The fight against cancer has seen major breakthroughs in recent years. More than a decade ago, tyrosine kinase inhibitors targeting constitutively activated signaling cascades within the tumor inaugurated a new era of oncological therapy. Recently, immunotherapy with immune checkpoint inhibitors has started to revolutionize the treatment of several malignancies, most notably malignant melanoma, leading to the renaissance and the long-awaited breakthrough of immunooncology. This review provides an overview of the basis of immunotherapy from its initial concepts of anti-tumor immunity and cell-based therapy to the development of immune checkpoint inhibitors and discusses published studies and the perspectives of immunooncology for the treatment of endocrine malignancies.

Recent scientific advances have expanded our understanding of the immune system and its response to malignant cells. The clinical goal of tumour immunotherapy is to provide either passive or active immunity against malignancies by harnessing the immune system to target tumours. Monoclonal antibodies, cytokines, cellular immunotherapy, and vaccines have increasingly become successful therapeutic agents for the treatment of solid and haematological cancers in preclinical models, clinical trials, and practice. In this article, we review recent advances in the immunotherapy of cancer, focusing on new strategies and future perspectives as well as on clinical trials attempting to enhance the efficacy of immunotherapeutic modalities and translate this knowledge into effective cancer therapies.

Whole-cell tumor vaccines have been investigated for more than 20 years for their efficacy in both preclinical models and in clinical trials in humans. There are clear advantages of whole-cell/polyepitope vaccination over those types of immunotherapy that target specific epitopes. Multiple and unknown antigens may be targeted to both the innate and adaptive immune system, and this may be further augmented by genetic modification of the vaccine cells to provide cytokines and costimulation. In this review, we give an overview of the field including the preclinical and clinical advances using unmodified and modified tumor-cell vaccines.

A vaccine is typically defined as any preparation used as a preventive inoculation to confer immunity against a specific disease. Vaccines for infectious diseases are highly effective, acting by inducing antigen-specific immunity that prevents subsequent infection. Unfortunately, the success of vaccines in infectious diseases has not been mirrored in oncology. This failure is the result of several challenges facing cancer vaccines, including the conceptual shift from disease prevention to disease treatment, tumor-induced immunosuppression and other mechanisms of immune escape, the similarity between tumor antigens and self antigens to which the patient is tolerant, unfavorable effector-to-target ratios in patients with established tumors, and financial and regulatory issues. Despite this, cancer remains a promising target for vaccine therapy. Melanoma in particular is known for its inherent immunogenicity on the basis of many anecdotal reports of spontaneous immune-based tumor regression, and thus has been the focus of immunotherapeutic approaches. Rare but significant vaccine-induced clinical regression of melanoma has spurred intensive investigations to augment vaccine efficacy. This review explores the many vaccine strategies that have been clinically tested for the treatment of melanoma and considers future approaches of cancer immunotherapy.

Sustained maintenance of therapeutic levels of angiostatic proteins in tumor tissues continues to represent a major challenge to antiangiogenesis therapy of cancer. In this study, we tested the hypothesis of utilizing gene transfer via replication-competent retroviral (RCR) vectors for chronic protein delivery. We now show that bioactive human interferon-inducible protein-10 (IP10) can be secreted from a variety of mammalian cells upon transduction with RCR vectors carrying the human IP10 gene. The production of IP10 from RCR-transduced cells could be maintained for at least three months in culture. The level and duration of IP10 expression in vivo was sufficient to inhibit growth of subcutaneous (s.c.) tumors as well as metastatic lesions in mice. This tumor inhibition was correlated to a marked reduction in tumor vascularization and mitotic activity. By conducting immunohistological studies, we have been able to show that IP10 vector-affected tumors evidenced elevated levels of IL-12p35 mRNA, with no sign of changes in the local inflammatory response, however, as determined by macrophage infiltration and the expression of proinflammatory cytokines. We are addressing the feasibility of using RCR vector-based gene therapy as a more convenient alternative tool to chronically deliver antiangiogenic proteins for cancer therapy.

Exploiting a naturally occurring defense system, the immunotherapeutic approach embodies an ideal nontoxic treatment for cancer. Despite the evidence that immune effectors can play a significant role in controlling tumor growth either in natural conditions or in response to therapeutic manipulation, the cascade of molecular events leading to tumor rejection by the immune system remains to be fully elucidated. Nevertheless, some recent tumor immunology advancements might drastically change the way to design the next generation of cancer vaccines, hopefully improving the effectiveness of this therapeutic approach. In the present work, we will focus on three main areas of particular interest for the development of novel vaccination strategies: (a) cellular or molecular mechanisms of immune tolerance to malignant cells; (b) synergism between innate and adaptive immune response; (c) tumor-immune system interactions within the tumor microenvironment.

As yet there are no FDA-approved cancer vaccines for malignant melanoma, but encouraging response rates and low toxicities reported in phase I/II trials suggest that antigen-based active immunotherapy may complement current treatment modalities. The cumulative data for Canvaxin therapeutic polyvalent cancer vaccine represent the largest phase II clinical trial of any cancer vaccine. Univariate and multivariate analyses of these data have demonstrated the prognostic significance of this allogeneic whole-cell preparation as a postoperative adjuvant treatment for patients with stage III and IV melanoma. The vaccine has also been shown promising results after resection of stage II melanoma and in patients with regional in-transit disease. The consistent correlation between immune and clinical responses to the vaccine suggests that immune parameters may be used to monitor a patient's response to vaccine therapy.

Interleukin-12 (IL-12) is one of the most effective cytokines for treating malignancy. Intratumoral delivery of the murine Il-12 gene, using electroporation, has been found effective in inducing regression of established tumors in mice, and more effective than intramuscular injection of this gene by electroporation, but what is not known is the molecular mechanism by which IL-12 exerts an antitumor effect. To define these candidate genes, the gene expression profiles of tumors treated with and without intratumoral Il-12 electroporation gene therapy were analyzed by cDNA array. Mig (Cxcl9), Stat1, and IRF7 are the three genes that are the most altered at the level of expression after administration of Il-12 via intratumoral electroporation, when subjected to further characterization by Northern blot, Western blot, and immunostaining. The results from Northern blot and immunostaining analyses indicate that intratumoral delivery of the murine Il-12 gene via electroporation induces accumulation of IRF7 in the nuclei of tumor cells and upregulates Mig and Stat1 expression by 15- and 5-fold, respectively, compared to intratumoral electroporation of control plasmid DNA. Intramuscular injection of the same Il-12 gene using electroporation upregulates Mig and Stat1 by only 6- and 2.9-fold, respectively, but does not induce any IRF7 accumulation in the nuclei. Further functional analyses of Mig indicate that expression in tumors can induce CD4+ but not CD8+ T cell infiltration. Further functional analysis of Stat1 indicates that a lack of Stat1 expression inhibits the Il-12-mediated induction of IP10, a known antiangiogenic gene. These data suggest that these three genes may positively correlate with the antitumor efficacy of intratumoral Il-12 electroporation gene therapy.

Melanoma continues to present a major therapeutic challenge to oncologists, oncologic surgeons, and dermatologists. Recent advances in molecular genetics and improvement in our understanding of immune responses to tumors have generated an interest in using gene-based treatment strategies to fight melanoma. Several basic strategies have emerged: (1) strengthening of the immune response against tumors by genetic modification of some target cell populations of the host using immunostimulatory genes such as cytokines and by genetic immunization with the genes coding for melanoma-associated antigens recognized by cytotoxic T cells; (2) interference with signaling cascades; and (3) suicide gene strategies. This article reviews these novel strategies and summarizes the most recent data generated by European groups either in experimental studies or in clinical trials.

Systemic IL-2 has shown some activity in metastatic melanoma, but its use is severely limited by toxicity. TG2001 is a product in which the human IL-2 cDNA was incorporated into the genome of Vero cells, a monkey fibroblast cell line. The goal of this intratumorally applied therapy was to create an antitumor immune response stimulated by xeno-antigens and local production of IL-2 in the close vicinity of tumor-specific antigens. TG2001 was reported to have a good safety profile in two previous dose-escalating phase I studies performed in 18 patients with various solid tumors, with encouraging clinical responses in three patients. The objectives of this study were to evaluate the tolerance and incidence of tumor regression in patients with metastatic melanoma, following repeated administration of Vero-IL-2 cells.

This was on open-label, randomized phase II study comparing two doses of Vero-IL-2, 5x10(5) and 5x10(6) cells. Twenty-eight patients with metastatic melanoma were enrolled in the study, 14 in each treatment group. Patients received TG2001 by intratumoral injection on days 1, 3, and 5 every 4 weeks for four cycles, and every 8 weeks thereafter, until evidence of progressive disease (PD). Criteria for patient selection included histologically proven metastatic melanoma, with one tumor accessible for product administration, and at least another tumor site for response assessment. Evaluation included tumor measurements, humoral and T cell-mediated local and systemic immune response, humoral response to Vero cells, adverse events and standard laboratory parameters.

None of the patients achieved a confirmed objective response. Stable disease (SD) was seen in six (43%) and eight patients (57%) at the 5x10(5) and the 5x10(6) dose level, respectively. Two patients, one in each group, died during the study (i.e., within 1 month after the last injection) due to PD. Three patients exhibited antibody responses to Vero cells. T-cell immunity, serum cytokine levels and cytokine mRNA expression in tumor biopsies did not show meaningful alterations after therapy, except for a trend toward an increase in intratumoral TH2 cytokine (IL-4 and/or IL-10) levels. The study drug was well tolerated at both dose levels and side effects mainly consisted of injection site pain and erythema, and pyrexia.

The intratumoral administration of TG2001 was generally well tolerated in patients with metastatic melanoma, and transient disease stabilization was observed in 50% of patients.

A phase I clinical trial using autologous, IL-7 gene-modified tumor cells in patients with disseminated melanoma has been recently completed. Although no major clinical responses were observed, increased antitumor cytotoxicity was measured in postvaccine peripheral blood lymphocytes in a subset of treated patients. To analyze the in situ immune response, the T cell receptor beta-chain variable region (BV) repertoire of T cells infiltrating postvaccine lesions was studied in two patients, and compared with that of T cells present in prevaccine ones, in peripheral blood lymphocytes, and, in one patient, in delayed type hypersensitivity (DTH) sites of autologous melanoma inoculum. A relative expansion of T cells expressing few BVs was observed in all postvaccine metastases, and their intratumoral presence was confirmed by immunohistochemistry. Length pattern analysis of the complementarity determining region 3 (CDR3) indicated that the repertoire of T cells expressing some of these BVs was heterogeneous. At difference, CDR3, beta-chain joining region usage, and sequence analysis enabled us to demonstrate, within a T-cell subpopulation commonly expanded at DTH sites and at the postvaccine lesion of patient 1, that both DTH sites contained identical dominant T-cell clonotypes. One of them was also expressed at increased relative frequency in the postvaccine lesion compared to prevaccine specimens. These results provide evidence for immunological changes, including in situ clonally expanded T cells, in metastases of patients vaccinated with IL-7 gene-transduced cells.