Interferons (IFN-α, -β, -γ) are glycoproteins that have antitumor and antiviral activity. Depending on dose, interferons may either enhance or decrease cellular and humoral immune functions. Interferons also inhibit division and certain synthetic processes in a variety of cells. Clinical trials have indicated that interferons have antitumor activity in various cancers, including hairy cell leukemia, chronic myelocytic leukemia, AIDS-associated Kaposi’s sarcoma, non-Hodgkin lymphoma (NHL), multiple myeloma, and ovarian carcinoma. However, interferons may have significant adverse effects, such as fever, malaise, leukopenia, alopecia, and myalgias.

Certain bacterial adjuvants (BCG and derivatives, killed suspensions of Corynebacterium parvum) have tumoricidal properties. They have been used with or without added tumor antigen to treat a variety of cancers, usually along with intensive chemotherapy or radiation therapy. For example, direct injection of BCG into cancerous tissues has resulted in regression of melanoma and prolongation of disease-free intervals in superficial bladder carcinomas and may help prolong drug-induced remission in acute myeloblastic leukemia, ovarian carcinoma, and NHL.

The poor immunogenicity of most tumor antigens largely reflects the nonconductive context in which these antigens are naturally presented, as well as tolerance resulting from most tumor antigens being normal proteins aberrantly expressed by the tumor. Therapeutic vaccines have attempted to circumvent these problems by presenting tumor antigens in a more enticing fashion, generally through activated DCs. This has been achieved either by:

The last category includes plasmid DNA containing the antigen and immunostimulatory CpG sequences as well as recombinant attenuated pathogens, such as adenoviruses or Listeria monocytogenes, that express the antigen and provide TLR ligands to trigger innate responses. However, most vaccinated patients exhibit only weak or undetectable T-cell responses to the tumor antigen and experience no clinical benefit. Therefore, methods to maintain APC activation and sustain immunogenic antigen presentation normally occurring during an encounter with a replicating foreign pathogen will likely be required before vaccines become more predictably beneficial.

An alternative to improving antigen presentation has been to mitigate negative checkpoint signals that limit the T-cell response. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a potent negative regulator of T-cell activation. Administration of blocking antibodies to CTLA-4 has had marked effects in murine models and recent clinical trials, with lymphocytic infiltration into tumors and significant antitumor responses, including complete regressions of advanced disease in a fraction of patients [16–18]. However, global in vivo CTLA-4 blockade predictably had effects beyond the antitumor response, causing significant autoimmunity. These studies again demonstrate the potent antitumor activity of T-cells and suggest that learning how to safely and effectively disrupt checkpoint signals should yield substantial therapeutic benefit.

There is now an emerging sense that cancer immunotherapy has the potential to effectively cure patients suffering from certain types of cancer. This hope and some of the data that supports one kind of immunotherapy (adoptive cell transfer or ACT) were recently summarized in a review article (Adoptive immunotherapy for cancer: harnessing the T-cell response) [19]. Furthermore, high-dose chemo-radiotherapy followed by rescue from the resulting ablation of normal bone marrow with an allogeneic hematopoietic stem cell transplant (HSCT) has also become standard therapy for many hematologic malignancies. One problem with this treatment is graft-versus-host disease (GVHD), due to allogeneic donor-derived T-cells injuring the “foreign” normal tissues of the host. However, malignant cells that survive chemoradiotherapy are also of host origin, and patients who develop GVHD have lower relapse rates from an associated graft-versus-tumor (GVT) effect. T-cells mediate this antitumor activity, as affirmed by the complete responses sometimes observed in patients who receive infusions of donor T-cells to treat relapse after HSCT and in recipients of a newly developed non-myeloablative allogeneic HSCT regimen in whom, because of the absence of high-dose chemoradiotherapy, all antitumor effects must result from GVT effects [20]. However, the GVT activity with these regimens is often associated with severe and life-threatening GVHD. Ongoing efforts to define antigenic targets with limited tissue distribution, permitting donor lymphocytes to preferentially target malignant cells and not critical normal tissues, coupled with methods to generate and/or select T-cells with such specificities, should provide a much-needed refinement to this approach [21].

An alternative to using allogeneic T-cells to mediate antitumor responses has been to isolate autologous tumor-reactive T-cells, expand the cells in vitro, and then reinfuse the cells back into the patient. This approach circumvents many of the obstacles to generating an adequate response in vivo, as the nature of the APCs and components of the microenvironment can be more precisely controlled in vitro. However, this strategy has required the recent development of methods to extensively manipulate T-cells in vitro with retention of specificity and function, such that after infusion the cells will survive and migrate to and eliminate tumor cells.

Initial therapies used tumor-infiltrating lymphocytes as an enriched source of tumor-reactive cells, but such cells can also usually be obtained from circulating blood lymphocytes. Although optimal methods for stimulating and expanding antigen-specific T-cells in vitro are still being defined, in general, DCs presenting the antigen are used to initially trigger reactive T-cells, which can then be selected and stimulated with antibodies to CD3. Supplemental cytokines are provided during cell culture to support lymphocyte proliferation, survival, and differentiation. With this approach, it has been possible to expand tumor-reactive T-cells to enormous numbers in vitro, infuse billions of specific cells without overt toxicity to achieve in vivo frequencies beyond that attainable with current vaccine regimens, and mediate regression and occasionally complete elimination of large disseminated tumor masses. However, despite the high in vivo frequencies of tumor-reactive effector cells achieved, only a fraction of patients respond, indicating the existence of additional hurdles. One essential requirement is that infused cells must persist to mediate an effective response. Analogous adoptive therapy trials for cytomegalovirus and Epstein-Barr virus infection in immunosuppressed hosts have demonstrated increased in vivo proliferation and persistence of CD8⁺ effector T-cells in the presence of specific CD4⁺ helper T-cells [22]. Such CD4⁺ T-cells likely provide many beneficial functions, including cytokine production and APC activation, which can improve the quality and quantity of the CD8⁺ cell responses, as well as direct effector activities against infected or tumor targets. However, unlike viral responses that induce robust CD4⁺ and CD8⁺ responses, identifying and characterizing the specificity of tumor-reactive CD4⁺ T-cells has proven considerably more difficult than with CD8 responses. Additionally, obstacles to safely maintaining a CD4⁺ response reactive with a potentially normal protein remain to be elucidated. Consequently, CD4 help is largely provided to transfer tumor-reactive CD8 cells in the form of surrogate exogenous cytokines. The largest experience is with IL-2, which prolongs persistence and enhances the antitumor activity of transferred CD8⁺ cells [23]. Alternative cytokines such as IL-15, IL-7, and IL-21, as well as activation of APCs with antibodies to CD40, are currently being evaluated in preclinical studies.

The infusion of T-cell clones, rather than polyclonal T-cell lines, represents an appealing refinement of adoptive therapy, because the specificity, avidity, and effector functions of infused cells can be precisely defined (Fig. 1.1). This facilitates subsequent analysis of requirements for efficacy, basis for toxicity, and rational design of improved therapies. The transfer of antigen-specific CD8⁺ T-cell clones has been shown to be effective for prevention of viral infections and treatment of malignant disease. Such studies have also formally demonstrated that low, nontoxic doses of IL-2 are sufficient to promote the in vivo persistence and antitumor activity of CD8⁺ T-cells.

Therapeutic cancer vaccines target the cellular arm of the immune system to initiate a cytotoxic T-lymphocyte response against tumor-associated antigens [24]. The development of human therapeutic cancer vaccines has come a long way since the discovery of MHC-restricted tumor antigens in the 1980s. The simplest model of immune cell-mediated antigen-specific tumor rejection consists of three elements: appropriate antigen, specific for the tumor, efficient antigen presentation, and the generation of potent effector cells. Moreover, the critical time when immune responses against the tumor are most important should also be determined. While eliminating some early transformed cells may be ongoing in an asymptomatic way as part of the immunosurveillance, if early elimination failed, equilibrium between small tumors and the immune system may be established. If the immune system is unable to maintain this equilibrium, tumors may escape and it is this last phase when they become symptomatic. Therapeutic cancer vaccines are applied in this last phase in order to reverse the lack of tumor control by the immune system. In addition to the increasing knowledge about how to optimize the elements of antitumor immunity in order to generate clinically relevant responses, there is an ever-increasing list of immune evasion mechanisms impeding the efforts of cancer vaccines. This indicates that the elements necessary for immune-mediated tumor rejection need to be optimized [25].

Potential tumor-associated antigens (TAAs) can be identified by the elution of peptides from MHC molecules on tumor cells [26] or with proteomic approaches such as two-dimensional gel electrophoresis, MALDI-MS and SELDI-MS (matrix-assisted or surface enhanced laser-desorption ionization mass spectrometry) [27]. Serological analysis of recombinant cDNA expression libraries (SEREX) is another widely used method; it utilizes sera of cancer patients to detect over expressed antigens from tumor cDNA libraries [28]. Furthermore, several RNA-based methods have also gained importance: transcriptome analysis that include DNA microarrays [29], serial analysis of gene expression (SAGE) [30], comparative genomic hybridization (CGH) [31], and massively parallel signature sequencing (MPSS) [32]. These methods provide an enormous amount of information and require complex computer-aided analysis and interpretation of the data, referred to as gene expression profiling. This is necessary in order to find gene expression patterns and to distinguish them from noise [33].

Following promising in vitro immunogenicity studies [34], multicenter vaccine trials have been organized with the sponsorship of the Cancer Vaccine Collaborative (NCI and Ludwig Institute for Cancer Research). These trials have provided some information about the optimum route of administration, type of vaccine, type of adjuvant, endpoints, etc. [35]. When testing the immunogenicity of candidate antigens and defining epitopes, it should be remembered that T-cells with high avidity for self-antigen undergo negative selection during T-cell development; thus, the new TAAs may only generate T-cell responses of intermediate or low affinity. Furthermore, the wide range of restriction elements in the human population means that due to the combination of tolerance and immunodominance, potentially ideal TAAs will not be equally immunogenic in all patients. Antigen loss may also occur during tumor progression, as TAAs which are not necessary for the maintenance of the transformed phenotype may be deleted and tumor cells in advanced disease may express antigens different from those in early stages [36]. Another promising approach to break this immune tolerance consists of the application of anti-idiotypic (anti-Id) mAbs, so called Ab2, as antigen surrogates. This vaccination strategy also allows immunization against nonprotein antigens (such as carbohydrates). In some clinical studies, anti-Id cancer vaccines induced efficient humoral and/or cellular immune responses associated with clinical benefit (see review by Ladjemi 2012) [37].

Despite the evidence that immune effectors play a significant role in controlling role in tumor growth under natural conditions or in response to therapeutic manipulation, it is well known that malignant cells can evade immunosurveillance [69]. This is in part due to the fact that peptides with sufficient immunogenic potential are not presented by malignant cells to antigen-presenting cells under molecular/cellular conditions conducive to an effective immune response. From a Darwinian perspective, the neoplastic tissue can be envisaged as a microenvironment that selects for better growth and resistance to the immune attack. Cancer cells are genetically unstable and can lose their antigens by mutation. This instability, combined with an immunological pressure, could allow for selective growth of antigen-loss mutants [70]. Mechanistically this could operate at several levels including loss of the whole protein or changes in immunodominant T-cell epitopes that alter T-cell recognition, antigen processing, or binding to the MHC. Antigen loss has been demonstrated in patients with melanoma and B-cell lymphoproliferative disease [71, 72]. Moreover, many cancer vaccines aim to induce a therapeutic CD8⁺ cytotoxic T-cell response against TAAs. This in turn is dependent on correct processing and presentation of TAAs by MHC class I molecules on tumor cells. This pathway is complex and involves multiple intracellular components. Defects in the components of the MHC class I antigen processing pathway are frequently found in human cancers and can occur in concert with the loss of tumor antigens [73, 74].

Other cancer-related mechanisms underlying tumor immune escape include loss of TAA expression [3], lack of co-stimulatory molecules expression [75], inactivating mutations of antigen presentation-related molecules [76], and production of soluble immunosuppressive factors, e.g., transforming growth factor beta (TGF-β), IL-10, reactive oxygen species (ROS), and nitric oxide (NO), produced by tumor cells. Furthermore, tumor-infiltrating immune cells such as suppressor immune cells, e.g., T regulatory (Treg) cells, macrophages, and myeloid-derived suppressor cells (MDSC), also influence this phenomenon and are now discussed in more detail.