Insufficient persistence, expression, and effector function of CARs in vivo has resulted in limited success in the trials testing first-generation CAR T cells (14
). Because preclinical modeling demonstrated enhanced persistence of CARs that incorporated a 4-1BB signaling molecule (7
), we developed second-generation CARs engineered with lentiviral vector technology—an approach that was previously found to be safe in the setting of chronic HIV infection (27
). Our results show that when second-generation CARs were expressed in T cells and cultured under conditions designed to promote engraftment of central memory T cells (28
T cell expansion after infusion was improved compared to previous reports. Moreover, CART19 cells induced a CD19-specific cellular memory response. These CART19 cells tracked efficiently to sites of tumor and became established as de facto “tumor-infiltrating lymphocytes” that exhibited CD19 specificity and retained effector function in vivo in patient blood and marrow specimens for months.
What drives the expansion and persistence of CART19 cells in vivo? Our study is one of few trials to have omitted IL-2 infusions (17
); thus, the CART19 cells likely expanded in response either to homeostatic cytokines or to CD19 expressed on leukemic targets and/or normal B cells. Indeed, the kinetics of cytokine release in serum and BM after the introduction of CART19 into patients correlated with peak CART19 numbers, which suggests that the decline in CART19 numbers may be initiated when cellular targets expressing CD19 become limiting. The extended survival of these cells may be due in part to the incorporation of the 4-1BB domain into the CAR itself or because of signaling through either the natural T cell receptor (TCR) or CAR. Another possibility is related to the presence of CART19 cells in BM specimens: CART19 cells could be maintained by encountering B cell progenitors in the BM. This potential stimulation of CART19 cells by normal B cells may provide a mechanism for CAR memory by means of “self-vaccination/boosting” and, therefore, long-term tumor immunosurveillance. Although the mechanisms driving CART19 homeostasis remain unclear, CAR therapy is clearly not always a transient form of immunotherapy, as has been previously supposed. Thus, CARs with optimized signaling domains may have a role both in remission induction and consolidation and in continued immunosurveillance in cancer patients.
Our studies indicate that persisting CART19 cells consist of both central and effector memory T cells, which likely contributes to their long-term survival compared to previous reports. Signaling of 4-1BB has been reported to promote the development of memory in the context of TCR signaling (30
). But whether CAR T cells can differentiate in vivo into a central memory–like state upon encounter and subsequent elimination of target cells expressing the surrogate antigen remains to be resolved.
We have observed potent antileukemic effects in all three patients examined (9
), including two patients with p53-deficient leukemia. Previous studies with CARs have had difficulty separating antitumor effects from lymphodepleting chemotherapy. However, the delayed cytokine release, combined with the kinetics of tumor lysis in fludarabine-refractory patients that was coincident, and possibly dependent, on in vivo CAR expansion in our study, indicates that CART19 cells mediate potent anti-tumor effects. The present results do not exclude a role for chemotherapy in potentiating the effects of CARs, and a number of studies suggest plausible mechanisms for coordinate effects of chemotherapy and CAR T cells (31
) in addition to the lymphodepleting aspects of chemotherapy, which promotes homeostatic expansion of T cells (33
), including, presumably, CART19 cells.
We have found that very low doses of CARs can elicit potent clinical responses. However, the results to date do not support an obvious dose-response relationship. This pilot study was not designed to determine optimal biologic dose with what is essentially a dynamic cell product, but to demonstrate safety of the CAR19 vector design. Nonetheless, the observation that doses of CART19 cells several orders of magnitude below those tested in previous trials can have clinical benefit may have important implications for future implementation of CAR therapy on a wider scale as well as for the design of trials testing CARs directed against targets other than CD19.
Although our second-generation CAR T cells led to considerable clinical effects, the lysis of at least a kilogram of tumor burden in all three patients was accompanied with the delayed release of potentially dangerously high levels of cytokines in two of the patients (9
). We did not observe classical cytokine storm effects; however, our trial was designed to mitigate this possibility by deliberate infusion of CART19 cells over a period of 3 days and by using signaling domains that did not promote secretion of IL-2 and TNF-α.
Humoral and cellular immunity against CAR-modified T cells has been reported in other studies (34
). The presence of cells that express surface CAR19 at 6 and 9 months after T cell infusion strongly suggests the absence of cellular and humoral immune responses against CART19 cells. The absence of antibody responses is not surprising because the therapy effectively eliminated B cells in patients. On the other hand, the absence of cellular immunity against CART19 cells is perhaps surprising because the CAR19 construct contains both murine sequences (the antibody determinants) and unique junctional fragments between the different components of the CAR19 construct. These results raise the possibility that B cell help may be required to prime T cell responses. It remains to be determined whether the severe immunosuppression at baseline in the heavily pretreated CLL patients might have contributed to the inability to reject the CART19 cells.
We used lentivirus vectors to transfer CAR19 into patient T cells. Lentiviral vectors have the potential to be safer than retroviruses from the perspective of insertional mutagenesis, and they have substantially higher efficiency for genetically engineering human T cells (36
). We conducted the first study using a lentiviral vector for gene transfer in HIV (27
). In that study, and in patients enrolled in subsequent lentiviral vector–engineered T cells studies (clinicaltrials.gov NCT00295477 and NCT00622232), no adverse events related to gene transfer have been observed in more than 280 patient-years of observation. Analysis of vector integration sites in several of these patients has not revealed any evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes (38
). Nonetheless, it will be important to continue to monitor patients enrolled in this and other lentiviral gene transfer clinical trials carefully, particularly when new constructs or targets are tested.
A thorough comparison of the vector, transgene, and cell manufacturing procedures with results from ongoing studies at other centers will be required to gain a full understanding of the key features required to obtain sustained function of CAR-expressing T cells in vivo. Unlike antibody therapies, CAR-modified T cells have the potential to replicate in vivo, and long-term persistence could lead to sustained tumor control and obviate the need for repeated infusions of antibody. The availability of an off-the-shelf therapy composed of non–cross-resistant killer T cells has the potential to improve the outcome of patients with B cell malignancies.