclusion: We demonstrate that rational design of GPC2 CAR T-cells results in potent preclinical activity in representative disease models, laying the groundwork for clinical trials and establishing a model to shed light on tumor escape mechanisms after GPC2 CAR T-cell immune pressure.
Citation Format: Sabine Heitzeneder, Kristopher R. Bosse, Zhongyu Zhu, Robbie G. Majzner, Johanna Theruvath, Peng Xu, Shaurya Dhingra, Hima Anbunathan, Anya Alag, Dimiter S. Dimitrov, John M. Maris, Crystal L. Mackall. Glypican-2 targeted CAR T cells designed to effectively eradicate endogenous site density solid tumors in the absence of toxicity [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A09.