Dendritic cell-based vaccination of patients with advanced pancreatic carcinoma: results of a pilot study

Background and aims

Dendritic cell (DC)-based vaccination can induce antitumor T cell responses in vivo. This clinical pilot study examined feasibility and outcome of DC-based tumor vaccination for patients with advanced pancreatic adenocarcinoma.

Methods

Tumor lysate of patients with pancreatic carcinoma was generated by repeated freeze?Cthaw cycles of surgically obtained tissue specimens. Patients were eligible for DC vaccination after recurrence of pancreatic carcinoma or in a primarily palliative situation. DC were generated from peripheral blood mononuclear cells (PBMC), loaded with autologous tumor lysate, stimulated with TNF-?? and PGE₂ and injected intradermally. All patients received concomitant chemotherapy with gemcitabine. Disease response was the primary endpoint. Individual immunological responses to DC vaccination were analyzed by T cell-based immunoassays using pre- and post-vaccination samples of non-adherent PBMC.

Results

Twelve patients received DC vaccination and concomitant chemotherapy. One patient developed a partial remission, and two patients remained in stable disease. Median survival was 10.5?months. No severe side effects were observed. Tumor-reactive T cells could be detected prior to vaccination. DC vaccination increased the frequency of tumor-reactive cells in all patients tested; however, the degree of this increase varied. To quantify the presence of tumor-reactive T cells, stimulatory indices (SI) were calculated as the ratio of proliferation-inducing capacity of lysate-loaded versus -unloaded DC. The patient with longest overall survival of 56?months had a high SI of 6.49, indicating that the presence of a pre-vaccination antitumor T cell response might be associated with prolonged survival. Five patients survived 1?year or more.

Conclusion

DC-based vaccination can stimulate an antitumoral T cell response in patients with advanced or recurrent pancreatic carcinoma receiving concomitant gemcitabine treatment.     

A gynecologic oncology group phase II trial of two p53 peptide vaccine approaches: subcutaneous injection and intravenous pulsed dendritic cells in high recurrence risk ovarian cancer patients

Purpose

Peptide antigens have been administered by different approaches as cancer vaccine therapy, including direct injection or pulsed onto dendritic cells; however, the optimal delivery method is still debatable. In this study, we describe the immune response elicited by two vaccine approaches using the wild-type (wt) p53 vaccine.

Experimental design

Twenty-one HLA-A2.1 patients with stage III, IV, or recurrent ovarian cancer overexpressing the p53 protein with no evidence of disease were treated in two cohorts. Arm A received SC wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells IV. Interleukin-2 (IL-2) was administered to both cohorts in alternative cycles.

Results

Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B developed an immunologic response as determined by ELISPOT and tetramer assays. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced the expansion of T regulatory cells. The median overall survival was 40.8 and 29.6?months for arm A and B, respectively; the median progression-free survival was 4.2 and. 8.7?months, respectively.

Conclusion

We found that using either vaccination approach generates comparable specific immune responses against the p53 peptide with minimal toxicity. Accordingly, our findings suggest that the use of less demanding SC approach may be as effective. Furthermore, the use of low-dose SC IL-2 as an adjuvant might have interfered with the immune response. Therefore, it may not be needed in future trials.     

Stratification according to HGG-IMMUNO RPA model predicts outcome in a large group of patients with relapsed malignant glioma treated by adjuvant postoperative dendritic cell vaccination

Purpose

Adult patients with relapsed high-grade glioma are a very heterogenous group with, however, an invariably dismal prognosis. We stratified patients with relapsed high-grade glioma treated with re-operation and postoperative dendritic cell (DC) vaccination according to a simple recursive partitioning analysis (RPA) model to predict outcome.

Patients and methods

Based on age, pathology, Karnofsky performance score, and mental status, 117 adult patients with relapsed malignant glioma, undergoing re-operation, and postoperative adjuvant dendritic cell (DC) vaccination were stratified into 4 classes. Kaplan–Meier survival estimates were generated for each class of this HGG-IMMUNO RPA model. Extent of resection was documented but not included in the prognostic model.

Results

Kaplan–Meier overall survival estimates revealed significant (p?<?0.0001) differences among the 4 HGG-IMMUNO RPA classes. Long-term survivors, surviving more than 24?months after the re-operation and vaccination, are seen in 54.5, 26.7, 11.5, and 0?% for the classes I, II, III, and IV respectively.

Conclusion

This HGG-IMMUNO RPA classification is able to predict overall survival in a large group of adult patients with a relapsed malignant glioma, treated with re-operation and postoperative adjuvant DC vaccination in the HGG-IMMUNO-2003 cohort comparison trial. The model appears useful for prognostic patient counseling for patients participating in DC vaccination trials. A substantial number of long-term survivors after relapse are seen in class I to III, but not in class IV patients.     

Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma

Background

Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets.

Patients and methods

This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS).

Results

Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR?+?PR?+?SD) more often showed SSTRs than patients with disease progression (p?=?0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6?months; p?=?0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p?=?0.013). The induction of SSTRs was associated with gender (female vs. male; p?=?0.014) and disease stage (M1a/b vs. M1c; p?=?0.010), but not with patient age, HLA type, performance status, or vaccination regimen.

Conclusion

Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma.     

Integration of autologous dendritic cell-based immunotherapy in the standard of care treatment for patients with newly diagnosed glioblastoma: results of the HGG-2006 phase I/II trial

Purpose

Dendritic cell (DC)-based tumor vaccination has rendered promising results in relapsed high-grade glioma patients. In the HGG-2006 trial (EudraCT 2006-002881-20), feasibility, toxicity, and clinical efficacy of the full integration of DC-based tumor vaccination into standard postoperative radiochemotherapy are studied in 77 patients with newly diagnosed glioblastoma.

Patients and methods

Autologous DC are generated after leukapheresis, which is performed before the start of radiochemotherapy. Four weekly induction vaccines are administered after the 6-week course of concomitant radiochemotherapy. During maintenance chemotherapy, 4 boost vaccines are given. Feasibility and progression-free survival (PFS) at 6?months (6mo-PFS) are the primary end points. Overall survival (OS) and immune profiling, rather than monitoring, as assessed in patients’ blood samples, are the secondary end points. Analysis has been done on intent-to-treat basis.

Results

The treatment was feasible without major toxicity. The 6mo-PFS was 70.1?% from inclusion. Median OS was 18.3?months. Outcome improved significantly with lower EORTC RPA classification. Median OS was 39.7, 18.3, and 10.7?months for RPA classes III, IV, and V, respectively. Patients with a methylated MGMT promoter had significantly better PFS (p?=?0.0027) and OS (p?=?0.0082) as compared to patients with an unmethylated status. Exploratory “immunological profiles” were built to compare to clinical outcome, but no statistical significant evidence was found for these profiles to predict clinical outcome.

Conclusion

Full integration of autologous DC-based tumor vaccination into standard postoperative radiochemotherapy for newly diagnosed glioblastoma seems safe and possibly beneficial. These results were used to power the currently running phase IIb randomized clinical trial.     

A multi-peptide, dual-adjuvant telomerase vaccine (GX301) is highly immunogenic in patients with prostate and renal cancer

Background

Anti-tumor vaccination is a new frontier in cancer treatment applicable to immunogenic neoplasms such as prostate and renal cancers. GX301 is a vaccine constituted by four telomerase peptides and two adjuvants, Montanide ISA-51 and Imiquimod.

Objective

The aim of this study was to analyze safety and tolerability of GX301 in an open-label, phase I/II trial. Immunological and clinical responses were also evaluated as secondary endpoints.

Experimental design

GX301 was administered by intradermally injecting 500 μg of each peptide (dissolved in Montanide ISA-51) in the skin of the abdomen. Imiquimod was applied as a cream at the injection sites. The protocol included 8 administrations at days 1, 3, 5, 7, 14, 21, 35, 63. Eligible patients were affected with stage IV prostate or renal cancer resistant to conventional treatments. Patients were clinically and immunologically monitored up to 6 months from the first immunization.

Results

No grade 3–4 adverse events were observed. Evidence of vaccine-specific immunological responses was detected in 100 % of patients. Disease stabilization occurred in 4 patients. Prolonged progression-free survival and overall survival were observed in patients showing a full pattern of vaccine-specific immunological responses.

Conclusion

GX301 demonstrated to be safe and highly immunogenic. Further studies are needed to determine its clinical efficacy.     

A case of spontaneous systemic immunity to melanoma associated with cure after amputation for extensive regional recurrence

Purpose

Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation.

Methods

A 71-year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELISPOT assay, and antibody responses to a panel of tumor antigens were assayed by ELISA.

Results

The patient’s tumor had minimal lymphocytic infiltrate (immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T-cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2–6 years after surgery.

Conclusion

The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, and then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.     

Laparoscopic ovarian transposition before pelvic radiation in rectal cancer patient: safety and feasibility

Background

Infertility due to pelvic radiation for advanced rectal cancer treatment is a major concern particularly in young patients. Pre-radiation laparoscopic ovarian transposition may offer preservation of ovarian function during the treatment however its use is limited.

Aim

The study investigates the safety, feasibility and effectiveness of pre-radiation laparoscopic ovarian transposition and its effect on ovarian function in the treatment o locally advanced rectal cancer.

Methods

Charts review of all young female patients diagnosed with locally advanced rectal cancer, underwent laparoscopic ovarian transposition, then received preoperative radiotherapy at king Faisal Specialist Hospital and Research Centre between 2003?C2007.

Results

During the period studied three single patients age between 21?C27?years underwent pre-radiation laparoscopic ovarian transposition for advanced rectal cancer. All required pretreatment laparoscopic diversion stoma due to rectal stricture secondary to tumor that was performed at the same time. One patient died of metastatic disease during treatment. The ovarian hormonal levels (FSH and LH) were normal in two patients. One has had normal menstrual period and other had amenorrhoea after 4?months follow-up however her ovarian hormonal level were within normal limits.

Conclusions

Laparoscopic ovarian transposition before pelvic radiation in advanced rectal cancer treatment is an effective and feasible way of preservation of ovarian function in young patients at risk of radiotherapy induced ovarian failure. However, this procedure is still under used and it is advisable to discuss and propose it to suitable patients.     

Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters

Purpose

Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.

Experimental design

Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.

Results

KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20?μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9?%, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.

Conclusions

We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.     

Immunoediting and persistence of antigen-specific immunity in patients who have previously been vaccinated with NY-ESO-1 protein formulated in ISCOMATRIX?

Background

NY-ESO-1 protein formulated in ISCOMATRIX? results in CD4+, CD8+ T cell and antibody-mediated immunity. We evaluated persistence of immunity, relapse-free survival and tumour antigen expression upon relapse in patients vaccinated in an earlier trial.

Methods

Immunity was measured in 28 patients with resected NY-ESO-1-expressing tumours (melanoma 25, breast 3) 252?C1,155?days (median?=?681) after vaccination. In the earlier vaccination, trial patients received NY-ESO-1 with ISCOMATRIX? adjuvant at three protein doses 10???g, 30???g or 100???g (n?=?14); 100???g NY-ESO-1 protein (n?=?8) or placebo (n?=?6), together with 1???g of intradermal (ID) NY-ESO-1 protein twice for DTH skin testing. Immune responses assessed in the current study included antibody titres, circulating NY-ESO-1-specific T cells and DTH reactivity 2?days after DTH skin testing with NY-ESO-1 protein (1???g) or peptides (10???g). Relapse-free survival was determined for 42 melanoma patients. On relapse NY-ESO-1 and HLA, class I was assessed by immunohistochemistry in 17.

Results

Persisting anti-NY-ESO-1 immunity was detected in 10/14 recipients who had previously received vaccine with ISCOMATRIX? adjuvant. In contrast, immunity only persisted in 3/14 who received 100???g un-adjuvanted NY-ESO-1 protein (3/8) or 2???g DTH protein (0/6) P?=?0.02. Hence, persisting NY-ESO-1 immunity was associated with prior adjuvant. Tumour NY-ESO-1 or HLA class I was downregulated in participants who relapsed suggesting immunoediting had occurred.

Conclusion

Immunoediting suggests that a signal of anti-tumour activity was observed in high-risk resected melanoma patients vaccinated with NY-ESO-1/ISCOMATRIX?. This was associated with measurable persisting immunity in the majority of vaccinated subjects tested. A prospective randomised trial has been undertaken to confirm these results.     

Clinical and immunological responses in metastatic melanoma patients vaccinated with a high-dose poly-epitope vaccine

Background

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.

Methods

Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-γ ELISPOT assays. Safety and clinical responses were monitored.

Results

Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit—one PR (24 months+), five SD (5 months+) and two mixed responses—seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.

Conclusions

DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.     

Mycophenolate mofetil inhibits the development of Coxsackie B3-virus-induced myocarditis in mice

Background

Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival.

Results

Observation of survival percentages over time and detection of LD₅₀s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice.

Conclusions

These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Immunological response induced by abagovomab as a maintenance therapy in patients with epithelial ovarian cancer: relationship with survival—a substudy of the MIMOSA trial

Purpose

To determine whether abagovomab induces protective immune responses in ovarian cancer patients in first clinical remission. The present analysis is a substudy of monoclonal antibody immunotherapy for malignancies of the ovary by subcutaneous abagovomab trial (NCT00418574).

Methods

The study included 129 patients, 91 in the abagovomab arm and 38 in the placebo arm. Circulating CA125-specific cytotoxic T lymphocytes (CTL) were measured by a flow cytometry-based interferon-γ producing assay. Human antimouse antibody and anti-anti-idiotypic (Ab3) were assessed by ELISA. Patients were evaluated before starting the treatment and at different time points during induction and maintenance phases.

Results

A similar percentage of patients in both the placebo and abagovomab arms had CA125-specific CTL (26.3 and 31.8 %, respectively; p = 0.673 by Fisher’s exact test). Patients with CA125-specific CTL in both arms tended to have an increased relapse-free survival (RFS, log-rank test p = 0.095) compared to patients without. Patients (n = 27) in the abagovomab arm without CA125-specific CTL but that developed Ab3 above the cutoff (defined as median Ab3 level at week 22) had a prolonged RFS compared to patients (n = 24) that did not develop Ab3 above the cutoff (log-rank test p = 0.019).

Conclusion

Abagovomab does not induce CA125-specific CTL. However, patients with CA125-specific CTL perform better than patients without, irrespective of abagovomab treatment. Abagovomab-induced Ab3 associate with prolonged RFS in patients without CA125-specific CTL. Further studies are needed to confirm these data and to assess the potential utility of these immunological findings as a tool for patient selection in clinical trial.     

Immunologic response to the survivin-derived multi-epitope vaccine EMD640744 in patients with advanced solid tumors

Purpose

Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses.

Experimental design

This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide^® ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 μg. Secondary objectives included safety, tolerability, and clinical efficacy.

Results

In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event.

Conclusions

Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.     

AAV2-mediated in vivo immune gene therapy of solid tumours

Background

Immunological therapies enhance the ability of the immune system to recognise and destroy cancer cells via selective killing mechanisms. DNA vaccines have potential to activate the immune system against specific antigens, with accompanying potent immunological adjuvant effects from unmethylated CpG motifs as on prokaryotic DNA. We investigated an electroporation driven plasmid DNA vaccination strategy in animal models for treatment of prostate cancer.

Methods

Plasmid expressing human PSA gene (phPSA) was delivered in vivo by intra-muscular electroporation, to induce effective anti-tumour immune responses against prostate antigen expressing tumours. Groups of male C57 BL/6 mice received intra-muscular injections of phPSA plasmid. For phPSA delivery, quadriceps muscle was injected with 50 μg plasmid. After 80 seconds, square-wave pulses were administered in sequence using a custom designed pulse generator and acustom-designed applicator with 2 needles placed through the skin central to the muscle. To determine an optimum treatment regimen, three different vaccination schedules were investigated. In a separate experiment, the immune potential of the phPSA vaccine was further enhanced with co- administration of synthetic CpG rich oligonucleotides. One week after last vaccination, the mice were challenged subcutaneously with TRAMPC1/hPSA (prostate cancer cell line stably expressing human PSA) and tumour growth was monitored. Serum from animals was examined by ELISA for anti-hPSA antibodies and for IFNγ. Histological assessment of the tumours was also carried out. In vivo and in vitro cytotoxicity assays were performed with splenocytes from treated mice.

Results

The phPSA vaccine therapy significantly delayed the appearance of tumours and resulted in prolonged survival of the animals. Four-dose vaccination regimen provided optimal immunological effects. Co - administration of the synthetic CpG with phPSA increased anti-tumour responses, preventing tumour occurrence in 54% of treated animals. Vaccination with phPSA resulted in anti-hPSA Abs production and a significant production of IFNγ was observed in immunised animals (p < 0.05). Immune responses were tumour specific and were transferable in adoptive T cell transfer experiments.

Conclusions

This phPSA plasmid electroporation vaccination strategy can effectively activate tumour specific immune responses. Optimisation of the approach indicated that a four-dose regimen provided highest tumour protection. In vivo electroporation mediated vaccination is a safe and effective modality for the treatment of prostate cancer and has a potential to be used as a neo-adjuvant or adjuvant therapy.     

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses

Introduction

Immunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH⁺ with respect to DTH^? unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood.

Materials and methods

Healthy donors and melanoma patient’s lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release.

Results/discussion

Peripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4⁺ TGF-β⁺) regulatory T lymphocytes compared with healthy donors. Notably, DTH⁺ patients showed a threefold reduction of Th3 cells compared with DTH^? patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH⁺ with respect to DTH^? patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response.

Conclusions

Our results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.     

Prognostic factors related to add-on dendritic cell vaccines on patients with inoperable pancreatic cancer receiving chemotherapy: a multicenter analysis

Objective

Dendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced pancreatic cancer. However, variations among clinical studies make it difficult to compare clinical outcomes. Here, we identified factors that determined the clinical benefits by analyzing data obtained at seven Japanese institutions that employed the same DC preparation and treatment regimens.

Methods

Of 354 patients who met the inclusion criteria, 255 patients who received standard chemotherapy combined with peptide-pulsed DC vaccines were analyzed.

Results

The mean survival time from diagnosis was 16.5 months (95 % CI 14.4–18.5) and that from the first vaccination was 9.9 months (95 % CI 8.0–12.9). Known prognostic baseline factors related to advanced pancreatic cancer, namely ECOG-PS, peritoneal metastasis, liver metastasis, and the prognostic nutrition index, were also representative. Importantly, we found that erythema reaction after vaccination was an independent and treatment-related prognostic factor for better survival and that OK-432 might be a good adjuvant enhancing the antitumor immunity during DC vaccination.

Conclusions

This is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of an add-on DC vaccine in patients with advanced pancreatic cancer who are undergoing chemotherapy. These findings need to be addressed in well-controlled prospective randomized trials.     

Gastric type I carcinoid: a pilot study with human G17DT immunogen vaccination

Context

Gastric type I carcinoid is a rare neoplasm, deriving from enterochromaffin-like cells (ECL), mainly affecting women with autoimmune gastritis. The approach to treatment, either endoscopic, medical or surgical, is not well defined, particularly in multifocal tumours or carcinoids with rapid growth/frequent recurrence.

Objective

To determine whether an anti-G17 vaccination might interfere on the natural history of gastric type I carcinoid.

Setting

Padua teaching Hospital, outpatient clinic.

Design and patients

Three patients with type I gastric carcinoid in autoimmune gastritis were administered, after informed consent and ethic committee approval, with a vaccine against gastrin 17 (G17), a synthetic peptide that stimulates specific and high-affinity anti-G17 antibodies, and followed up endoscopically and clinically for a mean of 36?months.

Main outcome measures

Gastric histology and specifically carcinoid growth/recurrence and trend in time in gastrin, G17, pepsinogens, chromogranin A and clinical parameters.

Results

Following vaccination, carcinoid regression was observed in 2/3 patients and, in one of the patients, even the disappearance of ECL hyperplasia, with a reduced ECL cells stimulation, confirmed by a significant reduction in chromogranin A levels. Regression was observed in the two patients that showed a more clear local response to the vaccine. Increased autoantibody titre was observed, but no appearance of new autoimmune diseases.

Conclusions

Anti-G17 vaccination induced regression of type I gastric carcinoid and could be considered for the treatment of this tumour, when endoscopic removal is not indicated.     

CTLA-4 blockade increases antigen-specific CD8+ T cells in prevaccinated patients with melanoma: three cases

Background

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.

Methods

To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100^209?C217 and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100^209?C217 (ITDQVPFSV), tyrosinase^369?C377 (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.

Results

Following vaccination, patients generated weak to no CD4⁺ or CD8⁺ T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7^loCD45RA^lo) tetramer⁺CD8⁺ T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.

Conclusion

Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially ??boosting?? the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.