Basic overview of current immunotherapy approaches in urologic malignancy

Urologic Oncology: Seminars and Original Investigations 24 (2006) 413– 418

Seminar article

Basic overview of current immunotherapy approaches in urologic malignancy Charles G. Drake, M.D., Ph.D.* Oncology, Immunology, and Urology, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21231, USA

Abstract The immune response to evolving prostate cancer is a complex and carefully orchestrated process. Such a response is initiated when immature dendritic cells take up and process tumor-associated antigens. These dendritic cells must then be activated in order to present peptides to helper (CD4) T cells. Cytolytic (CD8) T cells are next “licensed” to achieve full effector function by interacting with both antigen presenting cells and tumor-specific CD4 T cells. Finally, activated CD8 T cells traffic to sites of neoplasia and mediate killing by multiple mechanisms. This article provides a basic overview of these processes, and discusses the manner by which current clinical interventions seek to augment or initiate an antitumor immune response. Various compensatory mechanisms which serve to down-regulate an antitumor response are also examined. © 2006 Elsevier Inc. All rights reserved. Keywords: Tumor; Immunology; T cell; Vaccine; Prostate

Introduction

Antigen uptake—Monoclonal antibodies

In patients with urologic malignancies, clinical trials of cancer “vaccines” based on protein or peptide-pulsed dendritic cells, genetically modified viruses, naked deoxyribonucleic acid (DNA) vaccines, and tumor cells modified to secrete proinflammatory cytokines are currently underway. Although each of these approaches has unique advantages and disadvantages, they all endeavor to exploit a patient’s immune system to reject an evolving tumor burden. To appreciate the clinical relevance of these trials and data, a basic understanding of the immunologic mechanisms involved in tumor recognition and rejection is important. This section provides an introduction to the current immunologic understanding of a successful antitumor immune response and discusses how current immunotherapy strategies fit into this context. In addition, a number of the immunologic mechanisms that impede a successful antitumor response will be briefly outlined. It is noteworthy that this introduction is not intended to provide a comprehensive review of prior and ongoing clinical trails; these data will be presented in subsequent sections.

At the initiation of an antitumor immune response, dead and/or dying tumor cells are taken up locally by immature dendritic cells [1]. Although these specialized, antigen presenting cells are fully competent in terms of antigen uptake, immature dendritic cells have low levels of costimulatory molecules and surface class II major histocompatibility complex (MHC), and are not able to activate efficiently the T cells required for the generation of a successful antitumor response. Antigen uptake and presentation are markedly enhanced when the target is coated with antibody, and this process (known as opsonization) has been implicated in the mechanism of the monoclonal antibody Rituximab® (Genentech, Inc., South San Francisco, CA), which is widely used in lymphoma therapy [2]. In urologic malignancies, several monoclonal antibodies are under development. In particular, a humanized anti-prostate-specific membrane antigen (PSMA) antibody (HuJ591; BZL Biologics, Inc., Framingham, MA) has undergone a phase II trial; preliminary data indicate that the antibody is well tolerated and results in prostate-specific antigen (PSA) stabilization some patients [3]. To enhance the cytotoxic efficacy of a monoclonal antibody, it can be conjugated to a number of ␤-emitters, including 90Y and 111I. From an immunologic point of view,

* Tel.: ⫹1-410-502-7523; fax: ⫹1-410-614-0549. E-mail address: [email protected]. 1078-1439/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2005.08.013

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these conjugates may operate through more complex mechanisms, including direct toxicity in addition to the liberation of tumor antigens from necrotic tumor cells. Phase I trials of both 90Y and 177lutetium conjugated anti-PSMA antibodies have been recently reported, with evidence of biologic activity in both studies [4,5]. Thus, the first phase of an antitumor immune response is antigen uptake, and this phase of the immune response is probably most affected by monoclonal antibody administration. Monoclonal antibodies may also act through another mechanism known as antibody-dependent cell-mediated cytotoxicity (ADCC), which is discussed in detail later.

Dendritic cell activation—Cytokines Before dendritic cells can present antigen and activate T cells, the dendritic cells themselves must be activated. A variety of intracellular and extracellular pathways are involved in dendritic cell activation, and one or more of these pathways is targeted either directly or indirectly by most vaccination strategies. At one spectrum, vaccines based on protein or peptide pulsed dendritic cells, such as DCVax® (Northwest Biotherapeutics, Bothell, WA) or Provenge® (Dendreon Corp., Seattle, WA) directly mature and activate a patient’s dendritic cells ex vivo, before pulsing with a select target antigen [6]. One potential advantage of these approaches is that the ex vivo activation and maturation of dendritic cells avoids one layer of variability in the overall immunologic response to vaccination and may circumvent dendritic cell tolerance mechanisms [7]. The downside of ex vivo preparation of dendritic cells for vaccination is the complexity of the process, typically plasmapheresis is required, followed by some period of cell culture under sterile conditions. Thus, dendritic cell-based vaccines are a patientspecific product for which widespread dissemination may prove complex. In a phase III trial of Provenge® for patients with metastatic hormone-refractory prostate cancer, initial subgroup analysis suggested a clinical benefit for patients with an initial Gleason score of ⱕ7 [8]. Based on these data, an important confirmatory phase III trial is currently underway. Another major pathway for dendritic cell activation involves stimulation via cytokines. In particular, granulocytemacrophage colony-stimulating factor (GM-CSF) strongly promotes dendritic cell activation and migration [9], and provides the basis for cell-based vaccine approaches such as GVAX® (Cell Genesys, Inc., South San Francisco, CA). In these cell-based vaccine approaches, either autologous or allogeneic tumor cells are genetically modified to secrete cytokines such as GM-CSF or interleukin-12. A phase I/II trial of autologous prostate cancer cells transduced to secrete GM-CSF has been completed, and the vaccine was well tolerated with some evidence of an immune response [10]. However, this trial, as well as a similar trial in metastatic renal cell cancer [11], underscored the multiple vari-

ables involved in using autologous cells for vaccine generation. An alternative approach involves using cultured allogeneic tumor cells transduced to produce GM-CSF as a vaccine. Allogeneic cell-based vaccines avoid variability in vaccine preparation and GM-CSF secretion levels, and have the potential for large-scale biosynthesis. The downside of such allogeneic approaches is that at least some of the tumor-associated proteins must be shared between the patient’s tumor and the cultured cell lines for the vaccine to be effective. Phase II trials of allogeneic Prostate GVAX® have been completed, with some evidence of efficacy [12]. Two multisite, randomized phase III trials of Prostate GVAX® for patients with hormone-refractory metastatic prostate cancer are currently underway. It is also noteworthy that GM-CSF may also be administered as a single agent (i.e., without a vaccine). The hypothesis in such studies is that administration of exogenous GM-CSF may activate endogenous antigen presenting cells and foster an ongoing weak or tolerized immune response. Two clinical trials testing this hypothesis have been reported, with the most recent trial showing evidence of PSA slope changes as an outcome measure in patients with prostate cancer with biochemical-only relapse [13].

Dendritic cell activation—Toll-like receptors and heat shock proteins During the last decade, it has become increasingly clear that the natural immune response to common pathogens is characterized by the rapid and robust activation of resident antigen presenting cells. This activation is mediated in part by recognition of a series of conserved pathogen associated molecular patterns [14]. These patterns are recognized by a series of receptors on dendritic cells known as Toll-like receptors; the study of these receptors, their ligands, and their role in the immune response has become a major focus in basic immunology [15]. These data have direct relevance to immunotherapy for urologic malignancies. For example, although the precise mechanism of action of intravesical application of bacille Calmette-Guérin for superficial bladder cancer remains unclear [16], it is probable that this agent induces activation and differentiation of resident antigen presenting cells through recognition of bacterial lipopolysaccharide by Toll-like receptor-4 and Toll-like receptor-2 [17]. Another topic of intensive clinical and preclinical research involves Toll-like receptor-9, which responds to unique bacterial DNA sequences containing unmethylated cytosine-guanine (CpG) [18]. In preclinical models of rhabdomyosarcoma and colon cancer, administration of CpGs strongly potentiates an antitumor immune response [19,20]. From a clinical perspective, a phase I/II study of CpGs (ProMune®, Coley Pharmaceutical Group, Inc., Wellesley, MA) for stage IV renal cancer is currently underway. En-

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couraging preliminary data from trials in melanoma and non-small-cell lung cancer bolster interest in this study. Dendritic cells may also be activated and recruited when heat shock proteins released from necrotic cells bind to appropriate receptors [21]. Heat shock proteins are a family of molecules up-regulated in cells subject to a variety of stressors. A number of these proteins, particularly heat shock protein-96, appear to play a role in inducing an immune response by shuttling antigens from necrotic cells to antigen presenting cells. Cancer vaccine studies based on this approach have undergone clinical trials in renal cell cancer; a phase III trial is currently underway (i.e., Oncophage®, Antigenics Inc., New York, NY). Like dendritic cell vaccines, heat shock protein-based vaccines are a “personalized product.” A sufficient amount of tumor must be surgically removed and processed to produce vaccine. These vaccines also share the potential advantage of antigen selectivity, delivering a subset of antigens relevant to the patient’s individual tumor.

CD4 (helper) T cell activation Once dendritic cells are activated, they migrate to a draining lymph node where they engage CD4 (helper) and CD8 (killer) T cells in a complex interaction [22]. When dendritic cells are injected as a vaccine, similar migration seems to occur, although it is likely that only a small proportion of ex vivo activated dendritic cells actually arrive intact in the lymph node. In the lymph node, dendritic cells present antigen to CD4 T cells via class II major histocompatibility molecules. When activated dendritic cells are used as a vaccine, this presentation can occur “directly” (i.e., the dendritic cells can directly engage specific T cells though an interaction between the peptide/MHC and the T cell receptor). Other vaccination strategies rely on a process known as “cross-presentation” (i.e., antigen is taken up from a vaccine construct by resident host dendritic cells and presented to the host T cells by their own antigen presenting cells) [23,24]. Although well described in animal models, this process was only recently shown in a human vaccine study [25]. For CD4 T cells to become fully activated, they require 2 signals [26]. The first signal is provided by an interaction between the T cell receptor and the MHC/peptide complex. The second, or costimulatory, signal is delivered by an interaction between a molecule known as B7-1 on activated antigen-presenting cells and CD28 on the relevant T cell. If cognate T cells do not receive this second signal, they are rendered unresponsive, or “anergic” [27]. One vaccine strategy exploits this 2-signal dependence and directly activates CD4 T cells through a viral construct that expresses both costimulatory molecules and a relevant antigenic peptide. For prostate cancer, a vaccine (i.e., ProstVac®; Therion Biologics Corp., Cambridge, MA) that incorporates a triad of costimulatory molecules (B7-1, LFA-1, and intercellular

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adhesion molecule) into poxvirus based constructs along with the coding sequence for PSA has been developed. Extensive preclinical evaluation of this concept has been performed, and phase I and II clinical trials have been completed [28,29]. A phase III intergroup trial is in the planning stages.

CD8 (cytotoxic) T cell activation In a specific antitumor immune response, activated CD8 T cells are the ultimate effector cells; these cells traffic to tumors and specifically lyse target cells. CD8 T cells are activated in a manner similar to CD4 T cells, through an interaction between their cognate T-cell receptor and a complex of MHC class I and peptide on an antigen presenting cell. Once activated, CD8 T cells leave the lymph node and traffic to the tumor. Here, tumor cells are recognized in an extremely specific manner (i.e., through the same MHC class I /tumor peptide interaction that served to mediate CD8 T-cell activation in the lymph node). In a well-accepted model of antigen-presenting cell T-cell interaction, tumor-specific CD4 T cells may be necessary to “license” CD8 T cells and allow these effector cells to achieve full tumor-lytic capability [30]. This model postulates the transient existence of a tricellular construct, in which the CD4 and CD8 T cells interact with a common antigen presenting cell. Preliminary imaging data have visualized this tricellular complex directly, and the cellular signals that direct complex formation may have important implications for future vaccine development (A. Huang, personal communication, March 2005). Indeed, it is noteworthy that a large body of experimental data indicates that CD4 T cells are necessary for a successful antitumor response [31]. In support of this concept, recent experiments showed that low-affinity CD8 T cells have very little antitumor efficacy on their own but were able to mount a significant antitumor response in the presence of tumor-specific CD4 T cells [32]. Once activated CD8 T cells are generated, they may show powerful antitumor activity and can lyse even multidrug-resistant tumor cells [33]. From a clinical viewpoint, these data suggest that one method of generating a powerful antitumor CD8 T cell response might be to activate such cells ex vivo, and re-administer them intravenously. Approaches like this are known as adoptive immunotherapy and have been clinically tested in melanoma with encouraging response rates reported [34]. To date, specific adoptive immunotherapy for urologic malignancies has not undergone extensive clinical testing.

CD8 T cell effector function CD8 T cells lyse tumor cells by secreting granules containing perforins, which create pores in the target cell membrane, as well as granzymes, which induce apoptosis

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in the target cells. In general, the goal of cancer vaccine approaches has been to expand a population of antigenspecific CD8 cells. In clinical trials, the percentage of such cells can be assayed using tetramers, which consist of a specific peptide/MHC, and can be fluorescently conjugated for analysis using flow cytometry. Studies of antigen-specific CD8 T cells in patients with melanoma have been performed, with optimal vaccine approaches capable of generating an immune repertoire in which 1 of 200 T cells are tumor specific [35]. Surprisingly, many of these patients continued to progress, indicating that other factors beside the mere presence of antigen-specific CD8 T cells are involved in tumor regression. Identification of factors mediating CD8 effector cell trafficking and function is an area of active research. One cytokine that seems to affect CD8 trafficking to tumors is transforming growth factor-beta, which is increased in the serum of patients with advanced prostate cancer and has prognostic value [36,37]. In an interesting preclinical study, CD8 T cells were transformed ex vivo with a dominant negative transforming growth factor-beta receptor so they could no longer respond to signals mediated by this cytokine [38]. When adoptively transferred back into tumor-bearing hosts, these T cells were now able to home to and lyse evolving tumors. Clinical studies to translate these findings into the clinical setting are in the planning stages.

CD8 T cell memory generation

Natural killer cell effector function

Regulatory T cells

Natural killer cells are a lymphocyte subset that is generally considered part of the innate or nonspecific immune system. In this role, they recognize and lyse virally infected cells or cells that have lost class I MHC on their surface. These cells are also postulated to play a role in tumor surveillance [39]. However, natural killer cells can also act as surrogate members of the adaptive immune system by recognizing and lysing target cells that are coated with antibody. This mechanism is known as ADCC. ADCC has been implicated in the clinical activity of a monoclonal antibody directed at the cell surface molecule HER-2/neu, which is present on the surface of several tumor types, including breast cancer [40]. Based on data suggesting the presence of HER-2/neu on prostate cancer cells, a phase II trial of the monoclonal antibody trastuzumab (Herceptin®, Genentech, Inc.) in patients with hormone refractory prostate cancer was completed [41]. Although treatment was generally well tolerated, very little efficacy was noted with this particular agent. Nevertheless, it is noteworthy that the efficacy of unlabeled antibodies against PSMA or prostatic acid phosphatase may depend on ADCC and that future trials may combine monoclonal antibodies with either vaccines or cytotoxic approaches.

The T-cell arm of the immune response to cancer is further complicated by recent observations showing that not all tumor-associated CD4 T cells are “helpers.” In women with ovarian cancer, a large number of the CD4 T cells present in ascites possessed “regulatory function” and led to impressive decreases in CD8 T-cell responsiveness [46]. These CD4⫹ CD25⫹ regulatory T cells (Treg) have been shown in lung and breast cancer as well. Depletion or blockade of regulatory T cells strongly augments vaccine efficacy in a number of model systems [47], including prostate cancer [48]. In the clinical setting, attempts to block Treg have centered on blocking the Treg associated molecule cytotoxic T lymphocyte antigen (CTLA)-4 with a monoclonal antibody (MDX010; Medarex, Inc., Princeton, NJ) [49]. Although CTLA-4 blockade has shown promise in multiple experimental systems, clinical trials in patients with melanoma were tempered by the development of autoimmunity in a number of subjects [50]. A phase I trial treated 14 men with metastatic hormonerefractory prostate cancer with a single-dose of human antiCTLA-4 antibody [51]. A few patients had a PSA decline of ⱖ50%, suggesting activity and setting the stage for clinical trials involving vaccination in combination with CTLA-4 blocking antibody. Other investigational strategies to block

The generation of a long-term protective immune response depends on the generation and maintenance of a population of long-lived CD8 cells known as “memory” cells. Numerous experiments show that, in the presence of persistent antigen, CD8 T cells cannot achieve the final step required to acquire a memory phenotype [42]. This point is particularly relevant in the case of an immune response to cancer, in which it is challenging to eliminate effectively a substantial tumor burden. The form of tolerance induced by persistent antigen may depend on the level of antigen, with high levels of antigen leading to CD8 T cell deletion and lower antigen levels allowing persistence of anergic antitumor CD8 T cells. To surmount the problem of persistent antigen, some researchers have advocated initiating vaccination approaches in either the adjuvant setting or in the setting of minimal residual disease [43]. In prostate cancer, the initial efficacy of androgen ablation provides a unique opportunity for a window of immunotherapy. In a key observational clinical trial, androgen ablation alone induced trafficking of activated lymphocytes into the prostate gland [44]. In recent work using a murine model of prostate cancer, we showed that vaccine unresponsiveness could largely be mitigated after androgen ablation [45]. Thus, one potential immunotherapy strategy might involve vaccination of men with biochemically relapsed prostate cancer after androgen ablation.

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Treg function include depletion of CD4⫹ CD25⫹ T cells with an antibody targeted toxin (Denileukin diftitox; Seragen, Inc., Hopkinton, MA), blockade of the inhibitory molecule PD-1 [52], and blockade of the Treg associated molecule Lag-3 [53].

Target antigen selection One issue in the basic immunology of urologic cancer vaccines concerns the choice of target antigen(s). Initially, it was thought that tumor antigens would be molecules that were relatively uniquely expressed by a particular tumor. However, the identification a number of melanoma antigens showed that these antigens were not unique and represented nonmutated tissue-specific proteins [54]. Because a number of prostate tissue-specific proteins were already known, it was natural that these molecules were chosen as target vaccine antigens. Perhaps the most widely studied of these is PSA, which has been targeted by plasmid DNA, viral, and peptide/adjuvant approaches [55]. Additional prostate-tissue targets include prostatic acid phosphatase and PSMA. Other target antigens are preferentially expressed by tumors rather than prostate tissue, and include the carbohydrate moiety MUC1 and telomerase. It is noteworthy that most of these approaches target a single protein (or peptide) and are theoretically subject to the phenomena of antigenic escape, whereby the tumor down-regulates expression of the targeted protein [56]. Cell-based vaccine approaches represent one possible strategy to avoid this possibility by cross-presenting a variety of antigens. Nevertheless, rational target antigen selection represents one of the major challenges in antitumor immunity. In the future, such efforts are likely to be significantly augmented by an expanding array of data comparing gene expression patterns in normal and malignant prostate tissue [57].

Conclusions The immune response to urologic cancer vaccination is complex, and involves an intricate orchestration of elements of the innate and adaptive immune system. Tumor escape mechanisms such as antigen loss, regulatory T cells, T cell anergy, and dendritic cell dysfunction represent significant challenges to such approaches. As is the case for chemotherapy, it seems likely that combinatorial approaches coupling vaccines with strategies to decrease tumor burden and modulate regulatory pathways will be necessary for eventual clinical success.

References [1] Steinman RM. Some interfaces of dendritic cell biology. APMIS 2003;111:675–95.

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[2] Uchida J, Hamaguchi Y, Oliver JA, et al. The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptordependent mechanisms during anti-CD20 antibody immunotherapy. J Exp Med 2004;199:1659 – 69. [3] Nanus DM, Milowsky MI, Kostakoglu L, et al. Clinical use of monoclonal antibody HuJ591 therapy: Targeting prostate specific membrane antigen. J Urol 2003;170:S84 – 8. [4] Milowsky MI, Nanus DM, Kostakoglu L, et al. Phase I trial of yttrium-90-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 for androgen-independent prostate cancer. J Clin Oncol 2004;22:2522–31. [5] Bander NH, Milowsky MI, Nanus DM, et al. Phase I trial of 177lutetium-labeled J591, a monoclonal antibody to prostate-specific membrane antigen, in patients with androgen-independent prostate cancer. J Clin Oncol 2005;23:4591– 601. [6] Fong L, Engleman EG. Dendritic cells in cancer immunotherapy. Annu Rev Immunol 2000;18:245–73. [7] Gabrilovich D. Mechanisms and functional significance of tumourinduced dendritic-cell defects. Nat Rev Immunol 2004;4:941–52. [8] Schellhammer PF, Hershberg RM. Immunotherapy with autologous antigen presenting cells for the treatment of androgen independent prostate cancer. World J Urol 2005;23:47–9. [9] Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer 2004;4:11–22. [10] Simons JW, Higano C, Corman J, et al. A phase I/II trial of high dose allogeneic GM-CSF gene-transduced prostate cancer cell line vaccine in patients with metastatic hormone-refractory prostate cancer. Proc Am Soc Clin Oncol 2003;22:166 [abstract]. [11] Simons JW, Jaffee EM, Weber CE, et al. Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer. Cancer Res 1997;57:1537– 46. [12] Simons JW, Small EJ, Nelson WG, et al. Phase II trials of a GM-CSF gene-transduced prostate cancer cell line vaccine (GVAX) demonstrate anti-tumor activity. Proc Am Soc Clin Oncol 2001;20:292 [abstract]. [13] Rini BI, Weinberg V, Bok R, et al. Prostate-specific antigen kinetics as a measure of the biologic effect of granulocyte-macrophage colony-stimulating factor in patients with serologic progression of prostate cancer. J Clin Oncol 2003;21:99 –105. [14] Janeway CA Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol 1989;54: 1–13. [15] Germain RN. An innately interesting decade of research in immunology. Nat Med 2004;10:1307–20. [16] Bohle A, Brandau S. Immune mechanisms in bacillus CalmetteGuerin immunotherapy for superficial bladder cancer. J Urol 2003; 170:964 –9. [17] Begum NA, Ishii K, Kurita-Taniguchi M, et al. Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages. Infect Immun 2004;72:937– 48. [18] Krieg AM. CpG motifs: The active ingredient in bacterial extracts? Nat Med 2003;9:831–5. [19] Weigel BJ, Rodeberg DA, Krieg AM, et al. CpG oligodeoxynucleotides potentiate the antitumor effects of chemotherapy or tumor resection in an orthotopic murine model of rhabdomyosarcoma. Clin Cancer Res 2003;9:3105–14. [20] Baral RN, Saha A, Chatterjee SK, et al. Immunostimulatory CpG oligonucleotides enhance the immune response of anti-idiotype vaccine that mimics carcinoembryonic antigen. Cancer Immunol Immunother 2003;52:317–27. [21] Binder RJ, Anderson KM, Basu S, et al. Cutting edge: Heat shock protein gp96 induces maturation and migration of CD11c⫹ cells in vivo. J Immunol 2000;165:6029 –35. [22] Germain RN, Jenkins MK. In vivo antigen presentation. Curr Opin Immunol 2004;16:120 –5.

418

C.G. Drake / Urologic Oncology: Seminars and Original Investigations 24 (2006) 413– 418

[23] Adler AJ, Marsh DW, Yochum GS, et al. CD4⫹ T cell tolerance to parenchymal self-antigens requires presentation by bone marrowderived antigen-presenting cells. J Exp Med 1998;187:1555– 64. [24] Heath WR, Carbone FR. Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol 2001;19:47– 64. [25] Thomas AM, Santarsiero LM, Lutz ER, et al. Mesothelin-specific CD8(⫹) T cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients. J Exp Med 2004;200:297–306. [26] Bretscher P, Cohn M. A theory of self-nonself discrimination. Science 1970;169:1042–9. [27] Schwartz RH, Mueller DL, Jenkins MK, et al. T-cell clonal anergy. Cold Spring Harb Symp Quant Biol 1989;54:2:605–10. [28] Gulley J, Chen AP, Dahut W, et al. Phase I study of a vaccine using recombinant vaccinia virus expressing PSA (rV-PSA) in patients with metastatic androgen-independent prostate cancer. Prostate 2002;53: 109 –17. [29] Kaufman HL, Wang W, Manola J, et al. Phase II randomized study of vaccine treatment of advanced prostate cancer (E7897): A trial of the Eastern Cooperative Oncology Group. J Clin Oncol 2004;22: 2122–32. [30] Marzo AL, Kinnear BF, Lake RA, et al. Tumor-specific CD4⫹ T cells have a major “post-licensing” role in CTL mediated anti-tumor immunity. J Immunol 2000;165:6047–55. [31] Hung K, Hayashi R, Lafond-Walker A, et al. The central role of CD4(⫹) T cells in the antitumor immune response. J Exp Med 1998;188:2357– 68. [32] Lyman MA, Nugent CT, Marquardt KL, et al. The fate of low affinity tumor-specific CD8⫹ T cells in tumor-bearing mice. J Immunol 2005;174:2563–72. [33] Niethammer AG, Wodrich H, Loeffler M, et al. Multidrug resistance-1 (MDR-1): a new target for T cell-based immunotherapy. FASEB J 2005;19:158 –9. [34] Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol 2005;23:2346 –57. [35] Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 1998;4: 321–7. [36] Shariat SF, Shalev M, Menesses-Diaz A, et al. Preoperative plasma levels of transforming growth factor beta(1) (TGF- beta(1)) strongly predict progression in patients undergoing radical prostatectomy. J Clin Oncol 2001;19:2856 – 64. [37] Lee C, Sintich SM, Mathews EP, et al. Transforming growth factorbeta in benign and malignant prostate. Prostate 1999;39:285–90. [38] Zhang Q, Yang X, Pins M, et al. Adoptive transfer of tumor-reactive transforming growth factor-beta-insensitive CD8⫹ T cells: Eradication of autologous mouse prostate cancer. Cancer Res 2005; 65:1761–9.

[39] Smyth MJ, Thia KY, Street SE, et al. Differential tumor surveillance by natural killer (NK) and NKT cells. J Exp Med 2000;191:661– 8. [40] Spiridon CI, Ghetie MA, Uhr J, et al. Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved antigrowth activity of a human breast cancer cell line in vitro and in vivo. Clin Cancer Res 2002;8:1720 –30. [41] Ziada A, Barqawi A, Glode LM, et al. The use of trastuzumab in the treatment of hormone refractory prostate cancer; phase II trial. Prostate 2004;60:332–7. [42] Bevan MJ. Helping the CD8(⫹) T-cell response. Nat Rev Immunol 2004;4:595– 602. [43] Mitka M. Cancer vaccine research inches forward. JAMA 2004;291: 550 –2. [44] Mercader M, Bodner BK, Moser MT, et al. T cell infiltration of the prostate induced by androgen withdrawal in patients with prostate cancer. Proc Natl Acad Sci U S A 2001;98:14565–70. [45] Drake CG, Doody AD, Mihalyo MA, et al. Androgen ablation mitigates tolerance to a prostate/prostate cancer-restricted antigen. Cancer Cell 2005;7:239 – 49. [46] Curiel TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004;10:942–9. [47] Terabe M, Berzofsky JA. Immunoregulatory T cells in tumor immunity. Curr Opin Immunol 2004;16:157– 62. [48] Hurwitz AA, Foster BA, Kwon ED, et al. Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade. Cancer Res 2000;60:2444 – 8. [49] Chambers CA, Kuhns MS, Egen JG, et al. CTLA-4-mediated inhibition in regulation of T cell responses: Mechanisms and manipulation in tumor immunotherapy. Annu Rev Immunol 2001;19:565–94. [50] Phan GQ, Yang JC, Sherry RM, et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci USA 2003;100:8372–7. [51] Rini BI, Small EJ. The potential for prostate cancer immunotherapy. Crit Rev Oncol Hematol 2003;46(Suppl):S117–25. [52] Nishimura H, Honjo T. PD-1: An inhibitory immunoreceptor involved in peripheral tolerance. Trends Immunol 2001;22:265– 8. [53] Huang CT, Workman CJ, Flies D, et al. Role of LAG-3 in regulatory T cells. Immunity 2004;21:503–13. [54] Boon T, De Plaen E, Lurquin C, et al. Identification of tumour rejection antigens recognized by T lymphocytes. Cancer Surv 1992; 13:23–37. [55] McNeel DG, Malkovsky M. Immune-based therapies for prostate cancer. Immunol Lett 2005;96:3–9. [56] Zhou G, Lu Z, McCadden JD, et al. Reciprocal changes in tumor antigenicity and antigen-specific T cell function during tumor progression. J Exp Med 2004;200:1581–92. [57] Rhodes DR, Barrette TR, Rubin MA, et al. Meta-analysis of microarrays: Interstudy validation of gene expression profiles reveals pathway dysregulation in prostate cancer. Cancer Res 2002;62:4427–33.