In Silico Design and Evaluation of PRAME+FliCΔD2D3 as a New Breast Cancer Vaccine Candidate.

Background: The most prevalent cancer in women over the world is breast cancer. Immunotherapy is a promising method to effectively treat cancer patients. Among various immunotherapy methods, tumor antigens stimulate the immune system to eradicate cancer cells. Preferentially expressed antigen in melanoma (PRAME) is mainly overexpressed in breast cancer cells, and has no expression in normal tissues. FliCΔD2D3, as truncated flagellin (FliC), is an effective toll-like receptor 5 (TLR5) agonist with lower inflammatory responses. The objective of the present study was to utilize bioinformatics methods to design a chimeric protein against breast cancer. Methods: The physicochemical properties, solubility, and secondary structures of PRAME+FliCΔD2D3 were predicted using the tools ProtParam, Protein-sol, and GOR IV, respectively. The 3D structure of the chimeric protein was built using I-TASSER and refined with GalaxyRefine, RAMPAGE, and PROCHECK. ANTIGENpro and VaxiJen were used to evaluate protein antigenicity, and allergenicity was checked using AlgPred and Allergen FP. Major histocompatibility complex )MHC( and cytotoxic T-lymphocytes )CTL( binding peptides were predicted using HLApred and CTLpred. Finally, B-cell continuous and discontinuous epitopes were predicted using ABCpred and ElliPro, respectively. Results: The stability and solubility of PRAME+FliCΔD2D3 were analyzed, and its secondary and tertiary structures were predicted. The results showed that the derived peptides could bind to MHCs and CTLs. The designed chimeric protein possessed both linear and conformational epitopes with a high binding affinity to B-cell epitopes. Conclusion: PRAME+FliCΔD2D3 is a stable and soluble chimeric protein that can stimulate humoral and cellular immunity. The obtained results can be utilized for the development of an experimental vaccine against breast cancer.

Automated Good Manufacturing Practice-compliant generation of human monocyte-derived dendritic cells from a complete apheresis product using a hollow-fiber bioreactor system overcomes a major hurdle in the manufacture of dendritic cells for cancer vaccines.

BACKGROUND: Although dendritic cell (DC)-based cancer vaccines represent a promising treatment strategy, its exploration in the clinic is hampered due to the need for Good Manufacturing Practice (GMP) facilities and associated trained staff for the generation of large numbers of DCs. The Quantum bioreactor system offered by Terumo BCT represents a hollow-fiber platform integrating GMP-compliant manufacturing steps in a closed system for automated cultivation of cellular products. In the respective established protocols, the hollow fibers are coated with fibronectin and trypsin is used to harvest the final cell product, which in the case of DCs allows processing of only one tenth of an apheresis product. MATERIALS AND RESULTS: We successfully developed a new protocol that circumvents the need for fibronectin coating and trypsin digestion, and makes the Quantum bioreactor system now suitable for generating large numbers of mature human monocyte-derived DCs (Mo-DCs) by processing a complete apheresis product at once. To achieve that, it needed a step-by-step optimization of DC-differentiation, e.g., the varying of media exchange rates and cytokine concentration until the total yield (% of input CD14+ monocytes), as well as the phenotype and functionality of mature Mo-DCs, became equivalent to those generated by our established standard production of Mo-DCs in cell culture bags. CONCLUSIONS: By using this new protocol for the Food and Drug Administration-approved Quantum system, it is now possible for the first time to process one complete apheresis to automatically generate large numbers of human Mo-DCs, making it much more feasible to exploit the potential of individualized DC-based immunotherapy.

Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study.

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.

Dendritic cell immunotherapy: mapping the way.

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system, with the potential to either stimulate or inhibit immune responses. Exploiting the immune-regulatory capacities of dendritic cells holds great promise for the treatment of cancer, autoimmune diseases and the prevention of transplant rejection. Although early clinical trials indicate that DC vaccines can induce immune responses in some cancer patients, careful study design and use of standardized clinical and immunological criteria are needed.

Vitespen: a preclinical and clinical review.

Vitespen is a heat shock protein (gp96)-peptide complex purified from resected autologous tumors, developed as a means of capturing the antigenic 'fingerprint' of a specific cancer for use as a patient-specific vaccine. Vitespen has been extensively assessed in animal models, and clinically in a range of cancers, including Phase I and II trials in colorectal cancer, glioblastoma, lung cancer, melanoma and renal cell carcinoma, and two Phase III studies in melanoma and renal cell carcinoma. Vitespen has shown itself capable of inducing major histocompatibility class I-restricted immune responses in a range of tumor types, and clinical responses in patients with earlier-stage disease, in line with previously published data on cancer vaccines. Vitespen is almost devoid of side effects aside from minor injection-site reactions.

[Nobel price for vaccination against cervical cancer: current data and guidelines]. / Nobelpreis für die Impfung gegen Zervixkrebs: Aktuelle Daten- und Leitlinienlage.

Harald zur Hausen received the Nobel price for Medicine in 2008 for his seminal research on human papilloma viruses and their association with anogenital diseases. On the basis of his work highly effective prophylactic vaccines have been developed. Clinical studies have shown nearly 100% seroconversion and an excellent safety profile as well as greater than 96% efficacy against HPV infection and premalignant dysplasia, induced by HPV types covered by the vaccine. Due to the convincing data of phase II and III clinical trials the introduction of HPV vaccination was recommended by health authorities and scientific committees in Germany. The development and availability of guidelines and evidence-based recommendations should support the introduction of the vaccine and widespread vaccination.

GMP production of pDERMATT for vaccination against melanoma in a phase I clinical trial.

For the treatment of melanoma DNA vaccines are a promising therapeutic approach. In our institute a plasmid encoding a melanoma-associated epitope (MART-1) and an immunostimulatory sequence (tetanus toxin fragment-c) termed pDERMATT was developed. In a phase I study the plasmid will be administered intradermally using a newly developed tattoo strategy to assess the toxicity and efficacy of inducing tumor-specific T-cell immunity. To facilitate this study a Good Manufacturing Practice (GMP)-compliant plasmid manufacturing process was set up and a pharmaceutical dosage form was developed. Each batch resulted in approximately 200mg plasmid DNA of a high purity >90% supercoiled DNA, an A260/280 ratio 1.80-1.95, undetectable or extremely low residual endotoxins, Escherichia coli host cell protein, RNA, and DNA. In the manufacturing process no animal derived enzymes like RNase or potentially harmful organic solvents are used. After sterile filtration the concentration of the plasmid solution is approximately 1.1mg/mL. For the scheduled phase I study a concentration of 5mg/mL is desired, and further concentration of the solution is achieved by lyophilisation. The formulation solution is composed of 1mg/mL pDERMATT and 20mg/mL sucrose in Water for Injections. Upon reconstitution with a five times smaller volume an isotonic sucrose solution containing 5mg/mL pDERMATT is obtained. Lyophilised pDERMATT is sterile with >90% supercoiled DNA, an A260-280 ratio 1.80-1.95, content 90-110% of labeled, and residual water content <2% (w/w). The product yields the predicted profile upon restriction-enzyme digestion, is highly immunogenic as confirmed in an in vivo mouse model, and stable for at least six months at 5 degrees C. We have not only developed a reproducible process to manufacture pharmaceutical grade plasmid DNA but also a stable dosage form for the use in clinical trials.

Veterinary Oncology Immunotherapies.

The ideal cancer immunotherapy agent should be able to discriminate between cancer and normal cells, be potent enough to kill small or large numbers of tumor cells, and be able to prevent recurrence of the tumor. Tumor immunology and immunotherapy are among the most exciting and rapidly expanding fields; cancer immunotherapy is now recognized as a pillar of treatment alongside traditional modalities. This article highlights approaches that seem to hold particular promise in human clinical trials and many that have been tested in veterinary medicine.

Tumor-cell number and viability as quality and efficacy parameters of autologous virus-modified cancer vaccines in patients with breast or ovarian cancer.

PURPOSE: We investigated quality and efficacy criteria of an autologous, physically and immunologically purified, Newcastle disease virus (NDV)-modified, irradiated tumor-cell vaccine (ATV-NDV) by analyzing three independent cohorts (a through c) of patients vaccinated between 1991 and 1995. MATERIALS AND METHODS: Included were 63 patients with primary breast cancer (a), 27 with metastatic pretreated breast cancer (b), and 31 with metastatic pretreated ovarian cancer (c). In addition to vaccine, cohorts b and c received nonspecific immunotherapy as supportive treatment. After cryoconservation and purification, the vaccines varied in applied numbers of viable cells and dead cell contaminations. We retrospectively hypothesized that an immunogenic vaccine should contain at least 1.5 x 10(6) viable tumor cells and viability should be at least 33%. Each cohort was thus divided into two groups; one that received vaccine type A (A), fulfilling both criteria; and the other type B (B), missing one or both criteria. RESULTS: Conventional prognostic factors were wall balanced between A and B in cohorts a and c. In cohort a, there was a benefit in survival (P = .026) and disease-free survival (P = .089) for A. In addition, in cohort a, the relative risk of dying in the group that received A as compared with B was 0.2 (univariate Cox model). There were also survival trends in favor of A versus B (P = .18 and P = .09, respectively) in cohorts b and c, with relative risks of 0.5 and 0.42, respectively. In cohort b, the survival benefit could not be ascribed to vaccine quality alone, because of prognostic imbalance in favor of A. CONCLUSION: In cohort c, like in cohort a, the survival benefit for A may be ascribed to the ATV-NDV vaccine quality, since prognostic factors were not biased. This could imply clinical effectivity in breast and ovarian cancer with ATV-NDV high-quality vaccine. Furthermore, the data provide clinically relevant information for standardization and quality control of autologous tumor-cell vaccines. A randomized study is urgently needed.

Cancer vaccination progress.

The application of 'new' and 'recognized' tumour antigens in vaccination strategies that target CD8(+) and CD4(+) T-cell responses, and the mechanisms by which these and other effector cells are activated or respond, were discussed at the second 'Progress in Vaccination Against Cancer' meeting, held at the Nottingham Trent Djanogly Conference Centre, Nottingham, UK, from 18-20 July 2002.

The FDA guidance on therapeutic cancer vaccines: the need for revision to include preventive cancer vaccines or for a new guidance dedicated to them.

Cancer vaccines based on antigens derived from self molecules rather than pathogens have been under basic and clinical investigations for many years. Up until very recently, they had been tested primarily in the setting of metastatic disease with the goal to engage the immune system in slowing down disease progression. Many therapeutic vaccine trials, either investigator initiated or led by pharmaceutical companies, have been completed and many are currently ongoing, following the FDA Guidance on therapeutic cancer vaccines published in 2011. In recent years, the target of cancer vaccines is being shifted to early cancer and even premalignant disease with the goal of preventing cancer. Although some issues addressed in the FDA Guidance on therapeutic vaccines apply to preventive vaccines, many do not. Here, we discuss a set of recommendations for revising the current Guidance to also cover preventive vaccines, or to include in a new Guidance dedicated specifically to vaccines for cancer prevention.

Assessment of the impact of manufacturing changes on the physicochemical properties and biological activity of Her1-ECD vaccine during product development.

Vaccine preparations based on the extracellular domain of Her1 (Her1-ECD) have demonstrated, in vitro and in vivo, a potent antimetastatic effect on EGFR(+) Lewis lung carcinoma model, while associated side effects were absent. The Her1-ECD is a glycoprotein with a molecular weight of 105 kDa and has 11 potential sites for N-glycosylation. Currently Her1-ECD based vaccine has been evaluated in patients with hormone refractory prostate cancer. Her1-ECD molecule used for in clinical trials was obtained from culture supernatant of HEK 293 transfectomes used the protein free culture media and is purified by immunoaffinity chromatography. In order to increase the cell growth and productivity, new defined culture media have been developed (alternative culture media) in Her1-ECD vaccine production process. In this work, a comparability study was performed to evaluate the impact of process changes in the characteristics physic-chemical and biologicals of the Her1-ECD protein and the degree of similitude between both variants. Techniques such as: SDS-PAGE, SEC-HPLC, isoelectric point, peptide mapping, mass spectrometric, SCX-HPLC, oligosaccharide map, ELISA and flow cytometric were used with this aim. Results indicated that this process change decreases the degree of sialylation of the protein but does not affect its biological activity (measured as titers of Abs and recognition for A431 cell line).

Evaluation of tumour vaccine immunotherapy for the treatment of advanced non-small cell lung cancer: a systematic meta-analysis.

OBJECTIVES: Our meta-analysis performed a systematic evaluation on the therapeutic efficacy and safety of tumour vaccines for the treatment of advanced non-small cell lung cancer (NSCLC). DESIGN: Systematic review and meta-analysis of randomised controlled trials (RCT). DATA SOURCES: PubMed, the Cochrane Center Register of Controlled Trials, Science Direct and EMBASE were searched from January 1980 until January 2015. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: RCT were included; the control arm had to receive either placebo or chemotherapy or no treatment. MAIN OUTCOME MEASURES: The quality of the data from individual papers was assessed for overall survival (OS), clinical response rate and side effects. RESULTS: Overall, 11 RCT of advanced NSCLC with a total of 3986 patients were conducted for meta-analysis. The results showed that the vaccine arm significantly extended primary endpoint median overall survival compared with control group (p<0.00001) (HR 0.760; 95% CI 0.644 to 0.896; p=0.001). Three subgroup patients with tumour vaccine at 1-year, 2-year and 3-year survival rates also gained significant benefits compared with their corresponding control group (p=0.0004, 0.03 and 0.19, respectively). Besides, a significant improvement in median time to progression (TTP), median progression-free survival (PFS) and a trend of improvement in objective response rate were observed after tumour vaccine treatment (p=0.001, 0.005 and 0.05, respectively; median PFS HR 0.842; 95% CI 0.744 to 0.954; p=0.007). A few severe adverse effects occurred in the tumour vaccine group, but fewer side effects were observed in the vaccine group compared with the control group (p<0.00001). CONCLUSIONS: Taken together, NSCLC tumour vaccines markedly prolong median OS (p<0.00001), median TTP (p=0.001) and median PFS (p=0.005), improve clinical response rate (p=0.05) and lessen adverse side effects (p<0.00001). Our meta-analysis suggests tumour vaccines improve the efficacy of the treatment, and also provide superiority in treatment of patients with advanced NSCLC among a variety of immunotherapy strategies.

Melacine: an allogeneic melanoma tumor cell lysate vaccine.

Cancer vaccines have represented the holy grail of tumor immunology to many. In 2003, there are innumerable approaches to active specific immunotherapy for cancer. The identification of tumor antigens recognized by and able to activate human T-lymphocytes has heightened the enthusiasm for this approach. Melanoma is one of the diseases that has been primarily targeted by vaccine approaches. The identification of peptides and proteins associated with cancer has provided strategies to target specific components of the cancer cell. However, older vaccines utilizing products from whole cells may have a number of advantages. Melacine (Corixa Corp.) is an allogeneic melanoma tumor cell lysate combined with the adjuvant DETOX. Pioneered by Malcolm Mitchell, it has rare but confirmed antitumor activity in metastatic melanoma (5-10%). However, previous analysis of responses in advanced disease and a recent analysis in early-stage adjuvant trials suggest that Melacine may have its greatest benefit in a large subset of melanoma patients who express either the human leukocyte antigen (HLA) class I antigens A2 and/or HLA-C3. This finding must now be confirmed in a large perspective observation-controlled clinical trial to solidify the role of Melacine as an effective cancer vaccine in melanoma.

Granulocyte-macrophage colony-stimulating factor gene-transfected autologous tumor cell vaccine: focus[correction to fcous] on non-small-cell lung cancer.

Traditionally, non-small-cell lung cancer (NSCLC) is not thought of as an immunosensitive malignancy. However, recent clinical results with GVAX, a granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced autologous tumor vaccine, may suggest otherwise. This review summarizes immune-induced activity caused by GM-CSF protein and GM-CSF gene-transfected vaccines. Initial indication of use for GM-CSF protein (sargramostim) was to improve neutrophil recovery following cytotoxic chemotherapy. However, several trials involving patients with hematologic malignancy demonstrated improvement in survival related to delayed disease progression in patients receiving sargramostim in combination with chemotherapy. Subsequently, others explored potential antitumor activity with sargramostim in a variety of trials. Results did not consistently demonstrate sufficient antitumor activity to justify routine use of sargramostim as an anticancer agent. Preclinical work with GM-CSF gene-transfected vaccines, however, did demonstrate significant activity, thereby justifying clinical investigation. Patients with metastatic NSCLC who had previously failed chemotherapy demonstrated response to GVAX (3 of 33 complete responses) and dose-related improvement in survival (471 days vs. 174 days).