High-grade gliomas are aggressive tumors and respond poorly to all treatment modalities. Since these tumors are highly infiltrative, surgical resection leaves a residuum of cells responsible for recurrence. Malignant gliomas are almost uniformly lethal, with a median survival of about 15 months. This underscores the need for effective, non-toxic strategies that can eliminate the residual tumor cells and also, perhaps, immunize the endogenous immune system against the tumor.
Our immunotherapy approach originates from a series of studies, in which T cells have been redirected to EpCam on adenocarcinomas ; HER2/neu on prostate , breast , and ovarian cancer ; EGFR on a variety of tumor types ; and CD20 on malignant B cells . These studies were performed in vitro, in small animal models, and have progressed to clinical trials . In a recent study, we have shown the involvement of Granzyme B (GrzB) and IFN-γ signaling pathways in BiAb armed ATC mediated cytotoxicity of target cells .
This study shows that both long-term glioma lines and primary cultures of freshly-resected glioblastoma are efficiently killed by ATC armed with either HER2Bi or EGFRBi, indicating that both antigens are potential targets. We also showed that killing is not enhanced by using both BiAbs simultaneously. It may be that, in future studies, individual gliomas will show differential expression of HER2/neu and EGFR and that it will be prudent to both phenotype and functionally test each tumor as a target, in order to choose the best BiAb.
Cells with the stem-like property of self-renewal and the ability to differentiate into the bulk population of tumor cells have been identified in a number of different solid tumor types, amongst the best characterized of which are breast  and gliomas . CD133 was the initial marker identified as characterizing the glioma cancer stem cell, although there are subsequent reports of CD133– cells with similar behavior . The important additional features of stem cells are that they have been postulated to be both chemo- and radioresistant and to be responsible for the extensive infiltration seen in gliomas . The ability to kill CD133+ and CD133− cells, as shown above, indicates that these stem-like cells may be susceptible to killing in this system.
We showed that TMZ-resistant U251MG cells are also susceptible to targeted killing and that armed ATC still kill in the presence of TMZ. TMZ-resistant U251MG cells were <1% CD133+. This may be due to the length of time this line has been in culture, but their susceptibility does indicate that glioma cells that do not have stem-like properties but acquire chemoresistance are also suitable targets for BiAb-armed ATC. We also demonstrated the radioresistance of armed ATC effector function and an indication that irradiation of ATC may cause an increase in cytotoxicity. One possible interpretation is that there is a radiosensitive population of cells in the ATC that suppresses cytotoxic activity. Whether this involves active suppression or merely reflects death of the radiosensitive cells remains unclear. These results suggest that patients undergoing conventional chemoradiation may be suitable candidates for treatment with armed ATCs.
Some tumor types have been shown to produce soluble factors that inhibit immune effectors  and others to express membrane molecules, such as FasL that actually kill effectors [23–29]. We ruled-out the former by showing that long-term incubation of ATC in culture supernatants from immortalized malignant glioma lines and ex vivo gliomas and non-neoplastic astrocytes do not inhibit killing activity. We tested the latter, using long-term tumor lines and showed that these, at least, do not diminish the cytotoxic activity of armed ATC. That is, co-culture of armed ATC with glioma cells permits repeat killing .
Finally, we also showed that when armed ATC are incubated with targets, there is increased secretion of three Th1 cytokines (IFN-γ, GM-CSF, and TNF-α) and one Th2 cytokine (IL-13). The armed ATC are the presumed source of the Th1 cytokines and their secretion would serve to activate microglia (IFN-γ), act as an adjuvant for immunization of endogenous lymphocytes (GM-CSF) and potentially augment the killing of target tumor cells (TNF-α). The cellular origin of the IL-13 is probably also the ATC, since CD8+ T cells have been shown to secrete this cytokine . However, an IL-13 receptor is expressed on some glioma cells  and IL-13 has been shown to be an autocrine growth factor for both Reed-Sternberg cells in Hodgkin’s disease [32–35] and pancreatic cancer . The potential contribution of the glioma cells to the increased IL-13 is under investigation.
Several studies have shown promising results in glioblastoma using various immunotherapeutic approaches . Lymphokine-activated killer (LAK) cells generated from PBMC by co-culture with IL-2 have been reported to selectively kill glioma cells in vitro and when placed into the resection cavity with minimal systemic or neurological side effects [38, 39]. In the first study in which cells were targeted to WHO grade III/IV gliomas, LAK cells were treated with a conjugate of anti-CD3 cross-linked to the NE-150 monoclonal antibody which recognizes an epitope of NCAM . In the control group which received untreated LAK cells, 9/10 patients experienced recurrence within 1 year and 8/10 died within 4 years. In the experimental group, 2/10 showed no response, 4/10 showed regression and 4/10 had complete response. No recurrences occurred during 10—18 months of follow-up in the 8 patients showing partial or complete response. Subsequently, others have reported the use of different BiAbs both in vitro and early-stage clinical trials [41–46].