Over the past several years, there has been an increased understanding of the mechanisms behind transgene delivery. Efficient full gene (s) delivery to a mammalian cell involves several steps: delivery of genes to target cells, cellular entry, endosome escape, and integration into the nucleus [28, 29]. No single gene transfer system has been developed that can effectively fulfill the requirements for all of the features noted above. A combination of multifunctional nonviral gene transfer systems may satisfy the requirements for efficient gene delivery. Cationic gene carriers can facilitate the cellular entry of DNA by interacting with the negatively charged cell surface [29, 30]. Endosomal escape is achieved by including components that induce membrane fusion at low pH or block pH lowering inside the endosome in the DNA-carrier complex. PEI is a cationic polymer that serves as an effective vehicle for in vivo gene delivery in many tissues. It facilitates effective DNA binding and protection, combined with the capability to escape the endosome due to its proton buffering ability with resultant osmotic swelling and endosomal disruption [30]. PEI has been shown to be an efficient transfection agent for ovarian carcinoma cells by intraperitoneal injection [31]. Several studies indicated that PEI-mediated transgene expression was dose dependent and transient, with maximal transgene expression observed during 48 h immediately following transfection [32]. DNA was detectable in very small amounts after 72 h, indicating probable degradation and clearance [31]. This would indicate that development of nonviral gene transfer technologies that can support chromosomal integration and persistent gene expression in vivo is desirable. Transposon-mediated DNA delivery systems have shown to allow the development of a new generation of vectors for human gene therapy and mammalian [33]. Transposon-based systems have been proven to be effective integrating nonviral vectors that can mediate long-term in vivo transgene expression [34]. It has been shown that SB transposons are an efficient intratumoral gene transfer vector for glioblastoma multiforme and was delivered using PEI as gene transfer reagent. The investigators of these two studies observed a marked antitumor activity as reflected by reduced tumor vessel density, inhibition of tumor growth, and tumor elimination in up to 50 % of nude mice [35, 36]. In other studies, a more active transposon used [37–42], combined with PEI to deliver HSV-TK gene into ovarian cancer xenografts. Further studies are needed to determine how PB transposon performs in vivo in the long term. In addition, the potential safety issues from insertional disruption/deregulation in genome needed to be addressed.

Adenoviruses are well-understood vectors for gene therapy. Human adenovirus serotype 5 (Ad5) is a reasonable vector to use, as it is not associated with a serious disease [43]. It has several advantages: it is well characterized, it is not restricted to infecting only dividing cells, and it can be grown in high titers with relative biologic stability allowing it to be administered systemically. Early clinical trials utilizing no replicative adenoviruses demonstrated the feasibility and potential utility of adenoviral-based gene therapy strategies for the treatment of malignancy. While the initial adenoviral-based trials utilized no replicative vectors, subsequent studies investigated the potential of conditionally replicative adenoviruses (CRAd) for the treatment of cancer. These viral agents are designed to infect cancer cells specifically, replicate selectively within these cells, and then to spread laterally to other cancer cells. A key element of CRAd development is to replicate in cancer cells selectively, without damaging normal host cells. CRAds have thus been engineered to selectively replicate within tumor cells using two broad strategies. The first employs deletion of part of the E1A or E1B genome to prevent replication in normal cells but allow replication in tumor cells with genetic defects that complement the deleted viral genome functions. The second employs a tumor-selective promoter (TSP) to drive the expression of genes involved in viral replication selectively in tumor cells. An example of the first strategy is the E1B-negative adenovirus (dl1520), Onyx-015 [44–51]. In this approach, the E1B–55 k gene of Onyx-015 was deleted, resulting in a virus that would replicate selectively within p53-deficient tumor cells. These viruses expressed the E1A protein, which overrode the block imposed by p53 in infected cells, resulting in apoptosis of normal cells, in addition to tumor cells. ONYX-015 demonstrated notable results in both in vitro and in vivo studies but had limited effects in human clinical trials for ovarian cancer. Bischoff et al. demonstrated that ONYX-015 was capable of efficient, selective replication in p53-deficient human tumor cells [52]. Heise et al. demonstrated in vivo correlation in three nude mouse–human ovarian carcinomatosis xenograft models [53]. Clinical studies of patients with head and neck cancer demonstrated safety and efficacy, but Vasey et al. failed to demonstrate similar results in ovarian cancer patients. Fifteen of the 16 study patients were taken off of the study because they developed progressive disease [54].

The available scientific data suggest that immunotherapy for ovarian cancer (OC) could be effective. Ovarian cancer cells express tumor-associated antigens against which specific immune responses have been detected. The tumor immune surveillance plays an important role in clinical outcomes in OC, as manifested by the correlation between survival and tumor infiltration with CD3⁺ T cells.

Animal studies and human clinical trials have demonstrated that therapeutically induced tumor-specific immunity is an efficient methodology to treat human cancer based on facts that support this concept. Tumors generally can prevent tumor-specific immunity from eliminating tumors by inducing tumor-specific tolerance or inflammation. The effective immunotherapy for ovarian cancer will have to surmount these obstacles for optimal effectiveness.

Vaccines have been the main approach to ovarian cancer immunotherapy, as with many other types [91–94]. Vaccines have limited efficacy as monotherapy in patients with advanced and recurrent ovarian cancer. Some studies’ results provide encouragement for vaccine development and optimization for use. Sabbatini et al. [95] noted that patients vaccinated with monovalent or heptavalent vaccines against carbohydrate epitopes experienced significantly longer time to progression and higher progression-free survival rates. Also, vaccination with anti-idiotype ACA-125 resulted in CA-125-specific antibodies and was associated with prolonged survival [96]. Carcinoembryonic antigen–MUC-1–TRICOM poxviral-based vaccines were used in 16 patients, including three of them being ovarian cancer patients. Immune responses to MUC-1 and/or carcinoembryonic antigen were seen after vaccination in nine patients. A patient with clear-cell ovarian cancer and symptomatic ascites had a radiographically and biochemically clinical response [97]. The limitation in vaccine development in ovarian cancer is due to the lack of well-characterized rejection antigens and by notable molecular heterogeneity of the disease. HER2 is a rejection antigen, and it may also serve as a rejection antigen in ovarian cancer [98, 99]. Vaccination against HER2 has resulted in sustained antigen-specific T cell, humoral immunity, and epitope spreading in patients with ovarian cancer [100]. NY-ESO-1 is a bona fide ovarian cancer rejection antigen, and it is expressed in less than 30 % of ovarian cancers [101, 102]. A viable alternative to vaccines are whole-tumor antigen vaccines created using tumor cells, autologous tumor lysate, or tumor-derived RNA [103–105]. The advantages of these vaccines are the opportunity to induce immunity to a personalized and broad range of antigens, which could minimize the development of tumor escape variants; the inclusion of yet unidentified tumor rejection antigens; no HLA haplotype restriction; and the simultaneous administration of MHC class I and class II epitopes which could prove beneficial for immunologic memory. In a meta-analysis, it was found that 8.1 % of patients vaccinated with whole-tumor antigen had notable clinical responses, while 3.6 % of patients vaccinated with molecularly defined tumor antigens had objective clinical responses (P < .001, χ ² test) [106]. An intracavitary delivery of a viral oncolysate vaccine generated with ovarian cancer cell lines infected with influenza A virus showed objective clinical responses [107, 108]. Delivery of autologous tumor cells infected with Newcastle disease virus showed the same response [109]. HSV-infected tumor cells used directly or pulsed on dendritic cells have been shown to stimulate marked antitumor immune response in the mouse, which was better than utilizing ultraviolet-irradiated tumor cells [110–112]. Using mature dendritic cells (DCs) with whole autologous tumor lysate, 50 % of patients demonstrated remission inversion [113]. The use of DC/tumor cell fusion demonstrated to be able to induce antitumor cytotoxic T-lymphocyte activity in vitro [114]. There is a concern about the use of whole-tumor vaccination which relates to the inclusion of a large number of self-antigens, as it could drive tolerogenic responses (i.e., expand Treg) rather than cytotoxic lymphocyte responses. It has been shown that DCs can be polarized ex vivo with the use of interferons, Toll-like receptor agonists, or p38 mitogen-activated protein kinase inhibitors to stimulate cytotoxic lymphocytes and Th17. [115]. The limitation of cancer vaccines is due to the fact that it is unable to elicit an overwhelming T-cell response. In addition, it has been shown that immune modulation through blockade of the endothelin B receptor markedly increases the efficacy of weak vaccines by reversing the inhibitory function of tumor endothelium and enabling homing of tumor-reactive T cells [106, 116]. Moreover, it was observed that, depletion of Treg is a critical maneuver to enhance vaccine therapy [117].

Modulating immune checkpoints by activation of effector cells, depletion of Tregs, or activation of professional APCs could improve the therapeutic efficacy of vaccines or adoptively transferred T cells. The development of functional antibodies has enabled effective immune modulation as shown in Fig. 12.2.

The epithelial growth factor receptor (EGFR; also known as Her or ErbB) family of receptor tyrosine kinases has been shown to have oncogenic characteristics in several human cancers. It was suggested that members of the EGFR family, such as ErbB3, may have a role in promoting the growth and proliferation of human ovarian cancer cells. The EGFR family of receptor tyrosine kinases consists of four closely related family members: EGFR (Her1), ErbB2 (Her2), ErbB3 (Her3), and ErbB4 (Her4) [185]. These cytoplasmic membrane-bound receptors share a common extracellular ligand-binding domain and a single transmembrane domain, followed by an intracellular tyrosine kinase domain and a C-terminal non-catalytic signaling tail. Signaling through these receptors is typically mediated by homodimerization or heterodimerization with other family members. Binding of the ligand with the extracellular domain of its corresponding receptor induces a structural reconfiguration of the receptor, promoting exposure of dimerization domain [186, 187]. The EGFR family members can be activated by unphysiological stimuli (e.g., oxidative stress, UV, and γ-irradiation), by other receptor tyrosine kinases (MET, insulin-like growth factor-1 receptor, or tyrosine kinase receptor B), or by G protein-coupled receptors and adhesion proteins [188].