This concept was first put into practice by William Coley, who injected a cocktail of dead bacteria into tumors in the late 1800s, achieving cures in ≈30% of his patients with sarcoma and lymphoma (10, 11). The mechanism responsible for this seems to be LPS-induced IL-12 secretion triggering a robust bystander Th1-response against the tumor cells (12). Likewise, an attenuated *Salmonella* vaccine can induce a shift in the tumor milieu from an immunesuppressive to an immunogenic microenvironment (13). The most successful application derived from Coley's work is treatment of bladder cancer with the Bacillus Calmette–Guerin vaccine: it has become the standard therapy for superficial bladder cancer, eradicating existing tumors, reducing the frequency of tumor recurrence, delaying stage progression, and increasing survival (14). The advantage of such strategies is their lack of specificity. The immune response is not restricted to a single and, most likely, highly specific and selectable "tumor-antigen," but the presence of danger signals at the site of the tumor "uncloaks" the cancer cells, turning them into broad range immune targets. At this point, we can exploit a mechanism, which causes a break in self-tolerance in autoimmune diseases: transient autoimmunity accompanying any inflammatory process can, in the context of steady exposure to auto-antigens and danger signals, develop into stable autoimmunity. Following Polly Matzinger's ideas, the key to success of danger-based tumor vaccination strategies rests on repeated administration of the vaccine (15). Repeated immunization should help overcome transient tumor immunity and establish persistent protection.

One danger-based tumor vaccination approach conducts the immunization with dying tumor cells (16, 17). Certain kinds of dying or dead cells can trigger immune responses under the right conditions. The potential of dying/dead cells to induce autoimmunity can be seen in "systemic lupus erythematosus" (SLE), a chronic inflammatory disease, in which defective clearance of apoptotic cells leads to the accumulation of secondary necrotic cells, the release of danger signals, the presentation of auto-antigens and, finally, a chronic break in self-tolerance (18–20). Based on these observations, one can assume that, under the appropriate conditions, entities once considered to be nondangerous can become re-considered dangerous. We propose that one can learn from the processes which cause breaks of self-tolerance in patients with SLE and try to harness them to induce tumor (auto-) immunity.

In the context of tumor immunology, cell death is a double-edged sword. Tumor cells often modulate apoptotic pathways rendering them less responsive to death stimuli. Down-regulation of Fas expression or resistance to Fas-mediated apoptosis are common strategies of tumor cells to escape immunosurveillance (21) and are associated with resistance to therapy, metastatic capacity, and poor prognosis. For example, c-Jun and Stat-3 act as oncogenes by cooperatively repressing the transcription of *Fas*, rendering tumor cells insensitive to FasLinduced apoptosis (22). A complete loss of Fas expression is less common, possibly to low-level expression of Fas supporting tumor growth (23). Many other mechanisms to evade elimination by apoptosis, i.e., suppression of caspase-8 activity by CDK1/CYCLIN B1 dependent phosphorylation (24), *bcl-2* amplification (25), and loss of pro-apoptotic proteins like BAX (26) and PUMA (27), have been reported for a large variety of cancer types (28).

These findings are hard to reconcile with the observation that a high rate of tumor cell apoptosis is accompanied by poor prognosis in some types of cancer (29–31). It is known that cancer cells show many different changes to the apoptotic machinery (28, 32); but does this mean they have lost all capability to execute apoptosis? Apoptosis is necessary for tissue homeostasis, contributes to the maintenance of peripheral tolerance and might even play a role in the induction of the latter (33, 34). The fact that most chemotherapeutics at least initially induce tumor apoptosis confirms that cancer cells frequently retain their ability to execute apoptosis (35, 36). It is reasonable to assume that those parts of the apoptotic machinery involved in the induction of extrinsic apoptosis by the immune system preferentially experience negative selection. If other parts of the apoptotic pathway would also be a potential source of harm, why do they, in defiance of the exceptional adaptability of cancer cells, still function properly? We suggest that, in contrast to the oversimplified illustration, cancer cells do not completely lose their capability to undergo apoptosis, but that their apoptotic machinery can instead be "hijacked" in a way that not only sustains their existence, but also accelerates tumor formation (37–39): an "altruistic" death of limited amounts of cancer cells is a possible way to support the survival of the tumor on the whole.

Over the years, the tumor-supportive effects of apoptotic tumor cells have received greater recognition, and it is now assumed that apoptotic tumor cells and the corresponding phagocytes participate in forming and shaping the tumor microenvironment (40). Apoptotic cells release a diverse spectrum of molecules, which act as "keep-out," "find-me," "eat-me," and "tolerate-me" signals and ensure that the clearance of apoptotic cells is facilitated by defined groups of phagocytes, in particular by macrophages (41).

Of particular interest are lipid mediators, which are released from cells undergoing apoptosis: (I) lysophosphatidylcholine is a potent chemoattractant for macrophages and is released from cells executing apoptosis (42). (II) Upon proteolytic activation of sphingosine kinase 2, sphingosine-1-phosphate (S1P) is released from apoptotic cells (43). In addition to its role as a chemoattractant (44), S1P polarizes macrophages toward a noninflammatory phenotype (M2), characterized by decreased secretion of TNF-α and IL-12-p70 and increased formation of IL-8 and Il-10 (45).

The engulfment of apoptotic cells by macrophages induces their polarization toward the M2-phenotype (**Figure 1A**). These alternatively activated macrophages tune down inflammation and promote angiogenesis, tissue remodeling, and repair (46, 47). Furthermore, phagocytosis of apoptotic cells by M1-macrophages also triggers a shift toward alternative activation (48). Fittingly, a large number of macrophages at the site of the tumor are associated with a poor prognosis and these tumor-associated macrophages share many characteristics with M2 macrophages (49, 50). Their presence at the site of a tumor supports Dvorak's concept that tumors are "wounds that do not heal" (51).

In line with these findings is the observation that inhibiting the clearance of apoptotic tumor cells by administration of Annexin-A5 retards tumor growth in a colorectal carcinoma model and greatly enhances the effect of immunization with irradiated lymphoma cells in a lymphoma model (52, 53). The data presented suggests that this is due to the fact that the non-inflammatory clearance of apoptotic cells by macrophages is blocked so that the apoptotic cells get secondarily necrotic. The concomitant loss of membrane integrity is accompanied by the release of danger-associated molecular patterns (DAMP), which act as natural adjuvants. Phagocytosis of secondary necrotic cells by macrophages (**Figure 1B**) leads to an increased expression of TNF-α and IL-1β. In addition, several DAMPs released from secondary necrotic cells, like HMGB1 and HMGN1, are potent stimuli for dendritic cell maturation (54).

The close interaction between tumors, the immune system and cell death gives rise to new therapeutic approaches. Some aspects of this interaction may be exploited to support conventional cancer therapies. Systemic administration of Annexin-A5 or other phosphatidylserine ligands may help slow down tumor progression by blocking the tumor-supportive properties of apoptotic cells. In combination with radio- or chemotherapy, Annexin-A5 could be used as a natural adjuvant, which increases the immunogenicity of dying tumor cells and, thus, helps elicit an anti-tumor immune response (55). This may be especially helpful in targeting cancer cells, which have

resisted therapy and would possibly lead to a relapse.

Until recently, cell death was either characterized as programed and apoptotic, or accidental and necrotic. This paradigm has been undermined by the discovery of several other forms of cell death, ranging from immunogenic apoptosis (56) or necroptosis (57) to pyroptosis (58, 59). So, in addition to manipulating cell death induced by radio- or chemotherapy in a way to increase its immunogenicity, the direct induction of immunogenic tumor cell death pathways might become a promising approach in cancer therapy (17, 54, 60), especially, since our means of controlling the manner of cell death have greatly increased during recent years (61–63).

Surgical removal of malignant tissue plays an important role in modern cancer therapy. The cancer cells obtained in this process may be used as a vaccine to establish anti-tumor immunity, if treated and administered properly. The focus must be on cancer cells dying by immunostimulatory forms of cell death leading to necrotic cell corpses, whose deployment would activate antigen-presentingcells. This way, the specific autologous tumor cells can serve as reservoirs of tumor antigens, which, upon phagocytosis by inflammatory macrophages and dendritic cells, are effectively (cross-)presented. The impact of the vaccine could be optimized by repeated administration of the dying cells. However, we have to be very careful, since a recent study indicates that excessive immune responses against cancer can result in an increased risk of developing the autoimmune disease scleroderma (64), pointing out several parallels between the induction of autoimmunity and immunosurveillance. While this study actually supports the idea that mechanisms inducing autoimmunity can also be used to elicit tumor immunity, it also suggests that any agents used to recruit antitumor responses must be well-balanced. After all, nobody wants to escape cancer's fire by jumping into the frying pan of autoimmunity.

# **ACKNOWLEDGMENTS**

This project was supported by the German Research Foundation (SPP1468- IMMUNOBONE, collaborative research centers 643/TP-B5 and 796/TP-C4, GA 1507/1-1, and DFG-Graduiertenkolleg 1660: key signals of the adaptive immune response), by the Emerging Fields Initiative (EFI) of the FAU Erlangen-Nuremberg, by the German Federal Ministry of Education and Research (BMBF; m4

Cluster, 01EX1021R) and the K. und R. Wucherpfennig-Stiftung.

# **REFERENCES**

*Acta* (2011) **1808**:2638–45. doi:10.1016/j.bbamem. 2011.07.026

between STAT3 and c-Jun suppresses Fas transcription. *Mol Cell* (2001) **7**:517–28. doi:10.1016/ S1097-2765(01)00199-X

decisions. *Cancer Lett* (2013) **332**:202–5. doi:10. 1016/j.canlet.2011.12.021

for ischemic brain injury. *Nat Chem Biol* (2005) **1**:112–9. doi:10.1038/nchembio0905-234a

inhibit beclin 1-dependent autophagy. *Cell* (2005) **122**:927–39. doi:10.1016/j.cell.2005.07.002

64. Joseph CG, Darrah E, Shah AA, Skora AD, Casciola-Rosen LA, Wigley FM, et al. Association of the autoimmune disease scleroderma with an immunologic response to cancer. *Science* (2014) **343**:152–7. doi:10.1126/science.1246886

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 06 February 2014; accepted: 28 April 2014; published online: 13 May 2014.*

*Citation: Maueröder C, Munoz LE, Chaurio RA, Herrmann M, Schett G and Berens C (2014) Tumor immunotherapy: lessons from autoimmunity. Front. Immunol. 5:212. doi: 10.3389/fimmu.2014.00212*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Maueröder, Munoz, Chaurio, Herrmann, Schett and Berens. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Modulation of antigen-specificT-cells as immune therapy for chronic infectious diseases and cancer

#### **Suling Li 1 , Alistair L. J. Symonds 2 ,Tizong Miao2 , Ian Sanderson2 and PingWang2\***

1 Bioscience, Brunel University, London, UK

2 Blizard Institute (BICMS), Barts and the London School of Medicine and Dentistry, London, UK

#### **Edited by:**

Fang-Ping Huang, Imperial College London, UK

#### **Reviewed by:**

Jianping Huang, University of Florida, USA Alex Yee-Chen Huang, Case Western

Reserve University School of Medicine, USA

#### **\*Correspondence:**

Ping Wang, 4 Newark Street, London E1 2AT, UK e-mail<EMAIL_ADDRESS>
T-cell responses are induced by antigen presenting cells (APC) and signals from the microenvironment. Antigen persistence and inflammatory microenvironments in chronic infections and cancer can induce a tolerant state in T-cells resulting in hyporesponsiveness, loss of effector function, and weak biochemical signaling patterns in response to antigen stimulation. Although the mechanisms of T-cell tolerance induced in chronic infection and cancer may differ from those involved in tolerance to self-antigen, the impaired proliferation and production of IL-2 in response to antigen stimulation are hallmarks of all tolerant T cells. In this review, we will summarize the evidence that the immune responses change from nonself to "self"-like in chronic infection and cancer, and will provide an overview of strategies for re-balancing the immune response of antigen-specific T cells in chronic infection and cancer without affecting the homeostasis of the immune system.

**Keywords: tolerance induction, antigen-specificT cells, bystanderT-cells, nanoAPC, reverse tolerance**

#### **INTRODUCTION**

T cells are essential for robust adaptive immune responses against pathogen invasion, as well as maintaining immune tolerance to self-antigens. In the tolerant state, T cells generally fail to proliferate and produce IL-2 in response to antigen stimulation (1, 2). Anergy and immune regulation are two interconnected mechanisms that maintain peripheral tolerance to self-antigens *in vivo*. In contrast to the biochemical events induced during effective responses to pathogenic antigens, in anergy the biochemical signaling pathways in T cells are only partially activated. Activation of the calcium/calcineurin/nuclear factor of activated T cells (NFAT) pathway, but not AP1 and NFκB pathways (1–3), in anergic conditions results in the expression of tolerance associated genes such as E3-ligases (1–3). This partial TCR signaling is largely due to the lack of additional signals such as costimulatory signals and activating cytokines such as IL-2, or due to direct regulation by Treg (2). Therefore, altered expression of costimulatory signals and/or activating cytokines, or defective Treg function, results in full activation of TCR signals in response to self-antigens and may induce autoimmune responses. Recent studies have uncovered hyporesponsive phenotypes with partial activation of biochemical events in virus specific T cells in chronic infectious diseases (4, 5) and models mimicking chronic infectious conditions (5, 6). These findings indicate that during chronic infection the T-cells switch from mounting robust non-self responses to a state similar to self-tolerance due to antigen persistence and/or changes in the microenvironment. Similar to the immunological milieu of chronic infection, the tumor microenvironment contains a multitude of suppressive mechanisms that allow tumors to escape immune surveillance (4, 7). Immune hyporesponsive states have been studied in many different models *in vitro* and *in vivo* and have been categorized based on the phenotypes discovered in each tolerant state (8).

This review will briefly summarize the extracellular signals that affect self-tolerance or effector function of antigen-specific T cells. We will describe the application of these signals in therapeutic intervention and focus on the recently developed nano-technologies that can reverse the tolerant state of viral specific T cells by delivering costimulatory or cytokine signals to antigen-specific T cells.

# **ALTERED T-CELL RESPONSES DURING CHRONIC VIRUS INFECTION AND CANCER**

Chronic virus infections are associated with impaired anti-viral immunity, particularly in the infections caused by highly replicative viruses such as HIV, HBV, and HCV. In chronic infection, persistent viral antigen, and often chronic inflammation, renders T-cells dysfunctional. The mechanisms underlying dysfunctional immune responses in patients are largely unknown. Based on experimental systems studied *in vitro* and *in vivo*, different states of T-cell dysfunction have been discovered and are classified as exhaustion, tolerance, anergy, senescence, deletion, induced Treg, and ignorance based on the phenotypes, production of inhibitory cytokines such as IL-10 and TGFβ, impairment of T-cell receptor signaling molecules, and apoptosis of the T-cells in these models (**Figure 1**) (4–6, 8–10). These findings have been extensively reviewed (4–6, 8–10). Despite the differences in dysfunctional Tcells characterized in different model systems, the common feature is proliferative hyporesponsiveness, and impaired production of IL-2 following antigen stimulation *in vivo* or *in vitro* (4–6, 8–10). The chronic LCMV infection model resembles the observations from patients with chronic virus infections more closely than other models in terms of induction of dysfunctional T cells (4, 5). The phenotype of exhaustion of CD8 T cells in the chronic LCMV model is well-characterized, with hierarchical loss of effector cytokine production, including IL-2, TNFα, and IFNγ and

impaired proliferation in response to antigen receptor stimulation *in vitro* (4, 5). In addition to this hyporesponsive phenotype, increased expression of the inhibitory costimulatory molecule PD-1 and production of the repressive cytokine IL-10 are also found in T cells from chronic LCMV infected mice (9, 10). Notably, similar phenotypes have been found in T cells from HIV, HBV, and HCV patients (11–14). Under chronic infectious conditions, viral specific CD8 T cells often lose cytotoxic function (15, 16). At the late stages of exhaustion, viral specific CD8 T cells may be deleted (5, 6). However, in contrast to CD8 T cells, viral specific CD4 T cells can persist under chronic infectious conditions, but in a hyporesponsive state (17). Therefore, there is the potential to restore CD4 responses, which may thereafter help CD8 function. It has been reported that Treg cells are increased or induced in chronic infection (18, 19). The increased Treg cells can reduce chronic inflammation from persistent viral antigen stimulation, but may also contribute to the establishment of immune tolerance toward the virus (18, 19).

Comparable to chronic infection, high levels of tumor antigens and chronic inflammation can establish an immunosuppressive microenvironment. Tumor reactive T cells have been shown to respond to tumor antigens in a similar fashion to viral specific T cells in chronic infection with expression of high levels of inhibitory costimulatory molecules such as PD-1, CTLA-4, and LAG-3 and impaired production of effector cytokines including IFNg, TNFa, and IL-2 (7, 20–22). It has been shown that advanced tumors with high loads of tumor antigens cause functional exhaustion and rapid elimination of tumor reactive T cells (23). However, in contrast to chronic viral infections, tumor antigens are generally poorly antigenic. Therefore, the frequency and avidity of tumor reactive T cells are low.

## **IMPAIRED TCR SIGNALING DURING CHRONIC VIRUS INFECTION**

We have found that antigen persistence can impair TCR signaling resulting in hyporesponsiveness (24). This hyporesponsiveness is gradually induced during antigen persistence with reduction of NFkB and AP1 activation (2, 24). This characteristic phenotype of T-cell tolerance is similar to that observed in chronic HBV infection (25). Down-regulation of TCR proximal signaling molecules has been found in CD8 T cells from chronic HBV patients (25). The impaired TCR signaling in CD8 T cells from chronic HBV patients is partly due to the down-regulation of CD3ζ (25). The reduced expression of CD3ζ is associated with up-regulation of PD-1 and impaired production of IL-2, suggesting that it is part of the mechanism leading to exhaustion (25). Viral protein Nef of HIV and E2 and core protein of HCV directly modulate TCR signaling (26). HIV Nef protein interacts with a number of TCR signaling molecules including Lyn, Hck, and Lck (27). The interaction stimulates the TCR signaling pathways in the absence of antigens leading to maintenance of viral replication (26, 27). The altered TCR signaling induced by Nef negatively affects antigen-mediated TCR signaling (28). Similarly, viral proteins from HCV also modulate TCR signaling (29). E2 protein of HCV binds CD81 and promotes TCR signaling while the core protein inhibits JNK signaling and IL-2 expression (29). However, HCV does not infect T cells. Therefore, the altered TCR responses during chronic HCV infection are largely due to the persistence of viral antigens. Whether the persistent, but abnormal, TCR signaling induced by viral proteins causes the development of T-cell exhaustion is yet to be investigated.

It has been found that the tumor microenvironment impairs the formation of T-cell immunological synapses; supramolecular structures that assemble at the T cell-APC interface (30). Dysregulated synapse formation is associated with impaired activation of Rho-GTPases and can lead to partial activation or anergy of T cells.

#### **INDUCTION OF NEGATIVE COSTIMULATORY MOLECULES**

One of the important changes to the phenotype of CD8 T cells in chronic LCMV infection is the increased expression of the negative costimulatory molecules PD-1, 2B4, CTLA-4, and LAG-3 (5). A similar phenotype of increased negative costimulatory molecules has been discovered in CD4 and CD8 T cells from chronic HBV and HIV patients (5, 15, 31–35). The function of these negative costimulatory molecules is important in the maintenance of immune tolerance toward self-antigens. Although the mechanisms underlying the induction of negative costimulatory molecules in T cells during chronic infection are not clear, it may be part of a physiological protection mechanism to reduce immunopathology induced by viral persistence and chronic inflammation. These negative costimulatory molecules are transiently up-regulated in activated effector T cells in the early stages of acute infection. However, the sustained expression of PD-1 on virus-specific CD8 T cells is associated with chronic infection, both in LCMV mouse models and in HBV patients (5). Co-expression of multiple inhibitory molecules correlates with increased functional deficits in anti-virus responses and decreased control of viral loads. Similarly, increased expression of PD-1 and CTLA-4 has been found on tumor infiltrating T cells (TIL), which can be associated with E3-ligase expression and increased Treg cells (7). Thus, the overexpression of inhibitory molecules results in shifting the balance of the immune responses from effective anti-virus or -cancer responses toward tolerance.

#### **ALTERED CYTOKINE PRODUCTION IN T CELLS**

One of the most pronounced changes in T cells in chronic infectious conditions is the altered production of cytokines (4–6). In contrast to acute infection, antigen-specific T cells from chronic infectious diseases fail to produce IL-2 and TNFα, but express the regulatory cytokine IL-10 (4–6). We have discovered that antigen-specific CD4 T cells gradually alter their cytokine profile in response to antigen persistence *in vivo* (24). Initial antigen stimulation effectively induces IL-2 production in antigen-specific CD4 T cells *in vivo*, while repeated exposure to the same antigen yields CD4 T cells that produce both IL-2 and IL-10 (24). Antigen persistence can finally switch off the expression of IL-2 in T cells, but these cells still produce high levels of IL-10 (24). This altered cytokine profile is associated with impaired proliferative responses and reduced AP1 and NFκB activation in response to antigen stimulation *in vivo* (24). Impaired production of effector cytokines such as IL-2, TNFα, and IFNγ is also associated with the defective activation of TCR signaling pathways and effector function of viral-specific CD4 and CD8 T cells in chronic HBV, HCV, and HIV infections (5, 6, 25). The up-regulation of inhibitory molecules, especially PD-1, is closely associated with the production of IL-10 and/or TGFβ (5, 6). Thus, virus persistence skews the Tcell response from activation and differentiation into effector cells toward antigen-specific immune tolerance. However, the mechanisms whereby IL-10 and/or TGFβ result in tolerance in chronic infections are still undefined. In the LCMV model, the lack of IL-10 or a defect in IL-10 signaling improves CD8 T-cell responses and drastically enhances the control of the infection (36, 37). TIL also display an altered cytokine profile, which is similar to that seen in chronic infections. High levels of IL-10 producing Treg cells have been found in TILs, which is associated with impaired production of IL-2, TNFa and IFNg (7, 38).

## **THERAPEUTIC INTERVENTIONS TO REVERSE IMMUNE TOLERANCE IN CHRONIC INFECTION AND CANCER**

Therapeutic interventions for chronic viral infection and cancer aim to counter the effects of the immunosuppressive microenvironment and skew responses toward antigenic determinants that are highly immunogenic. Various approaches have been tried to increase antigen presentation quality via immunization with selected antigenic peptides, using methods such as recombinant vaccinia vaccines, DNA vaccines, peptide vaccines, and DC vaccines, to boost the anti-viral and -tumor responses (39). So far these therapeutic vaccines have not been successful. One of the possible explanations is that the hyporesponsiveness of T cells is not due to the lack of antigens, but to aspects of the chronic disease such as antigen persistence and chronic inflammation, which increase the activation threshold of T cells in response to antigen. Therefore, to overcome the high activation threshold of antigen-specific T cells in these conditions, immune therapy has to consider the antagonizing tolerogenic environment. Thus, therapeutic vaccines in combination with targeted immune modulation have been proposed as a more effective strategy to reverse the hyporesponsive state of T cells in chronic infections and cancer. In ovarian cancer, improved anti-tumor immune responses were observed after blockade of PD-1 (40). Similarly, in the LCMV model, immunization with LCMV GP33 encoding vaccinia virus coupled with administration of anti-PD-L1 blocking antibody significantly improved viral-specific CD8 T-cell responses and reduced viral load (41). Moreover, in chronic LCMV, combined therapy with a DNA vaccine and IL-10 neutralizing antibody effectively reversed viral specific CD8 T-cell tolerance (42). Immune tolerance induced by virus persistence is due to a network with multiple suppressive components. Blockade of multiple inhibitory receptors including PD-1,LAG-3,and CTLA-4 or combined blockade of inhibitory receptors and immunosuppressive cytokines achieves greater efficacy than blockade of a single inhibitory molecule in chronic LCMV models (43, 44). Although the increased T-cell function and concomitant decrease in viral load in these interventions are transient, these data support the hypothesis that reversing immune tolerance to the virus or tumor is the key for successful immunotherapy. While blockade of PD-1 and IL-10 resulted in restoration of viral specific CD8 T-cell function in a mouse model (45), the mechanisms underlying this recovery of effector function are still unknown. As many of these interventions do not specifically target the virus- or tumor-specific T cells and these pathways are important for maintenance of peripheral tolerance, it is essential to control the balance between restoration of anti-viral or -tumor responses and prevention of autoimmune diseases (5, 7). The ideal intervention will be to specifically reverse the tolerance of viral or tumor specific T cells, while maintaining the overall self-tolerance of the immune system.

# **RESTORING NON-SELF-RESPONSES OF VIRAL SPECIFIC T CELLS, WHILE MAINTAINING THE SELF-TOLERANCE OF BYSTANDER T CELLS IN CHRONIC INFECTION**

The differential responses of antigen-specific T cells result from biochemical signals induced in T cells following interaction with antigen-MHC complexes, costimulatory molecules, and cytokines. When the mitogenic biochemical signals break the activation threshold, the T cell will enter into the cell cycle and produce growth cytokines such as IL-2 to promote clonal expansion. Due to the persistence of viral antigen, the chronic inflammatory environment and the increased production of inhibitory molecules, the activation threshold of viral specific T cells is increased and the T cells are unable to enter the cell cycle following antigen stimulation (5, 24). However, chronic infection normally does not induce tolerance in T cells responding to antigens other than those derived from the virus itself. Therefore, systemic intervention may reverse the tolerance of viral-specific T cells, but also break the self-tolerance of bystander T cells potentially resulting in autoimmunity (5). Therefore, the ideal strategy is to reverse tolerance via modulations that increase positive and/or dampen negative costimulatory signals thereby breaking the activation threshold and driving clonal expansion of virus responding T cells, but importantly, without affecting bystander T cells. Cytokine modified and viral antigen pulsed DCs have been used to deliver antigen and positive costimulatory signals to viral specific T cells in chronic infection (46–48). However, DCs are unstable and very heterogeneous in terms of population and function. It is therefore difficult to target and deliver additional positive signals to antigen-specific T cells (49). We found that an increase in the amount of antigen presented by activated dendritic cells (DC) cannot reverse tolerance (50, 51). Although exogenous IL-2 can effectively overcome tolerance and restore the full activation of tolerant T cells in response to antigen stimulation in animal models and HBV specific CD8 T cells, systemic administration of high doses of IL-2 not only induces severe side effects, such as cardiovascular, pulmonary, hematological, hepatic, neurological, endocrine, renal, and/or dermatological complications (52), but may also promote Treg function, which can further increase the activation threshold of antigen-specific T cells (53).

In order to use IL-2 and/or anti-PD1 to overcome the hyporesponsiveness of viral specific T cells induced in chronic HBV infection while avoiding the side effects of systemic administration, we have developed a novel therapeutic vaccine (nanoAPC). These nanoAPC are derived from an APC line; the human B cell line 721.221. This cell line is MHC deficient, but expresses high levels of costimulatory molecules (51). The nanoAPC are prepared from the endoplasmic reticulum (ER) membranes of 721.221 cells (51), that are genetically engineered to express ER retained MHC class I alleles and membrane-bound IL-2. Therefore, MHC and IL-2 are synthesized physiologically in 721.221 human B cells and immobilized on ER-membranes (**Figure 2**) (51). After assembly with HBV antigenic peptide *in vitro*, the nanoAPC contain peptide-MHC complexes, costimulatory molecules, and IL-2. Unlike therapeutic DCs, the nanoAPC are homogeneous, stable, and can be stored at −80°C (50, 51). Equipped with defined viral-peptide-MHC complexes, the administered nanoAPC can directly interact with antigen-specific T cells *in vivo* (51). Due to the native structure of their membranes, nanoAPC effectively induce immune synapses and expression of the high affinity IL-2 receptor on T cells (**Figure 2**) (51). The IL-2 delivered by nanoAPC enhanced antigen-specific T-cell responses and effector function, but did not affect bystander T cells or Treg cells. When assembled with a pool of HLA A2 associated HBV peptides and HBV peptides associated with HLA DR and DP, IL-2-nanoAPC induced strong CD4 and CD8 T-cell responses in peripheral lymphocytes from chronic HBV patients (51). We demonstrated that IL-2 on nanoAPC is able to enhance TCR signaling and downregulate PD-1 expression on virus responding CD8 T cells from chronic HBV patients, which could effectively reverse tolerance as demonstrated by induction of IFNγ producing CD8 T cells in lymphocytes from chronic HBV patients (51). In

cells were stained by B220 (orange).

microscopy shows nanoAPC stained with anti-IL-2 (red) and anti-HLA A2

addition to TCR signaling, MAPK activation can result directly from IL-2R signaling (53). It has been found that the activation of MAPK and PI3K through Shc recruited by the IL-2R is independent of STAT5 signaling in effector T cells, which differs from that in Treg cells, and is important for the expansion of activated CD8 T cells (54). We have demonstrated that nanoAPC can induce CD25 expression and immune synapse formation, which not only enables the induction of T-cell activation but also brings engineered bio-adjuvants such as IL-2 stably into signalsomes of effector T cells (51). The increased expression of CD25 on CMV antigen-specific CD8 T cells by IL-2-CMVnlvA2-nanoAPC is consistent with the well-known observation that IL-2 can induce CD25 expression on pre-activated CD8 T cells (51). Thus, together with peptide-MHC complexes and costimulatory molecules, the selective delivery of IL-2 is important in inducing activation of HBV responding T cells in chronic HBV patients. As the overall pharmacological dose remains low, the IL-2-nanoAPC do not activate Treg cells indicating that this approach can be adapted for use with other bio-adjuvants. Our results demonstrate that IL-2-nanoAPC, which deliver both antigen and IL-2 to antigenresponding T cells, can significantly increase functional anti-viral responses, thereby overcoming the immune tolerance induced by persistent viral load.

Nano-particles prepared from synthetic materials or genetically engineered microbes have been used to deliver antigens to DC for induction of anti-viral or -cancer immune responses (55). In contrast to these particles, nanoAPC are prepared from the ERmembranes of bio-engineered APC. Therefore, they are not only more biocompatible than synthetic nano-particles or microbes, but also deliver therapeutic molecules that are physiologically synthesized by APC seed cells. Thus, the IL-2 on IL-2-nanoAPC is more stable than free IL-2 *in vivo*, and maintains its physiological conformation allowing optimal interaction with the IL-2 receptor (data not shown). Unlike other nano-particle based vaccines, we have demonstrated that nanoAPC can directly activate T cells. NanoAPC are derived from APC cells and contain high levels of costimulatory molecules (51). Therefore, the nanoAPC mimic live DC to induce lipid raft clustering on T cells and formation of an immunological synapse, which is essential for T-cell activation. Furthermore, using HLA I negative 721.221 cells as seed cells allows us to specifically express selected HLA alleles allowing construction of HLA allele matched nanoAPC for individual patient populations.

Previously, we observed nanoAPC homing to T-cell areas of peripheral lymphoid organs, largely due to the expression of homing receptors by the cells from which the nanoAPC are derived (50). We have now further demonstrated that nanoAPC are not efficiently endocytosed by DC *in vivo* (50, 51). This is important as it allows the nanoAPC to remain as free-particles in peripheral lymphoid organs. The absence of endocytosis may be due to the lack of molecules on nanoAPC recognized by DC pattern recognition molecules (56). Thus, nanoAPC effectively target viral specific T cells and deliver immune modulation to reverse their tolerant state.

#### **SUMMARY**

In chronic infection and cancer, T cells are continuously confronted with moderate to high levels of antigens, which, in combination with the induced immunosuppressive microenvironment resulting from high antigen load and dysregulated immune responses, leads to increased activation thresholds and, subsequently, a reduction in effector function resulting in a tolerant state. This tolerant state can be reversed by positive regulatory molecules such as IL-2, IL-7, and/or blockade of PD-1 and CTLA-4. However, systemic administration of positive regulatory cytokines, or blocking antibodies, may cause autoimmunity. Therefore, one of the major challenges for immunotherapy against chronic infectious diseases and cancer is to reverse the tolerance of antigenspecific T cells, without affecting bystander T cells, thereby maintaining immune homeostasis to self-antigens. The development of delivery vehicles targeting antigen-specific T cells allows the provision of not only antigen but also engineered bio-adjuvant(s), which can restore effector function.

#### **ACKNOWLEDGMENTS**

This work was supported by Arthritis Research UK and Medical Research Council UK.

# **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 03 January 2014; accepted: 04 June 2014; published online: 17 June 2014. Citation: Li S, Symonds ALJ, Miao T, Sanderson I and Wang P (2014) Modulation of antigen-specific T-cells as immune therapy for chronic infectious diseases and cancer. Front. Immunol. 5:293. doi: 10.3389/fimmu.2014.00293*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Li, Symonds, Miao, Sanderson and Wang . This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Immunotherapy for prostate cancer: lessons from responses to tumor-associated antigens

#### **HarmWestdorp1,2† , Annette E. Sköld1† , Berit A. Snijer 1 , Sebastian Franik 1 , Sasja F. Mulder 2 , Pierre P. Major 3 , Ronan Foley 3 ,Winald R. Gerritsen2 and I. Jolanda M. de Vries 1,2\***

1 Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands

2 Department of Medical Oncology, Radboud University Medical Center, Nijmegen, Netherlands

3 Juravinski Hospital and Cancer Centre, Hamilton, ON, Canada

#### **Edited by:**

Fang-Ping Huang, Imperial College London, UK

#### **Reviewed by:**

Masaaki Murakami, Osaka University, Japan

Fabio Grizzi, Humanitas Clinical and Research Center, Italy

#### **\*Correspondence:**

I. Jolanda M. de Vries, Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 26, 6525 GA Nijmegen, Netherlands

e-mail<EMAIL_ADDRESS>
†Harm Westdorp and Annette E. Sköld have contributed equally to this work.

Prostate cancer (PCa) is the most common cancer in men and the second most common cause of cancer-related death in men. In recent years, novel therapeutic options for PCa have been developed and studied extensively in clinical trials. Sipuleucel-T is the first cell-based immunotherapeutic vaccine for treatment of cancer. This vaccine consists of autologous mononuclear cells stimulated and loaded with an immunostimulatory fusion protein containing the prostate tumor antigen prostate acid posphatase. The choice of antigen might be key for the efficiency of cell-based immunotherapy. Depending on the treatment strategy, target antigens should be immunogenic, abundantly expressed by tumor cells, and preferably functionally important for the tumor to prevent loss of antigen expression. Autoimmune responses have been reported against several antigens expressed in the prostate, indicating that PCa is a suitable target for immunotherapy. In this review, we will discuss PCa antigens that exhibit immunogenic features and/or have been targeted in immunotherapeutic settings with promising results, and we highlight the hurdles and opportunities for cancer immunotherapy.

**Keywords: immunotherapy of cancer, prostate cancer, tumor-associated antigens, CRPC, immunotherapy**

#### **INTRODUCTION**

Prostate cancer (PCa) is the most commonly diagnosed noncutaneous cancer among men in the United States and is the second leading cause of death from cancer in men (1). In Europe, PCa is also the cancer type with the highest incidence in men apart from skin cancer, while it is the third most common type of cancer after lung cancer and colorectal cancer (2). PCa is usually diagnosed in men above 65 years of age. Depending on the severity of the disease, current treatment options for PCa consist of active surveillance, prostatectomy, radiation therapy, hormonal therapy, or chemotherapy. Up to one-third of patients with a localized tumor eventually fails on local therapy and progress to advanced-stage or metastatic PCa within 10 years. For advanced PCa, androgen deprivation therapy is the standard of care. Although the majority of patients initially respond, most tumors become resistant to primary hormonal therapy within 14–30 months (3). For men with metastatic castration-resistant prostate cancer (mCRPC), the median survival in phase III studies range from 15 to 19 months. For several years, the chemotherapeutic drug docetaxel was the only treatment option for mCRPC, resulting in a median overall survival benefit of 2–3 months compared with the previous treatment regimes mitoxantrone and prednisone (4–6). However, new agents targeting the androgen signaling pathway, immunotherapeutic options, radium-223 treatment, and the new chemotherapeutic treatment modality taxane cabazitaxel are emerging therapies with the ability to improve both the survival and the quality of life.

In 2010, the first cellular immunotherapy was approved as a treatment for mCRPC by the US Food and Drug Administration (FDA).  Especially, modulation of T-cell checkpoints via immune checkpoint inhibiting [anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonal antibodies and anti-programed death (ligand) 1 (PD-(L)1] monoclonal antibodies has been successful. Instead of tacking of the brake of the immune system, as is the case with checkpoint inhibitors, another challenge is out there: enhancement of immune responses to tumor-specific antigens. In this review, we discuss tumor antigens expressed by PCa, how they can be used to combat PCa via immunotherapy, and which hurdles need to be addressed and overcome. Other new treatment modalities are beyond the scope of this study.

### **INFLAMMATORY RESPONSES IN THE PROSTATE**

Inflammation is an innate response to harmful stimuli, such as infections, tissue damage, or tissue malfunction (8, 9). The main goal with the inflammatory process is to clear the potential threat and restore tissue homeostasis. This normally occurs in two phases – the recognition and elimination phase and the resolution and repair phase (8, 10). If the acute response fails in eliminating the inflammatory agents, the inflammation shifts toward a chronic state. Instead of initiating the resolution phase, additional macrophages and lymphocytes are recruited and, depending on the inflammatory inducer, act to remodel the local microenvironment to adapt to an altered tissue homeostasis.

Cancer has been described as a wound that refuses to heal (11), and today many cancers have been tightly correlated with preceding inflammatory responses (12, 13). Several lines of evidence support the theory that inflammation also precedes PCa (9). Proliferative inflammatory atrophy lesions are areas in the prostate with an increased infiltration of inflammatory cells. These regions can merge with prostatic intraepithelial neoplasia,which is considered to be a risk factor for the development of PCa (14, 15). Also, a correlation with regular intake of non-steroidal anti-inflammatory drugs and reduced PCa risk has been observed (16–18).

#### **AUTOIMMUNITY AND PCa**

Inflammatory response inducers in prostate vary from infections to life style factors, such as diet or smoking (19). Symptomatic prostatitis caused by bacterial infection has been correlated with an increased risk of PCa development (20, 21). However, the causing agents of the majority of symptomatic and asymptomatic prostatitis are not well characterized and are probably multifaceted events (22).

Several studies have reported autoimmune responses against both seminal proteins (23, 24) and prostate antigens causing prostatitis to become chronic (25–27). These finding are additionally verified in animal models, where a cytotoxic cellular response seems to be driving the autoimmune reaction (28). Androgen ablation in patients with PCa is shown to induce high levels of T-cell infiltration into both benign and cancerous prostate sites, indicating that autoimmune responses against prostate antigens might be hormonally regulated (29).

#### **ANTIGEN-BASED CANCER IMMUNOTHERAPY**

The increased knowledge of how specific immune responses are evoked and the development of tools to manipulate the immune system have enabled implementation of novel immune-based cancer therapies. The rationale of these immunotherapies is to induce anti-tumor immune responses, decrease tumor-load, and change the course of the disease. Recognition of target antigens by the immune system is crucial. Several types of immunotherapeutics have been developed, such as peptide vaccines, DNA/RNA vaccines, cell-based vaccines, and T-cell modulators. Although improving overall survival is the primary endpoint of most clinical studies, a better understanding of induced T-cell responses, boosting pre-existing immune responses, and the effect of the tumor microenvironment on the T cells is needed to further improve PCa immunotherapy.

Tumor-associated antigens in PCa can be proteins that are present on prostate cells and on their malignant counterparts. Examples are prostate-specific antigen (PSA), prostatespecific membrane antigen (PSMA), and the cancer/testis antigens (CTAs). In a steady state, these antigens are not provoking strong immune responses. Immunosuppressive mechanisms in the prostate microenvironment, such as transforming growth factor (TGF)-β, regulatory T cells (Tregs), or myeloid-derived suppressor cells, will maintain prostate infiltrating lymphocytes in an inactive state (30–32). In addition, PCa cells exploit several mechanisms to enhance immune tolerance (33). Despite the immunosuppressive microenvironment, several immunotherapeutic approaches are able to induce or enhance tumor-specific immune responses. In the following section, potential tumor antigens and their application as immunotherapeutic targets will be discussed. **Table 1** provides an overview of the antigens discussed and clinical results of antigen-based immunotherapy trials.

## **PROSTATE CANCER ANTIGENS PROSTATE-SPECIFIC ANTIGEN**

Prostate-specific antigen is a serine protease produced primarily in the epithelial cells lining the acini and ducts of the prostate gland (51–53). Physiologically, PSA is present at high concentrations in the seminal fluid. Its function is to cleave high molecular weight proteins into smaller peptides, which results in liquification of these peptides. This allows the spermatozoa to swim freely (51). Membrane-bound PSA is expressed by most PCa cells. Upon disruption of the prostate gland tissue by cancerous growth, PSA is released into the circulation. There, PSA can interact with several inflammatory cells, including fibroblasts and macrophages, which might cause chronic inflammation (9, 54, 55). PSA serum levels correlate with the extent of disease and are therefore a useful tumor marker, accurately reflecting tumor status and prognostic for clinical outcome. In case of relapse, PSA levels correlate with tumor recurrence (51, 56). Transcription of the PSA gene is positively regulated by the androgen receptor, which can partly explain the decline in PSA levels in response to androgen deprivation therapy (52). However, high PSA levels are also observed in patients with CRPC, due to the acquired ability of the tumor cells to maintain the androgen receptor function even in the androgen-ablated environment (57).

#### **PSA as tumor antigen**

Cellular autoimmune responses against PSA have been detected in both healthy men and patients suffering from chronic prostatitis (26, 27, 58), suggesting that PSA has immunogenic properties. It has been used as a target antigen in several immunotherapeutic constructs. Hodge et al. used a vector designated TRICOM, containing three co-stimulatory molecules B7-1, ICAM-1 and LFA-3, and a PSA peptide, for T-cell stimulation (59). Using a similar approach, Kantoff et al. studied a combination of PSA-expressing recombinant viral vectors, where treatment with a vaccinia-based priming vector was followed by six booster injections of a fowlpoxbased vector (PROSTVAC-VF). In the phase II, randomized controlled trial in patients with mCRPC, no significant difference in progression-free survival was detected between control group and the vaccinated group. However, vaccinated patients had a longer median overall survival, and a better 3-year survival (60). These clinically meaningful results have to be confirmed in an ongoing phase III trial (**Table 2**).

Other PSA-expressing vectors have been tested in phase I trials in patients with PCa with rising PSA levels. Vaccinations with vaccinia-based vectors expressing PSA resulted in stabilization of serum PSA levels and PSA-specific T-cell responses were observed (34). PSA-specific T cells were also detected after vaccination with a liposome-based PSA vaccine and a dendritic cell (DC)-based vaccine (35, 36). Treatment with a PSA encoding poxviral vector vaccine in combination with radiotherapy not only showed PSA-specific T-cell activation, but also T-cell responses against prostate-associated antigens not encoded by the vaccine. This is

#### **Table 1 | Antigens and their immunogenicity in prostate cancer**.

(Continued)

indicative for tumor cell killing and subsequent epitope spreading (37). Hence, PSA-targeted immunotherapy can boost conventional treatment strategies to induce stronger and broader effects. This was also shown in a recent study combining PSA-TRICOM treatment with the T-cell checkpoint inhibitor ipilimumab, where the majority of chemo-naive patients displayed a decline in serum PSA levels (61).

Despite thefact that PSA-based immunotherapeutic approaches can stimulate cytotoxic T lymphocytes (CTLs) both *in vitro* and *in vivo*, untreated patients with PCa often fail to induce a potent immune response against this antigen (62–64). Several factors might contribute to this phenomenon: (i) PSA activates TGF-β, which can suppress immune responses in the tumor microenvironment (65, 66); (ii) PSA has a negative effect on lymphocyte proliferation and differentiation (63, 64); (iii) PSA can inhibit the maturation, function, and survival of DCs (63, 64).

In summary, the serine protease PSA is expressed at high levels by most PCa. Targeting PSA might not only elicit a tumor-specific immune response, but also counteract the negative effect of PSA on both T cells and DCs. Therefore, PSA poses as a promising target antigen in immunotherapy, and this is underscored by the results of phase II trials using PSA in vector-based peptide vaccines (60, 67). The ongoing phase III clinical trial (NCT01322490) might provide more evidence on the clinical relevance of PSA-TRICOM/PROSTVAC-VF vaccinations (**Table 2**).

#### **PROSTATE ACID POSPHATASE**

Human prostate acid posphatase (PAP) is a secreted glycoprotein enzyme synthesized in the prostate epithelium (68). Only a few substrates have so far been identified for PAP, including adenosine monophosphate, phosphotyrosine, phosphocholine, phosphocreatine, and ErbB-2 (69, 70) Since PAP can act as a protein tyrosine phosphatase, many other yet to be identified substrates might be involved in the signal transduction of this protein. PAP is secreted by the prostate gland following puberty and its expression is correlated with testosterone. It is reported to enhance the mobility of sperm (71). Serum PAP levels are low in healthy individuals and increased levels are associated with PCa. For example, it is shown that PAP is aberrantly expressed in high Gleason score PCa (72, 73). Ozu et al. showed that serum PAP levels, like serum PSA, are significantly increased within the escalating PCa disease stages. PAP is also elevated in patients with bone metastasis, compared to those without bone metastasis (74). Elevation of PAP is associated with significantly shortened survival, while its decrease is correlated with responsiveness to therapy (75–77).

#### **PAP as tumor antigen**

Due to its elevated expression in PCa, PAP has been investigated as a possible target antigen for immunotherapeutic approaches. PAPspecific cytotoxic T cells (CTLs) can be found in blood of healthy donors and in patients with chronic prostatitis (26,78,79). In addition, patients with PCa vaccinated with DCs loaded with murine PAP showed responses against human PAP coinciding with significant clinical anti-tumor responses (80). Specific CTLs can also be generated by culturing with antigen presenting cells pulsed with a PAP-derived HLA-A2 binding peptide. The obtained CTLs can lyse peptide-loaded target cells in an antigen-specific manner, as

#### **Table 2 | Ongoing trials encompassing antigen-based immunotherapy**.

(Continued)

#### **Table 2 | Continued**

well as HLA-A2 positive prostate tumor cells *in vitro* (78). PAPspecific cytolytic T-cell responses have additionally been identified in HLA-A2 transgenic mice immunized with the PAP encoding DNA vaccine pTVG-HP (81). Moreover, PAP peptides with the ability to bind additional HLA-A alleles has also been described (82, 83). Also, small clinical studies using a PAP-derived peptide for different HLA-subclasses show promising results in patients with PCa (84, 85). Naturally occurring PAP-specific CD4+ T cells are only found in 7–11% of patients with PCa, but this can be augmented by immunotherapy. Overall, these data suggest that PAP-specific T-cell responses can be initiated, and that PAP is an interesting candidate to use in cancer immunotherapy (81, 83, 84).

#### **DNA-based PAP vaccine**

In a PAP-based DNA vaccine, patients with CRPC received six vaccinations with granulocyte-macrophage colony-stimulating factor (GM-CSF) biweekly. Both humoral and cellular immune responses were detected in 3 of the 22 patients, with an at least threefold increase in PAP-specific IFN-gamma secreting CD8+ T cells. Nine of 22 patients showed PAP-specific CD4+ and/or CD8+ T-cell responses, but no antibody responses were detected. Also, an increase in the PSA doubling time was observed (86). The results of two ongoing trials will shed light on the role of PAP-based DNA vaccines in PCa (**Table 2**).

cancer cells

# **APC-based PAP vaccine: sipuleucel-T**

After three phase III randomized controlled trials, the PAP-targeting vaccine sipuleucel-T, became the first cellular immunotherapy ever to be approved for any malignancy by the FDA (38, 39, 41). Sipuleucel-T is a peripheral blood mononuclear cell (PBMC)-based autologous vaccine. PBMCs are cocultured with a fusion protein, consisting of GM-CSF and PAP, for *ex vivo* activation of APCs and as tumor-associated antigen, respectively. The proposed mechanism of sipuleucel-T is inducing antigen-specific immune responses and thereby destroys PCa cells (40).

Sipuleucel-T treatment consists of three injections at 2-week intervals. In three phase III randomized controlled trials, an increase in overall survival of 4 months was noticed with no difference in progression-free survival. In general, treatment was well tolerated and only rigors and pyrexia were reported as adverse events (38, 39, 41). The trial by Kantoff et al. showed a trend of superior treatment outcome of sipuleucel-T in patients in the lowest PSA-level quartile (≤22.1 ng/mL). On the contrary, in the highest PSA-level quartile treatment with sipuleucel-T showed only 2.8 months overall survival benefit (41). This suggests that treatment with sipuleucel-T should be initiated directly after the diagnosis of mCRPC, when patients have a lower tumor load, hence less immune suppression.

To date, the OS benefit of sipuleucel-T cannot be fully explained by the recorded immune responses. An elevated T-cell stimulation index was observed in the sipuleucel-T treated group. Nevertheless, T-cell proliferation responses to the fusion protein (PA2024) or PAP did not show a survival difference. Increased antibody levels against PA2024 were observed in 66.2% of the sipuleucel-T treated patients and in 2.9% of the placebo-treated patients coinciding with a slight, although not significant, survival benefit (*P* = 0.08). Increased antibody levels against PAP were noticed in 28.5% of the sipuleucel-T treated patients and in 1.4% of the placebo-treated patients, not correlating with survival (41). Research is currently ongoing to define additional biomarkers that could be related to increased overall survival.

To conclude, sipuleucel-T is the first autologous cellular immunotherapy for the treatment of PCa. Three phase III trials demonstrated crucial clinical evidence for the worthiness of sipuleucel-T. However, although an increase in overall survival of 4 months is beneficial for the patients, it is not the breakthrough for immunotherapy many researchers were hoping for. Cellular immunotherapy might not be a monotherapeutic alternative for PCa. Instead, combination with standard or novel treatment modalities might be decisive. Currently ongoing trials are focusing on combination therapies with androgen deprivation therapy, chemotherapy, and immune checkpoint inhibitor antibodies (**Table 2**).

#### **PROSTATE-SPECIFIC MEMBRANE ANTIGEN**

Prostate-specific membrane antigen, also known as glutamate carboxypeptidase II, is a zinc metalloenzyme with folate hydrolase activity that is expressed in membranes of prostate epithelial cells (87, 88). Its function in the prostate is still unknown. Low expression of PSMA is also found in the kidneys, salivary glands, duodenum, and the central and peripheral nervous system.

# **PSMA as tumor antigen**

Prostate-specific membrane antigen is highly overexpressed in PCa and increased expression correlates with advanced disease and metastasis (89–91). It has also been shown that PSMA is involved in tumor angiogenesis of many solid tumors, and it is expressed in the endothelial lumen in tumors. Normal vascular endothelium in non-cancerous tissue is PSMA negative (92, 93). PSMA displays several features that qualify it as a suitable target for immunotherapy. In addition to its specific expression in the prostate, it is also a membrane-bound antigen that is presented on the cell surface, but not released into the circulation (94). PSMA has been exploited as a possible target for PCa treatment in different pre-clinical settings and in early-stage clinical trials (42, 43, 88, 95). Wolf et al. showed that the recombinant anti-PSMA-specific single-chain immunotoxin D7-PE40 was both specific and highly toxic for PSMA-expressing PCa cells *in vitro* and *in vivo* in prostate tumor-bearing mice (88). Usage of the 177lutetium radiolabeled anti-PSMA monoclonal antibody J591 induced a 50% PSA reduction in 4 of the 35 patients with mCRPC (95). A similar PSA decrease was seen in an early clinical trial with PSMA peptide-pulsed DCs, where 9 of 33 patients displayed a partial clinical response (43). However, not all studies targeting PSMA have showed encouraging results. The PMSA-derived HLA-A2-restricted peptide (LLHETDSAV) appeared to be poorly immunogenic compared with other HLA-A2-restricted peptides, both *in vitro* as well as in patients with PCa (42). This underscores the importance of pre-clinical studies before clinical testing.

In summary, based on the highly specific expression pattern of PSMA in patients with PCa, PSMA poses as a suitable target for immunotherapy. However, early clinical trials have shown varying results. Further research concerning PSMA-based immunotherapy is warranted. **Table 2** shows several ongoing clinical studies targeting PSMA as a tumor antigen.

# **PROSTATE STEM-CELL ANTIGEN**

Prostate stem-cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the cell surface of both basal and luminal cells in the normal prostate, but overexpressed by PCa cells (44, 96). It is shown that PSCA, like other GPI-anchored proteins,is involved in the survival of stem cells,in T-cell activation and proliferation, and in cytokine and growth factor responses (97, 98). Furthermore, several studies have connected the Ly-6 family of PSCA-like GPI-anchored proteins to tumor growth and metastazation (99–102).

#### **PSCA as tumor antigen**

Its distinct expression pattern and possible function in tumorprogression makes PSCA an interesting target for immunotherapy. It has already been exploited in several studies, with promising results (45, 103–105). Anti-PSCA monoclonal antibodies have been reported to inhibit tumor growth and prolong the survival of mice bearing human PCa xenografts (46, 106). Additionally, a chaperone complex vaccine made of PSCA and the heat-shock protein GRP170 was shown to enhance T-cell-mediated immune responses, inhibit tumor growth, and prolong the life span of PCa tumor-bearing mice (107). Two DC vaccination studies have been performed in humans (45, 105). In the study by Thomas-Kaskel

et al., patients with mCRPC were treated with DCs loaded with PSCA and PSA peptides. Endpoints were safety and induction of antigen-specific immunity. The vaccine was well tolerated in all patients, and 6 of 12 patients showed stable disease after four vaccinations. One patient had a complete response. Interestingly, this patient displayed an increase in serum PSA levels. Positive delayedtype hypersensitivity skin reactions were seen in four patients after four vaccinations. A positive delayed-type hypersensitivity test was associated with increased overall survival. HLA tetramer analysis detected high frequencies of peptide-specific T cells in one patient,who had an overall survival of 27 months (105). In another study, vaccinations were performed in three patients with mCRPC using multi-epitope (PSCA, PSMA, PAP, and PSA) pulsed DCs. The treatment was well tolerated, and significant CTLs responses against all PSAs were observed. In addition, DC vaccination was associated with an increase in PSA doubling time (45).

To conclude, PSCA has been used as a target for antigen-based immunotherapy in several clinical studies due to its role in tumor growth and metastases. Unfortunately, the study results were less impressive than expected. This might be the reason that to date there is no ongoing clinical trial with PSCA registered.

#### **MUCIN-1**

The mucin family members include proteins that enclose tandem repeat structures with a high proportion of prolines, threonines, and serines. The family consists of secreted and transmembrane forms, designated Mucin-1 (MUC-1) to MUC-21 (108). MUC-1 is a large cell surface glycoprotein found on the apical surface of most glandular and ductal epithelial cells, such as the lungs, intestines, and the prostate (109). In chronic inflammation,MUC-1 expression is induced by inflammatory cytokines like TNF-α, IFNγ, and IL-6. Overexpression contributes to oncogenesis by activation of growth and survival pathways (Wnt-β-catenin and nuclear factor-κB pathways), promoting receptor tyrosine kinase signaling and downregulation of stress-induced death pathways (108). MUC-1 overexpression is associated with colon, breast, lung, prostate, and pancreatic cancer. Moreover, it is associated with tumor-progression and correlated with advanced disease (110–112). MUC-1 has also been shown to have immunosuppressive effects in mice, and secreted MUC-1 has been shown to block T-cell activation (113, 114). Moreover, human monocyte-derived DCs cultured *in vitro* with MUC-1 peptide displayed a decreased expression of both co-stimulatory molecules and antigen presenting molecules upon activation (115). Similarly, depletion of soluble MUC-1 in tumor cell line supernatants abolished the antiproliferative effect of these supernatants on T cells, andMUC-1 has therefore been identified as a target in PCa (116). The inhibitory effect of MUC-1 has also been demonstrated *in vivo*, when synthetic MUC-1 decreased the immune response in patients vaccinated with an MUC-1 containing polyvalent peptide vaccine (117). In a recent phase I/II trial, an autologous DC vaccine loaded with an MUC-1 glycoproteine and KLH in patients with CRPC was studied. Patients received three injections biweekly followed by booster vaccinations at 6 and 12 months. The rate of PSA rise decreased in six of seven patients. The PSA doubling time increased from a median of 2.9 months prior to vaccination to 7.5 months during vaccination (118). Richman et al. also showed clinical

Taken together,MUC-1 is important in tumor-progression and therefore a very interesting tumor-associated antigen. Several trials focusing on MUC-1 as a target for cancer immunotherapy in PCa are ongoing (**Table 2**).

#### **CANCER/TESTIS ANTIGENS**

Cancer/testis antigens are normally only expressed in gametogenic tissue. However, this group of proteins is aberrantly expressed in several types of cancers, including PCa (48). CTAs have been shown to contribute to tumor formation and progression (119, 120). The CTAs NY-ESO-1, the MAGE family, and A-kinase Anchor Proteins (AKAP)-4 will be discussed here.

NY-ESO-1 is found to be expressed in a variety of malignancies. It is not expressed in normal adult tissue, with the exception of the testis. The expression of NY-ESO-1 is associated with level of disease, and higher NY-ESO mRNA and protein expression are observed in metastatic and advanced PCa, as compared to localized tumors (120–124). The function of NY-ESO-1 is unknown, but it is speculated to play a role in meiosis or in the assembly of the organelles that develops over the anterior half of the head in the spermatozoa (125, 126). The NY-ESO-1 is a promising candidate because of its tumor-restricted expression and the identification as one of the most immunogenic CTAs, eliciting spontaneous cytotoxic and antibody-mediated immune responses in patients with NY-ESO-1 + tumors (127–129). Humoral responses against NY-ESO-1 have been evoked by non-specific immune activation in patients with mCRPC treated with a combination of checkpoint inhibitor ipilimumab and GM-CSF, underscoring its immunogenicity (130). NY-ESO-1 has been used as target antigen in several clinical studies. Both MHC class I and II restricted T-cell epitopes specific for NY-ESO-1 are identified (131). MHC class I and/or II restricted NY-ESO-1 peptides were compared in a peptide-based vaccine trial in patients with mCRPC. The vaccine increased the PSA doubling time and yielded antigen-specific T-cell responses in all patients treated. The strongest results were seen in chemo-naive patients, most likely due to a lower tumor burden, thus less tumorinduced immune suppression (132). The immunogenic features of NY-ESO-1 are further supported by a study using a proteinbased vaccine with CpG as an adjuvant. This vaccine was able to prime antigen-specific B-cell responses and induced NY-ESO-1 specific, tumor-reactive CTLs in patients with metastatic PCa, independently of autologous NY-ESO-1 expression (49). Vaccination against a tumor-specific protein without it being present, repositions this clinical vaccination protocol toward a preventive setting.

Second, the MAGE CTA subfamily is also expressed in PCa. Upregulation of these CTAs is found in CRPC and is associated with resistance to chemotherapeutic agents (50). MAGE-A2 downregulates p53 transactivation function through histone deacetylase recruitment, a possible explanation how MAGE-A2 expression leads to resistance to chemotherapy (50). Indeed, silencing of MAGE-A2 increased sensitivity to docetaxel chemotherapy in PCa tumor cells (120). Expression of MAGE-C2/CT10, another member of the MAGE-A subfamily, is correlated with the degree of PCa malignancy. It is an indication of higher risk for biochemical recurrence after radical prostatectomy and represents a potential target for immunotherapy (133). Members of the MAGE-A subfamily and NY-ESO-1 are often co-expressed in prostate malignancies.

Third, the CTA AKAP are a family of scaffolding proteins capable of controlling intracellular signals. AKAP is involved in cytoskeletal regulation and organization by affecting cyclic AMPdependent protein kinase-A (134). In the prostate epithelium, the anchor proteins synthesize and secrete calcitonin. It has been shown that the calcitonin secretion from malignant prostates is several-fold higher than from benign prostates (135). The calcitonin receptor is expressed in malignant PCa, and its activation stimulates growth of PCa cells via activation of cyclic AMP as well as protein kinase C (136, 137). These mechanisms suggest a marked increase in the invasiveness of PCa cells (138). Modulation of protein kinase-A activation possibly interferes with the growth, tumor genicity, and metastatic potential of advanced tumors. First, AKAP-4 has been showed to be an immunogenic CTA in patients with multiple myeloma (139). Later, Chiriva-Internati et al. showed cytoplasmic and surface expression of AKAP-4 in the LnCAP PCa cell line. AKAP-4 expression in the prostate epithelial cells was shown in 13 of 15 patients with PCa, but not in healthy subjects. Cytotoxicity assays showed that AKAP-4-loaded DC-stimulated T cells were capable of killing autologous PCa cells *in vitro*. Neither killing of AKAP-4 negative PCa cells nor normal prostate epithelial cells was observed. This underscores the antigen specificity of the response and prevention of autoimmune reactions (140). This makes AKAP-4 a very interesting target for PCa anti-tumor vaccination.

To conclude, several CTAs, especially NY-ESO-1, the MAGE-A subfamily, and AKAP-4, could serve as therapeutic targets in the fight against PCa (120, 122, 140). Especially NY-ESO-1 is of major relevance in PCa and a target in different ongoing trials (see **Table 2**). Due to the tumor-restricted expression of CTAs, these antigens can also be used in an adjuvant or a preventive setting hindering the recurrence of CTA-positive tumors (49).

#### **MIXTURE OF TUMOR-ASSOCIATED ANTIGENS**

To date, many investigators underscore the importance of a personalized approach by selecting patient-specific mutations as target antigens for immunotherapy. The group of Noguchi took a first step in a personalized direction. They performed two phase II studies with a personalized peptide vaccine (PPV). The vaccine consisted of four peptides based on each patient's immunoreactivity profile. Peptides of a variety of tumor-associated antigens were tested, including PSA, PAP, PSMA, multidrug resistance protein, and a choice of different epithelial tumor antigens. The peptides included in the vaccine were selected on their capacity to induce CTL responses. In the first phase II trial, patients with CRPC were randomized to PPV combined with chemotherapy or chemotherapy only (141). Antibody responses were seen in 64% of the patients and cytotoxic T-cells responses in 72% of the patients. An increase in progression-free survival was observed in the PPV/chemotherapy group as compared with the patients who only received chemotherapy. However, immune responses did not correlate with clinical outcome in patients treated with PPV and

chemotherapy. Interestingly, the authors found that lower levels of IL-6 before PPV vaccination were favorable for overall survival. IL-6 have been associated with more aggressive cancer progression and decreased survival in PCa (142). In this perspective, IL-6 may be seen as an indicator of prognosis and a predictor of therapy effectiveness. It is also hypothesized that inhibiting IL-6 signaling may be beneficial in patients enduring other immunotherapeutic treatment.

The results of the PPV vaccinations are promising. A randomized trial with an appropriate control group before and after chemotherapeutic treatment is needed to fully identify a clinical benefit of PPV treatment. Currently, a vaccine consisting of 20 peptides is applied to patients with CRPC in an exploratory, randomized, open-label study (UMIN000008209, **Table 2**).

#### **PROSTATE CANCER CELL LINES GVAX**

GVAX is an allogeneic, cell-based immunotherapy consisting of the PCa cell lines LNCaP and PC-3. These cell lines are genetically modified with a recombinant GM-CSF adeno-based viral vector and irradiated before administration. Clinical results in patients with PCa are indicative for a favorable clinical outcome with no toxicities (143, 144). These results have led to phase III trials, using the most promising high-dose GVAX protocol. Unfortunately, due to an even increased mortality in the GVAX-treated group, and disappointing interim results the trials were abrogated (145, 146).

van den Eertwegh et al. combined the immune checkpoint inhibitor ipilimumab (anti-CTLA-4) with GVAX (147). More than 50% decline in PSA level was seen in 25% of the patients. All the responding patients got 3.0 or 5.0 mg/kg ipilimumab. There was dose-limiting toxicity in the 5.0 mg/kg group of patients, while the lower ipilimumab regimens were well tolerated. Markedly, all patients with immune-related adverse events showed a decrease in PSA levels. A small number of patients additionally displayed an anti-PSMA antibody response. These patients had a significant increase in median overall survival (46.5 months compared to 20.6 months for patients without this humoral response). T-cell monitoring studies were performed in 28 patients receiving the combination therapy of GVAX and ipilimumab. Compared with the control group, an increase in absolute lymphocyte counts and enhanced CD4+ and CD8+ T-cell differentiation was observed. These immune responses were associated with a significantly prolonged overall survival. In addition, an OS benefit was also seen in case of high pre-treatment levels of CD4+, CTLA-4+, CD4+/PD-1+, or non-naïve CD8+ T cells. Low pre-treatment frequencies of differentiated CD4+ or regulatory T cells resulted in a prolonged OS (148). This reveals perspectives for future biomarker research.

#### **mRNA-TRANSFECTED DCs**

An alternative approach for PCa cell lines is the use of PCa cell line-derived RNA or tumor antigen encoding mRNA. Kyte et al. transfected monocyte-derived DCs with mRNA derived from the PCa cell lines LnCAP, DU-145, and PC-3. Although the generation of mRNA-transfected DC is challenging, DC vaccination appeared feasible and safe (149, 150). Furthermore, PSA-specific T-cell responses were detected in 12 of 19 patients with PCa who underwent mRNA-DC vaccination (149). To date, patients are recruited in a phase I/II trial (NCT01197625, **Table 2**), studying the mRNA-transfected DCs in curative resected patients with PCa. More studies are needed to properly determine the strength of mRNA-transfected DCs. The usage of this immunotherapeutic modality within combination therapies might be of greater significance.

# **DISCUSSION**

In this review, we provided overview of PCa tumor-associated antigens and how they are used to target PCa via immunotherapy (**Table 1**). PSMA and PSCA are normally expressed in the prostate gland but upregulated during cancer development and they may play a role in tumor progression (44, 89, 96, 151). Increased serum levels of secreted tumor antigens, such as PSA and PAP, can be used as biomarkers for disease and disease progression (51, 73, 74). More general tumor antigens, like MUC-1, AKAP-4, and NY-ESO-1, can also be found in PCa and might be candidates for immunotherapeutic interventions (111, 123, 140). MUC-1 is expressed in normal tissue and upregulated on several tumors, where it can exert immunosuppressive effects and attain tumor growth (110). Hence, targeting MUC-1 could have a dual role – directing the immune response toward the tumor and reducing immune suppression. This might also be valid for other immunosuppressive antigens, such as the MAGE-A subfamily or PSA (54, 120). On the other hand, the NY-ESO-1 antigen is often immunogenic *per se*, and pre-existing immune responses directed against this antigen are common in treatment-naive patients (128). Pre-existing CTL responses against PSA and PAP in healthy individuals and patients with chronic prostatitis also support the definition of PCa as an immunogenic tumor (26, 27), where tolerance against self-antigens can be broken and the immune system can be harnessed against the tumors.

Today, the only registered product for antigen-targeted immunotherapy in PCa is sipuleucel-T (38, 39, 41). Although the significance of this intervention received criticism, sipuleucel-T proves an important point: autologous cellular immunotherapy is feasible and can indeed be developed as an approved treatment modality. To date, no convincing mechanism of action has been elucidated for sipuleucel-T. Increased immune responses were observed but no correlation with clinical outcome could be established. Clinical studies aiming at identifying immunological responses and thereby hopefully providing an in-depth understanding of the mode of action of sipulecuel-T are ongoing. Unraveling the mechanism might be beneficial for further development of sipuleucel-T and other immunotherapeutic approaches.

Effective immune responses induced by immunotherapeutic treatments are still not common, and probably vary depending on tumor type, somatic differences between tumor cells, and the tumor microenvironment (66). Several recent trials have shown promising results in both clinical and immunological responses. Constructs targeting the NY-ESO-1 antigen has led to significant immunological responses, which makes NY-ESO-1 an interesting antigen to target immunotherapeutic strategies in future (49, 132). Immunological responses are also induced by several PSA-targeting vaccines, supporting the usage of PSA as an immunogenic tumor antigen (34–36).

Insight in the localization of the tumor antigen (on/in cells, normal cells vs. tumor cells, in organs) and the specificity of the antigen facilitates a precise selection of target antigens with the intention of optimizing the translation of immunotherapeutic treatments to the clinic. However, despite significant T-cell responses, tumor progression is seen most frequently in patients treated with cancer immunotherapy. This is due to the complexity of human beings and the complexity of tumors and metastases (152). The complexity of cancer is also described by Fox et al. (153). This report of the collaborating immunotherapy organizations, known as the Society for Immunotherapy of Cancer (SITC), contains the identification of nine hurdles in cancer immunotherapy that significantly delays clinical translation of promising cancer immunotherapeutics. We here discuss the hurdles relevant for this review, for a complete overview, we refer to the original article (153). The first hurdle to overcome is the complexity of cancer, tumor heterogeneity, and immune escape. The immune signature of the tumor, distinguished by genetic or histological evaluation, can predict responders to cancer immunotherapy (154, 155). The second relevant SITC hurdle for this review is the lack of definitive biomarkers for the assessment of clinical efficacy of cancer immunotherapies. Biomarkers to distinguish between patients responsive to initial treatment, patients displaying immune inhibitory features, and patients with non-immunogenic tumors, are needed. Pre-existing anti-tumor responses or the expression of inhibitory markers are examples of suggested biomarkers that could be used to predict treatment outcome and individualize the treatment regime.

A correlation of immune parameters with clinical outcome after immunotherapy is not established in patients with PCa. This can be attributed to (i) a limited number of patients per immunotherapeutic approach; (ii) a variation in clinical features of patients with PCa before treatment; and (iii) the difference in clinical signs of tumor control between conventional toxic treatments and immunotherapeutic treatments. This last argument is also one of the hurdles identified by the SITC. Effective immunotherapy does not always display initial shrinkage of the tumor, but rather a pattern of tumor growth and progression followed by shrinkage when the tumor is recognized and destroyed by the immune system (156–158). This paradox has been illustrated by the negative outcomes on progression-free survival or PSA responses in the sipuleucel-T trials and PSA-TRICOM trial. Tumor swelling, increased release of PSA due to elevated tumor cell death, and initial detrimental symptoms might be associated with a favorable clinical outcome rather than with progressive disease, as stated in the WHO and Response Evaluation Criteria In Solid Tumors (RECIST) criteria (156, 157, 159). Although clinically responding patients might have been missed, some patients do not respond, neither clinically nor immunologically. Lack of immunogenicity of the antigens used might be an explanation, but a major factor is the immunosuppressive networks within cancer patients. Infiltrating lymphocytes can be regulated by a number of inhibitory pathways within the tumor and thereby shift the direction of the ongoing immune response toward a more tolerogenic one. Other patients might have "silent" tumors that do not display an inflammatory phenotype and hence do not attract lymphocyte infiltration (66).

Novel monoclonal antibodies targeted against inhibitory receptors on T cells (anti-CTLA-4 and anti-PD-1) are able to prolong their effector functions and prevent immune inhibition. These treatment strategies are tested in combination with other immunotherapeutic approaches and showed promising results in a subset of patients (61, 147, 160). There are still ongoing combination therapy studies with ipilimumab and sipuleucel-T which will hopefully overcome the immunosuppressive signals provided by immune evading tumors (NCT01804465 and NCT 01832870). Although antigen-based immunotherapy itself seldom gives rise to severe autoimmune reactions, the combination with an immune checkpoint inhibitor likely will enhance the risk of immunerelated adverse events, as recently shown in melanoma patients treated with ipilimumab (161–163).

#### **FUTURE CHALLENGES AND OPPORTUNITIES**

Until recently, patients with mCRPC had limited treatment options and a poor prognosis. With new sequential hormonal therapies, second-line chemotherapy, and new immunotherapeutic strategies, a new era has started. To date, PCa is one of the few tumor types in which immunotherapy is part of the current standard of care. Augmenting immune responses to PCa antigens is a valid therapeutic approach, and clinical responses with minimal toxic effects are observed.

In this review, we focused on commonly expressed tumorassociated antigens. Recently, patient-specific epitopes are identified as highly important to improve T-cell reactivity. Targeting these patient- and cancer-specific mutated epitopes holds promise for even better results and possibly cure of patients. By complete genome and transcriptome, sequencing and mass spectrometrymutated HLA-binding peptides, so-called neoantigens, might be identified (164). Vaccination with individually overexpressed tumor-specific peptides could result in a unique, personalized anticancer vaccine (165–167). Recently, the first two demonstrations of autologous cancer exome-based T-cell responses against patientspecific neoantigens in humans were published (167, 168). This knowledge is a major step forward for both the identification of new diagnostic strategies by tumor exome analysis, as well asfor the development of individualized immunotherapeutic approaches. Combination therapies harboring these patient-specific peptide vaccinations together with immune checkpoint inhibitors are likely to generate an even better immune control. There is no doubt that these are very exciting times for cancer immunotherapy.

#### **ACKNOWLEDGMENTS**

This work is supported by the Swedish Research Council and the "Koningin Wilhelmina Onderzoeksprogramma" (Queen Wilhelmina Research Program): KWF 2009-4402.

#### **REFERENCES**

**44**(1):49–54. doi:10.1002/1097-0045(20000615)44:1<49::AID-PROS7>3.3. CO;2-Z

hormone-refractory prostate carcinoma. *Cancer Immunol Immunother* (2006) **55**(12):1524–33. doi:10.1007/s00262-006-0157-3

epithelial cells and its activation by prostate-specific antigen. *J Cell Physiol* (2005) **202**(2):361–70. doi:10.1002/jcp.20147

**Conflict of Interest Statement:** Harm Westdorp, Annette E. Sköld, Berit A. Snijer, Sebastian Franik, Sasja F. Mulder, Pierre P. Major, Ronan Foley, and I. Jolanda M. de Vries declare no disclosures of potential conflicts of interest. Winald R. Gerritsen is a consult for Aglaia Biomedical Ventures, is on the supervisory board for PsytoBe, has received speakers fee from J&J and BMS, and is on the advisory boards for Amgen, BMS, Janssen-Cilag, Sanofi, Astellas, Bayer, and Merck.

*Received: 28 February 2014; accepted: 17 April 2014; published online: 06 May 2014. Citation: Westdorp H, Sköld AE, Snijer BA, Franik S, Mulder SF, Major PP, Foley R, Gerritsen WR and de Vries IJM (2014) Immunotherapy for prostate cancer: lessons from responses to tumor-associated antigens. Front. Immunol. 5:191. doi: 10.3389/fimmu.2014.00191*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Westdorp, Sköld, Snijer, Franik, Mulder, Major, Foley, Gerritsen and de Vries. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **Theofilos Poutahidis 1,2, Markus Kleinewietfeld3,4,5 and Susan E. Erdman1\***

#### **Edited by:**

Fang-Ping Huang, Imperial College London, UK

#### **Reviewed by:**

Graham Robert Leggatt, University of Queensland, Australia Yong Lu, Cleveland Clinic Foundation, USA

#### **\*Correspondence:**

Susan E. Erdman, Massachusetts Institute of Technology, 77 Mass Avenue, Cambridge, MA 02139, USA e-mail<EMAIL_ADDRESS>
It is recently shown that beneficial environmental microbes stimulate integrated immune and neuroendocrine factors throughout the body, consequently modulating regulatory T-lymphocyte phenotypes, maintaining systemic immune balance, and determining the fate of preneoplastic lesions toward regression while sustaining whole body good health. Stimulated by a gut microbiota-centric systemic homeostasis hypothesis, we set out to explore the influence of the gut microbiome to explain the paradoxical roles of regulatory T-lymphocytes in cancer development and growth. This paradigm shift places cancer prevention and treatment into a new broader context of holobiont engineering to cultivate a tumor-suppressive macroenvironment.

**Keywords: tumor macroenvironment, regulatoryT-cells, cancer immunotherapy, inflammation and cancer, probiotic bacteria**

# **INTRODUCTION**

The neoplastic process is characterized by overwhelming complexity. Cancer is comprised of a genetically unstable population of cells that proliferate at an extraordinarily high rate. Millions of cancer deaths each year make it obvious that the battle against cancer is asymmetric, with humankind often being the weaker element (1). To date, cancer research efforts directly confront malignancy by targeting properties of individual cancer cells. In 2000, Hanahan and Weinberg described that most of the research on origins and treatment of cancer had just contributed toward "adding further layers of complexity to a scientific literature that is already complex almost beyond measure" (2).

In the same landmark paper, however, the authors were optimistic enough to predict groundbreaking upcoming advances in the conceptual rather than the technical level (2). They were proven right. One such advancement was the increased awareness for the importance of the tumor microenvironment in the etiopathogenesis of neoplasia (3, 4). We now know that initially transformed cells are much less autonomous in their growth than previously thought (5, 6). Among the microenvironment elements, immune cells and factors have emerged as fundamental players (4–6). Accumulating evidence suggests that tumor-associated inflammatory cell accumulation, whether overt or smoldering, could be viewed as a tumor-promoting event (7–9). These inflammatory responses enhance mutagenesis by oxidative DNA damage and shape the tumor stroma in favor of cancer cell survival and expansion (6, 10, 11).

Will this knowledge base in the field of inflammation, immunity, and cancer lead to new, highly effective, and biologically safe cancer immunotherapy modalities? We assert that the outcome will depend upon the philosophy and the strategic goals that will dominate the bench-to-bedside research. We propose that research in this field should focus upon stimulating systemic innate immune balance and adaptive immune resiliency, making the mammalian host more powerful to resist its cancer challenger. One possible approach utilizes gut microbiota or microbial antigens to stimulate beneficial immune cells. On the other hand, existing immunotherapy aims to selectively interrupt immune factors to better recognize and exterminate cancer cells (12–17), an approach that may ultimately lead to host instability. To further explain this point of view, we will refer to the recently discovered paradoxical roles of regulatory T-cells (TREG) in cancer (10, 14).

# **TREG ARE CENTRAL IN PRESERVING SYSTEMIC IMMUNE HOMEOSTASIS AND GOOD HEALTH**

FOXP3+ CD4+ CD25+/high TREG are dominant cellular elements of the professional suppressor arm of the immune system and are important for orchestrating the control of peripheral immunological tolerance (18). The transcription factor FOXP3 is a fundamental regulator of TREG function in rodents and humans, and so far the most reliable phenotypic indicator of their identity. Recent studies on human TREG subpopulations, however, revealed that low but discernible levels of FOXP3 expression could be detected in non-suppressive TREG or even in activated effector T-cells. It is probable that this finding reflects the inherent plasticity of TREG; FOXP3+ cells co-expressing effector T-cell phenotypic markers or cytokines may be in stages of a progressive, epigenetically regulated, phenotypical, and functional shift process (14, 16, 19–22), ultimately favorable for healthful recovery of the host after environmental challenges. The role of TREG is central in preserving immune system homeostasis for health and the balance of beneficial inflammatory responses during infections while minimizing collateral tissue damage. In cancer, however, roles of TREG are traditionally considered to be negative (14–16, 23).

# **TREG GATHER NEAR TUMORS AND FAVOR CANCER SURVIVAL**

A large body of data suggest that TREG gather near tumors and suppress the anti-tumor inflammatory response, thus favoring cancer cell survival. To this end, tumor-associated TREG are thought as a major impediment of anti-tumor vaccines (13–16, 23). Clinical and experimental data suggest that tumor-associated TREG recognize both self and neoantigens expressed by tumor cells, counteracting antigen-specific effector T-cell responses. Consequently, immunotherapy strategies based on the vaccination with tumor-associated antigens fail to evoke an effective response against cancer cells due to the activation and expansion of tumor antigen-specific TREG (14–16). This potential interplay of TREG within tumors has been reviewed in detail elsewhere (12–16), and has led to the proposal of several anti-TREG regimens for cancer immunotherapy. These regimens aim to deplete TREG, inhibit their suppressive function, prevent their homing into tumor sites, or block their differentiation/proliferation (12–16).

Several of these TREG-targeting modalities have already been tested in the clinic, with mixed results (13, 16). Blocking TREG function by depleting the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) appears promising (24), due to the depletion of TREG from tumor tissues (25, 26). However, a similar regimen could lead to an opposite effect with the accumulation of TREG and CD8+ T-cells in tumors (27, 28). A phase III study of melanoma patients using a gp100 peptide vaccine with interleukin (IL)-2 administration led to equally promising results with discovery of TREG expansion in responding patients (29).

# **GUT MICROBIOTA INDUCE POTENT TREG WITH SYSTEMIC ANTI-NEOPLASTIC PROPERTIES**

As the results of these trials are anticipated, the literature reveals contradictory evidence. Indeed, the studies associating high densities of tumor-associated cells expressing TREG markers including FOXP3 with a poor prognosis in several types of human cancers are now challenged by similar studies on the very same types of cancer showing the opposite outcome (30–34). The different CD8+:TREG ratios and the presence of FOXP3+ cell subsets of undetermined identity in the tumor microenvironment have been proposed as probable explanations (16). Indeed, data from animal models show under certain conditions of microbial priming that TREG not only protect but also alter the tumor microenvironment to induce remission of already established intestinal, mammary, and prostate cancers (35–41). The hypothesis that the composition of the different subsets of FOXP3+, which may include effector Foxp3+ cells, is intriguing (16). Indeed, it was previously shown that IFN-γ levels were increased during TREG-mediated tumor regression in mice (37). Further, feeding of probiotic microbes to mice induces systemic oxytocin secretion that shifts immunity toward IFN-γ and CD25 for improved wound healing capacity and systemic good health (42). A question subsequently arising is whether gut microbiota may be engineered to harness an anti-neoplastic FOXP3+ cell milieu (5, 10, 41).

# **GUT-CENTRIC HYPOTHESIS: PRIOR EXPOSURES TO MICROBES EXPLAIN BENEFICIAL ROLES OF TREG**

Stimulated by a gut-centric systemic homeostasis hypothesis, we set out to explore and explain the paradoxical roles of TREG in cancer using several different mouse models of cancer and adoptive cell transfer methodologies (10). We found that TREG may suppress, promote, or have no effect in carcinogenesis depending upon their timing and prior exposure to gut bacterial antigens and presence of IL-10 (35–39, 41, 43, 44). Under some conditions, adoptive transfer of TREG rapidly led to apoptosis of emerging tumor cells (37, 45). Using as a model organism an opportunistic pathogen, *Helicobacter hepaticus*, commonly residing in the lower bowel of mice, we have shown in Rag2-deficient mice (otherwise lacking lymphocytes) that gut microbiota modulate inflammatory bowel disease and inflammation-associated colon cancer, a cancer process inhibited by properly functioning IL-10-dependent TREG (35, 36). Subsequently, by introducing *H. hepaticus* into the large bowel flora of mice lacking the APC tumor suppressor gene (*ApcMin/*+), we found that intestinal polypogenesis was greatly enhanced by bacteria and subsequently suppressed by immunecompetent TREG. Furthermore, adenomas of infected *ApcMin/*+ mice progressed into adenocarcinoma, a transition atypical of polyps of aged-matched uninfected controls (38, 41). Interestingly, *ApcMin/*+ mice having *H. hepaticus* in their gut flora were prone to develop cancer in tissues distant from intestine, such as prostate and the mammary glands (40, 41, 43, 46, 47). *H. hepaticus*-induced tumorigenic events were inhibited by supplementation with TREG from immune-competent wild type donor mice.

A potent treatment to counteract these local and systemic *H. hepaticus*-induced tumorigenic events was supplementation with TREG in an IL-10-dependent manner (10, 36, 38–40, 44, 46, 48). Purified TREG exhibited greatest anti-cancer potency when taken from donor mice previously colonized with *H. hepaticus*. By contrast, TREG taken from donor mice without prior *H. hepaticus* exposure were ineffective, and in some cases actually enhanced tumorigenesis (10). Based on these results, we theorize that the tumor microenvironment is subject to systemic inflammatory events arising from environmental exposures in the gastrointestinal tract (**Figure 1**). This microbe-inducible pro-inflammatory condition contributes to tumor trophic signaling. Interestingly, bacterial antigen triggered IL-10-dependent activities in the GItract impart sustained protection from the aforementioned events, resulting in immune cell recruitment, including TREG, which, by being more potent in their anti-inflammatory roles, work locally and systemically to suppress sepsis, myeloid precursor mobilization, and inflammatory signaling important in extra-intestinal cancer evolution (10, 43). These systemic events comprise the tumor macroenvironment.

The roles of intestinal microflora in promoting cancer development within the bowel have been well established (35, 49–52). Linking gut microbial flora and local and systemic effects that promote (38) or suppress (45) tumors throughout the body, expands this paradigm in a challenging manner. Recent findings show that gut flora imbalances considerably undermine the response to both immune (53, 54) and non-immune chemotherapeutic regimens, such as cisplatin and oxaliplatin (53).

# **A WEAKENED TREG FEEDBACK LOOP UNIFIES AUTOIMMUNE DISEASES AND CANCER**

These gut microbe-centric findings in mice are consistent with the "hygiene hypothesis," according to which insufficient microbial exposures earlier in life predispose to allergies, autoimmune disorders, and uncontrollable inflammation-associated pathologies later in life. We have shown that the basic principles of this

hypothesis may apply not only to auto-immunity, but also to neoplastic disease as well, and that TREG play a central role in this phenomenon (10, 41, 55). The ability of TREG to decrease risk for cancer and counteract established tumors depends upon microbe-triggered IL-10, which works to maintain immune system homeostasis and reinforce a protective anti-inflammatory, anti-neoplastic TREG phenotype (41). TREG display inherent phenotypic plasticity (10). Hygienic individuals with a weakened IL-10 and TREG feedback loop are prone to a re-direction of unstable resting peripheral TREG toward a T helper (Th)-17 proinflammatory process. As a result "hygienic" subjects are at higher risk to develop auto-immune diseases and cancer (10). It is tempting to postulate that this may explain why only a few people go on to develop cancer, while nearly everyone bears dysplastic and early neoplastic lesions throughout their body (56).

microbes that may be engineered for sustained good health.

Depending on composition of gut microbiota, the immune system of mice may acquire different subclinical characteristics, even in the absence of overt inflammatory processes. The clinically silent immune system status may determine the risk of developing sporadic cancer in epithelia throughout the body. Further, we found that consuming beneficial probiotic bacteria led to the

expansion of a Foxp3+ cell population in the periphery (42, 45, 57) conferring protection to diet-related and genetic predisposition to mammary cancer (45). Targeted oral challenge with such probiotic bacteria resulted in the activation of interrelated systemic inflammatory and metabolic pathways, either through blood circulation or via the vagus nerve (**Figure 1**). Consequently, there was an upregulation of systemic hormone levels, such as oxytocin, testosterone, and thyroxin. Oxytocin serves to sustain immune and integumentary homeostasis, biasing the immune system toward IL-10 and IFN-γ, without anergy, subsequently minimizing the deleterious systemic effects of IL-17 (57). This altered immune system and metabolic profile of mice imparted healthful phenotypes including shiny fur and youthful hair follicle cycling, accelerated skin wound healing capacity, and resistance to diet-induced obesity and senility (42, 47, 57, 58). Through tightly regulated immune activities, competent TREG permit brief beneficial host inflammatory responses to eliminate invading pathogens, and later inhibit chronic deleterious inflammatory tissue damage (43). The results of our wound healing assays further suggest that the probiotic microbe-induced enhancement of the TREG-dominated arm of the immune system did not compromise the ability of mice to respond to invading pathogens (42).

# **BENEFICIAL SYSTEMIC EFFECTS OF GUT MICROBES ARE TRANSPLANTABLE VIA FOXP3**+ **TREG INTO NAÏVE HOSTS**

Adoptive cell transfer models offer mechanistic insight as these beneficial effects were isolated to bacteria-primed TREG (42, 47, 57–59). In fact, healthful phenotypes were entirely reproducible in naive recipient mice by the adoptive transfer of highly purified TREG derived from probiotic-fed cell donors (42, 57, 59). These results suggest gut microbe-induced crosstalk with the host in a continuous and reciprocal manner. The fate of preneoplastic and neoplastic lesions arising in epithelia throughout the body depends upon this macroenvironment at the whole organism level. Consequently, the tumor macroenvironment is defined as the "holobiont," i.e., the mammalian organism plus the microbial symbionts it bears. The TREG population is a central player of the tumor macroenvironment connecting gut bacteria with reproductive fitness, youthful phenotypes, and anti-neoplastic properties.

# **MICROBIAL ENGINEERING OFFERS NEW STRATEGIES FOR PUBLIC HEALTH**

Taken together, microbial engineering strategies using food-grade bacteria highlight alternative directions in cancer immunotherapy. Modulating beneficial TREG via diet is a biologically safe and efficient approach, originating from genetic programs that have been shaped during the millions of years of co-evolution of mammals with their gut bacteria symbionts. These attributes remain largely inactive in individuals with a modern lifestyle, Westernized dietary habits, and stringent hygiene practices. Awakening these latent TREG-mediated capabilities may provide an alternative avenue to reduce cancer risk at a population level for public health. The perspectives presented here should be considered as an alternative paradigm – not only for fighting cancer – but also for promoting overall good health and longevity.

# **AUTHOR CONTRIBUTIONS**

Theofilos Poutahidis, Markus Kleinewietfeld, and Susan E. Erdman wrote the paper.

# **ACKNOWLEDGMENTS**

This work was supported by National Institutes of Health grants P30-ES002109 (pilot project award to Susan E. Erdman), U01 CA164337 (to Susan E. Erdman), and RO1CA108854 (to Susan E. Erdman).

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 February 2014; accepted: 25 March 2014; published online: 07 April 2014. Citation: Poutahidis T, Kleinewietfeld M and Erdman SE (2014) Gut microbiota and the paradox of cancer immunotherapy. Front. Immunol. 5:157. doi: 10.3389/fimmu.2014.00157*

*This article was submitted to Tumor Immunity, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Poutahidis, Kleinewietfeld and Erdman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Induced pluripotent stem cells: challenges and opportunities for cancer immunotherapy

#### **Patty Sachamitr † , Simon Hackett † and Paul Jonathan Fairchild \***

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

#### **Edited by:**

Fang-Ping Huang, Imperial College London, UK

#### **Reviewed by:**

Angel Porgador, Ben-Gurion University of the Negev, Israel Kerry Campbell, Fox Chase Cancer Center, USA

#### **\*Correspondence:**

Paul Jonathan Fairchild, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

e-mail<EMAIL_ADDRESS>
†Patty Sachamitr and Simon Hackett have contributed equally to this work. Despite recent advances in cancer treatment over the past 30 years, therapeutic options remain limited and do not always offer a cure for malignancy. Given that tumorassociated antigens (TAA) are, by definition, self-proteins, the need to productively engage autoreactive T cells remains at the heart of strategies for cancer immunotherapy. These have traditionally focused on the administration of autologous monocyte-derived dendritic cells (moDC) pulsed with TAA, or the ex vivo expansion and adoptive transfer of tumorinfiltrating lymphocytes (TIL) as a source of TAA-specific cytotoxic T cells (CTL). Although such approaches have shown some efficacy, success has been limited by the poor capacity of moDC to cross present exogenous TAA to the CD8+ T-cell repertoire and the potential for exhaustion of CTL expanded ex vivo. Recent advances in induced pluripotency offer opportunities to generate patient-specific stem cell lines with the potential to differentiate in vitro into cell types whose properties may help address these issues. Here, we review recent success in the differentiation of NK cells from human induced pluripotent stem (iPS) cells as well as minor subsets of dendritic cells (DCs) with therapeutic potential, including CD141+XCR1+ DC, capable of cross presenting TAA to naïve CD8+ T cells. Furthermore, we review recent progress in the use of TIL as the starting material for the derivation of iPSC lines, thereby capturing their antigen specificity in a self-renewing stem cell line, from which potentially unlimited numbers of naïveTAA-specificT cells may be differentiated, free of the risks of exhaustion.

**Keywords: cancer, immunotherapy, dendritic cell, cytotoxic T cell, NK cell, pluripotency, iPS cell**

# **CANCER AND THE IMMUNE SYSTEM: A HISTORICAL PERSPECTIVE**

Although it was Paul Ehrlich who,in 1909, first introduced the concept of the immune system as a means of controlling the incidence of cancer, it was 50 years later, with development of the field of cellular immunology and discovery of the role of the immune system in allograft rejection (1), that this notion first gained traction. In 1970, Burnet and Thomas introduced the concept of immunological surveillance and postulated that the immune system had a mechanism for eliminating potentially dangerous mutated cells and speculated that lymphocytes were actively involved in the process by the recognition of neo-antigens, either unique to the tumor (tumor-specific antigens; TSAs) or shared by other somatic cells (tumor-associated antigens; TAAs) (2, 3). This theory was met with skepticism, due in part, to the observation that the incidence of tumors in immune compromised *nude* mice did not differ substantially from their wild type counterparts (4, 5). These observations were, however, counter-balanced by the discovery that tumors may lack immunogenicity, not due to the absence of TAAs *per se* but rather their inability to activate the immune system (6). This subsequently gave rise to the modified concept of cancer immunoediting (7), which postulates that a developing tumor is under a constant immunological selection pressure, leading either to its elimination, the establishment of a dynamic equilibrium between the tumor and the immune system, or its escape from immune surveillance, resulting in unopposed growth. It is now accepted that one of the hallmarks of cancer is the lack of immune regulation (8) and that certain cancers therefore have the propensity to induce a state of autoimmunity in some individuals. While the underlying mechanisms remain to be clarified, mutations in specific TAAs may increase their immunogenicity, eliciting T and B cell responses that readily cross-react with the wild type protein, expressed in other cell types or anatomical locations. Indeed, a recent study has shown that certain cancer patients develop the chronic autoimmune rheumatic disease, systemic sclerosis (9), illustrating the important concept that immune surveillance harnesses elements of the autoreactive T-cell repertoire to elicit anti-tumor responses, sometimes at the cost of collateral damage to self-tissues: it is the same autoreactive repertoire that cancer immunotherapy seeks to recruit.

#### **APPROACHES TO CANCER IMMUNOTHERAPY**

Radiation, chemotherapy, and surgery are the three traditional methods for controlling the spread of cancer, which, although effective, may fail to completely eliminate neoplastic cells or the cancer stem cells that sustain a developing tumor. Additionally, the lack of specificity of these approaches and the damage to otherwise healthy tissues may lead to severe morbidity and, in extreme cases, mortality. Given its inherent specificity, adaptability, and capacity to generate a memory response, cancer immunotherapy promises to be more effective and durable than classical treatment modalities (10).

Cytokines, such as interleukin-2 (IL-2), interferon-α (IFNα), and tumor necrosis factor-α (TNF-α) have been used nonspecifically to stimulate an anti-tumor response. These cytokines act either by directly inhibiting growth of the tumor cells or by promoting proliferation and sustained cytokine production by T cells and NK cells, thereby increasing their ability to target tumor cells. Some cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), act on antigen-presenting cells (APCs), inducing upregulation of MHC and co-stimulatory molecules, which promote their capacity to activate lymphocytes. A number of cytokines, used singly or in combination, have proven effective in increasing the anti-tumor immune response and have, in recent years, entered clinical trials for the treatment of advanced cancer (11). IL-2 has, for instance, been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic melanoma and renal-cell carcinoma (12). However, given the nonspecificity of the approach, low response rates, and toxic side effects, additional understanding of cytokine signaling pathways and their function *in vivo* are still required (11).

The identification of a number of well-defined TAAs and Tissue Specific Antigens (TSAs), along with the development of hybridoma technology (13), has facilitated the production of monoclonal antibodies (mAbs) that either directly target these antigens or block central pathways involved in tumor function. mAbs have, for instance, been used to inhibit molecules such as CTLA-4 and PD-1, since upregulation of their ligands by tumor cells may inhibit T-cell function, enhancing their ability to evade immune surveillance (14, 15). In recent clinical trials of the PD-1 specific mAb, BMS-936558, objective and durable responses were observed in approximately one in four to one in five patients with non-small cell lung cancer, melanoma, or renal-cell cancer. Immunohistochemical staining of tumor specimens prior to the onset of treatment, revealed that intra-tumoral expression of PD-1 ligand (PD-L1) correlated with the induction of anti-tumor responses, providing a means of stratifying patients in order to identify those most likely to respond to treatment and greatly increasing the likely future clinical impact of mAb therapy (16). Although it was the exquisite specificity of mAbs that first earned them the name "magic bullets," response rates remain disappointingly low when used as a single therapy (17–19). One reason for this poor performance may lie in the fact that administration of therapeutic mAbs is inherently passive, failing to generate a memory response. Furthermore, the repeated administration required as a result, may elicit neutralizing anti-idiotypic responses, which greatly limit efficacy.

In contrast, the ultimate aim of cancer immunotherapy is to activate the immune system to recognize the tumor, thereby generating a specific and durable effector T-cell response. In order to achieve this goal, adoptive transfer of TAA-specific T cells has been explored, involving their expansion *ex vivo* and re-administration to the same patients from whom they were originally derived (20). Alternatively, the ability of DCs to present TAAs to T cells has been harnessed to generate an immune response against tumor cells. Whereas adoptive T-cell transfer may generate a burst of T-cell immunity that is short-lived, DC-based vaccines have the potential to induce a sustained immune response with the capacity for subsequent recall (21). It has been shown that DC vaccination, following adoptive T-cell transfer, may further boost anti-tumor responses, suggesting a rationale for combining the two therapeutic strategies (21). In this review, we shall discuss recent experience of harnessing DCs and T cells for cancer immunotherapy and obstacles hindering their success.We shall also focus on the emerging use of patient-specific induced pluripotent stem cells (iPSCs) for the differentiation of DCs, T cells, and NK cells and discuss how this novel source holds promise for overcoming some of the shortfalls of conventional cancer immunotherapy.

#### **CELL-BASED IMMUNOTHERAPIES AND THEIR OBSTACLES**

As sentinels of the immune system, DCs play a pivotal role in eliciting the primary immune response to antigen. The ability of DCs to process and present protein antigens via the canonical endocytic pathway is crucial to this process (22). However, some subsets of DCs also possess the ability to capture exogenous antigens and cross present them via MHC class I direct to CD8+ T cells, thereby eliciting a cytotoxic T-cell response (23–25). Since cancer cells are poor APCs due to constitutively low expression of MHC class I and II determinants, the generation of protective anti-tumor immunity depends upon the cross presentation of tumor antigens by DCs (22–24). Although various DC-based cancer vaccines have been exploited in the past and the properties, advantages, and disadvantages of each extensively reviewed (20, 26, 27), it is most commonly DCs cultured *ex vivo* from peripheral blood monocytes (monocyte-derived dendritic cells, moDCs) that have shown the greatest clinical benefit. Indeed, one such vaccine, Provenge, has entered the market for the routine treatment of prostate cancer and involves the co-culture of moDCs obtained by leukapheresis, with the TSA, prostatic acid phosphatase (PAP), to which T-cell responses have been detected following their reinfusion into patients (28).

The majority of the clinical trials involving cancer vaccination have used autologous moDCs cultured *ex vivo* and pulsed with soluble TAA before re-administration to patients with the hope of inducing a tumor-specific immune response (29). Although many of these trials have shown that immunotherapy based on the use of mature moDC is safe, well-tolerated, and able to elicit an immune response against the tumor (27), the overall results have been disappointing, showing significant inter-trial variability between outcomes (30–32). This may be due, in no small part, to the donor-to-donor variability in yield and quality of moDCs, which is further compounded by long-term exposure to chemotherapeutic agents. Furthermore, the poor capacity of moDCs to cross present exogenous TAAs to CTLs, limits the magnitude of the cell-mediated immunity required to clear established tumors.

The recent identification of cross presenting DCs in man, equivalent to the CD8α + subset that has long been recognized in the mouse, has rejuvenated interest in the use of DCs in cancer immunotherapy (33, 34). These cells are defined by their expression of the surface marker CD141 and the chemokine receptor XCR1 and are found in the peripheral blood, tonsils, and bone marrow (35). They display an unrivaled capacity to cross present exogenous antigen to CD8+ T cells, and hence to elicit effective cell-mediated immunity. While the properties of CD141+ XCR1+ DCs make them ideal candidates for immunotherapy against cancer, their trace numbers in peripheral blood (<0.1%) limit their therapeutic exploitation (25). Alternative sources of cross presenting DCs, including their isolation from the spleen or *in vitro* differentiation from hematopoietic progenitor cells, have so far failed to overcome these obstacles (36).

Since the holy grail of cancer immunotherapy is to stimulate tumor-specific T cells that will elicit a cytotoxic response with high specificity and minimal toxicity, adoptive transfer of TAAspecific T cells has gained popularity over the past few years (37, 38). Adoptive T-cell transfer involves the isolation of T lymphocytes from the patients and their reinfusion to treat disease. The adoptive transfer of T cells was first documented in rodents in 1955, where it was noted that T cells obtained from lymph nodes draining a tumor were able to confer immunity when transferred into the peritoneum of a secondary host, bearing a similar tumor (39). Almost three decades later, it was observed that the incubation of murine splenocytes with IL-2 generated large numbers of cells, called lymphokine-activated killer (LAK) cells, which were capable of lysing tumor cells with little effect on other somatic cells (40). These LAK cells were later shown to decrease tumor number and size in humans in a wide variety of tumors including pulmonary and hepatic metastases (41, 42). This work served as the basis for the use of tumor-infiltrating lymphocytes (TILs) in immunotherapy (43, 44). The combination of a lymphodepleting preparative regimen with adoptive transfer of TILs and administration of IL-2 has been shown to promote cancer regression in patients with metastatic melanoma, leukemias, and other types of tumor (44, 45).

The possibility of genetic modification of the T cells to overcome the immunosuppressive environment created by the tumor may lead to more effective therapies in the future, although current strategies for genetic modification are limited (46) and T cells are known to constitute particularly intractable targets. Nevertheless, the possibility of genetically engineering T cells to recognize specific TAAs makes it possible to target potentially any tumor using adoptive T-cell transfer (47), while leaving other tissues intact. The majority of clinical approaches use virus-based transduction of tumor antigen-specific T-cell receptor (TCRs) or chimeric antigen receptors (CARs) to generate T cells stably expressing tumor-specific transgenes which, although efficient, is expensive and risks insertional mutagenesis. Non-viral approaches to genetically engineer T cells have so far utilized transposon elements such as piggyback or zinc finger nucleases (46). TALEN and CRISPR/Cas-9-based-approaches, which allow for the insertion of transgenes into defined chromosomal loci, are, however, currently being actively explored (48, 49). Despite the attractiveness of using CAR technology to target cancer, only the treatment of B cell leukemia has so far proven successful using this approach. Furthermore, recent work has shown that the treatment of patients with myeloma or melanoma using T cells engineered to express affinityenhanced TCRs for an HLA-A\*01-restricted epitope of MAGE-A3 resulted in severe myocardial damage secondary to widespread Tcell infiltration leading ultimately to fatal cardiogenic shock. These findings have clearly shown how even altering the affinity of the TCR for its ligand may introduce unanticipated cross-reactivity with potentially fatal off-target toxicity (50).

Although much effort has been invested into the adoptive transfer of unmodified T cells in the treatment of cancer, outcomes have been disappointing. It is, for instance, sometimes challenging to identify tumor-specific T cells in patients with non-solid tumors. TILs can also be difficult to isolate from biopsies of most melanomas. *Ex vivo* expansion of tumor-specific CTLs can also prove difficult: in the case of EBV-specific CTLs, for instance, 3 months is required for the production of sufficient CTLs for re-administration to the patient, with obvious implications for disease progression. Often, reinfusion of T cells is required following adoptive transfer for the induction of a durable response due to the exhaustion of the expanded CTLs.

NK cells have likewise been used for adoptive transfer, due to their innate ability to recognize tumor cells deficient in MHC class I. NK cells have been isolated form peripheral blood, expanded *ex vivo*, activated using IL-2 or, more recently, the combination of IL-12, IL-15, and IL-18 (51), and re-administered to patients (52). Interestingly, although the use of autologous cells is normally preferred, several studies have demonstrated that the use of allogeneic NK cells is significantly more effective (53, 54). Accordingly, the results of several studies have shown NK cells to be well-toleratedfollowing adoptive transfer with encouraging results of up to 20 months' survival following their administration (55). Nevertheless, low circulating numbers of NK cells in peripheral blood, coupled with the difficulty in their expansion and their inability to stimulate a robust response *in vivo*, limits their use in immunotherapy.

Given the difficulty of obtaining sufficient numbers of cells to target tumors *in vivo,* the advent of induced pluripotency offers unrivaled opportunities. The proven ability to produce iPS cells from individuals in a patient-specific manner with the capacity for indefinite self-renewal and unrestricted differentiation potential, may facilitate the scale-up in production of critical hematopoietic cell types, for many of which, protocols have already been optimized. Below, we outline the history of induced pluripotency and discuss the properties that make them attractive candidates for use in immunotherapy.

# **BRIEF HISTORY OF PLURIPOTENCY**

Since their first description, embryonic stem (ES) cells have been regarded as the "gold standard" for pluripotency, displaying the capacity for indefinite self-renewal and differentiation into any somatic cell type, irrespective of its embryonic germ-layer of origin. Mouse ES cells were first isolated in 1981 by Martin Evans (56), work which later earned him the 2007 Nobel Prize for Physiology or Medicine. Nevertheless, it was not until 1998 that Thomson and colleagues succeeded in deriving ES cells from the inner cell mass of human blastocysts that were surplus to requirements following *in vitro* fertilization (57). Human ES cell lines, like their mouse counterparts, were found to be pluripotent, expressing embryonic markers such as SSEA-3, SSEA-4, TRA-1–60, and alkaline phosphatase and, following injection into immune compromised mice, forming teratomas containing cell types and tissues from all three embryonic germ layers.

Since their first derivation, there has been much interest in the use of human ES cells as a source of diverse cell types for drug discovery, regenerative medicine, and immunotherapy. However, their use has been highly controversial due to the ethical sensitivities surrounding their derivation from human blastocysts, as well as the inevitable scientific constraints of using an allogeneic source of cells. In 2006, Yamanaka and colleagues demonstrated the feasibility of deriving pluripotent stem cells from adult mouse fibroblasts by retroviral transduction with genes encoding Oct3/4, Sox2, c-Myc, and Klf4 (58). These so-called iPS cells are indistinguishable at the cellular level from conventional ES cells, acquiring the capacity for indefinite self-renewal, unrestricted differentiation potential and, following injection into mouse blastocysts, the ability to generate germline-competent chimeras. These findings were subsequently translated to human dermal fibroblasts in 2007 by two independent groups (59, 60), showing, in principle, the feasibility of generating iPS cells on an individual basis. This seminal work offered a means of "personalizing" pluripotency in a manner free of the ethical concerns, while simultaneously addressing the immunological issues that limit the effectiveness of allogeneic therapies. Indeed, the production of iPS cells in an autologous fashion has paved the way for harnessing the potential of pluripotency for immunotherapeutic intervention in the pursuit of treatments for numerous indications.