Abstract A09: GPR64-specific CAR-transgenic T cells selectively kill Ewing sarcoma in vivo
Authors: Schirmer David , Storz Isabel , Wisskirchen Karin , Feederle Regina , Schmidt Oxana , Abken Hinrich , Protzer Ulrike , Burdach Stefan , Richter Guenther H.S. 
Published: February 2019

Abstract
Introduction: Pediatric cancers, including Ewing sarcoma (EwS), are only weakly immunogenic and the tumor patient’s immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against the G-protein coupled receptor 64 (GPR64), normally expressed only in epididymal tissue, but specifically expressed in EwS and some other sarcoma, was examined.
Experimental Procedure: Two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second-generation CARs together with the scFv fragments of the respective mAbs were designed. CAR-constructs consisting of different spacers and costimulatory elements were compared (dIgG1-CD28-CD3z vs. CD8a-4-1BB-CD3z). Primary lymphocytes were transduced and tested in vitro via flow cytometry. Their activation profile and specificity was analyzed by micorarray analysis, ELISpot, and xCelligence assay as well as in immunodeficient xenograft mice.
Results: Antibodies specifically stained EwS cells as determined by flow cytometry and immune histology. The signal intensity was reduced after RNAi mediated downregulation of GPR64 in EwS cell lines confirming specificity of the isolated mAbs. Further immune histology detected GPR64 expression not only in EwS but also in epithelial cancers such as renal, prostate, and pancreas carcinoma. Following sequence determination of those mAbs, two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with high efficiency. The CAR transgenic T cells were enriched for CD8+CAR+ cells via microbead isolation and demonstrated strong proliferative capacities in vitro. Furthermore, GPR64 transmembrane cell surface target structures were specifically recognized as determined by ELISpot and xCelligence assays, but dIgG1-CD28 CAR T cells derived from one hybridoma revealed hints of early exhaustion via auto-activation. This exhaustion could be prevented by use of constructs containing CD8a-4-1BB-CD3z signaling domains for this particular hybridoma-derived scFv fragment, resulting in T-cell activation patterns more similar to TCR-transgenic T cells. Adoptive transfer of CAR-transgenic T cells into EwS tumor-bearing xenograft mice resulted in a significant suppression of tumor growth.
Conclusion: CAR-transgenic T cells targeting GPR64 are a promising approach to transfer the success of CARs in hematologic malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth. Since GPR64 expression is not restricted to EwS but also upregulated in a number of carcinomas derived from prostate, kidney, or pancreas, GPR64-specific CARs are a future treatment option for other tumor entities.
Citation Format: David Schirmer, Isabel Storz, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Ulrike Protzer, Stefan Burdach, Guenther H.S. Richter. GPR64-specific CAR-transgenic T cells selectively kill Ewing sarcoma in vivo [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A09.