Dcbased Immunotherapy Results Of Phase I And Ii Clinical Trials

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It is very difficult to therapeutically target every remaining individual tumor cell due to the disseminated nature of GBM. It is extremely important to eliminate all intracranial neoplastic foci left behind after surgical resection of the primary tumor (4). The use of the immune system to target residual tumor cells is one such strategy to enhance visibility of tumor cells to the immune system.

In a phase I study, Yu and colleagues describe the use of a DC vaccine in patients with newly diagnosed high-grade glioma (42). After surgical resection and external-beam radiotherapy, nine patients were given a series of three DC vaccinations using DCs cultured from patients' peripheral blood mononuclear cells (PBMC) pulsed ex vivo with autologous tumor cell-surface peptide isolated by means of acid elution. Each DC vaccination was given intradermally every other week over a six-week period. Four of the nine patients who had radiological evidence of disease progression underwent repeat surgery after receiving the third vaccination. Two of the four patients who underwent re-resection had robust infiltration of CD8+ and CD45RO+ T cells which was not apparent in the tumor specimen resected prior to DC trial entry (Fig. 1). Comparison of long-term survival data between the study group and matched controls demonstrated an increase in median survival of 455 days versus 257 days for the control group, conferring some survival benefit after DC vaccination.

Given the promising results and absence of observed autoimmune toxicity in the phase I study, Yu and colleagues expanded the study into a phase II trial (43). Fourteen patients with recurrent (12 patients) and newly diagnosed

Figure 1 (See color insert.) Immunohistochemical characterization of infiltrating cells in intracranial tumor before and after DC vaccination: Intratumoral CD8+ cells, pre- (A), and post-vaccination (B). Intratumoral CD4+ cells, pre- (C), and post-vaccination (D). Intratumoral CD45RO+ cells, pre- (E), and post-vaccination (F). Intratumoral CD8+ cells pre- (G) and post-recurrence (H) in a non-vaccinated patient.

Figure 1 (See color insert.) Immunohistochemical characterization of infiltrating cells in intracranial tumor before and after DC vaccination: Intratumoral CD8+ cells, pre- (A), and post-vaccination (B). Intratumoral CD4+ cells, pre- (C), and post-vaccination (D). Intratumoral CD45RO+ cells, pre- (E), and post-vaccination (F). Intratumoral CD8+ cells pre- (G) and post-recurrence (H) in a non-vaccinated patient.

(2 patients) malignant glioma, including anaplastic astrocytoma and GBM, were given three vaccinations with autologous DC pulsed with autologous tumor lysate every other week over a six-week period. In four out of nine patients, as part of an HLA-restricted tetramer staining assay, it was found that there were one or more tumor-associated antigen (TAA)-specific CTL clones against melanoma antigen-encoding gene-1, gp100, and human epidermal growth factor receptor (HER)-2 (Fig. 2). DC vaccination offered a significant survival benefit

Figure 2 Representative flow cytometry plots from a single glioma patient vaccinated with autologous tumor lysate pulsed DCs. PBMC isolated pre- (left column) and post-vaccination (right column) were stained with HLA restricted tetramers for HER-2, gp100, and MAGE-1 (y-axis). Additionally, cells were stained for the CD8 antigen (x-axis). Plots indicate a significant increase in the number of cells that registered as double positive (i.e. bound to antigen specific tetramers and positive for CD8). This demonstrates an expansion in the populations of CTL specific for these TAAs in this patient following DC vaccination.

Figure 2 Representative flow cytometry plots from a single glioma patient vaccinated with autologous tumor lysate pulsed DCs. PBMC isolated pre- (left column) and post-vaccination (right column) were stained with HLA restricted tetramers for HER-2, gp100, and MAGE-1 (y-axis). Additionally, cells were stained for the CD8 antigen (x-axis). Plots indicate a significant increase in the number of cells that registered as double positive (i.e. bound to antigen specific tetramers and positive for CD8). This demonstrates an expansion in the populations of CTL specific for these TAAs in this patient following DC vaccination.

as evidenced by an increase in median survival of 133 weeks for the study group versus 30 weeks for the control group.

In a phase I study by Kikuchi and colleagues (44), eight patients were treated with a series of three to seven intradermal vaccinations with DC-autologous glioma fusion cells. Glioma fusion cells were used as a strategy to improve DC-mediated TAA presentation by enhancing tumor cell-DC interaction. Although the ability to induce a tumor-specific immune response was demonstrated, only slight temporary responses to therapy were detected in two patients who had tumor progression on follow-up neuroimaging studies.

Kikuchi and colleagues reported a clinical trial using DC-glioma fusion cells and recombinant human IL-12 (45) after a mouse brain tumor model demonstrated systemic administration of recombinant IL-12 enhanced antitumor effect of this vaccine (46). The trial involved 15 patients who received vaccine therapy after progression of disease despite standard chemotherapy and/or radiation therapy. The vaccine of DC-autologous glioma fusion cells was given intradermally close to a cervical lymph node followed by recombinant IL-12 (30 ng/kg) injected subcutaneously at the same site on days 3 and 7. Two six-week courses of this regimen were completed with the second course starting two to five weeks after the last dose of IL-12.

However, results of this trial demonstrated limited success of the DC-glioma fusion cell vaccine. Only two patients demonstrated significant increase in cytolytic activity after vaccination, as shown in a Cr-releasing cytolytic assay (13) using peripheral blood lymphocytes and autologous glioma cells. Cytolytic activity was almost nonexistent in the remainder of patients in the study group. CD4+ T-cell subsets were not observed, although CD8+ T-cell infiltration was more robust in recurrent tumor specimens, with pathologic findings of larger tumor cells containing multiple nuclei and wide cytoplasm, when compared to primary tumors. Failure of tumor-specific T-helper 1 induction and/or the existence of tolerogenic CD4+ T-cell subsets may be a reason for the limited success of the DC-glioma fusion cell vaccine. The potential for T-helper 1 and resident APCs to stimulate each other lends to support TAA-specific CTL responses. The development of a successful antiglioma vaccine may depend on the helper activity of the antigen-specific T-helper subset which can interact with APCs to activate them in the tumor microenvironment (47).

A direct injection of DCs into tumor is a novel immunotherapeutic approach. DCs acquire and process tumor antigens in situ allowing migration to regional lymphoid organs via lymphoid vessels thereby initiating significant tumor-specific immune responses in the CNS (12,48). Yamanaka and colleagues described results of a phase I/II clinical trial in which five glioma patients received intradermal vaccination of autologous tumor lysate-pulsed DC vaccination, whereas another five patients underwent intratumoral injection of autologous immature DCs in addition to intradermal vaccination of tumor lysate-pulsed DCs (49). This study used immature DCs since the ability to capture, process, and traffic antigens have been demonstrated by DCs only in their immature state (50). Patients who received both the intratumoral and the intradermal vaccines demonstrated reduction in the size of contrast-enhancing tumor on neuroimaging. This indicates that immature DCs injected intratumorally can potentially induce an antitumor immune response by their ability to capture and process TAAs in situ. For patients with surgically unresectable tumors not allowing for sufficient tumor specimen and/or recurrent gliomas, this may be a novel strategy.

In a subsequent phase I/II clinical study, Yamanaka and colleagues, describe the clinical evaluation of malignant glioma patients vaccinated with DCs pulsed by an autologous tumor lysate (51). Twenty-four patients with malignant glioma (6 grade III malignant gliomas and 18 grade IV GBM) status postsurgical resection of tumor, external beam radiation therapy, and nitrosourea-based chemotherapy were enrolled in this study. These patients were monitored for recurrence via brain imaging (MRI or CT), and upon evidence of tumor recurrence, DC immunotherapy was initiated. Twelve patients received maintenance glucocorticoid therapy with prednisone 30 mg/day during DC therapy.

DCs were injected intradermally close to a cervical lymph node, or intradermally and intratumorally via an Ommaya reservoir. Patients received DC pulsed with autologous tumor lysate every 3 weeks and continued with up to 10 vaccinations depending on the clinical response. The mean number of administrations was 7.4 times intradermally and 4.6 times intratumorally. In the phase I section of the protocol, 17 patients received administration of immatured DCs pulsed by tumor lysate intradermally or both intradermally and intra-tumorally. Of the 17 patients, 2 had minor response, 6 had no change, and 9 had progressive disease. In the phase II section of the protocol, seven patients received administration of DCs matured with OK-432 pulsed by tumor lysate given intradermally and immatured DCs given intratumorally via an Ommaya reservoir. One out of the seven patients had partial response, one had minor response, four had no change, and one had progressive disease on MRI. Yamanaka and colleagues found that those 7 patients with GBM who received DCs matured with OK-432 had a significantly increased overall survival compared to the 11 patients who received DCs without OK-432 maturation. They also found that the GBM patients that received both intratumoral and intradermal DC vaccinations had a longer overall survival time than the patients who received intradermal administration alone. Survival of 18 DC-vaccinated patients was compared to 27 nonselected age-, gender-, and disease-matched controls that similarly underwent surgical resection, radiation, and nitrosourea-based chemotherapy. In the DC vaccinated group, results demonstrated a median overall survival time of 480 days with a percentage of overall survival 23.5% at 2 years versus 400 days in the control group with a percentage of overall survival 3.7% at 2 years, conferring DC vaccination is associated with prolonged survival.

In a phase I, dose-escalation study, Liau and colleagues enrolled 12 patients with GBM (7 newly diagnosed, 5 recurrent) and treated them with 1, 5, or 10 million autologous DCs pulsed with acid-eluted autologous tumor peptides (52). The newly diagnosed patients underwent surgical resection followed by standard external beam radiation therapy and then administration of DC vaccinations. The recurrent patients had undergone radiation therapy and/or chemotherapy previously before presenting with recurrent tumor and then underwent surgical resection before administration of DC vaccines. After DC vaccination for all 12 GBM patients, overall survival was 100% at six months, 75% at one year, and 50% at 2 years with two long-term survivors (>4 years). Median time to progression was 15.5 months and median overall survival was 23.4 months. For those five patients with ongoing progressive disease and bulky tumor, median overall survival was 11.7 months. For the seven patients with either gross stable disease or no measurable residual disease at baseline, overall survival was 18 to over 58 months. This resulted in an overall median survival benefit of 35.8 months after DC vaccination when compared to control population who had a median overall survival of 18.3 months.

Postvaccination using conventional CTL assays, six patients were found to have peripheral tumor-specific CTL activity. These patients did not have peripheral CTL activity prior to vaccination. Those who developed systemic antitumor cytotoxicity had longer survival time compared to those patients who did not. All of the patients who had stable/minimal residual disease at baseline generated a positive CTL response (100%), whereas those with active progressive disease at baseline did not produce statistically significant cell-mediated CTL responses (0%), suggesting that those with active tumor progression/ recurrence may have an impaired ability to mount an effective cellular antitumor immune response. Eight patients who developed tumor progression on follow-up MRI postvaccine therapy underwent repeat surgical resection or biopsy. A robust infiltration of CD3+ tumor-infiltrating lymphocytes (TIL), not present in tissue samples taken prior to DC vaccination, was found in four of the eight patients who survived >30 months. However, those patients who died within one year (3 patients) demonstrated no significant infiltration, demonstrating that accumulation of tumor-specific T cells locally within tumors is associated with positive clinical responses. CD8+/CD45RO+ memory T cells with lesser number of CD4+ helper T cells were the majority of TILs identified.

Liau and colleagues also found that patients who had minimal tumor burden prevaccination (4 of 4) demonstrated evidence of increased TIL, whereas those with progressive disease prevaccination (3 of 3) showed no detectable increase in TIL. The authors suggest that clinical benefit from DC vaccination may be limited by active tumor recurrence and/or bulky residual tumor, which can negatively influence T lymphocytes' ability to accumulate within the local tumor microenvironment. This study also looked at expression of TGF-p2 and IL-10 using reverse transcription-PCR and immunohistochemistry in the tumor tissue to demonstrate whether secretion of immunosuppressive cytokines by the tumors affected local accumulation of T cells. They found that those patients with detectable TIL had lower quantitative expression of TGF-p2 and had a longer survival (>30 months) than those with higher quantitative expression of TGF-p2. The authors suggest that a high expression of TGF-p2 may decrease the ability of TIL to accumulate within CNS gliomas to mount a clinical relevant local antitumor immune response in brain cancer patients.

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