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Clinical Cancer Research Vol. 11, 5900-5911, August 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Immunologic Evaluation of Personalized Peptide Vaccination for Patients with Advanced Malignant Glioma

Naoki Yajima1, Ryuya Yamanaka1, Takashi Mine3, Naoto Tsuchiya1, Jumpei Homma1, Masakazu Sano1, Terukazu Kuramoto2, Yayoi Obata3, Nobukazu Komatsu3, Yoshimi Arima3, Akira Yamada3, Minoru Shigemori2, Kyogo Itoh3 and Ryuichi Tanaka1

Authors' Affiliations: 1 Department of Neurosurgery, Brain Research Institute, Niigata University, Niigata, Japan and Departments of 2 Neurosurgery and 3 Immunology and Research Center of Innovative Cancer Therapy of the 21st Century COE Program for Medical Science, Kurume University School of Medicine, Kurume, Fukuoka, Japan

Requests for reprints: Ryuya Yamanaka, Department of Neurosurgery, Brain Research Institute, Niigata University, Asahimachi-dori 1-757, Niigata, Japan 951-8585. Phone: 81-25-227-0651; Fax: 81-25-223-5287; E-mail: ryaman{at}bri.niigata-u.ac.jp.


   Abstract
Top
 Abstract
Materials, Methods, and Patients
 Results
 Discussion
 References
 
Purpose: The primary goal of this phase I study was to assess the safety and immunologic responses of personalized peptide vaccination for patients with advanced malignant glioma.

Experimental Design: Twenty-five patients with advanced malignant glioma (8 grade 3 and 17 grade 4 gliomas) were evaluated in a phase I clinical study of a personalized peptide vaccination. For personalized peptide vaccination, prevaccination peripheral blood mononuclear cells and plasma were provided to examine cellular and humoral responses to 25 or 23 peptides in HLA-A24+ or HLA-A2+ patients, respectively; then, only the reactive peptides (maximum of four) were used for in vivo administration.

Results: The protocols were well tolerated with local redness and swelling at the injection site in most cases. Twenty-one patients received more than six vaccinations and were evaluated for both immunologic and clinical responses. Increases in cellular or humoral responses specific to at least one of the vaccinated peptides were observed in the postvaccination (sixth) samples from 14 or 11 of 21 patients, respectively. More importantly, significant levels of peptide-specific IgG were detected in the postvaccination tumor cavity or spinal fluid of all of the tested patients who showed favorable clinical responses. Clinical responses were 5 partial responses, 8 cases of stable disease, and 8 cases of progressive disease. The median overall survival for patients with recurrent glioblastoma multiforme in this study (n = 17) was 622 days.

Conclusions: Personalized peptide vaccinations were recommended for the further clinical study to malignant glioma patients.

Despite recent advances in cancer therapy, the prognosis of patients with malignant glioma is still very poor (1, 2). Therefore, the development of a new treatment modality is needed. Although immunotherapy is theoretically attractive due to the discovery of tumor-associated antigens and peptides capable of inducing specific immunity in patients with glioma (3–5), previously conducted immunotherapy trials failed to provide evidence of any definite clinical benefit to patients with glioma (6–8). One of the hurdles hindering the development of therapeutically effective immunotherapy for the treatment of gliomas would be the blood-brain barrier, but recent studies have shown that it does not always function in cases involving recurrent gliomas (9, 10). The other hurdle would be the lack of an immunotherapy protocol for malignant tumors. The clinical trials of active and specific immunotherapies conducted in the past decade have rarely obtained major clinical responses in cancer patients (11, 12). However, we recently conducted clinical trials of personalized peptide vaccination for advanced epithelial cancer patients (13–15). Consequently, the peptide-specific immune responses were raised in the postvaccination samples in the majority of the vaccinated patients with major tumor regression in several cases during the course of those trials. Here, we report the results of phase I clinical trials of personalized peptide vaccination administered to advanced malignant glioma patients.


   Materials, Methods, and Patients
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 Abstract
 Materials, Methods, and Patients
Results
 Discussion
 References
 
Protocols and eligibility criteria. Biweekly administration of 3 mg peptide per patient to a maximum of four peptides was initially conducted; then, the weekly administration of 2 mg peptide per patient to a maximum of four peptides was carried out primarily because of comparing the safety and immunologic responses among them. Both protocols were approved by the Ethical Committee of Niigata University and Kurume University. Complete written informed consent was obtained from all of the patients at the time of enrollment. The patients were required to be either HLA-A24+ or HLA-A2+ and had histologically proven glioblastoma multiforme (grade 4 glioma) or anaplastic astrocytoma (grade 3 glioma) according to the WHO criteria. After surgical resection of their tumor, patients had a course of external beam radiation therapy (standard dose, 40 Gy to the tumor with 3 cm margins, 20 Gy boost to the whole brain) and nitrosourea-based chemotherapy. Patients were diagnosed as recurrence or existence of their tumors even after the preceding various types of therapies by computed tomography (CT) scan or magnetic resonance imaging (MRI) examination. Patients did not undergo chemotherapy or radiotherapy during the 4 weeks before vaccination. Eligibility criteria included serum creatinine levels of <1.4 mg/dL, bilirubin levels of <1.5 mg/dL, a platelet count of ≥100,000/µL, hemoglobin levels of ≥8.0 g/dL, and a total WBC of ≥3,000/µL. Hepatitis B surface antigen and hepatitis C antigen were required to be negative. Exclusion criteria included pulmonary, cardiac, or other systemic disease, an acute infection, or a history of an autoimmune disease.

Peptides and vaccination. The peptides of the present study were prepared under the conditions for good manufacturing practice (GMP) as reported previously (13–15). The peptide sequences are shown in Table 2, in which the mother antigens, the cited references, and the frequencies used for vaccination during the first cycle and for the entire period are listed. These peptides exhibited the ability to induce HLA-A24- or HLA-A2-restricted and tumor-specific CTL activity in peripheral blood mononuclear cells (PBMC) of cancer patients as reported previously (16–36). The peptides in vials containing 3 mg/mL sterile solution were added in a 1:1 volume to Montanide ISA 51 adjuvant (Seppic, Inc., Franklin Lakes, NJ), and the solution was mixed in a Vortex mixer (Fisher, Inc., Alameda, CA). The resulting emulsion was injected s.c. into the upper back region. The six consecutive injections were done as the first cycle of vaccination to evaluate the patients for adverse reactions and immunologic responses. For patients with a favorable clinical course, PBMCs and plasma after the sixth vaccination were provided for the screening of the peptides, and the reactive peptides, to a maximum of four, were administrated as the second cycle of vaccination to further evaluate adverse reactions, immunologic responses, and clinical outcomes. The similar steps were taken for the third or fourth cycle.


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  		Table 2. Peptide list and sequences

 
Cellular and humoral responses to peptides. Peripheral blood (30 mL) was obtained before and after every three vaccinations for measurement of CTL precursor in PBMCs and IgG specific to a peptide in plasma according to previously reported methods (13–15). For the CTL precursor assay, the peptide-stimulated PBMCs were harvested and tested for their ability to produce IFN-{gamma} in response to CIR-A2402 cells for HLA-A24 or to T2 cells for HLA-A2 cases, which were preloaded with either a corresponding peptide or a HIV peptide as a negative control. The level of IFN-{gamma} was determined by ELISA in quadruplicate assays. A two-tailed Student's t test was employed for the statistical analysis. A well was considered positive when the level of IFN-{gamma} production in response to a corresponding peptide was significantly higher (P < 0.05) than that in response to a HIV peptide and also when the mean amount of IFN-{gamma} production in response to a corresponding peptide was >50 ng/mL compared with that to a HIV peptide. To assess delayed-type hypersensitivity (DTH) reactions, skin tests were done using 50 µg of each peptide separately injected i.d. followed by measurement of DTH reactions 48 hours after the injection. Cytotoxic activity was measured by a standard 6-hour 51Cr release assay as reported previously (13–15). The radiolabeled target cells were KNS-81, KALS-1 (HLA-A24+A2–), No. 11 (HLA-A24–A2+), KINGS-1, and Becker (HLA-A24–A2–) glioma cells and phytohemagglutinin-blastoid T cells (HLA-A24+ or HLA-A2+) as a negative control. Plasma levels of peptide-specific IgG were measured by the Luminex system as reported previously (37). The sample was considered positive when the median ± 2 SDs of fluorescence intensity (FI) of counting 100 beads in the sample in response to a corresponding peptide was significantly higher (P < 0.05) than that to a HIV peptide. Cutoff level sets as 10 FI.

Adverse events and clinical responses. Adverse events were monitored according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. The clinical response was evaluated based on clinical observations and radiological findings. All known sites of disease were evaluated on a monthly basis by CT scan or MRI examination before and after each cycle of vaccinations. The tumor size was estimated by the region of abnormal enhancement observed on CT scan or MRI examination via direct measurement. Patients were assigned a response category according to the Response Evaluation Criteria in Solid Tumors (RECIST). Survival time was estimated from the date of the initial vaccination. In addition, overall survival was estimated from the date of the first operation with histologic diagnosis of glioblastoma multiforme.


   Results
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 Abstract
 Materials, Methods, and Patients
 Results
Discussion
 References
 
Antigen expression in malignant gliomas and immune responses. We initially investigated the expression of antigens, which encode peptides for use in this clinical study, on glioma cells or cell lines. SART1 and SART2 expression in glioma cells has already been reported (16, 17), whereas cyclophin B is ubiquitously expressed in both malignant and normal proliferating cells (20). Subsequently, these antigens were excluded from the present analyses. The peptides derived from three (SART3, Lck, and MRP3) antigens were expected to be frequently vaccinated into the patients based on the previous results (13–15). Then, formalin-fixed brain tumor samples and the adjunct nontumorous brain tissues from the first nine patients were provided for the detection of SART3, Lck, and MRP3 antigens by immunohistochemical analysis (Fig. 1A). SART3 antigen was strongly expressed in the majority (>50%) of tumor cells from all nine tumor tissue samples tested, whereas it was dimly expressed in cells from nontumorous tissues. Both MRP3 and Lck antigens were expressed in a substantial proportion (10-50%) of tumor cells from six tumor tissue samples. In contrast, neither MRP3 nor Lck was detected in any cells from nontumorous tissue samples. One of the representative results is shown in Fig. 1A. Prevaccination PBMCs and the plasma of these patients were provided to investigate reactivity to the peptides derived from these antigens; this was done to ensure the correlation between antigen expression and immune reaction. CTL precursor or antibodies to the SART3 peptides were detected in the prevaccination PBMCs or plasma in all patients. Those to the Lck or MRP3 peptides were detected in all six patients whose tumors expressed the Lck or MRP3 antigen, respectively. A comprehensive listing of the results is not given due to space constraints.



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  		Fig. 1. Antigen expression in glioma cells. A, serial 10 µm paraffin sections of surgical intracranial tumor specimen were stained with 1:100 dilution mouse monoclonal antibodies against SART3 (40), Lck (Santa Cruz Biotechnology, Santa Cruz, CA), and MRP3 (Kamiya Biomedical Co., Seattle, WA) followed by standard immunohistochemical staining as reported previously (41). B, mRNA expression of the indicated genes was determined by semiquantitative RT-PCR analysis by the methods reported previously (42). Cell lines for positive control (PC) were as follows: LC-1 (lung cancer) for ART1 and ART4; MCF7 (breast cancer) for HER2; LNCaP (prostate cancer) for PTH-rP, EZH2, EGF-R, and PSCA; and Panc-l (pancreatic cancer) for WHSC2, HNRPL, UBE2V, and ppMAPkkk. C, To detect PTH-rP protein expression, tumor cells were stained with rabbit polyclonal anti-PTH-rP antibody (1:100 dilution; H-137; Santa Cruz Biotechnology) followed by staining with FITC-conjugated anti-rabbit IgG (1:200 dilution; Molecular Probes, Inc., Eugene, OR) and counterstained with propidium iodine (PI). D, For cell surface expression of PTH-rP protein in T98G and KALS-1 cells, tumor cells were stained with H-137 antibody followed by flow cytometric analysis.

 
The mRNA expression levels of the other 11 antigens (ART1, ART4, HER2, EZH2, WHSC2, HNRPL, ppMAPkkk, PSCA, UBE2V, PTH-rP, and EGF-R) in T98G and KALS-1 (glioblastomas), No. 10 and No. 11 (anaplastic astrocytomas), and KNS-81 (malignant glioma) cells and PBMCs as a negative control were investigated by semiquantitative reverse transcription-PCR (RT-PCR) analysis. All of these antigens, but not ppMAPkkk, were found to be expressed in at least three of five of the tumor cell lines tested (Fig. 1B). To further confirm the expression of these antigens at the protein level, the expression of PTH-rP in glioma cells was analyzed at the protein level. PTH-rP was expressed in the cytoplasmic area of about half of the T98G cells at the patty pattern (Fig. 1C). It was also expressed in 56% and 55% of the T98G and KALS-1 cells, respectively (Fig. 1D). Subsequently, we provided the peptides derived from these 16 different antigens, with the exception of ppMAPkkk, for the personalized peptide vaccination to HLA-A24 or HLA-A2 patients with malignant glioma.

Patient characteristics. Twelve (9 patients with grade 4 glioma and 3 patients with grade 3 glioma) or 13 (8 patients with grade 4 glioma and 5 patients with grade 3 glioma) patients were treated according to the biweekly or weekly administration protocol, respectively. The demographic details of the patient characteristics are provided in Table 1. Eight patients in the biweekly protocol and seven patients in the weekly protocol had a maintenance dose (10-45 mg/d prednisolone) of glucocorticoid therapy at the time of the initial vaccination, because patients suffered from local symptoms or increased intracranial pressure symptoms owing to peritumoral edema.


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  		Table 1. Patient characteristics

 
Peptide screening. Both prevaccination PBMCs and plasma were provided to investigate their reactivity to each of the 25 or 23 peptides in the HLA-A24 patients (n = 20) or HLA-A2 patients (n = 5), respectively. A summary of the peptide screening is shown in Table 2, in which the mean amount of IFN-{gamma} produced by the peptide-stimulated PBMCs in response to a corresponding peptide after the subtraction of that in response to a HIV peptide was considered as the CTL response, whereas the median score of the FI in the plasma IgG reacting to a corresponding peptide was obtained after the subtraction of FI reacting to a HIV peptide. A maximum of four peptides was administrated to each patient based on the results of screening for the following criteria: first priority, a peptide recognized by both PBMCs and plasma; second priority, a peptide recognized by PBMCs; and third priority, a peptide recognized by plasma. For example, in patient 1, SART2-93 and Lck-488 peptides were recognized by both PBMCs and plasma, whereas ART1-170 peptide was recognized by PBMCs alone. Subsequently, the above three peptides were administered to this patient. It was of note that the profiles of the vaccinated peptides were entirely different among these 25 patients.

Adverse reactions. All patients but one received more than three vaccinations; thus, these patients were eligible for the evaluation of adverse reactions. Grade 1 and 2 dermatologic inflammation at the injection site was observed in 6 and 4 patients, respectively, under the biweekly protocol, whereas this reaction was observed in 2 and 11 patients, respectively, under the weekly protocol. There was no clinical evidence of an autoimmune reaction as determined by symptoms, physical examination, or laboratory tests. Subsequently, each of the two protocols was evaluated as generally well tolerated.

Cellular and humoral immune responses. Twenty-one patients received vaccination six or more times and were eligible for the evaluation of both cellular and humoral responses. DTH reactions to at least one of the vaccinated peptides were induced in 3 of 10 and 8 of 11 patients under the biweekly and weekly protocols until the sixth vaccination, respectively (Table 3). Increased cellular responses to at least one of the vaccinated peptides were observed in the postvaccination (sixth) PBMCs from 5 of 10 and 9 of 11 patients under the biweekly and weekly administration protocols, respectively (Table 3). In contrast, neither the prevaccination nor the post-PBMCs from any of the 16 patients tested exhibited HLA class I–restricted cytotoxicity against glioma cells as measured by a 51Cr release assay (data not shown). Increased humoral responses to at least one of the vaccinated peptides were observed in the postvaccination (sixth) plasma from 4 of 10 and 7 of 11 patients under the biweekly and weekly administration protocols, respectively (Table 3). Collectively, an increase in either cellular or humoral responses specific to at least one of the vaccinated peptides was observed in the postvaccination (sixth) samples of 15 of 21 (71%) patients regardless of the administration of prednisolone to the majority of the patients (71%).


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  		Table 3. Immunologic responses and clinical outcomes

 
Clinical responses and prognostic marker analysis. Twenty-one patients received at least six vaccinations, and these patients were therefore eligible for an evaluation of their clinical responses. The clinical responses of 10 patients under the biweekly protocol were as follows: 2 partial responses (PR), 3 patients with stable disease (SD), and 5 patients with progressive disease (PD), all with a median time to progression (TTP) of 93 days (Table 3). The clinical responses of the 11 patients under the weekly protocol were 3 PRs, 5 patients with SD, and 3 patients with PD, with a median TTP of 133 days, but the median survival time (MST) was not determined because 6 patients are still alive (Table 3). Collectively, the clinical responses were 5 PRs (2 cases with grade 3 glioma and 3 cases with grade 4 glioma), 8 patients with SD, and 8 patients with PD. All clinical responses were confirmed by an independent review, and the radiological findings of the 3 PR cases are given in Fig. 2. The prognostic marker analysis revealed that the patients with a performance status (PS) of 0 to 2 (n = 15) at the time of enrollment had a significantly (P = 0.0002) longer survival time than those with a PS of 3 (n = 6; Fig. 3A). The patients administered ≤20 mg/d prednisolone (n = 17) at the initial vaccination had a longer (P = 0.0653) survival time than those who administered >20 mg/d prednisolone (n = 4; Fig. 3B). The patients whose postvaccination PBMCs exhibited increased levels of peptide-specific cellular responses to at least one of the vaccinated peptides (n = 14) had somewhat longer survival times (P = 0.1315) than the patients whose postvaccination PBMCs did not (n = 7; Fig. 3C). To determine a survival of the patients under personalized peptide vaccination, overall survival of 17 vaccinated patients with glioblastoma multiforme was assessed. In the study group of patients with glioblastoma multiforme from the time of the first operation, median overall survival time was 622 ± 87.1 days (range, 220-1,466 days) and percentage of overall survival was 82.3% at 1 year and 41.1% at 2 years (Fig. 3D).



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  		Fig. 2. Kinetic MRI of PR cases. Kinetic MRI examinations of three PR cases. Axial T1-weighted images with gadolinium enhancement show increased signal lesions. Patients were assigned a response category according to the RECIST.

 


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  		Fig. 3. Prognostic marker analysis. Survival time (A, B, C) and overall survival (D) were compared between the two groups as follows: A, PS 0-2 (n = 15) at the time of enrollment versus those with PS 3 (n = 6); B, patients under ≤20 mg/d prednisolone (n = 17) at the time of the initial vaccination versus those >20 mg/d prednisolone (n = 4); C, patients whose postvaccination PBMCs showed increased levels of peptide-specific cellular responses (n = 14) versus those did not (n = 7); D, Kaplan-Meier survival curve of study group of patients with glioblastoma multiforme from the time of the first operation.

 
Kinetic studies of immune reactions and clinical responses. Because major tumor regression was identified in four of five PR cases after the second cycle of vaccination, kinetic studies of the cellular and humoral immune reactions to the vaccinated peptides were conducted. As a control, the same studies were carried out using the samples from five SD patients and two PD patients who had received >10 vaccinations. Both cellular and humoral reactions to at least one of the vaccinated peptides showed an increase that was dependent on the increase in the number of vaccinations in all of the four PR cases tested but not in any of the other cases tested. In four of the five SD cases, either cellular or humoral responses to one of the vaccinated peptides increased. In contrast, neither cellular nor humoral responses increased in the remaining one SD case or the two PD cases. Representative results are given in Fig. 4.



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  		Fig. 4. Kinetic studies of immune reactions. Kinetic studies of the immune responses to the vaccinated peptides were conducted in four PR, five SD, and two PD cases that received >10 vaccinations. Representative results of the cases (four PR, two SD, and one PD case).

 
Analysis of cerebrospinal fluid and tumor cavity fluid. We examined whether antipeptide IgG would be detectable in the cerebrospinal fluid (CSF) and the tumor cavity fluid of the patients after the repeated vaccination. As expected, without peptide vaccination, no antipeptide IgG reactive to any of the 48 peptides listed in Table 2 was detected in the CSF from any of the 16 patients (3 patients without a brain tumor as a negative control; 4 patients with grade 3 glioma, including patient 11 in Table 1; and 9 patients with grade 4 glioma, including patients 3 and 8 in Table 1; data not shown). The postvaccination CSF harvested by lumbar puncture from three PR cases, three SD cases, and one PD case, along with the postvaccination plasma in these patients as a positive control, was simultaneously measured for the IgG levels to the vaccinated peptides. Significant levels of IgG to at least one of the vaccinated peptides were detectable in the CSF of all three PR cases and two of the three SD cases but not in the one PD case (Fig. 5A). The levels of antipeptide IgG varied largely among the patients, whose responses to particular peptides also differed markedly and were much lower in the CSF (0.4-52% FI of the plasma levels) than in the plasma in all cases. In contrast, the levels of antipeptide IgG in postvaccination tumor cavity fluids harvested from a reservoir at the tumor sites in PR (patient 16) and SD (patient 17) cases were much higher (160-223% FI of the plasma levels) than those in the plasma (Fig. 5B). More importantly, CD3+ T cells were detected in the cells from the tumor cavity fluid from this PR case (patient 16; Fig. 5C).



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  		Fig. 5. Analysis of CSF. A, IgG levels obtained from three PR, two SD, and one PD case in response to one representative peptide among six to eight peptides tested at a plasma dilution of 100:1. B, levels of antipeptide IgG in the postvaccination tumor cavity fluids harvested from a reservoir at the tumor site in PR (patient 16) and SD (patient 17) cases. C, cellular components of the tumor cavity fluid from patient 16 (a PR case) were provided for surface marker analysis. The majority of cells were determined to be CD3+CD14–CD20– cells. Result on CD3 expression.

 

   Discussion
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 Abstract
 Materials, Methods, and Patients
 Results
 Discussion
References
 
Several clinical trials of peptide-based immunotherapy for cancer have been conducted in the past decade, but major clinical responses were rarely obtained, including our studies (11, 12). One reason for this failure to obtain tumor regression could be an insufficient induction of antitumor responses in these vaccine regimens, in which peptide-specific memory T cells were not measured in prevaccination PBMCs. We have developed the personalized peptide vaccination from prevaccination measurement of peptide-specific CTLs and peptide-reactive IgG in cancer patients and then vaccinated only the CTL or antibody-directed peptides as a part of a phase I clinical study (13–15). This strategy is based on the assumption that initiation of immune boosting of CTLs through peptide vaccination could be more effective than immune priming of naive T cells with regard to induction of prompt and strong immunity. In this study, we had two protocols, namely, the biweekly administration of 3 mg peptide per patient to a maximum of four peptides and the weekly administration of 2 mg peptide per patient to a maximum of four peptides, primarily because of comparing the safety and immunologic responses among them.

We have chosen 25 or 23 peptides in HLA-A24+ or HLA-A2+ patients, respectively, as a list of candidate peptides for personalized peptide vaccination based on both basic (4, 5, 16–37) and translational (12–15) clinical researches. Our basic research showed that the majority of antigens encoding peptides used in this study were expressed in the malignant tumor cells regardless of different origins of tumor cells, including brain tumor cells. Those antigens were located in either nucleus or cytoplasm but not on the cell surface. Among the peptides, those from SART3, Lck, and MRP3 antigens were frequently chosen as the peptides for the cancer patients (12–15). Furthermore, preliminary results of peptide selection on prevaccination PBMCs of brain tumors showed that the peptides derived from these three antigens were frequently chosen as peptide candidates for personalized vaccination. Subsequently, we intensively carried out immunohistochemical analyses of these three antigens in brain tumor tissues.

Consequently, these three antigens were expressed in substantial proportions of tumor cells of >50% of brain tumor tissues tested.

There were no definite differences among the two protocols from the safety and clinical points of views. However, a clear trend toward a better immune response among the patients who underwent the weekly administration protocol was observed in all assays tested (2.7, 1.6, and 1.6 times higher frequencies of increase in the weekly protocol cases with regard to DTH response, peptide-reactive IFN-{gamma} production, and peptide-specific IgG level, respectively). Further clinical studies are needed to confirm this issue.

Increases in cellular or humoral responses specific to at least one of the vaccinated peptides were observed in the postvaccination (sixth) samples of 15 of 21 patients regardless of the administration of prednisolone to the majority of patients. DTH responses were also observed in 12 of 21 patients. In contrast, no HLA class I–restricted cytotoxicity against glioma cells was observed, as measured by a 6-hour 51Cr release assay, in either the prevaccination or the postvaccination PBMCs. These results suggest that the administration of steroid hormone did not greatly influence the peptide-induced humoral responses, DTH responses, or cytokine production in response to an antigen, although it suppressed HLA class I–restricted cytotoxicity. Although the modest levels of HLA-nonrestricted cytotoxicity were observed in both prevaccination and postvaccination PBMCs, these cytotoxicities could be a reflection of lymphokine-activated killer cells generated in in vitro culture with interleukin-2.

We detected significant levels of IgG specific to the vaccinated peptides in all available CSF and tumor cavity fluid samples from the PR cases and in those from two of three SD cases but not in the samples from patients with PD or unvaccinated patients. Furthermore, the levels of IgG to the vaccinated peptides at the tumor sites were higher than those in the same patient's plasma. We showed previously that an increase in peptide-specific IgG in the plasma of advanced epithelial cancer patients was a good laboratory marker for the prediction of the prolongation of overall survival in those patients (13–15), although the specific biological roles played by peptide-specific IgG are presently unclear. Our current studies revealed that antipeptide IgG was not found to react to the entire molecule encoding the peptide, nor it mediated antibody-dependent cell-mediated cytotoxicity against tumor cells. Regardless of such issues to be resolved in future, the present results revealed that IgG specific to vaccinated peptides, both in CSF and in plasma, could provide a good laboratory marker to predict the clinical outcome of glioma patients under the peptide vaccination.

Several reports have provided evidence that immunotherapy was clinically effective from the perspective of the overall survival of some grade 4 glioma patients, namely, those studies involved the vaccination of autologous tumor lysate-pulsed dendritic cells as reported by our group (10) and by others (38) and a virus-modified autologous tumor cell vaccine (39). However, such immunotherapies have several disadvantages: limited materials for vaccination, labor intensity for preparation, and difficulty to find a reliable laboratory marker. In contrast, peptide vaccination has several advantages, including easy supply of GMP levels of materials and a reliable laboratory marker for the prediction of clinical outcome as shown in the present study. More importantly, our present results suggest that personalized peptide vaccination can provide clinical benefits in terms of both tumor regression and prolongation of survival of patients with advanced grade 4 glioma. However, further clinical studies with a relatively large number of glioma patients shall be conducted to confirm the results of the present study.


   Footnotes
 
Grant support: Ministry of Education, Science, Sports, and Culture of Japan grant-in-aid 12213134 (K. Itoh), Research Center of Innovative Cancer Therapy of the 21st Century COE Program for Medical Science (K. Itoh and A. Yamada), and Ministry of Health and Welfare of Japan grant-in-aid H14-trans-002, 11-16 (K. Itoh).

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.

Received 3/16/05; revised 4/16/05; accepted 4/28/05.


   References
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 Abstract
 Materials, Methods, and Patients
 Results
 Discussion
 References
 

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