[CANCER RESEARCH 64, 4973– 4979, July 15, 2004] Vaccination with Tumor Lysate-Pulsed Dendritic Cells Elicits Antigen-Specific, Cytotoxic T-Cells in Patients with Malignant Glioma John S. Yu, 1 Gentao Liu, 1 Han Ying, 1 William H. Yong, 2 Keith L. Black, 1 and Christopher J. Wheeler 1 1 Maxine Dunitz Neurosurgical Institute and 2 Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, California ABSTRACT 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. Ad- verse 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 phenotypi- cally 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- expression by peripheral blood mononuclear cells in response to tumor lysate after vaccination. Using HLA-restricted tetramer staining, we identified a significant expan- sion 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. INTRODUCTION Glioblastoma multiforme (GBM) is the most aggressive and com- mon primary brain tumor, accounting for 50% of intracranial gliomas and 25% of intracranial tumors in adults (1). GBM diagnosis carries with it a median survival of 12–18 months (with 90 –95% of patients surviving 2 years), without the possibility of spontaneous remission (2). Current treatment, consisting of surgical resection followed by radiation therapy and chemotherapy, has not substantially changed the bleak prognosis for GBM patients, despite the efficacy of similar therapies for patients with non-glioma tumors (3, 4). Even the few treatments that have been found to be effective against GBM typically either exhibit only small increases in survival in large population studies or primarily benefit certain patient subpopulations, such as the young (5, 6). The overwhelming majority of patients with malignant glioma, composed of GBM and anaplastic astrocytoma (AA), expe- rience increasingly rapid tumor recurrence after surgical resection or other treatment and eventually succumb to such events. Thus, novel therapies for malignant gliomas and recurrent gliomas, in particular, are desperately needed. Cancer vaccines represent one novel form of therapy for recurrent malignant glioma (7). The clinical efficacy of therapeutic vaccination for any human tumor, however, remains controversial because con- sistent tumor destruction or extended life span is not observed in most vaccinated cancer patients (8 –11). In contrast, current cancer vaccines do reliably elicit tumor-reactive CTLs in most patients (8 –11). Con- sistent with this finding, we reported previously that therapeutic vaccination with autologous tumor peptide-pulsed dendritic cells (DCs) is sufficient to enhance peripheral tumor-reactive CTL activity and CD8+ T-cell infiltration into tumors in situ in newly diagnosed GBM patients (12). In the above-mentioned study, vaccine generation was constrained by the time required to obtain peptide antigen from each patient’s autologous cultured tumor cell line. The generation of such lines is particularly difficult in recurrent patients, whose tumors have been exposed to radiation. We therefore examined the ability of tumor lysate-pulsed DCs to activate peripheral tumor-reactive CTL activity and CD8+ T-cell infiltration into tumors in situ in recurrent malignant glioma patients. Here, we use autologous tumor lysate-pulsed DCs to introduce undefined tumor-associated antigens (TAAs) to the T cells of patients with recurrent as well as newly diagnosed malignant glioma. Three biweekly intradermal lysate-pulsed DC vaccinations were adminis- tered to nine patients with recurrent GBM and three patients with recurrent AA. One patient with newly diagnosed GBM and one patient with newly diagnosed AA were also treated. A potential risk of vaccine therapy, particularly for tumors within essential organs such as the brain, is destructive autoimmunity. Tra- ditionally, the concern over autoimmunity has been addressed by targeting vaccines to specific antigenic epitopes not present on vital host tissues. Vaccines using undefined mixtures of tumor antigens have been reported to support sustained antitumor responses more effectively than peptide-based vaccines. A balance, however, must be struck between specificity and the ability of the tumor to evade responses directed toward a limited set of antigens. In addition, as vaccines for brain tumors become increasingly effective for treating patients, it will be necessary to move from approaches at higher risk for destructive autoimmunity toward those specifically targeting tu- mor tissue. One such method would be to use glioma-associated antigens. We sought to characterize common tumor antigen epitopes to which malignant glioma patients responded after vaccination with undefined autologous tumor antigen. We recently demonstrated the expression of TAAs in glioblastoma and demonstrated the presenta- tion of some common TAA epitopes in a MHC-restricted fashion (13, 14). We used these findings to determine whether DC vaccination generated T-cell clones that recognized these epitopes. No significant adverse effects were associated with DC vaccination. One patient was tested for tumor-specific T-cell cytotoxicity through a conventional CTL assay and demonstrated robust cytotoxicity after vaccination. Ten additional patients had pre- and postvaccination CTL assays based on quantitative reverse transcription-PCR detection of IFN-  expression. Six patients demonstrated significant peripheral cytotoxicity postvaccination based on IFN-  expression. A subset of patients demonstrated the generation of not only tumor-specific T cells but also tumor antigen-specific T cells after DC vaccination. Six patients underwent reoperation for recurrence, and three of these patients demonstrated significant CD4+ and CD8+ tumor infiltra- tion. We also compared survival rates for the eight study patients with recurrent GBM with those of 26 control patients with recurrent GBM Received 11/8/03; revised 4/30/04; accepted 5/12/04. Grant support: This work was funded in part by NIH grant NS02232-01 to J. S. Yu. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: John S. Yu, 8631 West Third Street, Suite 800E, Los Angeles, CA 90048. Phone: (310) 423-0845; Fax: (310) 423-0810; E-mail: Yuj@cshs.org. 4973 Research. on July 28, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from