B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells

Abstract

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.

Trial registration: ClinicalTrials.gov NCT00924326.

Figure 1

Anti–CD19-CAR–transduced T-cell production and clinical treatment protocols. (A) PBMCs were stimulated with the anti-CD3 mAb OKT3 on day 0. The cells were transduced with gammaretroviruses encoding the anti-CD19 CAR on days 2 and 3. On day 10, a rapid expansion protocol was started, and the cells were ready for infusion on day 24. (B) Patients received 60 mg/kg cyclophosphamide chemotherapy daily for 2 days. Next, patients received 25 mg/m² fludarabine chemotherapy daily for 5 days. One day later, the patients received a single infusion of anti–CD19-CAR–transduced T cells. Starting on the same day as the T-cell infusion, the patients received IV IL-2 every 8 hours.

Figure 2

Anti–CD19-CAR–transduced T cells produced cytokines in a CD19-specific manner and recognized autologous leukemia cells. (A) Staining with an anti-Fab Ab revealed expression of the anti-CD19 CAR on the surface of T cells that were administered to patient 7. Staining with an isotype control Ab is also shown. Both plots were gated on CD3⁺ lymphocytes, which made up 99% of the cells in the culture. (B) On the day of infusion, T cells of patient 7 up-regulated CD107a expression after a 4-hour culture with the CD19⁺ target cell CD19-K562 but not the negative control cell NGFR-K562 that does not express CD19. (C) On the day of infusion, anti–CD19-CAR–transduced T cells of patient 7 produced IFNγ, TNF, and IL-2 when cultured for 6 hours with the CD19⁺ target cell CD19-K562 but not the negative control cell NGFR-K562 that does not express CD19. The results shown in panels A through C are representative of the results obtained for all of the patients on the protocol. (D) Anti–CD19-CAR–transduced T cells of patient 3 were cultured with either autologous pretreatment lymphocytes or autologous remission lymphocytes overnight, and an IFNγ ELISA was performed on the supernatant. Anti–CD19-CAR–transduced T cells of patient 3 specifically recognized pretreatment lymphocytes but not remission lymphocytes obtained 7 weeks after CAR-transduced T-cell infusion. Sixty-four percent of the pretreatment lymphocytes were CD19⁺ leukemia cells. The remission lymphocytes contained only 0.1% CD19⁺ cells. (E) Anti–CD19-CAR–transduced T cells of patient 6 were cultured with either pretreatment autologous lymphocytes or autologous remission lymphocytes overnight and an IFNγ ELISA was performed on the supernatant. Anti–CD19-CAR–transduced T cells of patient 6 specifically recognized pretreatment lymphocytes but not remission lymphocytes obtained 2 weeks after CAR–transduced T-cell infusion. Seventy-six percent of the pretreatment lymphocytes were CD19⁺ leukemia cells. The remission lymphocytes contained only 0.1% CD19⁺ cells. In both panels D and E, pretreatment lymphocytes cultured alone did not produce detectable quantities of IFNγ.

Figure 3

Normal and malignant B-lineage cells were eliminated from the blood and BM of patient 3. (A) Before treatment, the blood of patient 3 contained an elevated number of B cells, 96% of which were leukemia cells. After treatment, the blood B-cell count has remained below normal and patient 3 has been in complete remission for 67 weeks. B cells were quantitated by flow cytometry staining of CD19⁺ cells. (B) CD19 IHC staining of the BM of patient 3 is shown before treatment and 13 weeks after treatment. The BM contained large numbers of CD19⁺ cells before treatment. Thirteen weeks after treatment, CD19⁺ cells were nearly absent. (C) CD20 IHC staining of the BM of patient 3 is shown before treatment and 13 weeks after treatment. The BM contained large numbers of CD20⁺ cells before treatment. Thirteen weeks after treatment, CD20⁺ cells were nearly absent. (D) Flow cytometric results of a BM aspirate from patient 3 are shown. Plots are gated on lymphoid cells by forward and side scatter. A monoclonal population of B cells expressing the characteristic CD19⁺ and CD5⁺ phenotype of CLL (circled) was present before treatment but not 13 weeks after treatment.

Figure 4

Patients receiving infusions of anti–CD19-CAR–transduced T cells had reductions in adenopathy and elimination of normal B cells. CT scans of patient 7 showed extensive adenopathy before treatment. This adenopathy regressed by day 32 after CAR-transduced T-cell infusion (arrow). The enlarged lymph nodes continued to substantially regress between 32 and 132 days after CAR-transduced T-cell infusion. (B) Patient 8 had a normal number of polyclonal blood B cells before treatment. B cells were eliminated from the blood after treatment and had not recovered 26 weeks after anti–CD19-CAR–transduced T-cell infusion. B cell counts were determined by flow cytometry for CD19 and confirmed by flow cytometry for CD20. In contrast to the B cells, blood T-cell counts (C) and NK-cell counts (D) rapidly recovered after treatment.

Figure 5

Anti–CD19-CAR–transduced T cells can be detected in the blood of patients for up to 181 days after infusion. (A) The percentage of total PBMCs that contained the anti-CD19 CAR gene was determined by quantitative PCR. In patients 1 through 6, the peak levels and persistence of cells containing the CAR gene varied. (B) The percentage of total PBMCs from patient 7 and patient 8 that contained the anti-CD19 CAR gene was determined by quantitative PCR. Compared with the other patients, these patients had substantially greater persistence of cells that contained the CAR gene. (C) Anti-CD19 CAR expression was detected ex vivo on PBMCs of patient 7 by flow cytometry staining with labeled CD19 protein (CD19-DDK) 13 days after CAR-transduced T-cell infusion. Only background levels of cells expressing the anti-CD19 CAR were detected before treatment. (D) In patient 8, T cells expressing the anti-CD19 CAR were detected by flow cytometry staining with labeled CD19 protein 13 days after CAR-transduced T-cell infusion. Only background levels of cells expressing the anti-CD19 CAR were detected before treatment. (C-D) The plots are gated on CD3⁺ lymphocytes. (E) PBMCs from patient 7 were cultured for 4 hours with either CD19⁺ CD19-K562 cells or NGFR-K562 control cells that do not express CD19. Before treatment, CD107a was not up-regulated on T cells after culture with either CD19-K562 cells or NGFR-K562 cells. In contrast, T cells from 8 days after infusion up-regulated CD107a after a 4-hour culture with CD19-K562 cells but not NGFR-K562 cells. All plots are gated on CD3⁺ lymphocytes.

Figure 6

Elevations in serum levels of IFNγ and TNF occurred in some patients after infusions of anti–CD19-CAR–transduced T cells. (A) Patients 1, 2, 4, and 5 did not have prominent elevations of serum IFNγ after cell infusion. (B) Patients 3, 6, 7, and 8 had prominent elevations of serum IFNγ after cell infusion. (C) Patients 1,2,4, and 5 did not have prominent elevations of serum TNF after cell infusion. (D) Patients 3, 6, 7, and 8 had prominent elevations of serum TNF after cell infusion. For panels A through D, day 0 is the day of CAR-transduced T-cell infusion, and serum IFNγ and TNF were determined by standard ELISAs performed on serum samples. (E) The correlation between the total sequential organ failure assessment (SOFA) scores of each patient and the areas under the curves of each patient's serum IFNγ level (pg/mL)×(days) from the day of CAR-transduced T-cell infusion until day 11 after T-cell infusion is shown. The total SOFA scores and the areas under the curves of serum IFNγ were correlated (Pearson r = 0.8, P = .02). (F) The correlation between the total SOFA scores of each patient and the areas under the curves of each patient's serum TNF level (pg/mL)×(days) from the day before CAR-transduced T-cell infusion until day 12 after T-cell infusion is shown. The total SOFA scores and the areas under the curves of serum TNF were correlated (Pearson r = 0.9, P = .001).

Figure 7

Large numbers of T cells producing cytokines in a CD19-specific manner can be detected in the blood of patients after anti–CD19-CAR–transduced T-cell infusions. (A) PBMCs were collected from patient 3, 14 days after infusion of CAR-transduced T cells. When the PBMCs were cultured for 6 hours with CD19-K562 cells that expressed CD19, a population of CD4+ cells that produced IFNγ was detected; in contrast, when the PBMCs were cultured with the control cells NGFR-K562 that lack CD19 expression, T cells did not produce IFNγ. PBMCs collected before the anti–CD19-CAR–transduced T-cell infusion did not produce IFNγ after incubation with either CD19-K562 or NGFR-K562. Plots are all gated on CD3⁺ lymphocytes. (B) PBMCs were collected from patient 7, 18 days after infusion of CAR-transduced T cells. When the PBMCs were cultured for 6 hours with CD19-K562 cells that expressed CD19, T cells that produced IFNγ, TNF, and IL-2 were detected. When the PBMCs were cultured with the control cells NGFR-K562 that lack CD19 expression, T cells did not produce IFNγ, TNF, or IL-2. PBMCs collected before the anti–CD19-CAR–transduced T-cell infusion did not produce IFNγ, TNF, or IL-2 after incubation with either CD19-K562 or NGFR-K562. Plots are all gated on CD3⁺ lymphocytes.