The Chimeric Antigen Receptor Detection Toolkit

Hu, Yifei; Huang, Jun

doi:10.3389/fimmu.2020.01770

Cited by 46 publications

(60 citation statements)

References 92 publications

(129 reference statements)

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“…However, flow cytometry allows the identification and characterization of CAR-T cell subpopulations and of patient’s immune cells in a fast way and at a single-cell level. In addition, flow cytometry detects the CAR at the proteomic level and thus can provide information regarding CAR cell functionality ( 27 ). Therefore, flow cytometry together with upcoming next-generation sequencing (NGS) approaches ( 18 , 28 ) enables the comprehensive monitoring of CAR-T cell therapy ( 29 ).…”

Section: Discussionmentioning

confidence: 99%

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

et al. 2021

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Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

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Section: Discussionmentioning

confidence: 99%

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

et al. 2021

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“…Antigen-specific detection reagents bind to the antigen-binding domain of the CD19.CAR and are supposed to have a high specificity with a low background staining when compared to non-antigen specific detection reagents [18,19]. Yet, they are costly and can only be used for one particular scFv.…”

Section: Introductionmentioning

confidence: 99%

“…Both target IgG-like fragments. While they are more cost effective, they might on the other hand be characterized by a higher background staining [19].…”

Section: Introductionmentioning

confidence: 99%

Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR

Schanda

Sauer

Kunz

et al. 2021

Cells

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Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.

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“…However, as the performance of serial and multiple biopsies of metastatic sites is not feasible, the ability to interpret antitumor responses to immunotherapy is limited. Although flow cytometric-, immunohistochemical-, and polymerase chain reaction-based assays can detect the presence of CAR T cells in peripheral blood and tissue samples, 3 our understanding of CAR T-cell kinetics is limited by the lack of a dynamic imaging modality. To optimize the efficacy of CAR T-cell immunotherapy and gain insights into site-specific responses, it is critical to develop a noninvasive imaging modality that enables the monitoring of CAR T-cell trafficking in real time.…”

Section: Introductionmentioning

confidence: 99%

Imaging CAR T-cell kinetics in solid tumors: Translational implications

Skovgard

Hocine

Saini

et al. 2021

Molecular Therapy - Oncolytics

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Summary Success in solid tumor chimeric antigen receptor (CAR) T-cell therapy requires overcoming several barriers, including lung sequestration, inefficient accumulation within the tumor, and target-antigen heterogeneity. Understanding CAR T-cell kinetics can assist in the interpretation of therapy response and limitations and thereby facilitate developing successful strategies to treat solid tumors. As T-cell therapy response varies across metastatic sites, the assessment of CAR T-cell kinetics by peripheral blood analysis or a single-site tumor biopsy is inadequate for interpretation of therapy response. The use of tumor imaging alone has also proven to be insufficient to interpret response to therapy. To address these limitations, we conducted dual tumor and T-cell imaging by use of a bioluminescent reporter and positron emission tomography in clinically relevant mouse models of pleural mesothelioma and non-small cell lung cancer. We observed that the mode of delivery of T cells (systemic versus regional), T-cell activation status (presence or absence of antigen-expressing tumor), and tumor-antigen expression heterogeneity influence T-cell kinetics. The observations from our study underscore the need to identify and develop a T-cell reporter—in addition to standard parameters of tumor imaging and antitumor efficacy—that can be used for repeat imaging without compromising the efficacy of CAR T cells in vivo .

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The Chimeric Antigen Receptor Detection Toolkit

Cited by 46 publications

References 92 publications

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR

Imaging CAR T-cell kinetics in solid tumors: Translational implications

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