Design and Development of Anti-Linker Antibodies for the Detection and Characterization of CAR T Cells

Introduction: Chimeric antigen receptor (CAR) T cell therapy is a transformative treatment modality in B cell malignancies. As next generation CARs are developed to improve durable response rates and expand into other indications of unmet need, the proper tools to identify, characterize, and modulate CAR activity will play a pivotal role in the optimization of this therapeutic option. This study developed and evaluated one approach, that of using monoclonal antibodies (mAbs) against common elements utilized in approved and investigational CARs.Methods: The mAbs KIP-1 and KIP-4 were raised in rabbits against the linkers of 2 single-chain variable fragments, the Whitlow linker (Whitlow, et al. Protein Eng. 1993) and the G4S linker (Huston JS, et al. PNAS. 1988), respectively, to generate universal detection reagents without adding an exogenous peptide recognition motif. Specificity and sensitivity were assessed by flow cytometric analysis of CAR T cells vs nontransduced T cells, immunohistochemistry (IHC) staining of embedded cell pellets with or without a CAR carrying the relevant linker, and epitope mapping by ELISA. Activation was assessed in a competitive stimulation assay in which nontransduced T cells and CAR T cells were mixed at defined ratios and stimulated broadly with OKT3 or specifically with KIP-1 or KIP-4.Results: Flow cytometric analysis demonstrated that CAR T cells with the Whitlow linker could be detected by phycoerythrin-conjugated KIP-1 at ≤ 10 ng per million cells with no detectable staining on nontransduced T cells above background levels. To further demonstrate specificity, IHC data revealed that KIP-1 bound to T cell pellets expressing CARs containing the Whitlow linker, but not the G4S linker. Epitope mapping revealed that the minimal KIP-1 epitope was contained within the amino acid sequence SGKPGSGE. Furthermore, KIP-1 was able to effectively identify CAR T cells in patient samples by both flow cytometry and IHC. Having established that KIP-1 was specific and sensitive, activation assays were performed to determine its CAR T cell-specific activating capacity. KIP-1 specifically activated CAR T cells, but not nontransduced T cells, as demonstrated by CAR T cell expansion and upregulation of cell surface activation markers, including CD69 and 4-1BB. KIP-4 was also assessed for specificity, sensitivity, and activation capacity with similar results.Conclusions: Taking advantage of the linear epitopes within the commonly-used Whitlow and G4S linkers, KIP-1 and KIP-4, respectively, can be used in a wide variety of phenotypic and functional assays with CARs of various specificities that share these linkers. These tools provide the means to realize the full potential of investigational CAR T cell products by supporting research, clinical, and manufacturing efforts.Citation Format: Stuart A. Sievers, Keith A. Kelley, Stephanie H. Astrow, Adrian Bot, Jed J. Wiltzius. Design and development of anti-linker antibodies for the detection and characterization of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1204.