Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor

Abstract

T cells can be engineered to express the genes of chimeric antigen receptors (CARs) that recognize tumor-associated antigens. We constructed and compared 2 CARs that contained a single chain variable region moiety that recognized CD19. One CAR contained the signaling moiety of the 4-1BB molecule and the other did not. We selected the CAR that did not contain the 4-1BB moiety for further preclinical development. We demonstrated that gammaretroviruses encoding this receptor could transduce human T cells. Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced interferon-gamma and interleukin-2 specifically in response to CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. We transduced T cells from CLL patients that had been previously treated with chemotherapy. We induced these T cells to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer with a protocol consisting of an initial stimulation with an anti-CD3 monoclonal antibody (OKT3) before transduction followed by a second OKT3 stimulation 7 days after transduction. This protocol was successfully adapted for use in CLL patients with high peripheral blood leukemia cell counts by depleting CD19+ cells before the initial OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we generated a producer cell clone that produces retroviruses encoding the anti-CD19 CAR, and we produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice conditions.

Figure 1

A comparison of the design and transduction efficiency of two anti-CD19 CARs. (A) A diagram of the recombinant retroviral vector MSGV-FMC63-28Z is shown (LTR, long terminal repeat; Fv, variable regions; CD28, part of the extracellular region and all of the transmembrane and intracellular regions of CD28; TCR-ζ, the entire cytoplasmic region of the TCR-ζ molecule). (B) A diagram of the recombinant retroviral vector MSGV-FMC63-CD828BBZ is shown (LTR, long terminal repeat; Fv, variable regions; CD8, CD8 hinge region; CD28, CD28 cytoplasmic region; 4-1BB, 4-1BB cytoplasmic region; TCR-ζ, the cytoplasmic region of the TCR-ζ molecule. (C) PBMC were started in culture with OKT3 and IL-2 on day 0. The cells were transduced with retroviruses encoding FMC63-28Z on days 2 and 3. On day 8, expression of FMC63-28Z was detected on 45% of T cells when the cells were stained with anti-Fab antibodies and anti-CD3. Staining with isotype-matched control antibodies and anti-CD3 is also shown. (D) A PBMC culture from the same donor as in (C) was initiated on day 0. Cells were transduced with retroviruses encoding FMC63-CD828BBZ on days 2 and 3, and stained with either anti-Fab antibodies or isotype-matched control antibodies on day 8 in the same manner as in (C). Expression of FMC63-CD828BBZ was detected on 19% of T cells. (E) A PBMC culture from the same donor as in (C) and (D) was initiated on day 0. The cells were not transduced. On day 8 the cells were stained with either anti-Fab or isotype-matched control antibodies in the same manner as in (C) and (D). This experiment was performed using cells from the same cultures that were tested in the experiments described in Table 1 and Figure 2. The results presented in (C), (D), and (E) are representative of six experiments that used cells from six different donors.

Figure 2

A comparison of cytokine production by T cells transduced with different anti-CD19 CARs. PBMC cultures were initiated on day 0 and transduced on days 2 and 3 after culture initiation. On day 11 after the cultures were initiated, cells that were transduced with either FMC63-28Z or FMC63-CD828BBZ were stimulated with either CD19-K562 cells or NGFR-K562 cells for 5 hours and intracellular staining for IFNγ (A) and IL-2 (B) was performed. The transduced T cells produced IFNγ and IL-2 in a CD19-specific manner. The plots are gated on CD3⁺ lymphocytes, and the percentage of cells in each quadrant is shown on the plots. This experiment was performed using cells from the same cultures that were tested in the experiments described in Figure 1 and Table 1. This experiment is representative of six experiments that used cells from six different donors.

Figure 3

Expression of CARs is maintained in transduced T cells after extensive proliferation. (A) The design of the experiments reported in Figures 3 through 6 and Tables 2 and 3 is shown. PBMC were started in culture with OKT3 and IL-2 on day 0. On day 2, cultures were suspended in fresh medium with IL-2 and the first transduction was performed. On day 3 the transduction was repeated. On day 10, a rapid expansion protocol (REP) was initiated to generate large numbers of transduced T cells. (B) Ten days after initiation of a REP, FMC63-28Z expression was detected on 70% of T cells that had been transduced with FMC63-28Z when the T cells were stained with anti-Fab antibodies. Staining with isotype-matched control antibodies is also shown. This experiment is representative of three separate experiments using cells from three different donors. (C) Ten days after initiation of a REP, SP6-28Z expression was detected on 60% of T cells when T cells that had been transduced with SP6-28Z were stained with anti-Fab antibodies. Staining with isotype-matched control antibodies is also shown. The cells used in the experiments reported in (B) and (C) were from the same donor. The experiments reported in (B) and (C) were performed with cells from the same cultures tested in the experiments described in Figure 4, Figure 5, and Table 2.

Figure 4

Transduced T cells can produce IFNγ and IL-2 after rapid expansion. On day 14 after initiation of REPs, PBMC from the same donor that had been cultured identically and transduced with either FMC63-28Z or SP6-28Z were stimulated with either CD19-K562 cells or NGFR-K562 cells for 5 hours, and intracellular staining for IFNγ (A) and IL-2 (B) was performed. The transduced T cells produced IFNγ and IL-2 in a CD19-specific manner. The plots are gated on CD3⁺ lymphocytes, and the percentage of cells in each quadrant is shown on the plots. This experiment is representative of three experiments that used cells from three different donors.

Figure 5

FMC63-28Z-transduced T cells can kill primary CLL cells before and after a REP. (A) An example of the flow cytometry plots obtained when we performed a flow cytometry cytotoxicity assay is shown. The cytotoxicity of target cells is measured by comparing survival of target cells relative to the survival of negative control cells. The negative control cells and the target cells are combined in the same tube with effector T cells. Note that the percentage of CD19-expressing CLL cells relative to the control CCRF-CEM cells was less after incubation with FMC63-28Z-transduced T cells than after incubation with SP6-28Z-control transduced T cells. These data were part of the data used to construct the cytotoxicity plot shown in (B). (B) FMC63-28Z-transduced T cells could specifically kill allogeneic primary CLL cells 11 days after initial OKT3 stimulation. T cells from the same donor that were transduced with SP6-28Z caused only a low level of cytotoxicity against the same primary allogeneic CLL cells. Cytotoxicity is reported as the mean +/- the SEM of duplicate determinations. This experiment is representative of three similar experiments that used cells from three different donors. (C) Twenty-one days after initiation of a REP, FMC63-28Z-transduced T cells could kill primary allogeneic CLL cells. The FMC63-28Z-transduced T cells were from the same donor as in (B). The allogeneic CLL cells used in this experiment were from a different patient than the CLL cells that were used in the experiment presented in (B). T cells from the same donor that were transduced with SP6-28Z caused only a low level of cytotoxicity against the same primary allogeneic CLL cells. Cytotoxicity is reported as the mean +/- the SEM of duplicate determinations. This experiment is representative of three similar experiments that used cells from three different donors.

Figure 6

Large numbers of functional anti-CD19 T cells can be generated from the blood of CLL patients. (A) T cells from a patient with CLL that had received fludarabine plus rituximab were transduced with FMC63-28Z and induced to proliferate in a REP. On day 10 after initiation of the REP 75% of the T cells expressed the FMC63-28Z CAR as measured by staining with anti-Fab antibodies. Cells from six different CLL patients were transduced in a similar manner, and the percentage of T cells from each of these patients that expressed the FMC63-28Z CAR ten days after the initiation of a REP is reported in Table 3. (B) Ten days after initiation of a REP, cells from the same culture used for the experiment reported in (A) produced IFNγ upon stimulation with the CD19-expressing cell lines NALM6 and CD19-K562 but not the CD19-negative cell lines NGFR-K562 and CCRF-CEM. These results are representative of six experiments in which cells from six different CLL patients were used. (C) FMC63-28Z-transduced T cells from a CLL patient specifically killed autologous CLL cells while nontransduced T cells from the same patient exhibited minimal cytotoxicity 12 days after initiation of a REP. Cytotoxicity is reported as the mean +/- the SEM of duplicate determinations. These results are representative of two experiments in which cells from two different patients were used.