Fermentation, purification and immunogenicity of a recombinant tumor multi-epitope vaccine, VBP3.

The recombinant multi-epitope vaccine called VBP3 is designed to suppress tumor growth and angiogenesis through targeting both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA). We are aiming to produce VBP3 vaccine in a large scale and provide sufficient protein for pre-clinical study. High cost and potential toxicity are severe limitations of IPTG and we investigated whether lactose can mediate VBP3 induction. Firstly, we identified the biological characteristics and established a culture bank of VBP3 strains. The best-performing strains were selected and the fermentation mode of medium, bacterial growth and protein expression were optimized in shake flasks. We scaled up the VBP3 production in 10 L bioreactor using lactose as inducer and the protein yield was comparable with IPTG induction. Next, the target protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, with a SDS-PAGE purity over 90%. Further, the purified VBP3 vaccine was subcutaneously injected in BALB/c mice and elicited high-titer anti-bFGF (1:32,000) and anti-VEGFA (1:4000) antibodies. Take together, lactose was an applicable inducer for VBP3 production and the eligible product of VBP3 was harvested in the large-scale fermentation, supporting the industrial production and pre-clinical study in the future. The VBP3 vaccine with superior immunogenicity might be used as a potential therapeutic vaccine for tumor treatment.

Personalized cancer neoantigen vaccines come of age.

Cancer vaccines have encountered their ideal personalized partner along with evidence for great breakthroughs in the identification and synthesis of neoantigens. Individual cancer neoantigen vaccines are capable of eliciting robust T-cell responses and have been demonstrated to achieve striking clinical efficacy due to their high immunogenicity and central thymic tolerance escape of neoantigens. Two recent phase I clinical trials have provided support for the hypothesis and have heralded a nascent era of personalized vaccines in the field of immunotherapy. This review aims to address the identification of neoepitopes and describes advances made in personalized vaccines. In addition, this review discusses the challenges related to the exploitation of vaccine therapy, and provides potential thoughts for the improvement of vaccine design and applications.

Tumor lysate-loaded Bacterial Ghosts as a tool for optimized production of therapeutic dendritic cell-based cancer vaccines.

Cancer immunotherapy with dendritic cell (DC)-based vaccines has been used to treat various malignancies for more than two decades, however generally showed a limited clinical success. Among various factors responsible for their modest clinical activity is the lack of universally applied, standardized protocols for the generation of clinical-grade DC vaccines, capable of inducing effective anti-tumor immune responses. We investigated Bacterial Ghosts (BGs) - empty envelopes of Gram-negative bacteria - as a tool for optimized production of DC vaccines. BGs possess various intact cell surface structures, exhibiting strong adjuvant properties required for the induction of DC maturation, whereas their empty internal space can be easily filled with a source tumor antigens, e.g. tumor lysate. Hence BGs emerge as an excellent platform for both the induction of immunogenic DC maturation and loading with tumor antigens in a single-step procedure. We compared the phenotype, cytokine secretion profile, functional activity and ability to induce immunogenic T-cell responses in vitro of human monocyte-derived DCs generated using BG platform and DCs matured with widely used lipopolysaccharide (LPS) plus interferon-γ cocktail and loaded with tumor lysate. Both approaches induced DC maturation, however BG-based protocol was superior to LPS-based protocol in terms of the ability to induce DCs with a lower tolerogenic potential, resulting in a more robust CD8+ T cell activation and their functional activity as well as significantly lower induction of regulatory T cells. These superior parameters are attributed, at least in part, to the ability of BG-matured DCs to resist potential immunosuppressive and pro-tolerogenic activity of various tumor cell lysates, including melanoma, renal carcinoma and glioblastoma.

Cancer vaccines and immunotherapeutic approaches in hepatobiliary and pancreatic cancers.

Hepatobiliary and pancreatic cancers along with other gastrointestinal malignancies remain the leading cause of cancer-related deaths worldwide. Strategies developed in the recent years on immunotherapy and cancer vaccines in the setting of primary liver cancer as well as in pancreatic cancer are the scope of this review. Significance of orthotopic and autochthonous animal models which mimic and/or closely reflect human malignancies allowing for a prompt and trustworthy analysis of new therapeutics is underlined. Combinational approaches that on one hand, specifically target a defined cancer-driving pathway, and on the other hand, restore the functions of immune cells, which effector functions are often suppressed by a tumor milieu, are shown to have the strongest perspectives and future directions. Among combinational immunotherapeutic approaches a personalized- and individual cancer case-based therapy is of special importance.

Use of real-time cellular analysis and Plackett-Burman design to develop the serum-free media for PC-3 prostate cancer cells.

In this study, we developed a rapid strategy to screen a serum-free medium for culturing the anchorage-dependent PC-3 prostate cancer cells, which was going to be prepared in large scale to generate GM-CSF/TNFα-surface-modified whole cell prostate cancer vaccine. Automated real-time cellular analysis as a rapid and non-invasive technology was used to monitor the growth of PC-3 cells in 16-well plates. At the same time, Plackett-Burman design was employed to identify the most influential formulation by integrating relevant information statistically. The effects of the 16 selected factors were evaluated during exponential cell growth and three medium constituents (EGF, FGF and linoleic acid) were identified to have significant effects on the cell growth. Subsequently, the response surface methodology with central composite design was applied to determine the interactions among the three factors so that these factors were optimized to improve cell growth. Finally, the prediction of the best combination was made under the maximal response to optimize cell growth by Design-Expert software 7.0. A total of 20 experiments were conducted to construct a quadratic model and a second-order polynomial equation. With the optimized combination validated by the stability test of serial passaging PC-3 cells, the serum-free medium had similar cell density and cell viability to the original serum medium. In summary, this high-throughput scheme minimized the screening time and may thus provide a new platform to efficiently develop the serum-free media for adherent cells.

Prospects for the future of prostate cancer vaccines.

Cancer therapy is undergoing a revolution fueled by clinical data demonstrating that the immune system has significant anti-tumor capability. Although the main focus of this revolution currently rests upon immune checkpoint inhibitors in diseases such as melanoma, lung and bladder cancer, it was actually a therapeutic cancer vaccine in prostate cancer that provided the first data demonstrating that a modern immunotherapy, beyond cytokines, could enhance clinical outcomes. As immunotherapy is poised to take center stage among cancer therapies, the role of cancer vaccines remains somewhat undefined in prostate cancer, though emerging data suggest that vaccines could play a crucial therapeutic role.

Mapping T and B cell epitopes in sperm protein 17 to support the development of an ovarian cancer vaccine.

Ovarian cancer (OC) is the seventh most common cancer in women worldwide, and the leading cause of death from gynaecological malignancy. Immunotherapeutic strategies including cancer vaccines are considered less toxic and more specific than current treatments. Sperm surface protein (Sp17) is a protein aberrantly expressed in primary as well as in metastatic lesions in >83% of ovarian cancer patients. Vaccines based on the Sp17 protein are immunogenic and protective in animal models. To map the immunogenic regions and support the development of human Sp17 peptide based vaccines, we used 6 overlapping peptides of the human Sp17 sequence adjuvanted with CpG to immunise humanised HLA-A2.1 transgenic C57BL/6 mice, and assessed immunogenicity by ELISPOT and ELISA. No CD8 T cells were found to be induced to a comprehensive panel of 10 HLA-A2.1 or H-2K(b) binding predicted epitopes. However, one of the 6 peptides, hSp17111-142, induced high levels of antibodies and IFN-γ producing T cells (but not IL-17 or IL-4) both in C57BL/6 and in C57BL/6-HLA-A2.1 transgenic mice. C57BL/6 mice immunised with CpG adjuvanted hSp17111-142 significantly prolonged the life-span of the mice bearing the ovarian carcinoma ID8 cell line. We further mapped the immuno-dominant B and T cell epitope regions within hSp17111-142 using ELISPOT and competition ELISA. Herein, we report the identification of a single immuno-dominant B cell (134-142 aa) epitope and 2 T helper 1 (Th1) cell epitopes (111-124 aa and 124-138 aa). These result together support further exploration of hSp17111-142 peptide formulations as vaccines against ovarian cancer.

Current methods of epitope identification for cancer vaccine design.

The importance of the immune system in tumor development and progression has been emerging in many cancers. Previous cancer vaccines have not shown long-term clinical benefit possibly because were not designed to avoid eliciting regulatory T-cell responses that inhibit the anti-tumor immune response. This review will examine different methods of identifying epitopes derived from tumor associated antigens suitable for immunization and the steps used to design and validate peptide epitopes to improve efficacy of anti-tumor peptide-based vaccines. Focusing on in silico prediction algorithms, we survey the advantages and disadvantages of current cancer vaccine prediction tools.

Evolution of animal models in cancer vaccine development.

Advances in cancer vaccine development are facilitated by animal models reflecting key features of human cancer and its interface with host immunity. Several series of transplantable preneoplastic and neoplastic mouse mammary lesions have been used to delineate mechanisms of anti-tumor immunity. Mimicking immune tolerance to tumor-associated antigens (TAA) such as HER2/neu, transgenic mice developing spontaneous mammary tumors are strong model systems for pre-clinical vaccine testing. In these models, HER2 DNA vaccines are easily administered, well-tolerated, and induce both humoral and cellular immunity. Although engineered mouse strains have advanced cancer immunotherapy, basic shortcomings remain. For example, multiple mouse strains have to be tested to recapitulate genetic regulation of immune tolerance in humans. Outbred domestic felines more closely parallel humans in the natural development of HER2 positive breast cancer and their varying genetic background. Electrovaccination with heterologous HER2 DNA induces robust adaptive immune responses in cats. Importantly, homologous feline HER2 DNA with a single amino acid substitution elicits unique antibodies to feline mammary tumor cells, unlocking a new vaccine principle. As an alternative approach to targeted vaccination, non-surgical tumor ablation such as cryoablation induces anti-tumor immunity via in situ immunization, particularly when combined with toll-like receptor (TLR) agonist. As strategies for vaccination advance, non-invasive monitoring of host response becomes imperative. As an example, magnetic resonance imaging (MRI) and positron emission tomography (PET) scanning following administration of tryptophan metabolism tracer [11C]-alpha-methyl-tryptophan (AMT) provides non-invasive imaging of both tumor growth and metabolic activities. Because AMT is a substrate of indoleamine-pyrrole 2,3-dioxygenase (IDO), an enzyme that produces the immune regulatory molecule kynurenine, AMT imaging can provide novel insight of host response. In conclusion, new feline models improve the predictive power of cancer immunotherapy and real-time PET imaging enables mechanistic monitoring of host immunity. Strategic utilization of these new tools will expedite cancer vaccine development.

Vaccines for tumor prevention: a pipe dream?

Whether or not a tumor expresses peculiar antigens that differentiate it from normal cells was intensively investigated in the 1950s. A conclusive answer was provided in 1960 when George Klein showed that a tumor can be rejected by the immune response elicited by a vaccine administered to the same mouse in which the tumor was induced. Whether immunogenicity was a feature restricted only to tumors artificially induced by viruses or by high doses of chemical carcinogens was then hotly debated until Terry Boon showed, in the 1980s, that almost any tumor can be recognized by a syngeneic immune system triggered by an appropriate cancer vaccine. However, the therapeutic efficacy of vaccine-induced immunity against an advanced tumor is marginal. The combination of an anti-tumor vaccine with new sophisticated maneuvers to contrast tumor-induced suppression may yield new and effective therapeutic strategies. Also, the exploitation of tumor vaccines to prevent tumors in cohorts of people with a specific risk of cancer may become a fresh strategy with great potential to control tumor onset.

Design of universal cancer vaccines using natural tumor vessel-specific antigens (SANTAVAC).

Vaccination against endothelial cells (ECs) lining the tumor vasculature represents one of the most attractive potential cancer immunotherapy options due to its ability to prevent solid tumor growth. Using this approach, target antigens can be derived from ECs and used to develop a universal cancer vaccine. Unfortunately, direct immunization with EC preparations can elicit autoimmune vasculitis in normal tissues. Recently, tumor-induced changes to the human EC surface were described that provided a basis for designing efficient EC-based vaccines capable of eliciting immune responses that targeted the tumor endothelium directly. This review examines these data from the perspective of designing EC-based cancer vaccines for the treatment of all solid tumors, including the antigen composition of vaccine formulations, the selection ECs for antigen derivation, the production and control of antigens, and the method for estimating vaccine efficacy and safety. As the vaccine preparation requires a specifically derived set of natural cell surface antigens, a new vaccine preparation concept was formulated. Antigen compositions prepared according to this concept were named SANTAVAC (Set of All Natural Target Antigens for Vaccination Against Cancer).

Could mycobacterial Hsp70-containing fusion protein lead the way to an affordable therapeutic cancer vaccine?

Cancer vaccine development efforts have recently gained momentum, but most vaccines showing clinical impact in human trials tend to be based on technology approaches that are very costly and difficult to produce at scale. With the projected doubling of the incidence of cancer and its related cost of care in the U.S. over the next two decades, the widespread clinical use of such vaccines will prove difficult to justify. Heat shock protein-based vaccines have shown the potential to elicit clinically meaningful immunologic responses in cancer, but the predominant development approach - heat shock protein-peptide complexes derived from a patient's own tumor - face similar challenges of cost and scalability. New innovative modalities for deploying heat shock proteins in cancer vaccines may open the door to vaccines that can generate potent cytotoxic responses against multiple tumor targets and can be made in a cost-effective and scalable manner.

Portrait of Dr Michael G. Hanna, Jr. A pioneer in active specific immunotherapy of cancer.

While a PhD candidate, doing my thesis at the Oak Ridge National Laboratory, Biology Division under Dr. Charles Congdon, my introduction to the immune response was studying graft vs. host (GVH) disease as a consequence of bone marrow transplantation in mice. The sequalae of GVH was impressive, and demonstrated the potential of negative clinical consequences of the immune system. The idea of harnessing this immunological phenomena in cancer therapy was appealing even in the late 1960s. The problem was that at the time T-cells as a component of the immune system were identified but not defined. We moved to soluble antigen stimulation in mice and recognized and described the post antigen stimulation changes in lymphatic tissue germinal centers during the first 48 h after the induction of the humoral immune response. We described the extracellular localization of soluble antigens on the surface of dendritic reticular cells of the stroma, directing a response of B-cells to produce antibody against non-self. The ensuing reaction was the rapid proliferation of B-cells toward antibody secreting plasma cells.

Current status of autologous breast tumor cell-based vaccines.

Approximately nine out of ten breast cancer-related deaths are attributable to metastasis. Yet, less than 4% of breast cancer patients are initially diagnosed with metastatic cancer. Therefore, the majority of breast cancer-related deaths are due to recurrence and progression of non-metastatic disease. There is tremendous clinical opportunity for novel adjuvant strategies, such as immunotherapies, that have the potential to prevent progressive recurrences. In particular, autologous tumor cell-based vaccines (ATCVs) can train a patient's immune system to recognize and eliminate occult disease. ATCVs have several advantages including safety, multivalency and patient specificity. Furthermore, because lumpectomy or mastectomy is indicated for the vast majority of breast cancer patients, resected tumors offer a readily available, patient-specific source of tumor antigen. Disadvantages of ATCVs include poor immunogenicity and production inconsistencies. This review summarizes recent progress in the development of autologous breast tumor vaccines and offers insight for overcoming existing limitations.