Christopher J. Wheeler

Vaccination with Tumor Lysate-Pulsed Dendritic Cells Elicits Antigen-Specific, Cytotoxic T-Cells in Patients with Malignant Glioma

The primary goal of this Phase I study was to assess the safety and bioactivity of tumor lysate-pulsed dendritic cell (DC) vaccination to treat patients with glioblastoma multiforme and anaplastic astrocytoma. Adverse events, survival, and cytotoxicity against autologous tumor and tumor-associated antigens were measured. Fourteen patients were thrice vaccinated 2 weeks apart with autologous DCs pulsed with tumor lysate. Peripheral blood mononuclear cells were differentiated into phenotypically and functionally confirmed DCs. Vaccination with tumor lysate-pulsed DCs was safe, and no evidence of autoimmune disease was noted. Ten patients were tested for the development of cytotoxicity through a quantitative PCR-based assay. Six of 10 patients demonstrated robust systemic cytotoxicity as demonstrated by IFN-γ expression by peripheral blood mononuclear cells in response to tumor lysate after vaccination. Using HLA-restricted tetramer staining, we identified a significant expansion in CD8+ antigen-specific T-cell clones against one or more of tumor-associated antigens MAGE-1, gp100, and HER-2 after DC vaccination in four of nine patients. A significant CD8+ T-cell infiltrate was noted intratumorally in three of six patients who underwent reoperation. The median survival for patients with recurrent glioblastoma multiforme in this study (n = 8) was 133 weeks. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous tumor lysate-pulsed DC vaccine for patients with malignant glioma. We demonstrate for the first time the ability of an active immunotherapy strategy to generate antigen-specific cytotoxicity in brain tumor patients.

Vaccination elicits correlated immune and clinical responses in glioblastoma multiforme patients.

Cancer vaccine trials have failed to yield robust immune-correlated clinical improvements as observed in animal models, fueling controversy over the utility of human cancer vaccines. Therapeutic vaccination represents an intriguing additional therapy for glioblastoma multiforme (GBM; grade 4 glioma), which has a dismal prognosis and treatment response, but only early phase I vaccine trial results have been reported. Immune and clinical responses from a phase II GBM vaccine trial are reported here. IFN-gamma responsiveness was quantified in peripheral blood of 32 GBM patients given therapeutic dendritic cell vaccines. Posttreatment times to tumor progression (TTP) and survival (TTS) were compared in vaccine responders and nonresponders and were correlated with immune response magnitudes. GBM patients (53%) exhibited >or=1.5-fold vaccine-enhanced cytokine responses. Endogenous antitumor responses of similar magnitude occurred in 22% of GBM patients before vaccination. Vaccine responders exhibited significantly longer TTS and TTP relative to nonresponders. Immune enhancement in vaccine responders correlated logarithmically with TTS and TTP spanning postvaccine chemotherapy, but not with initial TTP spanning vaccination alone. This is the first report of a progressive correlation between cancer clinical outcome and T-cell responsiveness after therapeutic vaccination in humans and the first tracing of such correlation to therapeutically exploitable tumor alteration. As such, our findings offer unique opportunities to identify cellular and molecular components of clinically meaningful antitumor immunity in humans.

Clinical Responsiveness of Glioblastoma Multiforme to Chemotherapy after Vaccination

Purpose: Although the development of immune-based therapies for various cancers including malignant glioma has been heralded with much hope and optimism, objective clinical improvements in most vaccinated cancer patients have not been realized. To broaden the search for vaccine-induced benefits, we examined synergy of vaccines with conventional chemotherapy. Experimental Design: Survival and progression times were analyzed retrospectively in 25 vaccinated (13 with and 12 without subsequent chemotherapy) and 13 nonvaccinated de novo glioblastoma (GBM) patients receiving chemotherapy. Immune responsiveness and T-cell receptor excision circle (TREC) content within CD8+ T cells (CD8+ TRECs) was determined in vaccinated patients. Results: Vaccinated patients receiving subsequent chemotherapy exhibited significantly longer times to tumor recurrence after chemotherapy relative to their own previous recurrence times, as well as significantly longer postchemotherapy recurrence times and survival relative to patients receiving isolated vaccination or chemotherapy. Patients exhibiting objective (>50%) tumor regression, extremely rare in de novo GBM, were also confined to the vaccine + chemotherapy group. Prior tumor behavior, demographic factors, other treatment variables, distribution of vaccine responders, and patients with high CD8+ TRECs all failed to account for these differences in clinical outcome. Within all GBM patients receiving post-vaccine chemotherapy, however, CD8+ TRECs predicted significantly longer chemotherapeutic responses, revealing a strong link between the predominant T-cell effectors in GBM and tumor chemosensitivity. Conclusions: We propose that therapeutic vaccination synergizes with subsequent chemotherapy to elicit tangible clinical benefits for GBM patients.

HER-2, gp100, and MAGE-1 Are Expressed in Human Glioblastoma and Recognized by Cytotoxic T Cells

It has recently been demonstrated that malignant glioma cells express certain known tumor-associated antigens, such as HER-2, gp100, and MAGE-1. To further determine the possible utilization of these antigens for glioma immunotherapy and as surrogate markers for specific tumor antigen cytotoxicity, we characterized the presence of mRNA and protein expression in 43 primary glioblastoma multiforme (GBM) cell lines and 7 established human GBM cell lines. HER-2, gp100, and MAGE-1 mRNA expression was detected in 81.4%, 46.5%, and 39.5% of the GBM primary cell lines, respectively. Using immunoreactive staining analysis by flow cytometry, HER-2, gp100, and MAGE-1 protein expression was detected in 76%, 45%, and 38% of the GBM primary cell lines, respectively. HLA-A1-restricted epitope specific for MAGE-1 peptide (EADPTGHSY) CTL clone B07 and HLA-A2-restricted epitope specific for HER-2 peptide (KIFGSLAFL) CTL clone A05 and gp100 peptide (ITDQVPFSV) CTL clone CK3H6 were used in this study. The specificity of CTL clone was verified by HLA/peptide tetramer staining. Three CTL clones could efficiently recognize GBM tumor cells in an antigen-specific and MHC class I-restricted manner. IFN-γ treatment can dramatically increase MHC class I expression of GBM tumor cells and significantly increase CTL recognition of tumor cells. Treatment with the DNA hypomethylating agent 5-aza-2′-deoxycytidine induced and up-regulated the mRNA expression of MAGE-1 and epitope presentation by autologous MHC. These data indicate that HER-2, gp100, and MAGE-1 could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigen-specific active immunotherapy strategies for glioma patients.

Thymic CD8 + T Cell Production Strongly Influences Tumor Antigen Recognition and Age-Dependent Glioma Mortality

For unknown reasons, advanced age remains a dominant predictor of poor clinical outcome for nearly all cancers. A decrease in the production of T cells by the thymus accompanies normal aging and parallels the age-dependent increase in cancer progression, but the specific impact of immunity on tumor progression in general is unknown. Glioblastoma multiforme (GBM), the most common primary brain neoplasm, is characterized by rapid age-dependent rates of progression and death. In this study, we show levels of CD8+ recent thymic emigrants (RTEs) accounted for the prognostic power of age on clinical outcome in GBM patients. CD8+ RTEs, typically a tiny proportion of CD8+ T cells, remarkably accounted for the majority of tumor Ag-binding small precursor cells in PBMC from these patients and from healthy individuals. Large blasting tumor Ag-binding cells comprised of CD8+ RTEs and phenotypically related cells were predominantly expanded following experimental vaccination of GBM patients. Quantification of CD8+ RTE expansion in vivo correlated strongly with vaccine-elicited cytokine responses, and estimated numbers of expanding CD8+ RTEs were consistent predictors of clinical outcome in vaccinated GBM patients. Targeted mutant (CD8β−/−) mice specifically deficient in thymic CD8+ T cell production uniquely displayed an age-specific decrease in glioma host survival as well as a strong correlation between host survival and thymus cellular production. These findings suggest that levels and function of newly produced CD8+ T cells critically influence age-dependent cancer mortality and exert one of the strongest known influences on GBM outcome by predominantly mediating clinically beneficial antitumor immune responses.

DCVax®-Brain and DC vaccines in the treatment of GBM

Background: DCVax®-Brain (Northwest Biotherapeutics, Inc., Bethesda, MD, USA) is a personalized treatment for brain tumors. Its approach of administering autologous tumor antigen-bearing dendritic cells (DCs) has garnered hope for more effective and less toxic therapy for patients with malignant brain tumors including glioblastoma multiforme (GBM). DCVax-Brain composition and efficacy are not fully disclosed, although sponsors claim it is poised to critically test clinical DC vaccine efficacy in GBM patients. Objective: This review examines the efficacy of DC vaccine therapy in treating GBM patients. Review question: To determine if the approach of DC vaccination followed by DCVax-Brain shows ample clinical promise in GBM patients. Search strategy: All published reports of DC vaccination for GBM and press releases regarding DCVax-Brain findings were evaluated. Critical appraisal of reports and summary of outcomes: Published DC vaccine trials for high-grade glioma patients suggest favorable clinical outcomes not easily ascribed to non-treatment parameters. Evidence of possible selection bias exists in many reports, but efforts to account for this are evident in the most recent publications. Conclusion: DC vaccine trials provide evidence of low toxicity in GBM patients and effective induction of antitumor immunity in the latest publications correlate with clinical improvements. Preliminary reports on DCVax-Brain clinical outcomes seem to follow these trends.

Intratumoral T Cell Subset Ratios and Fas Ligand Expression on Brain Tumor Endothelium

SummaryIntroduction: T cell presence in TIL, and the ratio of CD8+ and CD4+ T cell subsets in particular, can correlate with tumor prognosis in some tumors, although the significance of such infiltration into glioma is controversial. However, gliomas represent a lower extreme in their extent of T cell infiltration, and are thus useful in assessing factors that can decrease T cell presence within tumor tissue. Fas ligand, a pro-apoptotic cell surface protein, may play a key role in reduction of T cells in tumor tissue. Objective: To assess the level of FasL expression on brain tumor endothelium and to correlate this with relative levels of CD4+ and CD8+ T cell subsets in TIL from brain tumors. Methods: CD3+, CD4+, and CD8+ cells were quantified in fresh TIL by flow cytometry. Paraffin embedded sections of tumors, including meningiomas and gliomas as well as extracranial malignancies, underwent immunohistochemical staining for FasL and Von-Willebrand’s factor (Factor VIII) to determine expression levels of endothelial FasL. Results: FasL expression was high in aggressive intracranial malignancies compared to more indolent neoplasms, and correlated inversely with CD8+/CD4+ TIL ratios in all tumor classes combined (ANOVA,p<0.05). Conclusion: Low levels of T cells within TIL, as well as low CD8+/CD4+ TIL ratios appear to be a property of parenchymal tumor presence. Together with the inverse correlation seen between FasL expression and CD8+/CD4+ TIL ratios, the high levels of endothelial FasL expression in gliomas suggests that FasL decreases T cell presence in brain tumors in a subset-selective manner, thus contributing to glioma immune privilege.

Characterization of Defective CD4−CD8− T Cells in Murine Tumors Generated Independent of Antigen Specificity

Immune-based therapy confers limited benefits to hosts bearing late-stage tumors. Mounting evidence points to local suppression of T cell function as the most substantial barrier to effective antitumor immunity in hosts with large tumor burdens. Despite this, events responsible for locally defective T cells and immune suppression in tumors remain unclear. We describe in this study a predominant T cell population localized within two murine tumors that is characterized by expression of apoptotic markers and TCRαβ/CD3, but not CD4, CD8, or NK-associated markers. These defective cells resembled double negative (DN) T cells in lpr mice, harbored defects in the expression of T cell signaling molecules, and produced the anti-inflammatory cytokine, IL-10. Conditions known to increase or decrease the accumulation of lpr DN T cells had corresponding effects on local DN tumor infiltrating lymphocyte (TIL) levels and inversely impacted host survival. Adoptive transfer into s.c. tumors demonstrated that naive CD8+ T cells were highly susceptible to becoming DN TIL, and local supplementation of tumors with nontumor Ag-bearing MHC class I-expressing fibroblasts decreased both this susceptibility and endogenous DN TIL levels. These findings identify a major defective T cell population with suppressive potential within tumors. The data also suggest that local T cell defectiveness is controlled by the tumor environment independent of cognate Ag specificity per se. Decreasing defective DN TIL levels by increasing noncognate peptide MHC class I availability, or modulating TCR or cytokine signaling may facilitate host survival by bolstering endogenous immunity to late-stage tumors, and may help improve therapeutic tumor vaccines.

T Cells Enhance Stem-Like Properties and Conditional Malignancy in Gliomas

Background Small populations of highly tumorigenic stem-like cells (cancer stem cells; CSCs) can exist within, and uniquely regenerate cancers including malignant brain tumors (gliomas). Many aspects of glioma CSCs (GSCs), however, have been characterized in non-physiological settings. Methods We found gene expression similarity superiorly defined glioma “stemness”, and revealed that GSC similarity increased with lower tumor grade. Using this method, we examined stemness in human grade IV gliomas (GBM) before and after dendritic cell (DC) vaccine therapy. This was followed by gene expression, phenotypic and functional analysis of murine GL26 tumors recovered from nude, wild-type, or DC-vaccinated host brains. Results GSC similarity was specifically increased in post-vaccine GBMs, and correlated best to vaccine-altered gene expression and endogenous anti-tumor T cell activity. GL26 analysis confirmed immune alterations, specific acquisition of stem cell markers, specifically enhanced sensitivity to anti-stem drug (cyclopamine), and enhanced tumorigenicity in wild-type hosts, in tumors in proportion to anti-tumor T cell activity. Nevertheless, vaccine-exposed GL26 cells were no more tumorigenic than parental GL26 in T cell-deficient hosts, though they otherwise appeared similar to GSCs enriched by chemotherapy. Finally, vaccine-exposed GBM and GL26 exhibited relatively homogeneous expression of genes expressed in progenitor cells and/or differentiation. Conclusions T cell activity represents an inducible physiological process capable of proportionally enriching GSCs in human and mouse gliomas. Stem-like gliomas enriched by strong T cell activity, however, may differ from other GSCs in that their stem-like properties may be disassociated from increased tumor malignancy and heterogeneity under specific host immune conditions.

Vaccines for glioblastoma and high-grade glioma.

Vaccination by administering tumor antigen plus cell-free or cellular adjuvant has garnered hope for more effective, less toxic therapy for patients with malignant brain tumors including glioblastoma multiforme. To determine if this approach demonstrates ample clinical promise, all published reports of vaccination for glioma were evaluated. These reports suggest vaccination is associated with low toxicity and favorable clinical outcomes. The possibility of selection bias is evident in many published vaccine trials, but several of the more recent ones appropriately attempt to account for bias. Effective induction of antitumor immunity is consistently observed, and, in the latest trials, correlates with significant clinical improvement.