Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia
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
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
View: Text | PDF
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

×

Figure 4

Cetuximab-mediated in vivo depletion of m1928E+ T cells allows for B cell recovery in vivo.

Options: View larger image (or click on image) Download as PowerPoint
Cetuximab-mediated in vivo depletion of m1928E+ T cells allows for B cel...
(A) Experimental layout of depletion experiments based on the transfer of m1928E-transduced cells targeting endogenous CD19+ B cells. (B) Thy1.1+ mouse splenocytes were retrovirally transduced with the m1928E CAR construct. After transfer of 1.2 × 106 m1928E-transduced (unsorted) T cells, EGFRt+ cells were detected by ex vivo EGFR staining in a blood sample on day 43 after infusion. (C) Mice were irradiated on day –1 and received m1928E+ T cells or mock T cells on day 0. Numbers of endogenous CD19+ B cells were monitored in the blood before T cell transfer (day –6) and 1 or 43 days after T cell transfer. Endogenous B cells and NK cells were detected in representative samples for each group with anti-CD19 and anti-NK1.1 mAbs, respectively. Gates were set on CD19+ B cells among Thy1.1– living lymphocytes, and numbers indicate B cell frequencies. (D) Numbers of endogenous CD19+ B cells per microliter blood were acquired for each mouse group over the time course. Time points of irradiation and the 2 mAb infusions are indicated. Means ± SD are shown; n = 6 per group. Two-way ANOVA followed by Bonferroni post-test was used for statistics. (E) In a separate long-term experiment, 8 × 106 m1928E-transduced, unsorted Thy1.1+ cells were transferred into irradiated WT C57BL/6 mice. Cetuximab infusions were performed at late time points (days 158 and 162). Numbers of endogenous CD19+ B cells were obtained over the course of the experiment as described in C and D. Means ± SD are plotted. n = 2 per group. (F and G) After recovery of normal B cell numbers, mice were immunized with OVA protein (100 μg/mouse) mixed 1:1 with Imject alum on day 245 after CAR T cell infusion. One mouse group had received mock T cells and was not immunized as a negative control. OVA-specific IgG levels in the blood plasma were measured by ELISA 7 days (F) and 14 days (G) after immunization. Means ± SD are shown for 2 independent experiments; P values were calculated by unpaired 2-tailed Student’s t test; n = 5 for immunized mock, Ctx, and Rtx groups; n = 7 for not immunized mock group. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.