Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia
Paulina J. Paszkiewicz, … , Stanley R. Riddell, Dirk H. Busch
Paulina J. Paszkiewicz, … , Stanley R. Riddell, Dirk H. Busch
Published October 17, 2016
Citation Information: J Clin Invest. 2016;126(11):4262-4272. https://doi.org/10.1172/JCI84813.
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Research Article Immunology

Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia

Abstract

The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies.

Authors

Paulina J. Paszkiewicz, Simon P. Fräßle, Shivani Srivastava, Daniel Sommermeyer, Michael Hudecek, Ingo Drexler, Michel Sadelain, Lingfeng Liu, Michael C. Jensen, Stanley R. Riddell, Dirk H. Busch

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Figure 2

Recombinant surface EGFRt is stably expressed over time and can be used for antibody-mediated T cell depletion in vivo.

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Recombinant surface EGFRt is stably expressed over time and can be used ...
(A) Experimental layout of depletion experiments in the OT-I mouse model. (B) GFP/EGFRt-transduced Thy1.1+ mouse splenocytes were sort-purified using EGFR Fab Streptamers, subsequently transferred into sublethally irradiated mice, and then tracked over time in the blood. Coexpression of GFP and EGFRt is shown for transduced cells before (left) and after sorting on the day of T cell transfer (middle; day 0), as well as in a blood sample on day 150 after infusion (right). Cells were stained with the EGFR-specific mAb, and the frequency of GFP/EGFRt double-positive cells among living Thy1.1+ cells is shown for samples obtained from representative animals. (C) Blood samples were obtained over 150 days after transfer of GFP/EGFRt-sort-purified cells. The level of GFP expression was acquired and EGFR expression was detected with EGFR-specific mAbs. The GFP and EGFR expression levels of living Thy1.1+ cells, as represented by the mean fluorescence intensity (MFI), were plotted over time for 4 individual animals. (D) Summary of flow cytometry analysis of GFP+/EGFRt+ double-positive cells pregated on Thy1.1+/EGFRt+ cells for 3 different time points. n = 12. (E) Two hundred fifty thousand GFP/EGFRt+Thy1.1+ OT-I cells were transferred into WT C57BL/6 mice. Blood samples were obtained before (pre–MVA-OVA) and after challenge with MVA-OVA (post–MVA-OVA), as well as after infusions of cetuximab or rituximab (post-mAb). The Thy1.1 congenic marker and the EGFRt were detected with Thy1.1- and EGFR-specific mAbs. Gates were set on all living transferred Thy1.1+ cells (larger gate) and EGFRt+Thy1.1+ cells (smaller gate), and numbers indicate respective frequencies. GFP versus EGFR staining of the Thy1.1+ pregated cells is also shown with an additional gate on GFP-high cells in red. A representative sample of the cetuximab and rituximab group is shown. (F) Frequencies of Thy1.1+ GFP-high cells for blood samples acquired on day 21 and day 27 (see scheme in A) from the cetuximab-treated (Ctx) and rituximab-treated (Rtx) mouse groups. Ctx group: n = 6; Rtx group: n = 5. Two-tailed Mann-Whitney test was used for statistical significance. **P ≤ 0.005. (G) Time course for the Thy1.1+ cell frequencies. Inset shows section from days 21–49 and a different y axis scale. Means ± SD are plotted.