A novel luciferase-based assay for the detection of Chimeric Antigen Receptors
| Autor: | Varun Sikri, Naman Sharma, Kenta Ito, Songjie Gong, Alberto Jeronimo, Venkatesh Natarajan, Sunju Choi, Magdalena Falat, Saurabh Deepak Chitnis, Nell Narasappa, Lalith Namburu, Queenie Qiu, Ankita Batra, Pooja Smruthi Keerthipati, Jason Braun, Hannalei Mae Zamora, Ramakrishnan Gopalakrishnan, Vishan Chaudhary, Hittu Matta, Michael Kahn, Rekha Prakash, Tomas Meza Stieben, Fatima Patel, Ruben Prins, Arta Zenunovic, Preet M. Chaudhary, Supriya Peshin, Allen Membreno, Katelyn Purvis, Dan Wang, Jyotirmayee Lenka, Andrei A. Kochegarov, Jae Han Lee |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Recombinant Fusion Proteins T-Lymphocytes Receptors Antigen T-Cell lcsh:Medicine Immunotherapy Adoptive Epitope Article Flow cytometry Cell Line 03 medical and health sciences 0302 clinical medicine Antigen Protein purification medicine Humans Luciferase Lymphocytes lcsh:Science Luciferases Multidisciplinary Receptors Chimeric Antigen medicine.diagnostic_test biology Chemistry lcsh:R Flow Cytometry Fusion protein Chimeric antigen receptor Receptors Antigen 030104 developmental biology Biochemistry biology.protein lcsh:Q Antibody 030217 neurology & neurosurgery |
| Zdroj: | Scientific Reports Scientific Reports, Vol 9, Iss 1, Pp 1-13 (2019) |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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