A novel luciferase-based assay for the detection of Chimeric Antigen Receptors

Ramakrishnan Gopalakrishnan,Jae Han Lee,Andrei Kochegarov,Sunju Choi,Michael Kahn,Saurabh Deepak Chitnis,Rekha Prakash,Hittu Matta,Allen Membreno,Jason Braun,Ruben Prins,Jyotirmayee Lenka,Venkatesh Natarajan,Hannalei Mae Zamora,Kenta Ito,Katelyn Purvis,Fatima Patel,Lalith Namburu,Songjie Gong,Alberto Jeronimo,Nupur Sharma ,Nell Narasappa,Supriya Peshin,Dan Wang,Pooja Smruthi Keerthipati,Ankita Sati Batra ,Arta Zenunovic,Magdalena Falat,Varun Sikri,Queenie Qiu,Tomas Meza Stieben,Vishan Chaudhary,Preet M Chaudhary

doi:10.1038/s41598-018-38258-z

Ramakrishnan Gopalakrishnan, Jae Han Lee + Show 31 more

Open Access

https://doi.org/10.1038/s41598-018-38258-z

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Journal: Scientific Reports	Publication Date: Feb 13, 2019
Citations: 14	License type: open-access

Affiliation: University of Southern California

Abstract

Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy has produced dramatic responses in hematologic malignancies. One of the challenges in the field is the lack of a simple assay for the detection of CARs on the surface of immune effector cells. In this study, we describe a novel luciferase-based assay, termed Topanga Assay, for the detection of CAR expression. The assay utilizes a recombinant fusion protein, called Topanga reagent, generated by joining the extra-cellular domain of a CAR-target in frame with one of the marine luciferases or their engineered derivatives. The assay involves incubation of CAR expressing cells with the Topanga reagent, a few washes and measurement of luminescence. The assay can detect CARs comprising either immunoglobulin- or non-immunoglobulin-based antigen binding domains. We further demonstrate that addition of epitope tags to the Topanga reagent not only allows its convenient one step purification but also extends its use for detection of CAR cells using flow cytometry. However, crude supernatant containing the secreted Topanga reagent can be directly used in both luminescence and flow-cytometry based assays without prior protein purification. Our results demonstrate that the Topanga assay is a highly sensitive, specific, convenient, economical and versatile assay for the detection of CARs.

Highlights

Chimeric Antigen Receptor (CAR) therapy is a revolutionary approach for the treatment of human malignancies
To develop Topanga assay for the detection of CD19 CARs, we made a fusion construct by joining in frame the extracellular domain (ECD) of human CD19 containing a signal peptide with Nluc via an intervening short Gly-Gly-Ser-Gly flexible linker
The CD19-CAR-expressing T and NK92MI cells showed marked increase in luminescence when bound by the CD19-ECD-Nluc fusion protein as compared to the control CAR (i.e. CD33-CAR)-expressing cells or parental cells. These results demonstrated the specificity of the CD19-ECD-Nluc Topanga reagent for detecting the expression of CD19-CAR expressing cells and confirmed that a luciferase-based assay can be developed for the specific detection of CARs

Summary

Introduction

Chimeric Antigen Receptor (CAR) therapy is a revolutionary approach for the treatment of human malignancies. Expression of CARs on effector cells is generally detected by flow cytometry using fluorochrome-tagged antibodies or ligands that bind to the extra-cellular domain of the CAR1–3. CD19-specific CARs have been detected following staining with an Alexa Flour 488-conjugated CD19-Fc fusion protein consisting of human CD19 extracellular domain and Fc region of human IgG13. The assay utilizes a recombinant fusion protein, called Topanga reagent, which is generated by joining the extra-cellular domain of a CAR target in frame with one of the marine luciferases. As they use marine luciferases, the assay and the reagent were named after the Topanga Beach in Los Angeles, California. The word Topanga is Native American in origin and means “where the mountain meets the sea”

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