Cancer Immunotherapy with Vaccines and Checkpoint Blockade

Cancer Antigens—the Difference between Tumor and Self

Tumors reflect the biologic and antigenic characteristics of their tissue of origin but also differ fundamentally from their normal-cell counterparts in both antigenic composition and biologic behavior. Both these elements of cancer provide potential tumor-selective or tumor-specific antigens as potential targets for cancer vaccination specifically and antitumor immune responses in general. Genetic instability, a basic hallmark of cancer, is a primary generator of tumor-specific antigens. The most common genetic alteration in cancer is mutation arising from defects in DNA damage repair systems of the tumor cell. Recent estimates from genome-wide sequencing efforts suggest that every tumor contains a few hundred mutations in coding regions. In addition, deletions, amplifications, and chromosomal rearrangements can result in new genetic sequences resulting from the juxtaposition of coding sequences that are not normally contiguous in untransformed cells. The vast majority of these mutations occur in intracellular proteins, and thus the “neoantigens” they encode would not be readily targeted by antibodies. However, the major histocompatibility complex (MHC) presentation system for T-cell recognition makes peptides derived from all cellular proteins available on the cell surface as peptide MHC complexes capable of being recognized by T cells. Based on analysis of sequence motifs, it is estimated that roughly one third of the mutations identified from genome sequencing of 22 breast and colon cancers were capable of binding to common human leukocyte antigen (HLA) alleles based on analysis of sequence motifs.

In accordance with the original findings of Prehn, the vast majority of tumor-specific antigens derived from mutation as a consequence of genetic instability are unique to individual tumors. The consequence of this fact is that antigen-specific immunotherapies targeted at most truly tumor-specific antigens would by necessity be patient specific. However, there are a growing number of examples of tumor-specific mutations that are shared. The three best studied examples are the Kras codon 12 G→A (found in roughly 40% of colon cancers and more than 75% of pancreas cancers), the BrafV599E (found in roughly 70% of melanomas), and the P53 codon 249 G→T mutation (found in about 50% of hepatocellular carcinomas). As with nonshared mutations, these common tumor-specific mutations all occur in intracellular proteins and therefore require T-cell recognition of MHC-presented peptides for immune recognition. Indeed, both the Kras codon 12 G→A and the BrafV599E mutations result in “neopeptides” capable of being recognized by HLA class 1– and class II–restricted T cells.

The other major difference between tumor cells and their normal counterparts derives from epigenetics. Global alterations in DNA methylation as well as chromatin structure in tumor cells result in dramatic shifts in gene expression. All tumors overexpress hundreds of genes relative to their normal counterparts and, in many cases, turn on genes that are normally completely silent in their normal cellular counterparts. Overexpressed genes in tumor cells represent the most commonly targeted tumor antigens by both antibodies and cellular immunotherapies. This is because, in contrast to most antigens derived from mutation, overexpressed genes are shared among many tumors of a given tissue origin or sometimes multiple tumor types. For example, mesothelin, which is targeted by T cells from vaccinated pancreatic cancer patients, is highly expressed in virtually all pancreatic cancers, mesotheliomas, and most ovarian cancers. Although mesothelin is expressed at low to moderate levels in the pleural mesothelium, it is not expressed at all in normal pancreatic or ovarian ductal epithelial cells.

The most dramatic examples of tumor-selective expression of epigenetically altered genes are the so-called cancer-testis antigens. These genes appear to be highly restricted in their expression in the adult. Many are expressed selectively in the testes of males and are not expressed at all in females. Expression in the testis appears to be restricted to germ cells, and in fact some of these genes appear to encode proteins associated with meiosis. Cancer-testis antigens therefore represent examples of widely shared tumor-selective antigens whose expression is highly restricted to tumors. Many cancer-testis antigens have been shown to be recognized by T cells from nonvaccinated and vaccinated cancer patients. From the standpoint of immunotherapeutic targeting, a major drawback of the cancer-testis antigens is that none appears to be necessary for the tumors’ growth or survival. Therefore, their expression appears to be purely the consequence of epigenetic instability rather than selection, and antigen-negative variants are easily selected out in the face of immunotherapeutic targeting.

A final category of tumor antigen that has received much attention encompasses tissue-specific antigens shared by tumors of similar histologic origin. Interest in this class of antigen as a tumor-selective antigen arose when melanoma-reactive T cells derived from melanoma patients were found to recognize tyrosinase, a melanocyte-specific protein required for melanin synthesis. In fact, the most commonly generated melanoma-reactive T cells from melanoma patients recognize melanocyte antigens. Although one cannot formally call tissue-specific antigens tumor-specific, they are nonetheless potentially viable targets for therapeutic T-cell responses when the tissue is dispensable (i.e., prostate cancer or melanoma).

From the standpoint of T-cell targeting, tumor antigens upregulated as a consequence of epigenetic alterations represent “self-antigens” and are therefore likely to induce some level of immune tolerance. However, it is now clear that the stringencies of immune tolerance against different self-antigens differ according to tissue distribution and normal expression level within normal cells. The mesothelin antigen described earlier is an example. In a recent set of clinical pancreatic cancer vaccine studies, mesothelin-specific T-cell responses were induced by vaccination with genetically modified pancreatic tumor cell vaccines, and induction of mesothelin-specific T cells correlated with ultimate disease outcome. Given that the immune system is capable of differential responsiveness determined by antigen levels, it is quite possible to imagine generating tumor-selective immune responses against antigens whose expression level in the tumor is significantly greater within normal cells in the tumor-bearing host. In addition, upregulated antigens that provide physiologically relevant growth or survival advantages to the tumor are preferred targets for any form of therapy because they are not so readily selected out.

Beyond the antigenic differences between tumor cells and normal cells, there are important immunologic consequences to the distinct biological behavior of tumor cells relative to their normal counterparts. Whereas uncontrolled growth is certainly a common biological feature of all tumors, the major pathophysiologic characteristics of malignant cancer responsible for morbidity and mortality are their ability to invade through natural tissue barriers and ultimately to metastasize. Both of these characteristics, never observed in nontransformed cells, are associated with dramatic disruption and remodeling of tissue architecture. Indeed, the tumor microenvironment is quite distinct from the microenvironment of normal tissue counterparts. One of the important consequences of tissue disruption, even when caused by noninfectious mechanisms, is the elaboration of pro-inflammatory signals. These signals, generally in the form of cytokines and chemokines, are potentially capable of naturally initiating innate and adaptive immune responses. Indeed, the level of leukocyte infiltration into the microenvironment of tumors tends to be significantly greater than the leukocyte component of their normal-tissue counterparts. Cancers are therefore constantly confronted with inflammatory responses as they invade tissues and metastasize. In some circumstances these inflammatory and immune responses can potentially eliminate a tumor—so-called immune surveillance. However, as discussed later, oncogenic pathways in the tumor appear to organize the immunologic component of the microenvironment in a fashion that not only protects the tumor from antitumor immune responses but can qualitatively shift immune responses to those that actually support and promote tumor growth. It is these elements of the cancer–immune system interaction that will be the central targets of future immunotherapeutic strategies.

Evidence Pro and Con for Immune Surveillance of Cancer

The fundamental tenet of the immune surveillance hypothesis, first conceived nearly a half century ago, is that a fundamental role of the immune system is to survey the body for tumors as it does for infection with pathogens, recognizing and eliminating them based on their expression of tumor-associated antigens. In animal models, carcinogen-induced tumors can be divided into those that grow progressively (termed progressor tumors ) and those that are rejected after an initial period of growth (termed regressor tumors ). The phenomenon of regressor tumors was thought to represent an example of the ongoing process of immune surveillance of cancer. A corollary to the original immune surveillance hypothesis is that progressor tumors in animals (presumed to represent clinically progressing cancers in humans) fail to be eliminated because they develop active mechanisms of either immune escape or resistance.

A fundamental prediction of the immune surveillance hypothesis is that immunodeficient individuals would display a dramatic increase in tumor incidence. After an extensive analysis of spontaneous tumor formation in immunodeficient nude mice, which have atrophic thymi and therefore significantly reduced numbers of T cells and T-cell–dependent immune responses, no increased incidence of tumors was observed. These studies were taken as a major blow to the immune surveillance hypothesis. However, a caveat to the interpretation of these results is that nude mice still produce diminished numbers of T cells via thymus-independent pathways and therefore can mediate some degree of T-cell–dependent immunity. In addition, nude mice frequently display compensatory increases in innate immunity that, as discussed later, may represent a potent form of antitumor immunity and could contribute to immune surveillance of cancer.

Epidemiologic studies of patients with heritable immunodeficiencies revealed a significantly increased risk of certain cancers that are distinct from the epithelial cancers commonly observed in normal immunocompetent adults. Many of these cancers are also observed in transplant patients on chronic pharmacologic immune suppression as well as in patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) whose immune system is depressed. The most common cancers in these individuals include lymphoblastic lymphomas as well as Kaposi’s sarcoma; however, certain epithelial cancers, such as stomach cancer, were also observed at increased frequency. A unifying theme for the majority of cancers observed in immunodeficient individuals is their microbial origin. The majority of lymphoblastic lymphomas are associated with Epstein-Barr virus (EBV), and Kaposi’s sarcoma is a result of infection with the herpesvirus KSHV (Kaposi’s sarcoma–associated herpesvirus). Other virus-associated cancers such as cervical cancer (from human papillomavirus) are also observed at increased frequency. It is now appreciated that stomach cancer is associated with ulcer disease related to infection with the bacterium Helicobacter pylori. From these studies, the notion emerged that immune surveillance indeed protects individuals against certain pathogen-associated cancers by either preventing infection or altering chronic infection by viruses and other microbes that can eventually induce cancer. These studies were taken to represent evidence that the common non–pathogen-associated cancers most commonly seen in adults in developed countries (i.e., prostate cancer, colon cancer, lung cancer, etc.) are not subject to immune surveillance.

Two caveats to this interpretation must, however, be noted. First, detailed epidemiologic analyses of immunodeficient individuals were performed at a time when these patients rarely lived beyond their 20s and 30s, when cancer incidence normally increases most significantly. It is therefore possible that a subtler cumulative increased incidence of common non–pathogen-associated cancers would have been observed had these individuals lived further into adulthood. Indeed, more recent analyses definitively demonstrate an increased incidence of some non–pathogen-associated cancers, particularly melanoma, in immunodeficient individuals. In addition to epidemiologic data, dramatic antidotal examples are difficult to ignore. There have been reports that patients receiving kidneys from a cadaver donor who had been in complete remission from a melanoma before organ donation each rapidly developed metastatic melanoma of donor origin after the transplant. These results indicate that at least for some non–pathogen-associated tumors, the immune system can play a significant role in maintaining the micrometastatic disease in a dormant state. Whether this principle applies to non–pathogen-associated human tumors besides melanoma remains to be demonstrated.

A number of recent studies reevaluating tumor immune surveillance in genetically manipulated mice has revealed clear-cut evidence that various components of the immune system can at least modify, if not eliminate, both carcinogen-induced and spontaneously arising cancers. A series of studies by Schreiber and colleagues reexamined cancer incidence in mice rendered immunodeficient via genetic knockout of the RAG2 gene (deficient in both B and T cells), the γ-interferon receptor gene, the STAT 1 gene, or the type 1 interferon receptor gene. When these knockout mice were either treated with carcinogens or crossed onto a cancer-prone P53 knockout background, the incidence of cancers was modestly but significantly increased relative to nonimmunodeficient counterparts when observed over an extended period (longer than 1 year). Transplantation studies demonstrated that direct γ-interferon insensitivity by the developing tumors played a significant role in the defect in immune surveillance. Interestingly, in contrast to γ-interferon receptor knockout mice, the mechanism for increased tumor incidence in tumors in type 1 interferon receptor knockout mice did not involve sensitivity by the tumor to type 1 interferons but rather reflected role of the type 1 interferons in the induction of innate and adaptive immunity. Even animals not crossed onto a cancer-prone genetic background or treated with carcinogens developed an increased incidence of invasive adenocarcinomas when observed over their entire life span. Furthermore, γ-interferon, RAG2 double knockout mice developed a broader spectrum of tumors than RAG2 knockout mice. All of the tumors that arise in these genetically manipulated immunodeficient animals behave as regressor tumors when transplanted into immunocompetent animals. These findings indeed suggest that tumors that arise in immunodeficient animals would have been eliminated had they arisen in immunocompetent animals. The relatively subtle effects on tumorigenesis, requiring observation over the life span of the animal, suggests that the original concept of immune surveillance of tumors arising on a daily basis is in fact not correct. Instead, it is clear that the presence of a competent immune system “sculpts” the tumor through processes that have been termed immunoediting.

The immunoediting hypothesis has been somewhat controversial, with differing outcomes in different animal models. One of the caveats in the interpretation of these studies comes from the work of Dranoff and colleagues, who studied mechanisms of increased tumor genesis in GM-CSF × γ-IFN double knockout mice. Although they observed an increase in gastrointestinal and pulmonary tumors, they noted that such animals harbored infection with a particular bacterium not normally observed in immunocompetent animals. Maintenance of these double knockout mice on antibiotics essentially eliminated the increased rate of tumor formation. Thus, it is possible that some of the increased tumor rates in genetically immunodeficient animals could be related to unappreciated chronic infections that develop in these animals, which are not housed under germ-free conditions. Nonetheless, although the classic concepts of immune surveillance of cancer remain unsupported by experimental evidence, studies on tumorigenesis in genetically manipulated immunodeficient mice indeed suggest that developing tumors must actively adapt themselves to their immune microenvironment in order to exist within the context of a competent immune system.

One of the approaches to test the immune surveillance and immunoediting of endogenously arising tumors has been to combine genetically engineered autochthonous tumor models with T-cell receptor transgenic models expressing defined marked T cells specific for a tumor antigen (either the transgenic oncogenic driver protein in the tumors or an antigen co-expressed with the oncogenic driver). In these models, tumor growth can be monitored in immunodeficient versus immunocompetent mice as well as expression of the cognate tumor antigen recognized by the transgenic T-cell receptor. In such a tumor model driven by Kras and p53 loss, tumors emerging in immunocompetent mice either lost antigen or MHC presentation capacity, whereas those emerging in immunodeficient mice did not. In contrast, in a mouse model of spontaneous random oncogene activation, antigen-specific tolerance was generated in immunocompetent mice without evidence for antigen loss. In some models, an intermediate result has been observed. For example, endogenous immune responses can, under some circumstances, establish an equilibrium state with the tumor in which the tumor is prevented from outgrowth in immunocompetent mice but is not completely eliminated. Ultimately, given the model dependence of outcome (i.e., tolerance, versus surveillance versus editing versus equilibrium) it will be important to ascertain which mechanisms are operative in particular human cancers.

Immune Tolerance and Immune Evasion—the Immune Hallmarks of Cancer

Although controversy over the ultimate role of immune surveillance in natural modulation of cancer development and progression will undoubtedly continue into the future, one can summarize the current state of knowledge as supporting the notion that natural immune surveillance plays a much smaller role than originally envisioned by Thomas and Burnet. However, developing tumors need to adapt to their immunologic milieu in a manner that either turns off potentially harmful (to the tumor) immune responses or creates a local microenvironment inhibitory to the tumoricidal activity of immune cells that could inadvertently become activated in the context of inflammatory responses associated with tissue invasion by the tumor. These processes—tolerance induction and immune evasion—have become a central focus of cancer immunology efforts and will undoubtedly provide the critical information necessary for the development of successful immunotherapies that break tolerance to tumor antigens and break down the resistance mechanisms operative within the tumor microenvironment.

Evidence from murine tumor systems as well as human tumors strongly demonstrates the capacity of tumors to induce tolerance to their antigens. This capacity to induce immune tolerance may very well be the single most important strategy that tumors use to protect themselves from elimination by the host’s immune system. Tolerance to tumors appears to operate predominantly at the level of T cells; B-cell tolerance to tumors is less certain because there is ample evidence for the induction of antibody responses in animals bearing tumors as well as human patients with tumors. However, with the exception of antibodies against members of the epidermal growth factor receptor family, there is little evidence that the natural humoral response to tumors provides significant or relevant antitumor immunity. In contrast, numerous adoptive transfer studies have demonstrated the potent capacity of T cells to kill growing tumors, either directly through cytotoxic T lymphocyte (CTL) activity, or indirectly through multiple CD4-dependent effector mechanisms. It is thus likely that induction of antigen-specific tolerance among T cells is of paramount importance for tumor survival.

The first direct evidence for induction of T-cell tolerance by tumors was provided by Bogen and colleagues, who examined the response of T-cell receptor (TCR) transgenic T cells specific for the idiotypic immunoglobulin expressed by a murine myeloma tumor. They first demonstrated induction of central tolerance to the myeloma protein followed by peripheral tolerance. Using influenza hemagglutinin (HA) as a model tumor antigen, Levitsky and colleagues demonstrated that adoptively transferred HA-specific TCR transgenic T cells were rapidly rendered anergic by HA-expressing lymphomas and HA-expressing renal carcinomas. Tolerance induction has been demonstrated in both the CD4 and CD8 compartment. In general, initial activation of tumor-specific T cells is commonly observed; however, the activated state of T cells is typically not sustained, with failure of tumor elimination as a frequent consequence. Tolerance induction among tumor antigen–specific T cells is an active process involving direct antigen recognition, although in some murine systems, tolerance to tumors appears to be associated with failure of antigen recognition by T cells—that is, the immune system “ignores” the tumor. Beyond studies on transplantable tumors, more recent analyses of immune responses to tumor antigens in tumor transgenic mice developing spontaneous cancer have further emphasized the capacity of spontaneously arising tumors to induce tolerance among antigen-specific T lymphocytes. In a model of prostate tumorigenesis, Drake and colleagues evaluated CD4 responses to HA and double transgenic animals expressing HA and SV40 T antigen under the control of the prostate-specific probasin promoter. The development and progression of prostate tumors did not result in enhanced activation of adoptively transferred HA-specific T cells. Tolerance to HA as a normal prostate antigen occurred largely through ignorance because there was no evidence for antigen recognition by HA-specific T cells. However, increased recognition was observed on either androgen ablation (which causes massive apoptosis within the prostate) or development of prostate cancer. Nonetheless, enhanced antigen recognition was not accompanied by activation of effector functions such as γ-interferon production. The consequences of transformation in additional tumor transgenic mouse systems have also been analyzed. As described earlier, Blankenstein and colleagues found that preimmunization of mice against the tumor-associated antigen prevented the development of tumors. However, non-immunized mice developed spontaneous tumors without any significant evidence of natural immune surveillance in the absence of preimmunization. They further demonstrated that an initial antigen-dependent activation of tumor-specific T cells could be observed at the time of spontaneous tumor induction, but that this recognition ultimately resulted in an anergic form of T-cell tolerance similar to that observed by Drake and colleagues in the prostate system.

The capacity of spontaneously arising tumors to tolerize T cells has not been uniformly observed. Ohashi and colleagues observed a contrasting result when LCMV GP33-specific TCR transgenic CD8 T cells were adoptively transferred into double transgenic mice expressing both SV40 T antigen and LCMV GP33 under the control of the rat insulin promoter. These animals develop pancreatic islet cell tumors that express GP33. These investigators found that as tumors progressed in the mice, enhanced T-cell activation occurred. CD8 T-cell activation was demonstrated through bone marrow chimera experiments to occur exclusively via cross presentation in the draining lymph nodes. Despite the activation of tumor-specific T cells, the tumors grew progressively, indicating that the degree of immune activation induced by tumor growth was insufficient to ultimately eliminate the tumors. These results suggest that developing tumors can induce immune responses but may titrate their level of immune activation to one that ultimately does not keep up with tumor progression. Such a circumstance is highly susceptible to the immune editing concept put forward by Schreiber and colleagues in which the tumor edits itself genetically to maintain a sufficient level of resistance to induced immune responses. In the case of the LCMV GP33 T antigen transgenic mice, because neither anergic nor deletional tolerance was observed, animals treated with the dendritic cell stimulatory anti-CD40 antibody demonstrated significant slowing of tumor growth. Thus, it may be possible under some circumstances to shift the balance between tumor immune evasion and tumor immune recognition by agents that affect the overall activation state of either antigen-presenting cells or T cells (see later discussion).

It has been more difficult to obtain definitive evidence that human cancers tolerize tumor-specific T cells, because humans cannot be manipulated the way mice are. However, T cells that are grown out of patients with cancer tend either to be of low affinity for their cognate antigen or to recognize antigens that bind poorly to their presenting HLA (human MHC) molecule, resulting in inefficient recognition by T cells. Recently, a crystal structure of the TCR-peptide-MHC trimolecular complex has been solved for an MHC class II–restricted human tumor antigen. Interestingly, the orientation of the TCR, which has low affinity for the peptide-MHC complex, is distinct from trimolecular complexes for viral (foreign) antigens and is partially similar to trimolecular complexes for a self-antigen. Thus, there may be fundamental structural features of tumor antigen recognition that lie between those of foreign antigen and self-antigen recognition.

As discussed later, one of the features of the tumor microenvironment that is likely central to the capability of tumors to tolerize tumor-specific T cells is the immature or inactive state of tumor-infiltrating dendritic cells (DCs). DCs are the major antigen-presenting cell that presents peptides to T cells to initiate adaptive immune responses. In the context of infection, microbial ligands or endogenous “danger signals” associated with tissue destruction activated DCs to a state in which they present antigens to T cells together with co-stimulatory signals that induce T-cell activation and development of effector function. However, in the absence of microbial products or danger signals, DCs remain in an immature state in which they can still present antigens to T cells but without co-stimulatory signals. These immature DCs function as “toleragenic” DCs, inducing a state of antigen-specific T-cell unresponsiveness (termed anergy ). It is thought that steady-state presentation of self-antigens by immature DCs is an important mechanism of peripheral self-tolerance. Thus, if a tumor is able to produce factors that inhibit local DCs from becoming activated in response to the endogenous danger signals associated with tissue invasion, it could shift tumor-specific T cells from a state of activation to one of tumor-specific tolerance.

Inhibition of Antitumor Immunity by Regulatory T Cells

Over the past 10 years, regulatory T cells (Tregs) have emerged as a central player in maintenance of the tolerant state as well as general downregulation of immune responses to pathogens. Not surprisingly, they appear to play a role in tolerance to tumor antigens as well as the resistance of tumors to immune-mediated elimination. In contrast to the ephemeral CD8 suppressor cells of the 1970s that failed to withstand experimental scrutiny, the more recently defined CD4 ⁺ Tregs are characterized by expression of a central master regulatory transcription factor—FoxP3—whose role in the gene expression programs of Treg is being actively studied. Although CD4 ⁺ Tregs selectively (but not specifically) express a number of cell-membrane molecules, including CD25, neuropilin, GITR, and LAG-3, their overall genetic program and inhibitory capacity are absolutely dependent on sustained expression of Foxp3. Mechanisms of immune suppression by Tregs vary and include production of inhibitory cytokines such as IL-10 and a recently described IL-12 family “hybrid” cytokine, IL-35, consisting of the alpha subunit of IL-12 and the beta subunit of IL-27. In keeping with the emerging appreciation that tumors are by nature highly tolerogenic, numerous murine studies have demonstrated that Tregs expand in animals with cancer and significantly limit the potency of antitumor immune responses—either natural or vaccine induced. For example, in a study by Sutmuller and colleagues, a combination of GM-CSF transduced tumor vaccine plus anti-CTLA-4 antibodies was much more effective at eliminating established tumors when animals were treated with anti-IL-2 receptor alpha antibodies to eliminate CD4 ⁺ Tregs. It is now appreciated that treatment with low-dose cyclophosphamide is a relatively simple and reasonably effective way to temporarily eliminate cycling Tregs. This appears to be a major mechanism by which pretreatment with low-dose cyclophosphamide before vaccination can significantly enhance the capacity of vaccines to break tolerance. As new cell membrane molecules that define Tregs are identified, the capacity to block regulatory T-cell activity with antibodies to these molecules presents new opportunities for immunotherapeutic strategies to break tolerance to tumor antigens. Because of their unusually high constitutive expression of high-affinity IL-2 receptors, IL-2 receptor–targeted antibodies and toxin-conjugated IL-2 molecules have been used therapeutically to eliminate Tregs in cancer patients. This continues to be pursued, although its ultimate value will likely come through combination of Treg elimination with other immunotherapy strategies such as vaccination and checkpoint inhibition (see later discussion).

Suppression of Antitumor Immunity by Immature Myeloid Cells in the Tumor Microenvironment

As shown in Figure 52-2 , the tumor microenvironment contains multiple inhibitory cells and molecules. Immature myeloid cells (iMCs), often termed myeloid-derived suppressor cells (MDSCs), represent a cadre of myeloid cell types, somewhat overlapping with tumor-associated macrophages (TAMs), which share the common feature of inhibiting both the priming and effector function of tumor-reactive T cells. It is still not clear whether these myeloid cell types represent distinct lineages or different states of the same general immune inhibitory cell subset. In mice, iMCs and MDSCs are characterized by coexpression of CD11b (considered a macrophage marker) and Gr1 (considered a granulocyte marker) while expressing low or no MHC class II or the CD86 co-stimulatory molecule. In humans, they are defined as CD33 ⁺ but lacking markers of mature macrophages, DCs, or granulocytes and are DR ⁻ . A number of molecular species produced by tumors tend to drive iMC/MSC accumulation. These include IL-6, CSF-1, IL-10, and gangliosides. IL-6 and IL-10 are potent inducers of STAT3 signaling, which has been shown to be important in iMC/MDSC persistence and activity.

In addition to inhibitory cytokine production, myeloid cells of multiple type in the tumor microenvironment express a number of enzymes whose metabolic activity ultimately results in inhibition of T-cell responses within the tumor microenvironment. These include the production of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). Nitric oxide (NO) production by iMC/MDSC as a result of arginase and iNOS activity has been well documented, and inhibition of this pathway with a number of drugs can mitigate the inhibitory effects of iMC/MDSC. ROS, including H ₂ O ₂ , have been reported to block T-cell function associated with the down-modulation of the ζ chain of the TCR signaling complex, a phenomenon well recognized in T cells from cancer patients and associated with generalized T-cell unresponsiveness.

Another mediator of T-cell unresponsiveness associated with cancer is the production of indoleamine 2,3-dioxygenase (IDO). IDO appears to be produced by DCs either within tumors or in tumor-draining lymph nodes. Interestingly, IDO in DCs has been reported to be induced via backward signaling by B7-1/2 on ligation with CTLA-4. The major IDO-producing DC subset is either a plasmacytoid DC (PDC) or a PDC-related cell that is B220 ⁺ ; however, IDO has been subsequently shown to be expressed by multiple cell types in the immune microenvironment, including tumor cells themselves. IDO appears to inhibit T-cell responses through catabolism of tryptophan. Activated T cells are highly dependent on tryptophan and are therefore sensitive to tryptophan depletion. Thus, Munn and Mellor have proposed a bystander mechanism, whereby DCs in the local environment deplete tryptophan via IDO upregulation, thereby inducing metabolic apoptosis in locally activated T cells. IDO has two isoforms, IDO-1 and IDO-2, encoded by distinct genes. The role of IDO-2 in human cancer is still unclear; a major IDO-2 polymorphism in humans encodes an inactive enzyme. A second tryptophan-metabolizing enzyme is TDO (tryptophan dioxygenase), which is upregulated commonly in human cancers—this may inhibit antitumor responses within the microenvironment similarly to IDO. Finally, there has been greater appreciation that a major product of IDO and TDO metabolism of tryptophan—kynurenine—has potent effects on T-cell differentiation. Under some circumstances, kynurenine can promote Treg development, and under other circumstances, it can promote development of a class of a subset of T cells termed Th17, known for its production of IL-17 and for its procarcinogenic properties (see later discussion). Ultimately, the relative role of tryptophan depletion versus kynurenine production in modulating the immune microenvironment remains to be determined.

A major inhibitory cytokine produced by iMC/MSC and by many other cell types that has been implicated in blunting antitumor immune responses is transforming growth factor beta (TGF-β), which is produced by a variety of cell types, including tumor cells, and which has pleiotropic physiological effects. For most normal epithelial cells, TGF-β is a potent inhibitor of cell proliferation, causing cell cycle arrest in the G ₁ stage. In many cancer cells, however, mutations in the TGF-β pathway confer resistance to cell cycle inhibition, allowing uncontrolled proliferation. In addition, in cancer cells, the production of TGF-β is increased and may contribute to invasion by promoting the activity of matrix metalloproteinases. In vivo, TGF-β directly stimulates angiogenesis; this stimulation can be blocked by anti-TGF-β antibodies. A bimodal role of TGF-β in cancer has been verified in a transgenic animal model using a keratinocyte-targeted overexpression. Initially, these animals are resistant to the development of early-stage or benign skin tumors. However, once tumors form, they progress rapidly to a more aggressive spindle-cell phenotype. Although this clear bimodal pattern of activity is more difficult to identify in a clinical setting, it should be noted that elevated serum TGF-β levels are associated with poor prognosis in a number of malignancies, including prostate cancer, lung cancer, gastric cancer, and bladder cancer.

From an immunological perspective, TGF-β possesses broadly immunosuppressive properties, and TGF-β knockout mice develop widespread inflammatory pathology and corresponding accelerated mortality. Interestingly, a majority of these effects seem to be T-cell mediated, as targeted disruption of T-cell TGF-β signaling also results in a similar autoimmune phenotype. Recent experiments by Chen and colleagues rather convincingly demonstrated a role for TGF-β in Treg-mediated suppression of CD8 T-cell antitumor responses. In these experiments, adoptive transfer of CD4 ⁺ CD25 ⁺ Treg inhibited an antitumor CD8 T-cell effector response, and this inhibition was ameliorated when the CD8 T cells came from animals with a dominant negative TGF-β1 receptor.

Beyond inhibitory cytokines and immune inhibitory metabolic enzymes, the therapeutically most relevant inhibitory pathways in the tumor microenvironment are the so-called checkpoints. Immune checkpoints generally refer to membrane ligands that interact with inhibitory receptors on lymphocytes (see later discussion). Many of the checkpoint ligands are upregulated in the tumor microenvironment, either by tumor cells themselves or by myeloid cells within the tumor stroma. The best studies of the checkpoint ligands fall into the B7 family (discussed later), but a number of additional checkpoint ligands do not. Because activated T cells commonly express cognate inhibitory receptors, they are inhibited from mediating antitumor responses, even if they enter the tumor in an activated state. Antibodies against two checkpoint receptors, CTLA-4 and PD-1, as well as against the major PD-1 ligand, PD-L1 (B7-H1), have demonstrated clinical success and are transforming cancer immunotherapy into an accepted cancer treatment modality. These and other checkpoints of potential relevance in tumor immune evasion are covered in the last sections of this chapter.

Therapeutic Cancer Vaccines

Therapeutic vaccines, initially introduced a half century ago, are the most investigated approaches to cancer immunotherapy. Although they are often lumped under one term, the diversity of cancer vaccine formulations is enormous, ranging from cell lysate vaccines, to genetically engineered whole-cell–based vaccines, to dendritic cell vaccines, to peptide- or protein-based vaccines, to engineered viral and bacterial vaccines. The activity in cancer vaccine development translated into many clinical trials beginning in the 1980s and continuing today. As discussed next, newer generations of vaccine design that incorporate scientific principles of dendritic cell biology and T-cell activation are demonstrating more significant clinical benefit after an initial run of failed randomized trials.

Dendritic Cells—The Key Target of Cancer Vaccines

The central theme among cancer vaccination strategies is enhancement of modulation of antigen-presenting cell (APC) function. This is based on the concept that the quantitative and qualitative characteristics of T-cell responses to antigen depend on the signals they receive from the APC. Among the major bone marrow–derived APC subtypes (B cells, macrophages, and dendritic cells), the DC has emerged as the most potent APC type responsible for initiating immune responses. As described earlier, DCs associated with cancer have altered properties that result in failure to activate T cells optimally. Cancer vaccines in essence seek to skew the function of DCs toward the generation of effector T-cell responses.

As virtually all phases of DC differentiation and function can be modulated by engineered vaccines, it is important to understand the molecular signals that regulate their role in activation of T-cell–dependent immunity. At sites of infection and inflammation, bone marrow–derived progenitor cells respond to both proliferative and differentiation signals. GM-CSF and other cytokines such as FLT-3L and IL-4 serve as mitogenic or comitogenic factors that induce an intermediate stage of DC differentiation, characterized by efficient antigen uptake and processing. Once they have ingested antigens at inflammatory sites in the tissue, immature DCs differentiate in response to a number of distinct “maturation” signals. Although many diverse molecules induce DC maturation, most appear to signal DCs via binding to two classes of receptors—the Toll-like receptors (TLR) and the TNF receptor (TNFR) family. TLRs are “pattern recognition receptors” (PRR), which bind common chemical moieties expressed by pathogens termed pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and unmethylated CpG DNA sequences. The two best characterized endogenous DC maturation factors are TNF-α itself and CD40L. In addition to TLRs, intracellular PRRs, including PKR, RIGI, MDA-5, and NOD1/2, recognize PAMPs from intracellular bacteria and viruses that invade the cytosol.

Maturation of DCs, which occurs as they traffic to draining lymph nodes, is characterized by transport of peptide-MHC complexes to the cell surface. In addition to provision of high densities of peptide-MHC complexes for T-cell stimulation (termed signal 1 ), DCs regulate T-cell activation and differentiation through the provision of co-stimulatory signals in the form of cytokines, such as IL-12, and membrane-bound ligands of the B7 and TNF family (collectively termed signal 2 ). The ever-expanding panoply of co-stimulatory signals used by DCs to instruct T cells as to their pathway of differentiation and effector function defines a high degree of complexity to the communications that occur between APC and T cell. When immature DCs present antigens to T cells in the absence of co-stimulatory signals, the outcome is tolerance induction ( Figure 52-3 ). This is a normal mechanism for the maintenance of tolerance to self-antigens. It is also a mechanism by which tumors can induce immune tolerance to their own antigens. As discussed throughout this chapter, tumor-induced immune tolerance is a major barrier to successful vaccination of established cancers. Each of the molecular events involved in proliferation, antigen presentation, and co-stimulation represents potential targets that are being exploited in the design of immunotherapy approaches.

Prior to the molecular identification of tumor-specific antigens, investigators used tumor cells themselves as a source of tumor antigen ( Table 52-1 ). Efforts to modify tumor cells as vaccines date back roughly half a century. Whole-cell tumor vaccines have been generated through mixing with adjuvants aimed at enhancing “immunogenicity” of tumor-specific or tumor-selective antigens incorporated therein, with clinical testing of these mixtures dating back to the 1980s. Another approach has been to hapten modify whole tumor cells with chemicals such as dinitrophenol or infect them with a virus. The general concept is that increasing the immunogenicity of tumor cells using either adjuvants or expression of foreign antigens will enhance immune responses to the endogenous tumor antigens, thereby allowing the immune system to kill metastatic tumor deposits.

Table 52-1

General Formulations for Cancer Vaccines

Vaccine Type	Advantages	Disadvantages
Cell-based vaccines
Autologous Allogeneic	Highly polyvalent antigenic content Vaccine antigens match those of patient’s tumor Generic vaccine for all patients	Contains mostly self-antigens Individualized patient formulation Relies solely on shared antigens
Dendritic cell (DC)
Peptide loaded Protein loaded Tumor lysate loaded RNA transduced	Can manipulate DC type and direct Ag loading Specific antigen and easy to synthesize Specific antigen and no need for HLA match Highly polyvalent antigenic content Can amplify from tiny amount of tumor	Individualized cell culture Must match patient’s HLA type Loading of HMC I less efficient Contains mostly self-antigens Complex individualized process
Peptide + adjuvant	Very easy to produce	Limited immunogenicity
Protein + adjuvant	Moderately easy to produce	Potency adjuvant-dependent
DNA vaccine	Easy to produce, versatile construction	Limited immunogenicity
Viral vaccine	Moderately easy to produce, immunogenic	Neutralizing Ab limit revaccination
Bacterial vaccine	Easy production, immunogenic, incorporate many Ag	Regulatory challenges

More recently, a new era in genetically engineered whole-cell vaccination has involved the modification of tumor cells through the transfer of genes encoding cell membrane immunostimulatory molecules or cytokines. Although most of the clinical activity related to adjuvanted whole-cell vaccines is diminishing significantly, active clinical investigation continues for cytokine gene-modified whole-cell vaccines, particularly with the GM-CSF gene (described later). Adjuvanted whole-cell tumor vaccines have been tested extensively in patients with melanoma, renal cell carcinoma, and colorectal carcinoma. Most of these vaccine strategies have involved the co-injection of either autologous or allogeneic tumor cells with adjuvants such as bacille Calmette-Guérin (BCG) and Corynebacterium parvum . Although BCG and C. parvum were long known to represent reasonable vaccine adjuvants for the generation of antibody responses, a limitation of this vaccination approach has been their relatively poor capacity to generate T-cell responses, particularly in the face of established tolerance. Initially, nonrandomized clinical trials were performed that demonstrated hints of promise. In some of these studies that reported antitumor responses, the responses were shown to correlate with the return of delayed-type hypersensitivity (DTH) responses to recall antigens and more importantly with the development of DTH responses to autologous tumor cells.

The application of BCG-adjuvanted tumor cell vaccines to patients with bulky metastatic cancer demonstrated an insignificant clinical response rate. However, given the plethora of studies in animal models suggesting that cancer vaccination might be more effective in the setting of minimal residual disease, a number of studies employing BCG-adjuvanted tumor vaccines in clinical trials were undertaken in the minimal residual disease setting after resection of the primary tumor. Initial enthusiasm for a BCG-adjuvanted, autologous colon cancer vaccine in patients with resected stage II/III colon cancer as well as a melanoma vaccine consisting of a mixture of irradiated allogeneic human melanoma lines with BCG used in melanoma patients with stage III and resected stage IV disease was based on Phase II studies and limited single-institution Phase III studies. The concern in the interpretation of clinical outcomes of these Phase II studies is that it was unclear whether the untreated “historical controls” were truly comparable to the population of patients treated in the Phase II studies. In the absence of careful case-controlled comparisons, the ultimate acceptance of these vaccines depended on pivotal randomized Phase III studies in which both progression-free survival and overall survival were the relevant clinical endpoints. In the case of the autologous BCG-adjuvanted colon cancer vaccine, an initial randomized single-institution study in The Netherlands claimed a longer overall survival in patients with stage II but not stage III colon cancer. Unfortunately, these findings were not reproduced in expanded multicenter trials, possibly owing in part to technical difficulties in consistent autologous tumor preparation as part of the patient-specific vaccine formulation. After 20 years of Phase I and II studies with an allogeneic BCG-adjuvanted melanoma vaccine, a randomized Phase III clinical trial of BCG-adjuvanted allogeneic melanoma cells versus BCG control demonstrated no evidence of enhanced overall survival for the BCG plus tumor vaccine arm. Although the Phase II studies claimed to have demonstrated significant survival benefit relative to case-matched controls, the case-matched controls demonstrated suspiciously short overall survival times relative to melanoma patients of similar stage from multiple other clinical studies. There were encouraging reports of responses to vaccination with BCG-adjuvanted DNP-modified allogeneic melanoma vaccines. However, definitive randomized Phase III trials have not been completed at the time of this writing. Although a number of these studies reported that patients with enhanced DTH responses post vaccination had better disease outcomes than patients who did not, these studies were largely devoid of analyses of antigen-specific T-cell responses, and it is unclear whether the association between DTH responses and enhanced survival had anything to do with the vaccination. A similar fate befell the melanoma vaccine Melacine, a mixture of lysates from multiple allogeneic melanoma cells admixed with the “detoxified” LPS derivative monophosphoryl lipid A (MPL) plus mycobacterial cell wall extracts. Despite encouraging reports from Phase II studies, a definitive Phase III study in patients with stage II/III operated melanoma failed to demonstrate a statistically significant effect on overall survival. A retrospective subset analysis suggested that HLA-A2 ⁺ and HLA-C1 ⁺ patients had greater benefit, but this result has not been confirmed in a prospective trial.

One of the limitations of these trials is that none demonstrates definitive enhancement of T-cell responses against relevant antigens. In the case of melanoma, many tumor antigens recognizable by T cells are indeed well defined and responses to them should be measured as part of the development process. As described earlier, a more limited set of “immunorelevant” antigens are defined for other human cancers. In summary, the age of adjuvanted whole-cell or lysate tumor vaccines appears to be slowly drawing to a close and will likely be a historical footnote in the development of cancer immunotherapies.