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

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

doi:10.1038/s41598-018-38258-z

Cited by 14 publications

(16 citation statements)

References 25 publications

(50 reference statements)

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“…Incubation of the Topanga reagent with CAR-T cell mixtures facilitated luminescent detection. The Topanga reagent's exceptional sensitivity allows it to detect CAR-binding in a mixture of 0.001% CAR-T cells out of 1 million PBMCs (55). Although the Topanga reagent cannot determine percentage of CAR-expressing cells, it might be useful for quality control during manufacturing and testing the binding functionality of the CAR.…”

Section: Biochemical Assaysmentioning

confidence: 99%

The Chimeric Antigen Receptor Detection Toolkit

Huang

2020

Front. Immunol.

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Chimeric antigen receptor-T (CAR-T) cell therapy is a promising frontier of immunoengineering and cancer immunotherapy. Methods that detect, quantify, track, and visualize the CAR, have catalyzed the rapid advancement of CART cell therapy from preclinical models to clinical adoption. For instance, CAR-staining/labeling agents have enabled flow cytometry analysis, imaging applications, cell sorting, and high-dimensional clinical profiling. Molecular assays, such as quantitative polymerase chain reaction, integration site analysis, and RNA-sequencing, have characterized CAR transduction, expression, and in vivo CART cell expansion kinetics. In vitro visualization methods, including confocal and total internal reflection fluorescence microscopy, have captured the molecular details underlying CAR immunological synapse formation, signaling, and cytotoxicity. In vivo tracking methods, including two-photon microscopy, bioluminescence imaging, and positron emission tomography scanning, have monitored CART cell biodistribution across blood, tissue, and tumor. Here, we review the plethora of CAR detection methods, which can operate at the genomic, transcriptomic, proteomic, and organismal levels. For each method, we discuss: (1) what it measures; (2) how it works; (3) its scientific and clinical importance; (4) relevant examples of its use; (5) specific protocols for CAR detection; and (6) its strengths and weaknesses. Finally, we consider current scientific and clinical needs in order to provide future perspectives for improved CAR detection.

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Section: Biochemical Assaysmentioning

confidence: 99%

The Chimeric Antigen Receptor Detection Toolkit

Huang

2020

Front. Immunol.

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“…This approach could also be applied to other platform used for the selection of TCR-like binders, like nanobodies, DARPins, and anticalins, that can be expressed on Jurkat cells in a CAR-T like fashion. 45 …”

Section: Discussionmentioning

confidence: 99%

CAR-J cells for antibody discovery and lead optimization of TCR-like immunoglobulins

Jöst

Darowski

Challier

et al. 2020

mAbs

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T-cell bispecific antibodies (TCBs) are a novel class of engineered immunoglobulins that unite monovalent binding to the T-cell receptor (TCR) CD3e chain and bivalent binding to tumor-associated antigens in order to recruit and activate T-cells for tumor cell killing. In vivo, T-cell activation is usually initiated via the interaction of the TCR with the peptide-HLA complex formed by the human leukocyte antigen (HLA) and peptides derived from intracellular proteins. TCR-like antibodies (TCRLs) that recognize pHLA-epitopes extend the target space of TCBs to peptides derived from intracellular proteins, such as those overexpressed during oncogenesis or created via mutations found in cancer. One challenge during lead identification of TCRL-TCBs is to identify TCRLs that specifically, and ideally exclusively, recognize the desired pHLA, but not unrelated pHLAs. In order to identify TCRLs suitable for TCRL-TCBs, large numbers of TCRLs have to be tested in the TCB format. Here, we propose a novel approach using chimeric antigen receptors (CARs) to facilitate the identification of highly selective TCRLs. In this new so-called TCRL-CAR-J approach, TCRL-candidates are transduced as CARs into Jurkat reporter-cells, and subsequently assessed for their specificity profile. This work demonstrates that the CAR-J reporter-cell assay can be applied to predict the profile of TCRL-TCBs without the need to produce each candidate in the final TCB format. It is therefore useful in streamlining the identification of TCRL-TCBs.

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“…Malibu reagents carry the epitope tags (4xFLAG-2xStreptagII-8xHis) at their C-termini. We purified the CD19-scFv1-Gluc Malibu reagent by affinity chromatography using a Strep-Tactin column 23 . The purified Malibu reagent was used to determine the half-maximal effective concentration (EC50) for binding to 1 × 10 4 Raji and RS411 cells.…”

Section: Figurementioning

confidence: 99%

“…For this purpose, we employed Topanga assay. This assay was recently developed in our lab for the detection of CAR and utilizes CD19-extracellular domain (ECD)-Nluc fusion protein 23 . Equal number of JNG cells expressing the different CD19-CAR were incubated with supernatants containing CD19-ECD-Nluc for 45 min on ice, washed and cell associated luminescence was measured.…”

Section: Malibu-glo Assay Allows For Selection Of Optimal Scfvs For Cmentioning

confidence: 99%

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors

Natarajan

Gopalakrishnan

Matta

et al. 2020

Sci Rep

Self Cite

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Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modifiedwith chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferasebased assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

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

Cited by 14 publications

References 25 publications

The Chimeric Antigen Receptor Detection Toolkit

The Chimeric Antigen Receptor Detection Toolkit

CAR-J cells for antibody discovery and lead optimization of TCR-like immunoglobulins

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors

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