In this study we investigated HER2 targeted T-cells as a potential therapeutic agent for GBM and showed that T cells from GBM patients can be readily genetically engineered to be rendered HER2-specific. These effector cells recognized autologous HER2-positive GBMs including their CD133-positive stem cells in vitro and had potent antitumor activity in an orthotopic, xenograft model.
It has proved difficult to generate tumor-specific T cells in cancer patients using DC vaccines, most likely because tumor specific T cells are present at low frequency and in an anergic state due to the immunosuppressive environment induced by malignancies such as GBMs (10
). Adoptive T-cell transfer of ex vivo
expanded tumor-specific T cells may overcome these limitations and forced expression of antigen-specific CARs or transgenic α/β T-cell receptors (TCR) have been used to generate such effector T lymphocytes (23
). CARs combine the antigen-binding property of monoclonal antibodies with the lytic and self-renewal capacities of T cells and have several advantages over transgenic α/β TCR receptors (11
). CAR-expressing T cells recognize and kill tumor cells in an MHC nonrestricted fashion, so that target cell recognition by CAR T cells is unaffected by some of the major mechanisms by which tumors avoid MHC-restricted T-cell (α/β TCR) recognition, such as downregulation of HLA class I molecules and defective antigen processing (11
). In addition, most tumors do not express costimulatory molecules so that α/β TCR engagement is followed by incomplete T-cell activation. We (27
) and others (28
) have shown that CARs can overcome this limitation if they incorporate costimulatory endodomains within the chimeric receptor sequence or if they are expressed in virus specific cytotoxic T lymphocytes. Our current HER2-specific CAR with a CD28.ζ signaling domain induces immunostimulatory cytokine release, including IL-2, from transduced cells of 8 of 9 GBM patients following CAR engagement.
Several cell surface antigens have been identified as potential targets for GBM directed CAR T-cell therapies including HER2, IL-13Rα2, the EPH receptor A2 (EphA2) and EGFRvIII (14
). We chose HER2 since this antigen is expressed by a high percentage of tumors (>70%) and signaling through HER2 deregulates cell proliferation, inhibits apoptosis, and increases the metastatic potential of cancer cells (33
). Moreover, HER2 expression increases with the degree of anaplasia in glial tumors, and mutations in the HER2 signaling pathway have been identified in gliomas (35
) Our own results confirmed a high frequency of HER2 expression, showing the antigen on 9 of 10 GBM tumors by two independent methods (IHC and FACS analysis). As with other tumor associated antigens expressed by GBMs, there was moderate heterogeneity in the expression pattern of HER2 within individual tumors (36
), and optimum killing in a clinical setting may require targeting more than one tumor associated antigen or the induction of epitope spreading beyond the original targets to prevent immune escape (20
Despite the biological significance of HER2 signaling, the expression of HER2 on GBMs is low in comparison to breast cancer, rendering HER2 monoclonal antibodies ineffective (19
). HER2-specific T cells allow targeting of the relatively low levels of HER2 expressed by GBMs because the overall avidity of receptors arrayed on a T-cell is greater than the avidity of a bivalent antibody, and engagement of even a limited number of T cell receptor molecules is sufficient to trigger a cytotoxic effector response (39
). One concern of targeting HER2-expressing malignancies with T cells are “ off target” effects since the administration of HER2 monoclonal antibodies has been associated with side effects, the most concerning of which is the poorly understood cardiac toxicity. If HER2-specific T cells are long lived, this problem could be accentuated in severity and persistence. Potential cardiac toxicities are difficult to model in mice since T cells that are specific for human HER2 do not recognize murine HER2. Nonetheless, we have shown that HER2-negative cells are not killed by HER2-specific T cells. In addition, primary endothelial and epithelial cells (Supplemental Figure 4
) do not activate HER2-specific T cells and 5 patients who received HER2-specific T cells had no dose limiting toxicities (41
). Finally, HER2 vaccines are well tolerated, and no cardiac toxicities were observed in patients, who developed HER2-specific T-cell responses (43
CD133 expression has identified a population with stem-cell-like properties in normal and cancerous tissues of the central nervous system (4
). In GBMs these cells are chemo- and radiotherapy resistant and most likely contribute to the ineffectiveness of conventional therapies. For example, while temozolomide was reported to preferentially deplete CD133-positive stem cells in primary GBMs in vitro
, this effect was absent in O(6
)-methylguanine-DNA-methyltransferase (MGMT)-non-methylated tumors which represent 50-70% of primary GBMs and carry a worse prognosis (44
). The use of temozolomide in a randomized prospective clinical trial in patients with GBM has thus only resulted in a marginal survival advantage (14.6 versus 12.1 months) (3
). We show here that CD133-positive GBM stem cells express HER2 and are killed by autologous HER2-specific T cells in vitro and in vivo similar to CD133-negative GBM cells. Hence immune targeted therapies may eradicate malignant stem cells that are resistant to conventional therapy.
Several studies have reported the infusion of activated T lymphocytes systemically or into resection cavities of recurrent or progressive malignant gliomas (45
). These infusions were safe and resulted in disease stabilization and in some instances in partial regression. We injected HER2-specific T cells directly into autologous GBM xenografts in tumor bearing mice. Tumors from patients 2 and 3 regressed completely without the need for exogenous cytokines, whereas untreated tumors and tumors treated with non-transduced T cells continued to grow. Tumors from patient 5 only had a partial response. Initial analysis revealed no significant differences in the level of HER2 expression as well as the in vitro effector function of HER2-specific T cells from these three patients. We are now investigating other biological differences beteween the tumors that may account for variability in effector cell sensitivity.
Tumors recurred in a number of animals most likely due to limited T cell persistence in vivo
. Kahlon et al.
reported complete regression and no recurrence of U87 gliomas in an orthotopic, xenogeneic SCID model after intratumoral injection of T cells expressing an IL-13Rα2-specific CAR (48
). In their model, the U87 glioma cell line was genetically modified to secrete IL-2, a cytokine critical for T-cell survival and expansion in vivo
. Tumor recurrence in our model may be due to inadequate persistence of human HER2-specific T cells in the xenograft, a known limitation of SCID mouse models.
In summary, this study shows that HER2-specific T cell can be readily generated from GBM patients by gene transfer with HER2-specific CARs. HER2-specific T cells recognized and killed autologous HER2-positive GBM, including CD133-positive stem cells, ex vivo, and induced regression of experimental GBMs in vivo. Hence, the adoptive transfer of HER2-redirected T-cells may be an attractive immunotherapeutic approach for GBM.