This chapter describes the most common method for evaluating cytotoxicity of chimeric antigen receptor (CAR) T cells, the xCELLigence real-time cell analysis (RTCA) platform (Agilent Technologies, Inc., Santa Clara, CA). Though there are a variety of assays used to evaluate conventional and engineered T cell cytotoxicity, the benefit of the xCELLigence platform is the depth of real-time data collected. This chapter begins by providing information on the conceptual basis underlying the xCELLigence assay, followed by a detailed protocol for the application of this assay to evaluate your own CAR-T cells, as well as specific insight and helpful tips for assay design, usage, and data analysis. Application of the information and methods discussed within this chapter will provide a greater understanding for evaluating cytotoxicity of CAR-T cells using this in vitro model system.

ABSTRACT Introduction Colorectal cancer (CRC) is one of the most-deadly malignancies worldwide. Current therapeutic regimens for CRC patients are relatively generic, based primarily on disease type and stage, with little variation. As the field of molecular oncology advances, so too must therapeutic management of CRC. Understanding molecular heterogeneity has led to a new-found promotion for precision therapy in CRC; underlining the diversity of molecularly targeted therapies based on individual tumor characteristics. Areas covered We review current approaches for the treatment of CRC and discuss the potential of precision therapy in advanced CRC. We highlight the utility of the intestinal protein guanylyl cyclase C (GUCY2C), as a multi-purpose biomarker and unique therapeutic target. Here, we summarize current GUCY2C-targeted approaches for treatment of CRC. Expert opinion The GUCY2C biomarker has multi-faceted utility in medicine. Developmental investment of GUCY2C as a diagnostic and therapeutic biomarker offers a variety of options taking the molecular characteristics of cancer into account. From GUCY2C-targeted therapies, namely cancer vaccines, CAR-T cells, and monoclonal antibodies, to GUCY2C agonists for chemoprevention in those who are at high risk for developing colorectal cancer, the utility of this protein provides many avenues for exploration with significance in the field of precision medicine.

Background Esophageal cancer is the fifth most common cause of cancer-related death in the world1 with a 5-year survival rate of <20%.2 Current therapies result in high toxicity and low efficacy, with as many as 60% of esophageal cancer patients not responding to therapeutics.3 CAR-T cell therapy is a therapeutic that can selectively and robustly target cancer cells and eliminate bulky metastatic disease. Previous studies have shown preclinical success with CAR-T cell therapy targeting the human colorectal cancer antigen guanylyl cyclase C (GUCY2C).4 5 Interestingly, esophageal cancers arising from premalignant metaplasia resembling intestine (intestinal metaplasia, also known as Barrett’s esophagus) are highly prevalent and ectopically express GUCY2C. Thus, we hypothesize that GUCY2C will serve as an effective CAR-T cell therapy target in many esophageal cancer patients. However, the paucity of intestine-like human esophageal cancer models limits exploration of this hypothesis, necessitating development of suitable esophageal cancer models (figure 1). Methods To develop esophageal cancer models for GUCY2C immunotherapy testing, esophageal cancer samples were collected at Thomas Jefferson University Hospital by endoscopic biopsy of treatment-naïve patients or by esophagectomy, primarily in patients previously treated with standard neoadjuvant therapy. Patient-derived xenograft (PDX) models were initiated from samples to establish in vivo models for immunotherapy testing. qRT-PCR, immunoblot, and immunofluorescence were performed to test for GUCY2C expression in primary and PDX specimens. Histopathology was performed to confirm retention of primary tumor features. Results GUCY2C was present in only 2 of 6 esophagectomy samples. Interestingly, those patients with detectable GUCY2C were treatment-naïve, while neoadjuvant-treated patients lacked viable tumor, revealing neoadjuvant therapy as a significant barrier to esophageal cancer model generation. In contrast, of the 3 adenocarcinoma specimens collected by endoscopic biopsy in treatment-naïve patients, 2 express GUCY2C. To date, PDX models have been initiated from 6 total samples and successfully established for 3 samples. This 50% success rate may improve over time as PDX formation is often delayed in many models (>150 days). Importantly, established esophageal adenocarcinoma PDX models were histologically similar to their matched primary tumors and retained GUCY2C expression, integral to their validation as models of GUCY2C immunotherapy testing. Conclusions Several human esophageal adenocarcinoma models were successfully established, primarily from endoscopic biopsy of treatment-naïve patients as neoadjuvant therapy proved to be a significant barrier. These models will be useful to explore GUCY2C-directed CAR-T cell therapies and other novel therapies targeting intestine-like esophageal cancer, prior to testing in early-phase clinical trials. Acknowledgements The authors thank the Translational Research & Pathology Core Facility and the Office of Animal Resources at Thomas Jefferson University for their continued support to make this research possible. The authors would also like to thank the Clinical Research Unit at Thomas Jefferson University for their assistance in the collection of patient specimens. This work was supported by a DeGregorio Family Foundation Award and by the Department of Defense Congressionally Directed Medical Research Programs (W81XWH-17-1-0299, W81XWH-191-0263, and W81XWH-19-1-0067) to AES. SAW is supported by the National Institutes of Health (NIH) (R01 CA204881, R01 CA206026, and P30 CA56036), the Defense Congressionally Directed Medical Research Program W81XWH-17-PRCRP-TTSA, and Targeted Diagnostic & Therapeutics. SAW and AES were also supported by a grant from The Courtney Ann Diacont Memorial Foundation. SAW is the Samuel M.V. Hamilton Professor of Thomas Jefferson University. AZ and MC were supported by NIH institutional award T32 GM008562 for Postdoctoral Training in Clinical Pharmacology. Ethics Approval The study was approved by the Thomas Jefferson University Institutional Review Board (#18D.495) and Institutional Animal Care and use Committee (#01529). References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin2018;68:394–424. doi:10.3322/caac.21492. Yousefi MS, Sharifi-Esfahani M, Pourgholam-Amiji N, Afshar M, Sadeghi-Gandomani H, Otroshi O, et al. Esophageal cancer in the world: incidence, mortality and risk factors. Biomed Res Ther 2018;5:2504–17. doi:10.15419/bmrat.v5i7.460. Huang F-L, Yu S-J. Esophageal cancer: risk factors, genetic association, and treatment. Asian J Surg 2018;41:210–5. doi:10.1016/j.asjsur.2016.10.005. Magee MS, Abraham TS, Baybutt TR, Flickinger JC, Ridge NA, Marszalowicz GP, et al. Human GUCY2C-targeted chimeric antigen receptor (CAR)-expressing T cells eliminate colorectal cancer metastases. Cancer Immunol Res 2018;6:509–16. doi:10.1158/2326-6066.CIR-16-0362. Magee MS, Kraft CL, Abraham TS, Baybutt TR, Marszalowicz GP, Li P, et al. GUCY2C-directed CAR-T cells oppose colorectal cancer metastases without autoimmunity. Oncoimmunology 2016;5:e1227897. doi:10.1080/2162402X.2016.1227897.

Background Gastric cancer is the sixth most common cancer and second-leading cause of cancer-related mortality worldwide.1 The heterogenous and genetically complex nature of this disease underlies the challenges in developing effective therapies for metastatic gastric cancer. In the majority of cases, stomach tumors evolve from intestinal metaplasia resulting in ectopic expression of the enterocyte differentiation antigen guanylyl cyclase C (GUCY2C) by ~50% of primary and metastatic gastric cancers.2–4 In the context of the efficacy of GUCY2C-directed chimeric antigen receptor (CAR)-T cells against metastatic colorectal cancer in animal models,5,6 we hypothesized that this adoptive cell therapy may be effective against metastatic gastric cancer. Methods Here, we explored the efficacy of GUCY2C-directed CAR-T cells for gastric cancer in a patient derived xenograft (PDX) tumor model. Also, we interrogated translational GUCY2C biomarker assays using RT-qPCR, immunoblot analysis, and immunohistochemistry (IHC) for the intended purpose of identifying patients whose tumors express GUCY2C and could benefit from GUCY2C-directed CAR-T cell therapy. Results GUCY2C-directed CAR-T cells significantly reduced subcutaneous T84 colorectal tumor growth, producing a 5-fold reduction in tumor volume, compared to control treated tumors. GUCY2C-directed CAR-T cells produced no response in tumors produced from the GUCY2C-deficient colorectal cancer cell line, SW480. Importantly, GUCY2C-directed CAR-T cells controlled gastric cancer PDX growth, maintaining a >12-fold reduction in tumor volume compared to control and in some cases produced complete tumor elimination. Furthermore, IHC based assays, indicate that antibodies developed in our laboratory may be suitable for development of a companion diagnostic for GUCY2C-directed CAR-T cells. Indeed, the commercial polyclonal antibody demonstrated robust, non-specific staining regardless of tissue type or GUCY2C mRNA profile, while novel monoclonal antibodies produced in our laboratory primarily detected protein localized to the membrane of glandular epithelial cells, demonstrating antigen specificity, and indicating their potential for further development in diagnostic companion assays to identify gastric cancer patients who may benefit from GUCY2C-directed CAR-T cell therapy. Conclusions GUCY2C-directed CAR-T cells prevented the growth of, and at times eliminated, a subcutaneous gastric cancer PDX model. In the context of previously established safety in mouse models, additional studies defining the efficacy of GUCY2C-directed CAR-T cells in gastric cancer models may allow future translation of this therapy to patients with advanced gastric cancers. Concurrent development of a novel companion diagnostic IHC assay would permit identification of the ~50% of gastric cancer patients whose tumors express GUCY2C and could benefit from this therapy. Acknowledgements This work was supported by a DeGregorio Family Foundation Award and by the Department of Defense Congressionally Directed Medical Research Programs (W81XWH-17-1-0299, W81XWH-191-0263, and W81XWH-19-1-0067) to AES. SAW is supported by the National Institutes of Health (NIH) (R01 CA204881, R01 CA206026, and P30 CA56036), the Defense Congressionally Directed Medical Research Program W81XWH-17-PRCRP-TTSA, and Targeted Diagnostic & Therapeutics. SAW and AES were also supported by a grant from The Courtney Ann Diacont Memorial Foundation. SAW is the Samuel M.V. Hamilton Professor of Thomas Jefferson University. AZ was supported by NIH institutional award T32 GM008562 for Postdoctoral Training in Clinical Pharmacology.The authors thank the NCI Patient-Derived Models Repository for their support and resources to make this research possible. The authors also thank the Sidney Kimmel Cancer Center Translational Research & Pathology Core Facility, and the Office of Animal Resources at Thomas Jefferson University for their continued technical assistance and support in this research. Ethics Approval This study was approved by the Thomas Jefferson University Institutional Review Board (#14.0204) and the Instituational Animal Care and Use Commitee (Protocol #01529). References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin2018;68:394–424. doi:10.3322/caac.21492. Park J, Schulz S, Haaf J, Kairys JC, Waldman SA. Ectopic expression of guanylyl cyclase C in adenocarcinomas of the esophagus and stomach. Cancer Epidemiol Biomarkers Prev 2002;11:739–44. Birbe R, Palazzo JP, Walters R, Weinberg D, Schulz S, Waldman SA. Guanylyl cyclase C is a marker of intestinal metaplasia, dysplasia, and adenocarcinoma of the gastrointestinal tract. Hum Pathol. 2005;36:170–9. doi:10.1016/j.humpath.2004.12.002. Mathur D, Root AR, Bugaj-Gaweda B, Bisulco S, Tan X, Fang W, et al. A Novel GUCY2C-CD3 T-Cell Engaging Bispecific Construct (PF-07062119) for the Treatment of Gastrointestinal Cancers. Clin Cancer Res 2020;26:2188–202. doi:10.1158/1078-0432.CCR-19-3275. Magee MS, Kraft CL, Abraham TS, Baybutt TR, Marszalowicz GP, Li P, et al. GUCY2C-directed CAR-T cells oppose colorectal cancer metastases without autoimmunity. Oncoimmunology 2016;5:e1227897. doi:10.1080/2162402X.2016.1227897. Magee MS, Abraham TS, Baybutt TR, Flickinger JC, Ridge NA, Marszalowicz GP, et al. Human GUCY2C-Targeted Chimeric Antigen Receptor (CAR)-Expressing T Cells Eliminate Colorectal Cancer Metastases. Cancer Immunol Res 2018;6:509–16. doi:10.1158/2326-6066.CIR-16-0362.