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A promising area of clinical investigation is the use of cancer immunotherapy to treat cancer patients. Dendritic cells (DCs) operate as professional antigen-presenting cells (APCs) and play a critical role in the induction of antitumor immune responses. Thus, DC-based cancer immunotherapy represents a powerful strategy. One DC-based cancer immunotherapy strategy that has been investigated is the administration of fusion cells generated with DCs and whole tumor cells (DC-tumor fusion cells). The DC-tumor fusion cells can process a broad array of tumor-associated antigens (TAAs), including unidentified molecules, and present them through major histocompatibility complex (MHC) class I and II pathways in the context of co-stimulatory signals. Improving the therapeutic efficacy of DC-tumor fusion cell-based cancer immunotherapy requires increased immunogenicity of DCs and whole tumor cells. We discuss the potential ability of DC-tumor fusion cells to activate antigen-specific T cells and strategies to improve the immunogenicity of DC-tumor fusion cells as anticancer vaccines.

Background/aim: Chemoimmunotherapy has been used to treat intrahepatic cholangiocarcinoma (ICC). However, little is known about the phenomena underlying the immunomodulation of ICC cells elicited by chemoimmunotherapy., Materials and Methods: Primary ICC cells from a patient with ICC who received gemcitabine followed by 5-fluorouracil (5-FU), both combined with dendritic cells pulsed with Wilms' tumor 1 (WT1) peptides were cultured. ICC cells were treated with gemcitabine, 5-FU or interferon (IFN)-γ in vitro. The phenotype of the ICC cells was examined by flow cytometry and quantitative reverse transcription polymerase chain reaction., Results: Stimulation of the ICC cells with gemcitabine resulted in up-regulation of WT1 mRNA, programmed death receptor ligand-1 (PDL1) and calreticulin. Gemcitabine, 5-FU and IFN-γ induced up-regulation of mucin-1. Moreover, human leukocyte antigen (HLA)-ABC, HLA-DR and PDL1 were extremely up-regulated by IFN-γ., Conclusion: Chemoimmunomodulating agents alter the immunogenicity of ICC cells, resulting in complex clinical efficacy results., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The incidence of colorectal cancer (CRC) is on the rise, and the prognosis for patients with recurrent or metastatic disease is extremely poor. Although chemotherapy and radiation therapy can improve survival rates, it is imperative to integrate alternative strategies such as immunotherapy to improve outcomes for patients with advanced CRC. In this review, we will discuss the effect of immunotherapy for inducing cytotoxic T lymphocytes and the major immunotherapeutic approaches for CRC that are currently in clinical trials, including peptide vaccines, dendritic cell-based cancer vaccines, whole tumor cell vaccines, viral vector-based cancer vaccines, adoptive cell transfer therapy, antibody-based cancer immunotherapy, and cytokine therapy. The possibility of combination therapies will also be discussed along with the challenges presented by tumor escape mechanisms.

In the current study, exposure of mammary tumor cells derived from mice transgenic for the polyomavirus middle T oncogene to ionizing radiation resulted in the generation of a tumor cell population that preferentially expressed cancer stem cell markers. In addition, these cells were more resistant to subsequent radiation treatments and appeared to acquire an enhanced capacity for dissemination to the lungs of mice. Therefore, we tested an immunotherapy approach to the treatment of local and disseminated mammary tumor cells in a murine model using a recently developed molecular chaperone-based vaccine that specifically targets the radioresistant subpopulation of tumor cells. Heat shock protein 70-peptide complexes (Hsp70.PC-F) were extracted from fusions of dendritic cells and radiation-enriched tumor cells, and the resulting chaperone vaccines were used to treat mice with pre-existing lung metastases. Immunization of mice with the Hsp70.PC-F vaccine resulted in a T cell-mediated immune response, including a significant increase in CD4 and CD8 T cell proliferation and the induction of effector T cells capable of targeting radioresistant tumor cells. Importantly, the growth of primary tumors was inhibited, and the number of tumor cells metastasizing to lung was reduced significantly by combining chaperone vaccine with radiotherapy. These results indicate that Hsp70.PC-F vaccine can induce specific immunity to radioresistant populations of mammary tumor cells and, thus, can complement radiotherapy, leading to synergistic killing.

The molecular chaperone heat-shock protein 70 (Hsp70) possesses immune stimulatory properties that have been employed in the preparation of anticancer vaccines. Hsp70 binds antigenic peptides in the cytoplasm of cancer cells. Hsp70 thus serves as a convenient, non-discriminating transporter of antigens and can protect the peptides during internalization by APC and cross presentation to T lymphocytes. We describe a method for purifying Hsp70-peptide complexes that can be used to prepare molecular chaperone-based vaccines, involving sequential gel filtration, ion exchange, and affinity chromatography.

Pancreatic cancer is a highly aggressive and notoriously difficult to treat. As the vast majority of patients are diagnosed at advanced stage of the disease, only a small population is curative by surgical resection. Although gemcitabine-based chemotherapy is typically offered as standard of care, most patients do not survive longer than 6 months. Thus, new therapeutic approaches are needed. Pancreatic cancer cells that develop gemcitabine resistance would still be suitable targets for immunotherapy. Therefore, one promising treatment approach may be immunotherapy that is designed to target pancreatic-cancer-associated antigens. In this paper, we detail recent work in immunotherapy and the advances in concept of combination therapy of immunotherapy and chemotherapy. We offer our perspective on how to increase the clinical efficacy of immunotherapies for pancreatic cancer.

Fetal calf serum (FCS)-independent pancreatic cancer cells were established in plasma protein fraction (PPF)-supplemented medium that is an agent of good manufacturing practice (GMP) grade. Dendritic cells (DCs) were activated with the Toll-like receptor agonist, penicillin-inactivated Streptococcus pyogenes (OK-432) that is also a GMP grade agent. Therefore, sufficient amounts of FCS-independent fusions were successfully generated with decreased potential hazards of FCS. The FCS-independent fusions expressed tumor-associated antigens, HLA-DR, costimulatory molecules, IL-12, and IL-10. Stimulation of T cells with fusions from healthy donors resulted in proliferation of T cells with high expression levels of perforin/granzyme B and IFN-gamma and efficient induction of antigen-specific cytotoxic T lymphocytes (CTLs). Selection and expansion of T-cell clones were confirmed by TCR Vbeta analysis. However, fusions from patients with metastatic pancreatic cancer induced increased expression levels of TGF-beta1 in CD4+ CD25high T cells and low levels of CTLs with decreased IFN-gamma production., (Copyright 2010 Elsevier Inc. All rights reserved.)

The deployment of dendritic cell (DC) and tumor cell fusions is increasing in tumor immunotherapy. In animal and human studies, fusion cell vaccines have been shown to possess the elements essential for processing and presenting tumor antigens to host immune cells, for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens. Moreover, fusion cell vaccines provide protection against challenge with tumor cells and mediate regression of established tumors. Despite these unique features of fusion cell vaccines and the observation of tumor eradication in animal studies, limited success has occurred in clinical trials. This article reviews the methods used for optimizing the preparation and selection of DC-tumor fusion cells and analyzes factors influencing the success or failure of fusion cell-mediated immunotherapy. In addition, we discuss the challenges facing effective fusion cell vaccine production, including factors in preparation, selection and quality control of fusion cell vaccines, as well as approaches for enhancing anti-tumor immunity.

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2(-)/HLA-24(-)), carcinoembryonic antigen (CEA)(+), and MUC1(+) as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2(+) and/or HLA-A24(+)) and allogeneic COLM-6 resulted in MHC class I- and MHC class II-restricted proliferation of CD4(+) and CD8(+) T cells, high levels of IFN-gamma production in both CD4(+) and CD8(+) T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.

Presentation of AML antigens by dendritic cells (DC) could potentially induce a T cell-mediated anti-leukemic immune response. In the present study, we generated DC from adherent (AD-DC) and non-adherent (NAD-DC) myeloblasts obtained from bone marrows of AML patients. Both cell populations displayed morphological, phenotypic and functional properties of DC. The functions of NAD-DC were compared to AD-DC that had been fused with autologous AML blasts (FC/AML). The FC/AML induced greater T cell proliferation and CTL activity against autologous AML blasts (9/10 cases) as compared to NAD-DC. FC/AML may thus represent a promising strategy for DC-based immunotherapy of patients with AML. [Copyright &y& Elsevier]

Multiple myeloma (MM) cells express certain tumour-associated antigens (TAAs) that could serve as targets for active-specific immunotherapy. The aim of the present study was to test the MM/dendritic cell (DC) fusion as a vaccination strategy. We fused MM cells with DC to generate fusion cells (FCs) and tested their antigen presenting cell (APC) function in mixed lymphocyte reactions and cytotoxicity assays. First, the HS Sultan and SK0-007 HAT sensitive human MM cell lines and DCs generated from peripheral blood of normal donors were fused in the presence of 50% polyethylene glycol to form FCs. Next, tumour cells freshly isolated from patients were similarly fused with autologous DCs to generate FCs. The FCs demonstrated a biphenotypic profile, confirmed both by flow-cytometry and dual immunofluorescence microscopy. These FCs induced MM-specific cytotoxicity. FCs, but not MM cells or DCs alone, were potent stimulators of autologous patient T cells. More importantly, FC-primed autologous peripheral blood mononuclear cells demonstrated major histocompatibility complex-restricted MM-specific cytolysis. These studies therefore demonstrated that MM/DC FC can trigger an autologous immune response to MM cells and formed the framework for a clinical trial currently underway. [ABSTRACT FROM AUTHOR]

We designed a phase 1 study using dendritic cells (DCs) pulsed with a mixture of three types of Wilms’ tumor 1 (WT1) peptides, including MHC class I/II restricted epitopes (DC/WT1-I/II). Our recent work reveals that the combination of DC/WT1-I/II and chemotherapy induced long-term WT1-specific CD4+and CD8+T cell responses. [ABSTRACT FROM AUTHOR]

Summary The tumour-associated antigen mucin 1 (MUC1) is a multifunctional protein involved in protection of mucous membranes, signal transduction, and modulation of the immune system. More than 70% of cancers overexpress MUC1, making MUC1 a potential target for immunotherapy. In the present study, MUC1 transgenic mice were crossed with syngeneic strains that express the polyomavirus middle-T oncogene (PyMT) driven by the mouse mammary tumour virus promoter long-terminal repeat (MMTV-LTR). The resultant breed (MMT mice) developed spontaneous MUC1-expressing mammary carcinomas with 100% penetrance at 8–15 weeks of age. As found in human breast cancer, the mammary carcinoma in MMT mice arose in multiple stages. Immunization with fusions of dendritic cells and MUC1-positive tumour cells (FC/MUC1) induced MUC1-specific immune responses that blocked or delayed the development of spontaneous breast carcinomas. In contrast, there was no delay of tumour development in MMT mice immunized with irradiated MC38/MUC1 tumour cells. The efficacy of fusion cells was closely correlated with the timing of initial immunization. Immunization with FC/MUC1 initiated in MMT mice at < 1, 1–2 and 2–3 months of age rendered 33, 5 and 0% of mice free of tumour, respectively, up to 6 months. Whereas mice immunized in the later stage of tumour development succumbed to their disease, immunization resulted in control of tumour progression and prolongation of life. These results indicate that immunization with FC/MUC1 can generate an anti-MUC1 response that is sufficient to delay the development of spontaneous mammary carcinomas and control tumour progression in MMT mice. [ABSTRACT FROM AUTHOR]

Abstract: We have reported that fusions of patient-derived dendritic cells (DC) and autologous breast cancer cells induce T-cell responses against autologous tumors. However, the preparation of fusion cells requires patient-derived tumor cells, and these are not always available in the clinical setting. In the present study, we explore an alternative approach to constructing DC-breast cancer fusion vaccine by using breast caner-cell lines. DC generated from HLA-A*0201-positive donor were fused to HLA-A*0201+ allogeneic MCF7 breast cancer cells. These fusion cells co-expressed tumor-associated antigens and DC-derived costimulatory and MHC molecules. Both CD4 and CD8 T cells were activated by the fusion cells as demonstrated by the production of IFN-γ. The fusion cells induced strong antigen-specific cytotoxic T lymphocytes (CTL) activity against their parent cells. The lysis of targets was restricted by HLA-A*0201, since killing was blocked by the anti-HLA-A2 mAb. Similar CTL activity against HLA-A*0201-positive targets was induced when T cells were cocultured with fusions of DC and HLA-A*0201-negative allogeneic BT20 breast cancer cells. In addition, administration of T cells stimulated by DC-breast cancer fusion cells regressed 7-day-old tumors and rendered mice free of disease up to 90 days. These results suggest that tumor-cell lines can be used as a fusion partner in the construction of DC-tumor fusion vaccine. Such fusion cells hold promise since they can be used as a vaccine for active immunotherapy or as stimulators to activate and expand T cells for adoptive immunotherapy. [Copyright &y& Elsevier]