Oncologist. The. Genitourinary Cancer. Immunotherapeutics in Development for Prostate Cancer. The Oncologist 2009;14:

Transcription

1 The Oncologist Genitourinary Cancer Immunotherapeutics in Development for Prostate Cancer ANDREA L. HARZSTARK,ERIC J. SMALL Department of Medicine, University of California San Francisco, San Francisco, California, USA Key Words. Castration-resistant prostate cancer GM-CSF Immunotherapy Phase II PROSTVAC GVAX Ipilimumab Sipuleucel-T Disclosures: Andrea L. Harzstark: None; Eric J. Small: None. The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, and free from commercial bias. No financial relationships relevant to the content of this article have been disclosed by the authors or independent peer reviewers. ABSTRACT Whereas chemotherapy is the standard of care for metastatic castration-resistant prostate cancer and is associated with a survival benefit, there remains a need for alternative approaches. Extensive work has been done evaluating multiple immunotherapies for the treatment of prostate cancer. This review discusses clinical results INTRODUCTION Although prostate cancer has not been classically considered a cancer amenable to immunologic therapeutic maneuvers like melanoma or renal cell carcinoma, there are multiple characteristics of prostate cancer that make immune-based therapy a promising approach. Prostate cancer grows relatively slowly, which may allow the immune system, when stimulated, the necessary time to generate an antitumor immune response [1]. In addition, the nonessential nature of the prostate gland makes immune-based therapy a potentially safer treatment for prostate cancer than for other malignancies, in the event of tissue-specific, but not cancerspecific, targeting. Recent evidence has also suggested that prostate cancer is more immunogenic than previously understood, with the ability to induce spontaneous autoantibodies in patients [2]. Immune approaches offer the hope of for the most promising developments. These include cytokine-based therapy with GM-CSF; vaccines; antibody-based immunotherapies, including anti cytotoxic T lymphocyte associated antigen 4 therapy and antibodies against additional targets; and dendritic cell-based immunotherapy. The Oncologist 2009;14: less toxicity than chemotherapy for advanced disease, although this has not yet been borne out in clinical testing. As metastatic prostate cancer afflicts an elderly group of patients, the possibility of less toxicity is a particularly important goal. CYTOKINES GM-CSF is a growth factor capable of enhancing antitumor activity by means of the activation of dendritic cells (DCs) as well as through indirect T-cell activation via interleukin (IL)-1 release [3]. In addition, GM-CSF activates macrophages and induces the release of tumor necrosis factor (TNF). Myeloid growth factors are widely known to shorten the period of neutropenia associated with chemotherapy, allowing dose intensification. However, they were first studied in hematologic malignancies and have direct Correspondence: Andrea L. Harzstark, M.D., Department of Medicine, University of California San Francisco, 1600 Divisadero Street, Box 1711, San Francisco, California 94115, USA. Telephone: ; Fax: ; Andrea.harzstark@ucsf. edu Received November 5, 2008; accepted for publication February 28, 2009; first published online in The Oncologist Express on April 2, AlphaMed Press /2009/$30.00/0 doi: /theoncologist The Oncologist 2009;14:

2 392 Immunotherapeutics for Prostate Cancer therapeutic value as well. For example, GM-CSF promotes differentiation of hematopoietic stem cells toward the production of DCs, which are the most effective antigen-presenting cells (APCs). In addition, GM-CSF shifts the cell cycle of myeloid leukemic cells from the G 0 phase to S phase in vitro and in vivo, resulting in greater sensitivity to cell cycle dependent cytostatic agents [4, 5]. Exogenously administered GM-CSF has been shown to modulate prostate-specific antigen (PSA) levels in men with castration-resistant prostate cancer (CRPC) [6]. When administered to 14 patients with metastatic CRPC for 14 consecutive days, followed by maintenance three times per week, all but one patient had a PSA decline (median decline, 32%), with one patient experiencing a 99% decline accompanied by an improvement in bone scan lasting 14 months. Although PSA modulation is not a universally accepted marker of response, these data suggest the possibility that GM-CSF may have biologic and antitumor effects. Although this study did not definitively determine whether the effects of GM-CSF on PSA were immunologic in nature, they form the basis for later immunotherapeutics employing GM-CSF. However, the use of GM-CSF, as well as other cytokines, is challenged by counterregulatory immune responses that aim to decrease the expansion of cytotoxic T cells, thereby limiting antitumor activity. It is necessary to control this response in order to take advantage of the immune benefits of GM-CSF. In addition, GM-CSF is associated with the presence of CD34 myeloid suppressor cells, which are capable of infiltrating head and neck carcinomas [7]. These suppressor cells can release transforming growth factor, which inhibits T-cell functions [8]. Head and neck cancers that secreted GM-CSF and contained CD34 tumor suppressor cells were found to have a higher rate of recurrence metastasis than those that did not secrete GM- CSF [9]. It is important to use this knowledge to determine the best use of GM-CSF as an adjuvant to vaccines. In these studies, doses have been in the range of g, with dosing as infrequently as once to as often as daily for 7 days [10 12]. Generally, low doses of GM-CSF are associated with greater stimulation of the immune response, whereas higher doses are not associated with additional stimulation of the immune response. It is theorized that higher doses may activate myeloid suppressor cells, creating a counterproductive immune response. Therefore, it is critical that the use of GM-CSF be optimized, in order to improve, rather than hinder, the immune response. Several other cytokines are being evaluated for their role as possible therapeutics for prostate cancer. IL-6 may cause growth of hormone-sensitive prostate cancer via activation of the androgen receptor. Blockade of IL-6 could potentially provide a hormone-sparing therapy for hormone-sensitive disease [13]. IL-8, the release of which is induced by chemotherapy, has been found to reduce the sensitivity of prostate cancer cells to undergoing apoptosis, suggesting that targeted inhibition of IL-8 signaling may be a potential therapeutic approach to improving chemotherapy sensitivity for castration-resistant disease [14]. Interferon (IFN)- induces apoptosis and decreases the adhesiveness and invasiveness of prostate cancer cell lines [15]. Agents like insulin growth factor binding protein-1 and rapamycin may be able to sensitize cells to the proapoptotic actions of IFN- [16]. Other cytokines being evaluated include TNF, IL-7, IL-12, and IL-13. VACCINES Several vaccine approaches, some of which use whole tumor cells, have been used. These approaches offer the potential advantage of stimulating endogenous immune function. Gene-modified tumor cells have been used, but the most explored approach thus far has been with GM- CSF modified tumor cell vaccines. GVAX (Cell Genesys, South San Francisco, CA) is a cellular vaccine that uses exogenous tumor cells engineered to secrete GM-CSF, which is thought to increase DC presentation of antigens to the immune system. Unlike with sipuleucel-t (see below), in which the antigen source is prostatic acid phosphatase (PAP), with GVAX whole tumor cells are used as the antigen source [17]. Two cell lines are used for GVAX : the PC-3 cell line, which was derived from a prostate cancer bone metastasis in a patient with CRPC, and LNCaP, which was derived from a prostate cancer metastasis to a lymph node in a patient with hormone-sensitive disease, although the androgen receptor is mutated. The panel of antigens these cell lines supply, in concert with the high concentration of GM-CSF, has been shown to induce DC antigen presentation and activation of effector cells, such as T cells and macrophages [18]. GVAX was evaluated in a total of 114 patients with metastatic CRPC in two phase II clinical trials. The median survival time was compared with the survival duration predicted by the CRPC pretreatment nomogram devised by Halabi and colleagues [19]. In the G9803 trial, the median survival time was 26.2 months, compared with the 19.5 months (p.01) predicted by the pretreatment nomogram [20]. In the G-0010 trial, a study in which the vaccine was re-engineered to secrete a higher dose of GM-CSF, the median survival time for the group receiving the phase III dose was 35.0 months [21 23]. Based on these encouraging results, two phase III studies evaluating GVAX in comparison with or in addition to

3 Harzstark, Small docetaxel-based chemotherapy in patients with metastatic CRPC were initiated. In one study (VITAL-1), therapy with GVAX was compared directly with docetaxel plus prednisone; accrual was completed, but the study was closed in October of 2008 when a futility analysis revealed a 30% likelihood of the study meeting its primary endpoint of longer overall survival. In the second study (VITAL-2), the treatment arm consisted of GVAX plus docetaxel (without prednisone) and was compared with docetaxel plus prednisone alone. VITAL-2 was halted in August of 2008 when an interim analysis revealed a higher number of deaths in the GVAX arm; further evaluation has not yet been released. GVAX has been combined with other immunotherapeutics (as discussed below), although, given these recent developments, further development of this agent has been halted and its future is unclear. Poxviral vectors have also been used to treat patients with recombinant vaccinia PSA inserted into a viral vector in an attempt to stimulate an immune response. In a phase II study, 64 patients with nonmetastatic hormone-sensitive prostate cancer and a rising PSA level after definitive local therapy were randomized to receive four vaccinations with fowlpox-psa (rf-psa), three rf-psa vaccines followed by one vaccinia-psa (rv-psa) vaccine, or one rv-psa vaccine followed by three rf-psa vaccines [24]. The primary endpoint was PSA response at 6 months. At a median follow-up of 19.1 months, 45.3% of men remained free from PSA progression, with a trend favoring the group that received a priming dose of rv-psa. No significant increases in anti-psa antibody titers were detected, but 46% of patients demonstrated an increase in PSA-reactive T cells. Toxicity was minimal, with two grade 3 hyperglycemic events in diabetic patients being the only grade 3 or 4 toxicities observed. No autoimmune events were observed. These data suggest that the use of poxviral vectors is safe and capable of generating a T-cell immune response. PROSTVAC (BN ImmunoTherapeutics, Inc., Sunnyvale, CA) consists of these constructs of fowlpox and vaccinia vectors and contains a triad of costimulatory molecule transgenes, including intercellular adhesion molecule 1, B7.1, and leukocyte function associated antigen 3, which has been designated TRICOM. In preclinical studies, use of all three of these costimulatory molecules resulted in superior clinical responses and was safe [25, 26]. A phase I study of one dose of PROSTVAC -V (a partially attenuated vaccinia virus with gene sequences for PSA and TRICOM ), followed by a booster dose of PROSTVAC -F (the fowlpox virus with gene sequences for PSA and TRICOM ) after 4 weeks was performed in 10 chemotherapy-naïve patients with metastatic or nonmetastatic CRPC [27]. No grade 3 or 4 toxicities were experienced, with the most common adverse events being injection site reactions and fatigue. Four of the 10 patients in the study had a stable PSA level through the 8-week study period. No patients mounted an anti-psa antibody response by enzyme-linked immunosorbent assay, but antivaccinia titers increased by day 15 of treatment. This off-target response raises the issue of a serious potential problem with viral vectors: the potential to generate antiviral immunity that may focus the immune response on other targets. In this way, they may actually be counterproductive. Further clinical activity and immune evaluations of the poxviral approach are currently being conducted in phase II studies, but these data demonstrate that the viral vector approach is a promising one. Additional vaccine approaches use peptides, either alone or loaded onto DCs (see below). The challenge is to identify an immunogenic peptide. PSA, prostate-specific membrane antigen (PSMA), PAP, and prostate stem cell antigen (PSCA) are all expressed primarily in prostate cells and the majority of prostate cancer tissues, making them optimal targets for an immune response. However, because these antigens are present in self-cells, it is necessary to break immune tolerance. For example, human leukocyte antigen A2 restricted PSCA peptides able to generate an in vitro tumor-reactive cytotoxic T lymphocyte (CTL) response in vitro have been generated [28]. CD8 T cells recognizing some of these peptides have been detected in the blood of patients with prostate cancer [29]. ANTIBODY-BASED IMMUNOTHERAPY Several antibody-based immunotherapies have been evaluated. CTL-associated antigen (CTLA)-4 targeted therapy has undergone the most extensive evaluation. T-cell activation depends on the T-cell receptor s ability to recognize specific antigenic peptides in the context of major histocompatibility complex molecules expressed by APCs, such as DCs. Additional costimulatory signals are also required to generate a T-cell response. T cells express two related receptors, CD28 and CTLA-4, on their cell surface, and both bind to the same ligands present on APCs. Whereas engagement of the B7 molecule by CD28 stimulates T cells, interactions between CTLA-4 and CD28 inhibit T-cell stimulation. It is hypothesized that the prevention of an interaction between CTLA-4 and its ligand CD28 with an antibody to CTLA-4 will augment immune responses against antigens that would not generally stimulate a robust immune response. Thus, blockade of CTLA-4 represents an important mechanism to potentiate T-cell stimulation and, potentially, antitumor T-cell responses. Tremelimumab (Pfizer, New York) and ipilimumab (Bristol-Myers Squibb, New York) are humanized anti CTLA-4 antibodies that have been tested in multiple malignancies, including melanoma, renal cell carcinoma, colon

4 394 Immunotherapeutics for Prostate Cancer cancer, and prostate cancer. In the first human phase I trial of an anti CTLA-4 antibody in patients with prostate cancer, 14 patients with progressive metastatic CRPC, 50% of whom had received prior chemotherapy, were given one dose of ipilimumab. Two patients demonstrated PSA declines of 50% and two patients demonstrated prolongation of their PSA doubling times [30]. These data suggest that some degree of immune autopriming exists, with an ongoing low-level presentation of antigen, perhaps from basal apoptotic rates, which can then be enhanced with CTLA-4 blockade. In contrast to CTLA-4 targeting antibodies, antibodies focused on other, tumor antigen, targets have also been evaluated. For example, PSMA is a protein that is preferentially expressed on the surface of prostate cancer cells. Several monoclonal antibodies targeting this protein have now been developed. Bander et al. [31] reported results of a phase I study using the 177 lutetium-labeled anti-psma monoclonal antibody J591. Of 35 patients receiving the agent, no patient had a detectable antibody response; however, four patients demonstrated a 50% decline in PSA, suggesting that further evaluation of these agents is warranted. However, the best target on which to focus these efforts has not yet been determined. COMBINATION THERAPY Basal autopriming is unlikely to be enough to stimulate a sufficient immune response to generate enough antitumor activity to control disease. Therefore, multiple different approaches to enhancing the immune response are being evaluated. As discussed above, GM-CSF is thought to expand the DC population, thereby enhancing antigen presentation. Currently, a phase I trial combining ipilimumab with GM- CSF, as a means of putatively enhancing antigen presentation, is under way [32]. At the highest dose of ipilimumab reported to date (3 mg/kg), three of six patients had 50% PSA responses, and one patient had a partial response in liver metastases. No patients at lower dose levels had responses. Taken together these data suggest that the combination of GM-CSF and ipilimumab has clinical activity. However, two of the three patients who had PSA responses also had some evidence of autoimmunity, with National Cancer Institute Common Toxicity Criteria grade 3 panhypopituitarism and rash in one and grade 3 colitis in another. The patient with panhypopituitarism responded to replacement hormone therapy and the patient with colitis responded to pulsed high-dose steroids. Such episodes of autoimmunity, which have been termed immune-related adverse events (IRAEs), appear to correlate with responses in other malignancies [33]. Multiple additional IRAEs have been observed in other studies involving ipilimumab, including hepatitis, uveitis, and vitiligo. Although these events are generally manageable with immunosuppression and replacement hormone therapy, they can be severe and represent a challenge both to the clinical use of immunotherapy and to the dogma that immune-based therapy represents a less toxic anticancer therapeutic approach. A phase I trial of the combination of ipilimumab and GVAX (see above) has also been initiated [34]. The results of that study have been promising, with five of six patients treated at the highest dose in the phase I study achieving a 50% decline in PSA level and four of those responses being durable for 2 6 months after the completion of therapy. However, of the 10 patients in the expansion cohort for which data are reported, only one PSA response was observed. The minimal single-agent activity of ipilimumab observed thus far suggests that combination therapy is likely indicated, but it is not yet clear whether GVAX will play this role. Radiotherapy has also been combined with immunotherapy because radiation has been found to augment the immune response to prostate cancer [35]. Transgenic mice with prostate cancer that express influenza hemagglutinin, a unique tumor-associated antigen, under the control of a prostate-specific promoter, were given local radiation in combination with CD4 T cells and a recombinant vaccinia vaccine as immunotherapy. Neither radiation nor immunotherapy alone elicited an antitumor response in animals with evolving tumors. However, the combination was able to incite antitumor T-cell activation, although only with specific timing of the administration of immunotherapy 3 5 weeks following radiation. This suggests that a combination approach may be needed to take full advantage of the potential of immunotherapy. As such, ipilimumab is being combined with radiation therapy to sites of bony disease in a phase I dose escalation study. This is done with the theory that it takes advantage of the poorly understood immune, or abscopal, effects of radiation as well as possibly causing release of an antigen source from the bone [36]. Initially, the dose of ipilimumab was escalated, with later cohorts receiving a single dose of radiation to up to three bony sites of disease. In this study, radiation is delivered hours prior to the first dose of ipilimumab, and ipilimumab is repeated every 3 weeks for four doses. Seven of 33 patients in the dose-escalation portion of the study experienced a confirmed 50% PSA decline, with two of 13 responses occurring in patients who received radiation. In a recently published study in a transgenic adenocarcinoma of the mouse prostate model, prostate cancer specific CD4 T cells were only generated when the radiation was delivered 3 5 weeks prior to ipilimumab; there was no detectable immune response if these

5 Harzstark, Small 395 therapies were delivered earlier or later [35]. These results suggest that some additional refinement of the timing of delivery of these therapies is necessary. Chemotherapy has also been added to CTLA-4 blockade with the theory that it results in the release of dying prostate cancer cells, which release antigen to stimulate an immune response, although there is no confirmation of this presumed mechanism. In a study of 43 chemotherapy-naïve patients with CRPC, all patients received ipilimumab at a dosage of 3 mg/kg every 4 weeks for four doses [37]. Patients on one arm also received a single infusion of docetaxel at a dosage of 75 mg/m 2 on day 1 of therapy. Six patients, three in each arm, experienced a PSA decline 50%. Three patients, one of whom experienced a PSA response, had events that were thought to be IRAEs. Although this small study is certainly not definitive evidence to suggest that chemotherapy does not have the potential to enhance the activity of CTLA-4 blockade, it does not suggest a benefit. Efforts have focused on the evaluation of more promising adjuvants to CTLA-4 blockade therapy. Standard therapies, such as chemotherapy, have also been added to other immunotherapies. Although there were theoretical concerns that the administration of chemotherapy would blunt the immune response, rendering immunotherapy less effective, these concerns have not been borne out in studies completed to date. For example, a randomized phase II study was performed in which patients with metastatic CRPC were administered a recombinant vaccinia virus vaccine expressing the PSA gene alone or with concurrent docetaxel [38]. In that study, T-cell precursors increased in response to PSA by the same amount in both arms, suggesting that docetaxel does not inhibit vaccinespecific T-cell responses. Similar work done in a murine melanoma model suggested that docetaxel does not impede T-cell activation in response to a GM-CSF secreting tumor cell vaccine [39]. DC-BASED IMMUNOTHERAPY DCs, which are the most potent APCs, are known to be deficient in number and functional activity in patients with cancer [40]. Presentation of antigen under ex vivo conditions is an effective method of improving the T-cell immune response [41]. In addition, as noted above, breaking tolerance is a significant challenge with immunotherapies that use self-antigens, and DCs are capable of breaking peripheral tolerance, which is a significant potential advantage to their use in immunotherapy. DCs have been loaded with several different tumor antigens, including PSMA, PAP, and PSA, to attempt to stimulate a T-cell response [42, 43]. The most promising treatment to date has been with sipuleucel-t (Dendreon, Inc., Seattle WA), a treatment that uses PAP-loaded DCs. To produce sipuleucel-t, human PAP is fused to a GM-CSF cassette, which targets the protein to cells expressing the GM-CSF receptor. This facilitates internalization and processing of PAP antigen. The results of a randomized, placebo-controlled phase III study of sipuleucel-t in 127 men with metastatic CRPC (both chemotherapy-naïve and chemotherapy-treated patients were eligible) have been reported [44]. The primary endpoint was time to progression (TTP), which did not include PSA progression. The median TTP for sipuleucel-t was 11.7 weeks, compared with 10.0 weeks for placebo (p.052). Survival was not an endpoint; however, despite the fact that the primary endpoint of the study was not met, a survival benefit was observed. The 3-year overall survival rate was 34% in the sipuleucel-t group, versus 11% in the placebo group (p.0046). Treatment was well tolerated, with the only statistically significant differences in toxicities between the two groups being more rigors, pyrexia, tremors, and feeling cold in the sipuleucel-t group. Based on the above results, the U.S. Food and Drug Administration (FDA) s Office of Cellular, Tissue and Gene Therapies Advisory Committee recommended to the FDA that there was substantial evidence supporting both the efficacy and safety of sipuleucel-t. However, the FDA concluded that additional evidence, either from an interim or final survival analysis of an ongoing study, would be required before approval. There is, therefore, an ongoing phase III study of sipuleucel-t versus placebo in patients with metastatic CRPC, which has a primary endpoint of overall survival. Interim results were released in October of 2008 and reported a hazard ratio of 1.20 for survival favoring the sipuleucel-t arm. This did not meet prespecified stopping criteria and the study is ongoing. OTHER IMPORTANT ISSUES TO CONSIDER Evaluation of Immune-Based Therapies Evaluating immune-based therapies poses additional challenges for clinical trial design, similar to the challenges posed by other biologic therapies. As discussed in the recent PSA Working Group (PSAWG) 2 Criteria, biologic agents may require a particularly long period of time to demonstrate their benefit, given the time required to develop an immunologic response [45]. As such, studies evaluating immunotherapy are less likely to miss a biologic effect (if present) if a minimum exposure to drug of 12 weeks is specified in the protocol, with early progression in PSA being excluded as a criterion for study removal in the absence of symptomatic progression. In the ongoing study of radiation combined with ipilimumab, one patient s initial

6 396 Immunotherapeutics for Prostate Cancer response did not occur until 23 weeks into treatment [36]. Therefore, it was critical that, in the absence of symptomatic progression, the patient was allowed to continue on study. In addition, the PSAWG 2 Criteria recommend continuation of therapy in equivocal cases, with patients removed from therapy only for evidence of clear, symptomatic progression. This allows for the possibility of late responses and avoids removing a patient from a potentially effective therapy before it has been given a chance. Evaluating for evidence of clear, symptomatic progression creates multiple obstacles in patients with advanced disease. For example, there is a risk for progression while patients are being monitored for response over a period of months. This is in contrast to chemotherapy, which most often demonstrates benefit over a period of weeks. For this reason, immunotherapy is unlikely to be an appropriate therapeutic option for patients with bulky or rapidly progressive disease. In addition, it is often difficult for patients, as well as their practitioners, to patiently wait for evidence of clear objective progression in the face of a rising PSA level. Considerable time and effort from the practitioner are required to ensure patients understand the plan and why it is crucial to evaluate the patient rather than the PSA level. With the current culture, it may be difficult to not check the PSA level regularly, but this would likely decrease patient anxiety in immunotherapy studies in which a change in PSA is not a criterion for removal from study therapy. This is beginning to make its way into clinical trial design but has not yet become a standard part of clinical practice. In addition, evaluating therapies in prostate cancer is always hampered by the predominance of bone metastases, and a response in bone metastases cannot be adequately measured using current imaging modalities. PSA may be a less reliable measure of outcome for immune-based therapies. Additionally, the Response Evaluation Criteria In Solid Tumors, which are the most widely used criteria to evaluate response in clinical studies, may have less utility because immune-based therapy may be associated with more stable disease than clinical responses. Finally, there is no validated gold standard for immune monitoring in immunotherapy studies [46]. Delayed-type hypersensitivity, enzyme-linked immunosorbent spot, and tetramer analysis/t-cell proliferation have all been used as measurements of the immune response. However, it is important to note that the clinical significance of these responses is unclear. Rare studies have revealed a correlation between an immune response and TTP and survival [44, 47]. Although evidence of an immune response is necessary to an immunotherapy, it is far from an acceptable surrogate for clinical response, and the optimal method of measurement of this response remains to be determined. Duration of Effect The greater death rate on the GVAX and sipuleucel-t arms of clinical studies raise important concerns that the use of immunotherapy, which has offered the hope of a less toxic alternative to chemotherapy, may in fact be limited by its toxicity. The possibility of more durable responses associated with immunotherapy than with chemotherapy still remains, with durable responses demonstrated in multiple clinical studies of immunotherapies. For example, of 139 patients with melanoma who were reported to have undergone treatment with ipilimumab, three patients had complete responses, which were durable for 29,52, and 53 months [48]. Although cure continues to remain an unlikely goal, long-term control may be possible and is certainly the hope. Neoadjuvant Therapy Most of these therapies are being tested in the metastatic setting, when disease burden is high and disease is resistant to standard therapies. Although this makes sense from a regulatory standpoint, it may not be the way these therapies achieve their highest efficacy. In the above-noted radiation plus ipilimumab study, responses occurred in six of 23 (26%) chemotherapy-naïve patients and only one of 10 (10%) chemotherapy-experienced patients. Although the results from this phase I study cannot be directly compared, they suggest that the effect may not be greatest in patients with more resistant disease. Evaluation in the neoadjuvant setting, when disease burden is low and disease is least resistant, may be the most appropriate biologically. This also offers the advantage of obtaining tissue for correlative work to better understand the activity of these agents. The potential toxicity of some of these therapies, such as ipilimumab, as discussed above, may limit the activity of these agents if their toxicity cannot be uncoupled from their activity. However, agents such as GM-CSF and sipuleucel-t, which are fairly well tolerated, may be very appropriate in this setting. These agents are both currently being evaluated in neoadjuvant studies with radical prostatectomy specimens by flow cytometry and immunohistochemistry for immune activity [49]. CONCLUSIONS Much remains unknown about the best use of the immunebased therapies in prostate cancer, including the optimal disease state for their usage, the timing of their usage, whether the best antigens to target have been identified, and how to uncouple toxicity from activity. In addition, the role of the tumor microenvironment in the efficacy of immunotherapy is being explored. The tumor microenvironment provides multiple factors vital for the growth of tumors, including survival signals, growth factors, angiogenic fac-

7 Harzstark, Small 397 tors, and adhesion molecules [50]. Control of these factors will be vital in the clinical application of immunotherapy. The future of immune-based therapy will likely be dependent on a better understanding of the biology of these agents, which will allow their activity to be focused. Although the hope for these agents has been that their toxicity profile will be better than the toxicity profile of chemotherapy, this may not prove to be true. Studies of both GVAX and ipilimumab have demonstrated higher death rates and/or incidences of toxicity in the treatment arms, suggesting that the application of these agents may be limited by their toxicity as well as their efficacy. In addition, a greater emphasis on animal models with which to evaluate these agents, as well as a better understanding of the immune effects of currently existing therapies, like androgen-deprivation therapy and possibly bevacizumab, will likely play a role in the development of these agents. AUTHOR CONTRIBUTIONS Conception/design: Andrea L. Harzstark, Eric J. Small Administrative support: Andrea L. Harzstark, Eric J. Small Manuscript writing: Andrea L. Harzstark, Eric J. Small Final approval of manuscript: Andrea L. Harzstark REFERENCES 1 Sanda MG, Ayyagari SR, Jaffee EM et al. Demonstration of a rational strategy for human prostate cancer gene therapy. J Urol 1994;151: Wang X, Yu J, Sreekumar A et al. Autoantibody signatures in prostate cancer. N Engl J Med 2005;353: Fagerberg J. Granulocyte-macrophage colony-stimulating factor as an adjuvant in tumor immunotherapy. Med Oncol 1996;13: Schulz G, Frisch J, Greifenberg B et al. New therapeutic modalities for the clinical use of rhgm-csf in patients with malignancies. Am J Clin Oncol 1991;14(suppl 1):S19 S26. 5 Lanza F, Rigolin GM, Castagnari B et al. Potential clinical applications of rhgm-csf in acute myeloid leukemia based on its biologic activity and receptor interaction. Haematologica 1997;82: Small EJ, Reese DM, Um B et al. Therapy of advanced prostate cancer with granulocyte macrophage colony-stimulating factor. Clin Cancer Res 1999; 5: Young MR, Wright MA, Lozano Y et al. Mechanisms of immune suppression in patients with head and neck cancer: Influence on the immune infiltrate of the cancer. Int J Cancer 1996;67: Young MR, Wright MA, Pandit R. Myeloid differentiation treatment to diminish the presence of immune-suppressive CD34 cells within human head and neck squamous cell carcinomas. J Immunol 1997;159: Young MR, Wright MA, Lozano Y et al. Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34 natural suppressor cells. Int J Cancer 1997;74: Gjertsen MK, Buanes T, Rosseland AR et al. Intradermal ras peptide vaccination with granulocyte-macrophage colony-stimulating factor as adjuvant: Clinical and immunological responses in patients with pancreatic adenocarcinoma. Int J Cancer 2001;92: Dillman RO, Wiemann M, Nayak SK et al. Interferon-gamma or granulocyte-macrophage colony-stimulating factor administered as adjuvants with a vaccine of irradiated autologous tumor cells from short-term cell line cultures: A randomized phase 2 trial of the cancer biotherapy research group. J Immunother 2003;26: Simmons SJ, Tjoa BA, Rogers M et al. GM-CSF as a systemic adjuvant in a phase II prostate cancer vaccine trial. Prostate 1999;39: Malinowska K, Neuwirt H, Cavarretta IT et al. Interleukin-6 stimulation of growth of prostate cancer in vitro and in vivo through activation of the androgen receptor. Endocr Relat Cancer 2009;16: Wilson C, Wilson T, Johnston PG et al. Interleukin-8 signaling attenuates TRAIL- and chemotherapy-induced apoptosis through transcriptional regulation of c-flip in prostate cancer cells. Mol Cancer Ther 2008;7: Hastie C, Masters JR, Moss SE et al. Interferon-gamma reduces cell surface expression of annexin 2 and suppresses the invasive capacity of prostate cancer cells. J Biol Chem 2008;283: Fang P, Hwa V, Little BM et al. IGFBP-3 sensitizes prostate cancer cells to interferon-gamma-induced apoptosis. Growth Horm IGF Res 2008;18: Simons JW, Sacks N. Granulocyte-macrophage colony-stimulating factortransduced allogeneic cancer cellular immunotherapy: The GVAX vaccine for prostate cancer. Urol Oncol 2006;24: Warren TL, Weiner GJ. Uses of granulocyte-macrophage colony-stimulating factor in vaccine development. Curr Opin Hematol 2000;7: Halabi S, Small EJ, Kantoff PW et al. Prognostic model for predicting survival in men with hormone-refractory metastatic prostate cancer. J Clin Oncol 2003;21: Simons JW, Small E, Nelson W et al. Phase II trials of a GM-CSF genetransduced prostate cancer cell line vaccine (GVAX) demonstrate antitumor activity. Proc Am Soc Clin Oncol 2001;20: Simons J, Nelson W, Nemunaitis J et al. Phase II trials of a GM-CSF genetransduced prostate cancer cell line vaccine (GVAX) in hormone refractory prostate cancer. Proc Am Soc Clin Oncol 2002;21: Small EJ, Higano C, Smith D et al. A phase II study of an allogeneic GM- CSF gene-transduced prostate cancer cell line vaccine in patients with metastatic hormone-refractory prostate cancer. Presented at the 2005 Prostate Cancer Symposium, Orlando, FL, February 17, Cell Genesys, Inc. Cell Genesys Completes Patient Recruitment for First Phase 3 Clinical Trial of GVAX Immunotherapy for Prostate Cancer. Available at &p irolnewsarticle&id &highlight, accessed February 1, 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: Freund YR, Mirsalis JC, Fairchild DG et al. Vaccination with a recombinant vaccinia vaccine containing the B7 1 co-stimulatory molecule causes no significant toxicity and enhances T cell-mediated cytotoxicity. Int J Cancer 2000;85: Hodge JW, Schlom J, Donohue SJ et al. A recombinant vaccinia virus expressing human prostate-specific antigen (PSA): Safety and immunogenicity in a non-human primate. Int J Cancer 1995;63: DiPaola RS, Plante M, Kaufman H et al. A phase I trial of pox PSA vaccines (PROSTVAC-VF) with B7 1, ICAM-1, and LFA-3 co-stimulatory molecules (TRICOM) in patients with prostate cancer. J Transl Med 2006;4:1.

8 398 Immunotherapeutics for Prostate Cancer 28 Kiessling A, Schmitz M, Stevanovic S et al. Prostate stem cell antigen: Identification of immunogenic peptides and assessment of reactive CD8 T cells in prostate cancer patients. Int J Cancer 2002;102: Matsueda S, Kobayashi K, Nonaka Y et al. Identification of new prostate stem cell antigen-derived peptides immunogenic in HLA-A2( ) patients with hormone-refractory prostate cancer. Cancer Immunol Immunother 2004;53: Small EJ, Tchekmedyian NS, Rini BI et al. A pilot trial of CTLA-4 blockade with human anti-ctla-4 in patients with hormone-refractory prostate cancer. Clin Cancer Res 2007;13: Bander NH, Milowsky MI, Nanus DM et al. Phase I trial of 177 lutetiumlabeled J591, a monoclonal antibody to prostate-specific membrane antigen, in patients with androgen-independent prostate cancer. J Clin Oncol 2005;23: Fong L, Kavanagh B, Hou Y et al. Combination immunotherapy with GM- CSF and CTLA-4 blockade for hormone refractory prostate cancer: Balancing the expansion of activated effector and regulatory T cells. J Clin Oncol 2007;25(suppl 18): Reuben JM, Lee BN, Li C et al. Biologic and immunomodulatory events after CTLA-4 blockade with ticilimumab in patients with advanced malignant melanoma. Cancer 2006;106: Gerritsen W, van den Eertwegh AJ, de Gruijl T et al. Expanded phase I combination trial of GVAX immunotherapy for prostate cancer and ipilimumab in patients with metastatic hormone-refractory prostate cancer (mhprc). J Clin Oncol 2008;26(18 suppl): Harris TJ, Hipkiss EL, Borzillary S et al. Radiotherapy augments the immune response to prostate cancer in a time-dependent manner. Prostate 2008;68: Beer TM, Slovin SF, Higano CS et al. Phase I trial of ipilimumab (IPI) alone and in combination with radiotherapy (XRT) in patients with metastatic castration resistant prostate cancer (mcrpc). J Clin Oncol 2008;28(suppl 15): Small E, Higano C, Tchekmedyian N et al. Randomized phase II study comparing 4 monthly doses of ipilimumab (MDX-010) as a single agent or in combination with a single dose of docetaxel in patients with hormonerefractory prostate cancer. J Clin Oncol 2006;24(suppl 18): Arlen PM, Gulley JL, Parker C et al. A randomized phase II study of concurrent docetaxel plus vaccine versus vaccine alone in metastatic androgenindependent prostate cancer. Clin Cancer Res 2006;12: Prell RA, Gearin L, Simmons A et al. The anti-tumor efficacy of a GM- CSF-secreting tumor cell vaccine is not inhibited by docetaxel administration. Cancer Immunol Immunother 2006;55: Tas MP, Simons PJ, Balm FJ et al. Depressed monocyte polarization and clustering of dendritic cells in patients with head and neck cancer: In vitro restoration of this immunosuppression by thymic hormones. Cancer Immunol Immunother 1993;36: Carlsson B, Totterman TH, Essand M. Generation of cytotoxic T lymphocytes specific for the prostate and breast tissue antigen TARP. Prostate 2004;61: Simons JW, Mikhak B, Chang JF et al. Induction of immunity to prostate cancer antigens: Results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer. Cancer Res 1999;59: Tjoa BA, Simmons SJ, Elgamal A et al. Follow-up evaluation of a phase II prostate cancer vaccine trial. Prostate 1999;40: Small EJ, Schellhammer PF, Higano CS et al. Placebo-controlled phase III trial of immunologic therapy with sipuleucel-t (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. J Clin Oncol 2006;24: Scher HI, Halabi S, Tannock I et al. Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: Recommendations of the Prostate Cancer Clinical Trials Working Group. J Clin Oncol 2008;26: Thomas-Kaskel AK, Waller CF, Schultze-Seemann W et al. Immunotherapy with dendritic cells for prostate cancer. Int J Cancer 2007;121: Thomas-Kaskel AK, Zeiser R, Jochim R et al. Vaccination of advanced prostate cancer patients with PSCA and PSA peptide-loaded dendritic cells induces DTH responses that correlate with superior overall survival. Int J Cancer 2006;119: Downey SG, Klapper JA, Smith FO et al. Prognostic factors related to clinical response in patients with metastatic melanoma treated by CTL-associated antigen-4 blockade. Clin Cancer Res 2007;13: Fong L, et al. Neoadjuvant immunotherapy for prostate cancer with GM- CSF and tumor infiltration by antigen presenting cells ASCO Annual Meeting Proceedings, 2008;28(suppl 15): Disis ML, ed. Immunotherapy of Cancer. Totowa, NJ: Humana Press, 2006:1 517.