Artificial chimeric immunoreceptors offer the possibility of reprogramming T cells for efficient targeting of tumors in an HLA-independent fashion. However, while initial clinical studies demonstrate feasibility with the retargeted T cells, poor in vivo
persistence and low expression of the transgene have been documented, and these limitations have reduced potential clinical activity [2
]. To address these issues, our studies have used a robust pre-clinical model, and we demonstrate that a single infusion of as few as 2 million engineered T cells could control and in some cases, eliminate pre-established disseminated leukemia. Surprisingly, expression of the CD137 signaling domain rather than the CD28 domain was most correlated with reprogramming T cells for persistence in vivo
Previous in vitro
studies have characterized the incorporation of CD137 domains into CARs [10
]. Our results represent the first in vivo
characterization of these CARs and uncover several important advantages of CARs that express CD137 that were not revealed by the previous in vitro
studies. We demonstrated that CARs expressing the CD137 signaling domain could survive for at least 6 months in mice bearing tumor xenografts. This may have significant implications for immunosurveillance, as well as for tumor eradication. For example, in a mouse prostate cancer xenograft model, survival of CAR+ T cells for at least a week was required for tumor eradication [30
Long term survival of the CARs did not require administration of exogenous cytokines, and these results significantly extend the duration of survival of human T cells expressing CARs shown in previous studies [17
]. To our knowledge, this is the first report demonstrating elimination of primary leukemia xenografts in a pre-clinical model using CAR+ T cells. Furthermore, complete eradication was achieved in some animals in the absence of further in vivo
therapy, including prior chemotherapy or subsequent cytokine support.
The long-term control of well established tumors by immunotherapy has rarely been reported. Most pre-clinical models in a therapeutic setting have tested tumors that have been implanted for a week or less before initiation of therapy [32
]. After establishing leukemia two to three weeks before T cell transfer, we found that many animals had long-term control of leukemia for at least 6 months. The effect was specific for the chimeric receptor, as animals injected with an equivalent number of unmodified T cells had high blast counts within 3weeks (). The efficacy of targeted, adoptive immunotherapy in this xenograft model of primary human ALL compares favorably to our prior experience testing the antileukemic efficacy of single cytotoxic [27
, and unpublished data] or targeted agents [26
], where we have observed extension of survival but not cure of disease. Additionally, we have not previously observed the ability to control xenografted ALL for a period of as long as 6 months.
It is likely that several mechanisms account for the enhanced efficiency of the redirected T cells observed in the present report. First, previous studies have generally used T cells after a culture for a month or longer [2
]. In the present work we have used an efficient bead based artificial APC, which shortens the culture to approximately 10 days, and permits the use of the T cells early at a time when we have shown previously that the average telomere length of the cultured T cells is actually longer than at the start of culture [33
]. We attribute this to the previous demonstration that the anti-CD28 driven culture system induces telomerase activity [34
]. Furthermore, the addition of CD28 to culture conditions promotes transduction of central memory T cells [35
]. CD28 bead-based cell expansion has the capacity to routinely generate >1010
CAR+ T cells in approximately 10 days using FDA compliant manufacturing procedures already in use for clinical trials in humans [36
Second, previous studies have generally used murine retroviruses or electroporation to introduce the chimeric receptor [2
]. We have used lentiviral gene transfer which permits highly efficient engineering of T cells with >85% successful gene transfer [24
]. As shown in our study, CAR expression was maintained for at least 6 months in vivo
with no evidence of silencing using the EF-1α promoter. In comparison, murine retroviral vectors have been shown to exhibit significant silencing of gene expression over time, despite the incorporation of elements such as chromatin-insulator sequences.
Similar to other groups who have evaluated CARs incorporating costimulatory domains [9
], we confirmed that the addition of the CD28 intracellular domain into CARs enhances the in vitro
proliferation and cytokine production of T cells stimulated through these receptors. Interestingly, the αCD19-BB-ζ CAR appears to antagonize the production of the type II cytokines, IL-4 and IL-10, but it remains unclear whether this was due to direct signaling or to selective outgrowth of Th1-like cells.
While the CD28 cytoplasmic domain has been reported to significantly enhance the anti-tumor efficacy of CAR-expressing T cells in a number of models, the present study is the first to show that incorporation of the CD137 cytoplasmic domain into a CAR augments in vivo
efficacy. In other studies using CAR+ T cells and trans costimulation of CD28 and CD137 through genetic expression of CD80 and 4-1BBL, redirected T cells were also found to have potent antitumor effects [31
]. The significantly enhanced anti-leukemic activity in vivo
is associated with the improved persistence of the CAR+ T cells. Our results indicate that enhanced survival and/or proliferation of CAR+ T cells contribute to the increased antitumor effects.
CD137 plays an important role in T cell proliferation and survival, particularly for T cells within the memory T cell pool [42
]. CD137 mediates its effects on T cell survival and proliferation through activation of the AKT/mTOR pathway [43
] and the upregulation of the anti-apoptotic genes, Bcl-XL
and BFL-1 [44
]. Surprisingly, the αCD19-28-ζ modified T cells failed to show a significant improvement in anti-leukemic efficacy in vivo
using our primary pre-B ALL model compared with the αCD19-ζ modified T cells. This is in contrast to other models using solid tumors including B cell-derived tumors and a recent study by our laboratory using a mesothelin-directed CAR where CD28 domain-containing CARs show enhanced anti-tumor efficacy [45
]. Together, these studies suggest that the optimal signals produced by CARs may be dependent upon the particular tumor being targeted and/or the nature of the particular scFv antibody.
Our studies are the first to reveal antigen-independent effects of the αCD19-BB-ζ receptor on T cells. This receptor, while capable of triggering cytotoxicity in an antigen-dependent fashion, also significantly prolonged the initial blast-phase of T cell activation. There are several possible mechanisms by which the CARs could deliver antigen-independent signals. CARs, like some natural receptors, may deliver tonic ligand-independent signals. Impairment of the regulatory mechanisms that normally extinguish receptor signaling such as the SHP-1 and PTPH1 phosphatase that dephosphorylate the TCR-ζ ITAMs might be impaired, leading to the antigen-independent effects observed in this study. While CARs appear to exist predominantly as homodimers, these artificially-constructed receptors might also spontaneously aggregate into oligomers, especially at the high levels of expression possible with the EF-1α promoter.
The enhanced growth effects of the αCD19-BB-ζ receptor are consistent with the antigen-independent growth effects that are observed in T cells stimulated through the natural CD137 receptor by agonist monoclonal antibody [23
]. Normally, CD137 expression is tightly regulated on T cells with expression limited to a window of a few days following T cell activation or following IL-15 treatment [47
]. The 4-1BB/4-1BBL interaction has been proposed as one mechanism by which IL-15 mediates its effect on memory T cells under limiting CD137 expression [42
]. The antigen-independent signals derived from the CD137 domain within the CAR may be critical to the anti-leukemic effects observed in our study, analogous to the continued presence of a 4-1BB agonist antibody, and ectopic trans expression of 4-1BBL, both of which have been shown to promote antitumor effects in vivo
]. Although previously unreported, these antigen-independent effects of CARs have important implications for the clinical use of these receptors.