Gene expression profile correlates with T-cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy.

PURPOSE: To assess the feasibility, safety, and toxicity of autologous tumor lysate-pulsed dendritic cell (DC) vaccination and toll-like receptor (TLR) agonists in patients with newly diagnosed and recurrent glioblastoma. Clinical and immune responses were monitored and correlated with tumor gene expression profiles. EXPERIMENTAL DESIGN: Twenty-three patients with glioblastoma (WHO grade IV) were enrolled in this dose-escalation study and received three biweekly injections of glioma lysate-pulsed DCs followed by booster vaccinations with either imiquimod or poly-ICLC adjuvant every 3 months until tumor progression. Gene expression profiling, immunohistochemistry, FACS, and cytokine bead arrays were performed on patient tumors and peripheral blood mononuclear cells. RESULTS: DC vaccinations are safe and not associated with any dose-limiting toxicity. The median overall survival from the time of initial surgical diagnosis of glioblastoma was 31.4 months, with a 1-, 2-, and 3-year survival rate of 91%, 55%, and 47%, respectively. Patients whose tumors had mesenchymal gene expression signatures exhibited increased survival following DC vaccination compared with historic controls of the same genetic subtype. Tumor samples with a mesenchymal gene expression signature had a higher number of CD3(+) and CD8(+) tumor-infiltrating lymphocytes compared with glioblastomas of other gene expression signatures (P = 0.006). CONCLUSION: Autologous tumor lysate-pulsed DC vaccination in conjunction with TLR agonists is safe as adjuvant therapy in newly diagnosed and recurrent glioblastoma patients. Our results suggest that the mesenchymal gene expression profile may identify an immunogenic subgroup of glioblastoma that may be more responsive to immune-based therapies.

Central nervous system tumor immunity generated by a recombinant listeria monocytogenes vaccine targeting tyrosinase related protein-2 and real-time imaging of intracranial tumor burden.

OBJECTIVE: Previously, we demonstrated that a recombinant Listeria monocytogenes (rLM) vector encoding the melanoma-associated antigen, tyrosinase related protein (TRP)-2, could successfully treat subcutaneous B16 melanomas. The purpose of the present study was twofold: 1) to test whether this rLM-nucleoprotein (NP)/TRP-2 could generate antitumor immunity to a B16 tumor challenge in the immunologically privileged central nervous system (CNS) and 2) to develop a noninvasive imaging modality to monitor tumor progression in the brain after immunotherapy. METHODS: Mice were vaccinated with either a control rLM strain expressing only a viral antigen (rLM-NP) or a strain expressing both the viral epitope and TRP-2 (rLM-NP/TRP-2). These mice were then analyzed for their ability to mount tumor-specific T-cell responses, to generate protective antitumor immunity to a CNS tumor challenge, and for the localization of T cells at the tumor site. To noninvasively measure tumor growth within the CNS in vivo, we developed a B16 cell line expressing firefly luciferase that could be readily detected via bioluminescent imaging. RESULTS: Vaccination with rLM-NP/TRP-2 induced a robust, tumor-specific CD8 T-cell response to the dominant cytotoxic T lymphocyte epitope of TRP-2 and selective interferon-gamma secretion when cocultured with B16 melanoma cells in vitro. Significant decreases in CNS tumor sizes were easily visualized in mice vaccinated with rLM-NP/TRP-2 compared with mice that received a control rLM expressing the NP epitope alone (rLM-NP). The subsequent decreased tumor size and extension of survival induced by rLM-NP/TRP-2 was similarly associated with an early increase of tumor infiltrating T cells. CONCLUSION: The ability to treat tumors arising within the CNS is difficult because of the nature of the anatomic confines of the brain and a microenvironment that may not promote immune responsiveness. These studies describe an in vivo bioluminescent imaging system to monitor CNS tumor growth in mice, which we successfully used to document decreased intracranial tumor progression and size after vaccination with rLM-NP/TRP-2. The results suggest that metastatic tumors in the CNS can be targeted immunotherapeutically without overt autoimmune toxicity.

Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment.

PURPOSE: We previously reported that autologous dendritic cells pulsed with acid-eluted tumor peptides can stimulate T cell-mediated antitumor immune responses against brain tumors in animal models. As a next step in vaccine development, a phase I clinical trial was established to evaluate this strategy for its feasibility, safety, and induction of systemic and intracranial T-cell responses in patients with glioblastoma multiforme. EXPERIMENTAL DESIGN: Twelve patients were enrolled into a multicohort dose-escalation study and treated with 1, 5, or 10 million autologous dendritic cells pulsed with constant amounts (100 mug per injection) of acid-eluted autologous tumor peptides. All patients had histologically proven glioblastoma multiforme. Three biweekly intradermal vaccinations were given; and patients were monitored for adverse events, survival, and immune responses. The follow-up period for this trial was almost 5 years. RESULTS: Dendritic cell vaccinations were not associated with any evidence of dose-limiting toxicity or serious adverse effects. One patient had an objective clinical response documented by magnetic resonance imaging. Six patients developed measurable systemic antitumor CTL responses. However, the induction of systemic effector cells did not necessarily translate into objective clinical responses or increased survival, particularly for patients with actively progressing tumors and/or those with tumors expressing high levels of transforming growth factor beta(2) (TGF-beta(2)). Increased intratumoral infiltration by cytotoxic T cells was detected in four of eight patients who underwent reoperation after vaccination. The magnitude of the T-cell infiltration was inversely correlated with TGF-beta(2) expression within the tumors and positively correlated with clinical survival (P = 0.047). CONCLUSIONS: Together, our results suggest that the absence of bulky, actively progressing tumor, coupled with low TGF-beta(2) expression, may identify a subgroup of glioma patients to target as potential responders in future clinical investigations of dendritic cell-based vaccines.

Modulation of major histocompatibility complex Class I molecules and major histocompatibility complex-bound immunogenic peptides induced by interferon-alpha and interferon-gamma treatment of human glioblastoma multiforme.

OBJECT: Little is known about the quantitative modulation of major histocompatibility complex (MHC) Class I expression on human gliomas that is effected by interferons; even less is known about the immunogenic peptides that are accommodated in the peptide-binding motifs of MHC Class I alleles in these brain tumors. In this article the authors investigated the ability of interferon (IFN)alpha and IFNgamma to upregulate MHC Class I expression and to modulate acid-eluted Class I-bound peptides on human glioblastoma multiforme (GBM) cells in vitro. METHODS: Early-passage primary human GBM cell cultures and U87MG GBM cells were incubated with varying doses of INFalpha or IFNgamma ranging between 0 and 2000 U/ml. Upregulation of MHC Class I expression was assayed by immunocytochemical analysis, flow cytometry, and Western blot analysis. Modulation of acid-eluted MHC Class I-bound peptides from the IFN-treated GBM cells was examined with the aid of mass spectroscopy. The in vitro expression of the MHC Class I molecule was upregulated by both IFNalpha and IFNgamma in a dose-dependent manner. Interferon-gamma exhibited a more potent effect on MHC Class I upregulation, peaking at 10 U/ml; whereas the effect of IFNalpha was less marked, reaching a plateau at 500 U/ml. In addition, a native peptide eluted from MHC Class I molecules of human GBM cells was identified and found to be consistently upregulated by IFN treatment. CONCLUSIONS: Interferon-alpha and IFN-gamma can significantly upregulate the MHC Class I molecules that are expressed on the cell surface of human GBM cells as well as the potentially immunogenic peptides bound to the MHC. These results may help explain the molecular basis for increased immunogenicity with IFN treatment of human GBMs and might provide added insight into the design of future antitumor vaccines for human brain tumors.

Immunotherapeutic targeting of shared melanoma-associated antigens in a murine glioma model.

Immune-based treatments for central nervous system gliomas have traditionally lagged behind those of more immunogenic tumors such as melanoma. The relative paucity of defined glioma-associated antigens that can be targeted by the immune system may partially account for this situation. Antigens present on melanomas have been extensively characterized, both in humans and in murine preclinical models. Melanocytes and astrocytes are both derived embryologically from the neural ectoderm. Their neoplastic counterparts, malignant melanomas and gliomas, have been shown in humans to share common antigens at the RNA level. However, little is known concerning whether gliomas can be targeted by immune-based strategies that prime T cells to epitopes from melanoma-associated antigens (MAAs). In this study, we provide evidence that two common murine glioma cell lines (GL26 and GL261) express the melanoma antigens gp100 and tyrosinase-related protein 2 (TRP-2). To understand the immunogenicity of murine gliomas to CD8(+) T cells, we examined the ability of a MAA-specific CTL cell line to lyse the glioma cells, as well as the in vivo expansion of MAA-specific CD8(+) T cells in animals harboring gliomas. Both glioma cell lines were lysed by a human gp100-specific CTL cell line in vitro. Mice harboring s.c. GL26 gliomas possessed TRP-2-specific CD8(+) T cells, providing further evidence that these gliomas express the protein products in the context of MHC class I. Furthermore, MAA peptide-pulsed dendritic cells could prime T cells that specifically recognize GL26 glioma cells in vitro. Lastly, mice that were prevaccinated with human gp100 and TRP-2 peptide-pulsed dendritic cells had significantly extended survival when challenged with tumor cells in the brain, resulting in >50% long-term survival. These results suggest that shared MAAs on gliomas can be targeted immunotherapeutically, pointing the way to a new potential treatment option for patients with malignant gliomas.

MART-1 adenovirus-transduced dendritic cell immunization in a murine model of metastatic central nervous system tumor.

Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially. Genetic modifications of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector. Sixty-two C57BL/6 mice were immunized subcutaneously with AdVMART-1-transduced DCs (n = 23), untransduced DCs (n = 17), or sterile saline (n = 22). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes from representative animals in each group were harvested for standard cytotoxicity (CTL) and enzyme-linked immunospot (ELISPOT) assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with an adenoviral vector encoding the MART-1 antigen elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared to control-immunized mice with intracranial B16 tumors, AdMART-1-DC vaccination was able to elicit partial protection against central nervous system tumor challenge in vivo.

Tumor immunity within the central nervous system stimulated by recombinant Listeria monocytogenes vaccination.

Tumors arising within the central nervous system (CNS) present the immune system with a challenging target, given the heterogeneous nature of these neoplasms and their location within an "immunologically privileged" site. We used the lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a pseudotumor antigen to investigate recombinant Listeria monocytogenes as a tumor vaccine against s.c. and intracerebral challenges with a NP-expressing glioma, 9L-NP. Using Fischer 344 rats, we demonstrate that vaccination with recombinant L. monocytogenes-NP stimulates protection against s.c., but not intracerebral, 9L-NP tumor challenge in an antigen-specific, CD8(+) T-cell-dependent manner. After s.c. tumor rejection, enhanced antitumor immunity is achieved via epitope spreading that permits complete resistance against lethal intracerebral challenge with 9L-NP and with the untransfected parental 9L tumor. Unlike the CD8(+)-dependent immune responses against s.c. 9L-NP tumors, this expanded intracerebral immunity against endogenous tumor-associated antigens is dependent on both CD4(+) and CD8(+) T cells. Taken together, these results demonstrate that the mechanisms of tumor immunity within the brain are different from those elicited against non-CNS tumors. Furthermore, vaccination approaches exploiting the concept of epitope spreading may enhance the efficacy of antitumor immune responses within the immunologically privileged CNS, potentially mediating tumor cell killing through both CD4(+)- and CD8(+)-dependent effector pathways.