Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma

Abstract

We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4(+) cells and central memory cells (CD45RO(+)CD62L(+)) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.

Figure 1

Composition of GD2–T-cell products. The cellular composition of GD2-CTL (A) and GD2-ATC (B) lines was analyzed using flow cytometry. Both products were polyclonal with a predominance of CD8⁺ T cells, followed by a smaller percentage of CD4⁺ cells and NK cells within each product line. Furthermore, as expected, GD2-CTL lines were terminally differentiated and composed of mostly effector memory cells (C), whereas GD2-ATCs (D) had a higher percentage of naive and central memory cells within each T-cell line.

Figure 2

First-generation GD2-CAR T cells can be detected in the peripheral blood of patients for a prolonged period of time. Real-time quantitative PCR was used to assess the presence and persistence of CAR–T cells. GD2-ATCs or GD2-CTLs could be detected, at or after 6 weeks after infusion, in the circulation of 11 of 19 (58%) patients. Although the levels of detection were low, transgenic signals could be identified at 96 weeks for CAR-CTLs and 192 weeks for CAR-ATCs after infusion. The estimate of frequency of CAR– T cells was obtained using standard curves of DNA from tumor cell lines transduced with the retroviral vectors encoding each CAR. Integrant analysis showed between 6 and 8 proviral integration sites per cell. Sensitivity assays in which transduced T cells were diluted with nontransduced T cells showed unequivocal detection ability when 1 transduced cell was diluted in 1000 to 6000 nontransduced cells, corresponding to 0.0001% to 0.002% of the tumor cell lines used for the standard curve.

Figure 3

Prolonged detection of GD2–T cells within the peripheral blood of patients was highly concordant with the percentage of helper (CD4⁺ cells) and central memory cells (CD45RO⁺CD62L⁺) in the infused product. Using regression analysis, it was determined that each 1-unit increase in the number of CD4⁺ cells within the infused product was associated with a 2.28 increase in the log (duration) of CAR-CTL (A) and a 9.83 increase in the log (duration) of CAR-ATC (B). Further, each 1-unit increase in the percentage of central memory cells was associated with a 6.1 increase in the log (duration) of CAR-CTL (C) and a 6.63 increase in the log (duration) of CAR-ATC (D) in the T-cell product.

Figure 4

Detection of CAR–T cells for 6 weeks or more was associated with prolongation of TTP. (A) With a median follow-up of 329 days, the median OS was 931 days. (B) Patients with active disease at the time of infusion had a shorter OS compared with those with no evidence of disease. (C) In addition, for those with active disease at the time of infusion, the lack of CAR-ATCs or CAR-CTLs at or beyond 6 weeks was associated with a significantly shorter TTP.