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

Ulrich Blache,Andreas Boldt,Ulrike Koehl,Ronald Weiss,Tewfik Miloud,Andrea Quaiser,Mégane Burgaud,Stephan Fricke,Michael Kapinsky,Annabelle Pohl,Vladan Vucinic,Ulrich Sack,Uwe Platzbecker,André-René Blaudszun

doi:10.3389/fimmu.2021.658314

Abstract

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.

Highlights

Adoptive immunotherapy using immune effector cells engineered ex vivo to express chimeric antigen receptors (CARs) that mediate the lysis of cancer cells has become an innovative approach in cancer therapy [1]
In order to establish a standardized flow cytometry method for CAR-T monitoring, we developed assays that are based on preformulated dry antibody panels usefull at all steps of CAR-T cell therapy (Figure 1)
These flow cytometry assays are produced so that the antibodies are present in dried form at the bottom of the reagent tube and the sample can be directly added to the pre-formulated antibody mixtures

Summary

Introduction

Adoptive immunotherapy using immune effector cells engineered ex vivo to express chimeric antigen receptors (CARs) that mediate the lysis of cancer cells has become an innovative approach in cancer therapy [1]. CD19 CAR-T cell treatment led to impressive remission rates in patients with precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL), Diffuse Large B-Cell Lymphoma (DLBCL), Primary Mediastinal B-Cell Lymphoma (PMBCL) and Mantle Cell Lymphoma (MCL) [2,3,4,5] Spearheaded by this success, the potential of CAR-T cell therapy is currently being investigated in hundreds of clinical trials – the large majority in B cell malignancies [6]. CAR-T cell therapy comes with severe, toxic and potentially life-threating adverse effects, which have been observed in a high proportion of patients [2,3,4,5, 7] These side effects are caused by cytokine secretion (e.g. IL-1, IL-6) due to immune cell and target interactions, which initiate cytokine release syndrome (CRS), macrophage activation syndrome (MAS) and neurotoxic symptoms [8,9,10,11,12]. The expression profiles of immune checkpoint molecules, co-inhibitory and co-stimulatory receptors, and differentiation markers of T lymphocytes are of high interest, because ligand interactions with these molecules can modulate endogenous T cell and CART cell efficacy

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Conclusion