Dataset Information

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

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.

SUBMITTER: Gopalakrishnan R

PROVIDER: S-EPMC6374361 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Gopalakrishnan Ramakrishnan R Matta Hittu H Choi Sunju S Natarajan Venkatesh V Prins Ruben R Gong Songjie S Zenunovic Arta A Narasappa Nell N Patel Fatima F Prakash Rekha R Chaudhary Vishan V Sikri Varun V Chitnis Saurabh Deepak SD Kochegarov Andrei A Wang Dan D Falat Magdalena M Kahn Michael M Keerthipati Pooja Smruthi PS Sharma Naman N Lenka Jyotirmayee J Stieben Tomas Meza TM Braun Jason J Batra Ankita A Purvis Katelyn K Ito Kenta K Lee Jae Han JH Jeronimo Alberto A Zamora Hannalei Mae HM Membreno Allen A Qiu Queenie Q Peshin Supriya S Namburu Lalith L Chaudhary Preet M PM

Scientific reports 20190213 1

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-targe ...[more]

PMID: 30760795

Similar Datasets

Project description:Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T cell signaling domains. CD30 is a cell surface protein expressed on Hodgkin's lymphoma, some T cell lymphomas, and some B cell lymphomas. CD30 has a restricted expression pattern in normal cells, so CD30 has good potential as a clinical target for CAR T cells. We compared three different anti-CD30 CAR designs incorporating a single-chain variable fragment derived from the 5F11 fully human monoclonal antibody. 5F11-28Z has hinge, transmembrane, and costimulatory domains from CD28 and a CD3ζ T cell activation domain. 5F11-CD828Z has hinge and transmembrane domains from CD8α, a CD28 costimulatory domain, and a CD3ζ T cell activation domain. 5F11-CD8BBZ is identical to 5F11-CD828Z, except for the replacement of the CD28 moiety with a 4-1BB moiety. We found that T cells expressing 5F11-CD8BBZ had lower levels of CD30-specific degranulation and cytokine release compared with CD28-containing CARs. When compared to the CD28-containing CARs, T cells expressing 5F11-CD8BBZ had higher levels of nonspecific functional activity, including degranulation, cytokine release, and proliferation, when stimulated with CD30-negative target cells. We established tumors in nod-scid common gamma-chain deficient (NSG) mice and treated the tumors with T cells expressing different CARs. T cells expressing 5F11-28Z were most effective at eradicating tumors. T cells expressing 5F11-CD828Z had intermediate effectiveness, and T cells expressing 5F11-CD8BBZ were least effective. CD30+ T cells are lost from cultures of T cells containing 5F11-28Z-expressing T cells. This indicated the killing of CD30+ T cells by the 5F11-28Z-expressing T cells. Despite this, the number of T cells in the cultures consistently accumulated to numbers needed for use in a clinical trial. Based on all in vitro and murine experiments comparing the different CARs, we selected 5F11-28Z for further development, and we have initiated a clinical trial testing 5F11-28Z T cells.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets