Preparation Of Cancer Vaccines Research Articles

Establishing safe high hydrostatic pressure devitalization thresholds for autologous head and neck cancer vaccination and reconstruction

High hydrostatic pressure specifically devitalizes cells and tissues without major changes in their molecular structure. Hence, high hydrostatic pressure may enhance the development of whole-cell anti-tumor vaccines, representing tumor heterogeneity and thus (neo-) antigen diversity. Moreover, safe devitalization of tumor-infiltrated supporting tissue may facilitate reimplantation for functional reconstruction. However, precise high hydrostatic pressure thresholds for safe cancer cell killing are unknown. Here, we show that high hydrostatic pressure of at least 450 MPa is necessary to safely devitalize head and neck squamous cell cancer. A pressure of 300 MPa, which has been used frequently in cancer vaccine preparation, resulted in partial devitalization with 27% live cells in flow cytometry and 4% remaining autofluorescence in cell culture after one week. The remaining cells could form vital tumors in the chorioallantoic membrane assay. In contrast, 450 MPa killed all cells in vitro and prevented tumor outgrowth in ovo. The effectiveness of 450 MPa was attributed to the induction of DNA double-strand breaks, independent of apoptosis, autophagy, or methuosis. Furthermore, 450 MPa continued to induce immunogenic cell death. Our results demonstrate that 450 MPa of high hydrostatic pressure induces safe and sustained devitalization of head and neck cancer cells and tissues. Because of the heterogeneity in pressure resistance, we propose our approach as a starting point for determining the precise thresholds for other cancer entities. Further studies on head and neck cancer should focus on immunological co-cultures, combinations of immune checkpoint inhibition, and accurate anatomical reconstruction with pressure-treated autografts.

A novel rapid modularized hepatitis B core virus-like particle-based platform for personalized cancer vaccine preparation via fixed-point coupling

Personalized cancer vaccine which targets neoepitopes shows great promise for cancer treatment. However, rapid preparation is a critical challenge for clinical application of personalized cancer vaccine. Genetic recombination and chemical modification are a time-consuming “trial and error” pattern for making vaccines. Here we first constructed a platform for peptide vaccine preparation by inserting SpyCatcher into the major immunodominant region (MIR) of hepatitis B core protein (HBc) (1-183). The resulted recombinant protein HBc(1-183)-SpyCatcher (HBc(1-183)-S) assembled to virus-like particles (VLPs) and readily bound to SpyTag conjugated with OVA epitope peptides by just mixing, forming HBc(1-183)-S-OVA. HBc(1-183)-S-OVA VLPs effectively induced dendritic cell maturation. Our further results indicated that HBc(1-183)-S-OVA VLPs vaccination inhibited tumor growth in both prophylactic and treatment ways in E.G7-OVA tumor bearing mice by generating significant OVA-specific cytotoxic T lymphocyte responses. Our study provides a simple, rapid, efficient and universal HBc-based platform for the preparation of personalized cancer vaccine.

Melanoma antigen family A4 protein produced by transgenic silkworms induces antitumor immune responses.

Recent clinical trials with the aim of developing tumor antigen (TA)-specific cancer vaccines against a number of malignancies have focused on the identification of TAs presented by tumor cells and recognized by T cells. In the present study, the TA melanoma antigen family A4 (MAGE-A4) protein was produced using a transgenic (TG) silkworm system. Using in vitro stimulation, it was subsequently determined whether MAGE-A4 protein induced MAGE-A4-specific T cells from peripheral blood mononuclear cells of healthy donors. TG silkworm lines expressing a MAGE-A4 gene under an upstream activating sequence (UAS) were mated with those expressing a yeast transcription activator protein (GAL4) at the middle silk glands (MSGs) and embryos that harbored both the GAL4 and UAS constructs were selected. Recombinant MAGE-A4 protein was extracted from the MSGs of TG silkworms and evaluated using SDS-PAGE and western blot analysis. It was observed that MAGE-A4 produced by the TG silkworm system successfully induced MAGE-A4-specific CD4+ T cell responses. Furthermore, MAGE-A4-specific CD4+ T cells recognized antigen-presenting cells when pulsed with a MAGE-A4+ tumor cell lysate. The present data suggests that recombinant tumor antigen production using the TG silkworm system may be a novel tool in the preparation of cancer vaccines.

In vivo antitumor activity evaluation of cancer vaccines prepared by various antigen forms in a murine hepatocellular carcinoma model.

Cancer cell vaccines with strong specificity and low tolerance have been revealed to be a promising option for oncology treatment. Various antigen forms, including tumor cell lysate and glutaraldehyde-fixed tumor cells, have been intensively used in cancer vaccine preparation. However, the most effective antigen form has not yet been identified. In the present study, the antitumor efficiency of vaccines prepared by these two antigen forms was systematically investigated. Murine H22 hepatocellular carcinoma cell lysate and glutaraldehyde-fixed H22 hepatocellular carcinoma cells were conjugated with Freund's adjuvant to prepare vaccines, H22-TCL and Fixed-H22-CELL, respectively. H22-TCL and Fixed-H22-CELL were administrated by subcutaneous immunization in prophylactic and therapeutic strategies. The results of the present study revealed that H22-TCL immunization induced more significant inhibition on tumor growth and metastasis compared with Fixed-H22-CELL injection. Furthermore, histopathological observation demonstrated that H22-TCL vaccine induced larger areas of continuous necrosis within tumors compared to the Fixed-H22-CELL vaccine, which was associated with the extent of tumor inhibition. More importantly, the H22-TCL vaccine injection elicited more evident antigen-specific antibody responses compared with the Fixed-H22-CELL injection. Splenocytes from H22-TCL vaccinated mice also exhibited a more significant T lymphocytes proliferation compared with that from Fixed-H22-CELL-treated mice. All the results indicated that whole tumor cell lysate may be a more effective antigen form in cancer vaccine preparation compared with glutaraldehyde-fixed tumor cells, which elicited more marked antigen specific humoral and cellular immune responses resulted with a superior antitumor efficiency. This would have important clinical signification for cancer vaccine preparation and serve a role in prompting this to other researchers.

Delivery of Synthetic mRNA Encoding FOXP3 Antigen into Dendritic Cells for Inflammatory Breast Cancer Immunotherapy.

Dendritic cell (DC)-based vaccines are commonly used for cancer immunotherapy. To prepare vaccines, DCs are pulsed or transfected with either: (a) defined peptides of tumor-associated antigens, (b) total protein isolated from the tumor cell, (c) autologous total RNA isolated from the tumor cell, (d) synthetic tumor-antigen-encoding mRNA, or (e) genes that encode for specific tumor-associated antigens. Introduction of tumor-associated antigen(s) and subsequent generation of mature DCs that can stimulate tumor-antigen-specific cytotoxic T lymphocytes comprise the critical steps of cancer vaccine preparation. Here, we described a method of: (a) preparing and delivering synthetic FOXP3 mRNA into human DCs, (b) generating mature DCs,

A microfluidic study of mouse dendritic cell membrane transport properties of water and cryoprotectants

Dendritic cells (DCs) are antigen presenting cells that have been increasingly used in immunotherapy treatment of various diseases and have a great potential for cancer vaccine preparation. Cryopreservation and banking of DCs is critical to facilitate flexible and effective immunotherapy treatment. Therefore, determination of water and cryoprotectant transport properties of DC membrane is indispensable for developing optimal conditions for DCs cryopreservation. Using mouse DCs (mDCs), membrane transport properties were investigated using a microfluidic perfusion system which has recently been developed and fabricated by soft lithography. Cells in the microfluidic system were perfused with various hyperosmotic solutions in the presence or absence of cryoprotectant. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and analyzed using a biophysical model to determine water and cryoprotectant transport properties of the cell membrane as well as associated activation energies. It was also shown from this study that the microfluidic perfusion system readily allowed us to: (1) quickly change or manipulate extracellular environment of cells; (2) confine cells in a monolayer channel which prevents imaging ambiguity; and (3) study cell membrane transport kinetics and properties.

A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumor cell hybrid vaccines.

Fusion of tumor cells with antigen-presenting cells (APCs) has been proposed for the preparation of cancer vaccines. However, generation of these hybrids, using physical or chemical methods such as electrofusion or polyethylene glycol (PEG), has been difficult to standardize. Characterization of cell fusion has also been problematic because of difficulties in differentiating fusion from cell aggregation, leakage of cellular dyes and dendritic-cell (DC) phagocytosis of tumor material. In this report, we describe a new method to generate hybrid cell vaccines, based on gene transfer of a viral fusogenic membrane glycoprotein (FMG) into tumor cells, and incorporate a genetic method by which true hybrid formation can be unambiguously detected. We describe a new class of tumor cell-DC hybrid that can be rapidly isolated after cell fusion. These hybrids are highly potent in in vitro antigen presentation assays, target lymph nodes in vivo and are powerful immunogens against established metastatic disease.

Cancer immunotherapy: potential involvement of mediators

The description of a cell-free soluble anti-tumour factor by Carswell et al. in 1975 (Proc Natl Acad Sci USA, 72: 3666–3670) was followed by a long series of experimental and clinical investigations into the role of cell-free mediators in cancer immunotherapy. These investigations included research on the effects of macrophage–derived eicosanoids (cycloxygenase and lipoxygenase derivates of arachidonic acid) and of monokines such as tumour necrosis factor-α, interleukin-1 and granulocyte–monocyte–macrophage–colony stimulating factor) and of lymphocyte products: interleukins and interferons. The investigations yielded information on the effects of various factors on macrophage and T-cell activation in vitro, determination of direct anti-tumour properties on animal and human tumour cells in vitro and on therapeutic effectiveness in tumour-bearing individuals either alone or in combination with other therapeutic factors and their production by tumour cells. During recent years much effort has been dedicated towards the use of the tumour cells transfected with cytokine genes in the preparation of cancer vaccines. Cycloxygenase products (prostaglandins) were usually assumed to inhibit expression of anti-tumour activity by macrophages and an increase in their production in cancer patients was considered as a poor prognostic index. Lipoxygenase (leukotrienes) products were assumed to exhibit antitumour activity and to induce production of IL-1 by macrophages. Interleukins 2, 4, 6, 7, 12 and the interferons were extensively tested for their therapeutic effectiveness in experimental tumour models and in cancer clinical trials. The general conclusion on the use of cell-free mediators for cancer immunotherapy is that much still has to be done in order to assure effective and reproducible therapeutic effectiveness for routine use in the treatment of human neoplasia.