Abstract 3774: A high-throughput process for the development and characterization of chimeric antigen receptors (CARs)
Authors: Radhakrishnan Sabarinath Venniyil , Miles Adam , Atanackovic Djordje , Luetkens Tim 
Published: July 2017

Abstract
INTRODUCTION: Chimeric antigen receptors (CARs) expressed by T cells bind to surface antigens via a single-chain variable fragment (scFv) and are able to elicit potent anti-tumor efficacy. To our knowledge no rapid CAR development platforms exist to facilitate the high-throughput generation and characterization of novel CARs to allow head-to-head comparisons and to determine the influence of scFv properties on CAR T cell activation, phenotype, and persistence.
EXPERIMENTAL PROCEDURES: We have established a rapid process for the development of functional novel CARs using fully human antibody phage display, vector systems for bacterial and mammalian expression, as well as high-throughput functional and biochemical characterization assays. We validated individual parts of the workflow using different tumor antigens and completed the entire workflow for one model antigen expressed on the surface of multiple myeloma cells. An initial population of scFvs against the model antigen was selected from a large naïve fully-human antibody phage display library by panning. The reactivity of the polyclonal phage population was then confirmed by time-resolved fluorescence before reformatting into various monoclonal binder formats. We established a high-throughput flow cytometry assay simultaneously determining CAR surface expression and antigen binding. In addition, we adapted protocols for small-scale lentiviral transduction and expansion of primary CAR T cells followed by a sensitive, high-throughput luciferase-based cytotoxicity assay. Finally, in collaboration with Wasatch Microfluidics we performed high-throughput surface plasmon resonance measurements as well as epitope binning of candidate binding domains.
RESULTS: We obtained 1,323 scFvs after two rounds of antibody phage selection. The subsequent bacterial screen of 163 clones identified 23 unique monoclonal binders. In a comparison of all 23 binders in different formats we determined that none of the traditional screening formats, including soluble scFv or scFv-Fc fusion constructs, correlated with CAR binding. CAR constructs with high expression and antigen binding were found to have affinities in the low nanomolar range (18-22nM) and evidenced strong killing of human cancer cell lines spontaneously expressing the antigen (53-79% killing at an effector:target ratio of 10:1), but not healthy cells expressing lower levels of the antigen. Importantly, in a murine xenograft model of multiple myeloma we observed specific killing of tumor cells and no overt toxicities.
CONCLUSION: We show that our process allows the generation, screening, as well as functional and biochemical characterization of novel CARs within 2 months. Our approach enables researchers to easily carry out head-to-head comparisons allowing comprehensive lead identification and the determination of binding domain properties which may shape CAR T cell function and phenotype.
Citation Format: Sabarinath Venniyil Radhakrishnan, Adam Miles, Djordje Atanackovic, Tim Luetkens. A high-throughput process for the development and characterization of chimeric antigen receptors (CARs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2017-3774