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Cancer and Metastasis Reviews 18: 437–449, 1999. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Target antigens for prostate cancer immunotherapy Douglas C. Saffran1 , Robert E. Reiter2∗, Aya Jakobovits1 and Owen N. Witte3† UroGenesys, Inc., Santa Monica, CA, USA; 2 Department of Urology, 3 Departments of Microbiology and Molecular Genetics and Molecular Biology Institute and Howard Hughes Medical Institute, University of California, Los Angeles, CA, USA 1 Key words: prostate cancer, monoclonal antibody therapy, cancer vaccines, prostate antigens Abstract The detection and treatment of prostate cancer has been markedly improved by the use of Prostate-Specific Antigen (PSA) as a serological biomarker for disease. However, even after surgical intervention and hormone ablation therapy, a significant proportion of patients progress to advanced metastatic disease, for which there is no cure. An important goal has become the identification of antigens in advanced stage prostate cancer that represent targets for therapy. Recently, great progress has been made to utilize immunological therapies to treat cancer. Monoclonal antibody therapy has been successfully approved for the treatment of breast cancer and B-cell lymphoma, and multiple clinical trails are currently in progress in a variety of cancers, including prostate cancer. Pre-clinical and clinical studies are also underway to evaluate cancer vaccine approaches directed against antigens that are highly expressed in prostate and other cancers. This article describes several target antigens expressed in prostate cancer and immunological approaches directed against them that may be effective for treating prostate cancer patients. Introduction Prostate cancer (CaP) is the most commonly diagnosed cancer and is the second leading cause of cancer related deaths in American males [1]. Although several curative therapies exist for localized disease, such as radical prostatectomy, radiation therapy, and cryotherapy, approximately one-third of treated patients will relapse [2,3]. Since CaP is dependent upon androgens for growth, treatment for advanced, metastatic disease is systemic androgen deprivation therapy. Initially, a large percentage of patients show clinical improvement, but androgen-independent (AI) clones eventually develop in most patients at which time the disease is incurable [3]. Clearly, new therapeutic approaches need to be considered in addition to conventional therapy for patients at this stage of disease. One alternative approach to treat late-stage CaP is immunotherapy. The earliest immunological approach ∗ R.E.R. is supported by grants from the Cancer Research Institute and CapCure and is the recipient of an NIH K08 award. † O.N.W. is supported by grants from the Cancer Research Institute and CapCure. to treat advanced CaP was adjuvant immunotherapy with Bacillus Calmette-Geurin, BCG [4,5]. This has since been followed by many additional approaches including vaccination with mucins and carbohydrate antigens, use of dendritic cell vaccines, use of genetically-engineered tumor cells as vaccines, and administration of cytokines (reviewed in [2]). Recently, molecular biological approaches have been used to identify genes that are expressed during CaP progression. Many novel antigens have been identified which are prostate-specific and represent targets for immunotherapy. This review will focus on several of those targets and will describe their utilization in the development of either monoclonal antibody (MAb) or vaccine therapies for advanced CaP. Target antigens expressed in prostate cancer The identification of target antigens in CaP, especially in advanced disease, is critical to the development of immune-based therapies. Target antigens should exhibit one or more of the following features to make 438 them ideal candidates for immunological therapy: (1) The antigen should be prostate-specific, expressed at high levels in CaP, and not be expressed in essential organs; (2) The antigen should be expressed on the cell surface, where it is susceptible to recognition by either naked or conjugated antibodies; (3) Cell surface proteins which play a potential role in cell growth are desired targets for antibody-mediated inhibition; and (4) The antigen needs to be accessible to antigenpresenting cells for processing into MHC Class I and II molecules. Table 1 highlights several antigens that are overexpressed in CaP, some of which are currently being evaluated in pre-clinical and clinical studies. These proteins fulfill some, but not all of the criteria for an ideal target antigen. The two most promising cellsurface antigens for CaP are Prostate-Specific Membrane Antigen (PSMA) and Prostate Stem Cell Antigen (PSCA). These antigens represent attractive candidates for immunotherapy mainly due to their ability to be targeted by antibodies. MAb therapy is the only immunological approach confirmed to date to be effective for treating cancer, examples being anti-CD20 (Rituxan) in B-cell lymphoma and anti-HER-2/neu (Herceptin) in breast cancer. However as will be described even secreted proteins, such as Prostate-Specific Antigen (PSA) and Prostatic Acid Phosphatase (PAP), have been used as targets for antibody-directed drug therapies in experimental studies. It is likely that the most effective immune-based therapies will be able to activate or utilize both the humoral and cellular arms of the immune system to induce the greatest anti-tumor effect. Table 1. Target antigens for CaP immunotherapy Target antigen (a) Cell surface proteins PSMA PSCA HER-2/neu (b) Secreted proteins PSA PAP (c) Intracellular (nuclear) proteins PAGE GAGE Function Potential immune therapy Homology to NAALADase Unknown MAb Cancer vaccine MAb Cancer vaccine MAb Cancer vaccine Activated tyrosine kinase receptor Serine protease Acid phosphatase Unknown Unknown MAb conjugate Cancer vaccine MAb conjugate Cancer vaccine Cancer vaccine Cancer vaccine Also, it may not be a single antigen but a combination of the prostate antigens described below which are most effective at inducing therapeutic immune responses against CaP. A. Cell surface proteins Prostate-Specific Membrane Antigen PSMA was originally identified as a result of the generation of specific MAbs against membrane preparations of the CaP cell line, LNCaP [6]. One of the resulting hybridoma clones, 7E11-C5.3, was specific for the cell surface of LNCaP cells as well as the epithelium of normal and malignant prostate tissue sections. No staining of a large panel of normal and cancerous cell lines or tissues was detected, except normal kidney where 2/14 sections stained positive. PSMA was eventually cloned from a cDNA library of LNCaP cells using degenerate oligonucleotide primers generated by microsequencing of protein fragments identified by the MAb [7]. PSMA mRNA expression agreed with antibody staining of CaP tissue sections and expression levels increased with advanced disease [8]. The PSMA gene encodes for a 750 amino acid (aa) protein that is a type II integral membrane protein with a short amino-terminal cytoplasmic domain and a large, extracellular carboxyl-terminal domain containing N-acetylated α-linked acidic dipeptidase (NAALADase) hydrolase activity [9]. The presence of a hydrolase domain suggests that PSMA might function by hydrolyzing specific peptides in prostatic fluid or the surrounding environment. The original paper by Horoszewicz et al. also demonstrated that circulating PSMA could be detected in the serum of approximately 50% of CaP patients but not in normals [6]. This suggests that PSMA shed from the membrane of malignant prostate epithelial cells may serve as a serum-based marker for diagnosis or prognosis of CaP in combination with PSA. An alternative splice variant of PSMA, called PSM0 , has been identified in which the 50 end, including the cytoplasmic and transmembrane domains, is deleted [8,10]. PSM0 was believed to be cytoplasmic since it lacks an apparent signal sequence. This has been confirmed using a combination of the 7E11-C5.3 MAb, which recognizes an intracellular epitope consisting of the first six aminoterminal aa, and a second MAb against the extracellular portion of the protein [11]. This study and one other using the 7E11-C5.3 MAb reported that the PSMA protein was located in plasma membrane fractions of 439 LNCaP cells, although staining could also be detected in mitochondrial fractions [12]. Since the 7E1l-C5.3 MAb recognizes an intracellular epitope, there is some controversy as to whether it detects cell-surface PSMA expression on viable or apoptotic/necrotic cells. Troyer et al. demonstrated that 7E11-C5.3 could only bind to permeabilized and fixed but not viable LNCaP cells [12]. In contrast, Barren et al. reported that the 7E11-C5.3 MAb could stain the surface of either viable or fixed LNCaP cells equally well [13]. The technical differences to account for this discrepancy are unclear. In humans the 7E1l-C5.3 antibody has been evaluated and approved for in vivo imaging of CaP recurrences after surgical, hormonal, or radiation treatment [14,15]. This product, referred to as ProstaScint, is comprised of the 7E11-C5.3 MAb conjugated with 111 Indium. In spite of the fact that ProstaScint is a murine MAb, human anti-mouse antibody (HAMA) responses have only been reported in less than 5% of imaged patients. Troyer et al. argue that since the PSMA epitope that ProstaScint recognizes is cytoplasmic, only necrotic tissue but not viable micrometastases will be detected [12]. Although the 7E1l-C5.3 MAb can recognize PSMA under different circumstances, it is likely that MAbs that recognize the extracellular domain of PSMA may be more effective at either in vivo imaging or MAb directed therapy. Several groups have recently developed second generation MAbs against the extracellular portion of PSMA [11,16–18]. In these cases mice were immunized with preparations of LNCaP cell membranes as was done for the generation of the 7E1l-C5.3 MAb. Murphy et al. derived five anti-PSMA MAbs that recognized distinct regions of the PSMA extracellular domain [17]. These antibodies stained the surface of unfixed LNCaP cells and also recognized baculovirusprepared PSMA protein in a sandwich ELISA format. Bander and colleagues derived four anti-PSMA MAbs that recognized distinct extracellular PSMA epitopes [16,18,19]. Viable LNCaP cells were stained on the cell surface with all of the MAbs. Internalization studies on LNCaP cells demonstrated endocytosis of the MAbs via clathrin-coated pits [19]. In addition cell surface biotinylation experiments showed that PSMA underwent constitutive endocytosis in LNCaP cells, although not to as great a level as mediated by the MAbs. The fact that the MAbs are internalized after incubation with LNCaP cells suggests a potential mechanism to deliver toxic drugs or radioisotopes into prostate tumors. This panel of MAbs was also evaluated for the ability to recognize PSMA in CaP and other tissues by immunohistochemistry [18]. The MAbs recognized PSMA on several normal tissues including benign prostate epithelial cells, duodenal epithelium, renal tubular epithelium, colonic ganglion cells, and benign breast epithelium. Interestingly, the MAbs recognized not only CaP epithelial cells (12/12 clinical specimens), but also the tumor vasculature of prostate, kidney, bladder, testicular, colon, brain, melanoma, pancreas, lung, soft tissue sarcoma, and breast cancers [16,18]. This suggests that in addition to using anti-PSMA antibodies to target CaP epithelial cells, a broader application may be targeting to the neovasculature of multiple tumor types. Murphy and colleagues have evaluated the immunogenicity of PSMA in vitro that has led to the initiation of clinical trials in CaP. They demonstrated that dendritic cells (DC) from a CaP patient pulsed with an HLA-A2 specific PSMA peptide could stimulate autologous T-cell proliferation [20]. They later reported that either intact PSMA derived from LNCaP membranes or baculovirus-derived PSMA could also stimulate proliferation of T-cells from either healthy donors or CaP patients [21]. A Phase I clinical trial was initiated to assess administration of autologous DC pulsed with PSMA peptides on patients with advanced, hormoneresistant disease [22]. Seven partial responders were observed out of 51 patients in the trial that had durable responses of at least 100–200 days after treatment [23]. The criteria for clinical responsiveness included lymphocyte and hematocrit levels as well as alkaline phosphatase and prostate marker (PSA, percent free PSA, and PSMA) levels. A Phase II trial followed that included patients with either metastatic disease or patients that had locally recurrent disease after failure of primary treatment [24,25]. In both cases, about 30% of the patients showed either a partial or complete response at the end of the study. The average duration of the responses was > 150 days and 58% of the responders were still responsive at the end of the study. The combined results of this study suggests that PSMA peptide-pulsed DC provide an alternative therapy for advanced CaP, and also should facilitate the commencement of trials with other CaP specific antigens. Prostate Stem Cell Antigen PSCA was discovered using the recently described LAPC-4 xenograft model in an effort to identify genes associated with CaP progression [26,27]. The LAPC-4 xenograft was originally derived from a lymph 440 node metastasis of a patient with advanced, hormonerefractory disease. This model consists of sub-lines that display progression from androgen-dependent (AD) to AI growth with associated micrometastases [27]. PSCA was identified using representational difference analysis (RDA) comparing differential gene expression in the LAPC-4 AD and AI sublines. The PSCA gene encodes for a 123 aa protein with an aminoterminal signal sequence and a carboxyl-terminal GPIanchor sequence. PSCA is 30% homologous to the SCA-2 gene, also called RIG-E, that is a member of the Ly-6 family of GPI-anchored cell-surface proteins [28]. A mouse homologue of PSCA was also identified with 70% identity to the human gene. In normal tissues, PSCA mRNA is expressed in prostate, and at lower levels in placenta. Although PSCA was identified from an LAPC-4 AD/AI comparison, mRNA levels are up-regulated in both xenografts and clinical specimens, and expression is significantly higher than seen in normal prostate. In situ hybridization analysis performed on multiple normal tissue sections revealed PSCA mRNA expression was restricted to the basal cell layer of epithelial cells, the precursor population for the more differentiated secretory cells. A similar analysis of CaP tissue sections demonstrated that PSCA mRNA is expressed in malignant epithelial cells in 102/126 (81%) specimens analyzed, and that expression was consistently higher in cancerous glands than in normal glands. The fact that PSCA remains expressed at all stages of disease suggests its utility as a target for early or late stage CaP. Polyclonal antibodies derived against human PSCA demonstrated the cell-surface expression of the protein [26]. More recently, a panel of monoclonal anti-PSCA antibodies was derived that recognized both the native and denatured protein [29]. Similar to the polyclonal Ab, the anti-PSCA MAbs recognize PSCA on the surface of either transfected cells or LAPC xenografts that overexpress PSCA. These antibodies have been used to examine PSCA expression by immunohistochemistry on normal and CaP tissues. PSCA protein was expressed on normal prostate basal and epithelial cells and also on transitional epithelial cells in normal bladder (R. Reiter, personal communication). CaP expression was observed on 10/10 (100%) advanced cases and on 9/9 (100%) bone metastases, with a higher level of expression correlating with advanced disease [29]. An extended panel of CaP specimens has also been evaluated and PSCA expression was confirmed in 93/112 (84%) of the cases (R. Reiter, personal communication). An example of staining of CaP is shown in Figure 1. Especially striking was the intense homogeneous staining seen in the bone metastatic sample, which was characteristic of all nine bone metastatic samples analyzed. Recent studies suggest that PSCA protein overexpression in advanced CaP is associated with co-amplification of sequences on chromosome 8q, where the PSCA and MYC genes are located [30]. Amplification of PSCA occurred in 5/7 cases where there was also amplification of MYC. In addition, overrepresentation of PSCA at the chromosomal level correlated with overexpression of PSCA protein by immunohistochemical analysis of patient samples. The ability of MAbs to recognize PSCA on the cell surface suggests its utility as target for MAb directed therapies of CaP. The fact that PSCA, like PSMA, is highly expressed in advanced disease including AI CaP is especially important since no effective therapy is available. Currently the function of PSCA is unknown, although work is in progress using transgenic and knock-out strategies in mice to address the issue (O. Witte, R. Reiter, T. Watabe, personal communication). The fact that a mouse homologue exists would also allow for testing of immunogenicity of PSCA as a cancer vaccine in syngeneic murine models of CaP such as TRAMP-C [31]. HER-2/neu Immunotherapy to HER-2/neu has recently gained much attention resulting in FDA approval of a MAb (Herceptin) to treat HER-2 positive tumors in patients with advanced breast cancer. HER-2/neu, also referred to as erbB2, is an oncogenic protein that is a member of the epidermal growth factor receptor (EGFR) family [32]. HER-2/neu is overexpressed in 20–30% of human breast cancer and 60–80% of ductal carcinomas-in situ (DCIS), and in 20–30% of ovarian cancers [33,34]. In normal adult tissues, HER-2/neu is expressed at low levels in skin, digestive tract epithelium, breast, ovary, hepatocytes, and alveoli [35]. Additionally, HER-2/neu is also expressed and has been shown to play a role in fetal development [35]. More recently, expression of HER-2/neu has been examined in normal and cancerous prostate tissues. HER-2/neu expression was found in both normal and cancerous prostate epithelial cells, although conflicting results have been obtained with respect to the frequency of overexpression [36–40]. Two groups have recently described a potential function of HER-2/neu in the acquisition of androgenindependence in CaP [41,42]. In patients treated with hormone ablation therapy, AI tumors arise which continue to express androgen receptor (AR) and also 441 Advanced Prostate Cancer (Gleason Grade 9) Bone Metastasis Figure 1. Examples of PSCA protein expression in CaP by immunohistochemical staining using an anti-PSCA MAb. The panel on the left demonstrates staining (arrows) of a section from a patient with locally advanced CaP. The panel on the right is a representative section from a bone metastasis. Note the intense staining of PSCA in the bone metastatic lesions (arrows). AD genes such as PSA. Investigation of LAPC CaP xenografts revealed that AI sublines expressed higher levels of HER-2/neu than their AD counterparts [41]. Overexpression of HER-2/neu cDNA in the AD cell line LNCaP allowed AI growth and induced expression of PSA through the AR pathway. Similarly, induction of hormone-independent tumor growth in breast cancer cell lines has also been observed as a result of HER-2/neu overexpression [43]. In LNCaP cells, activation of AR function involved the MAP kinase pathway, via phosphorylation of specific tyrosine residues, and promoted interaction between AR and the ARA 70 co-activator [42]. These studies demonstrated that HER-2/neu may play an important functional role in progression to androgen-independence. Since HER-2/neu is overexpressed in a subset of advanced, hormone-refractory CaP it represents a target for immunotherapy. Pre-existing immunity to HER-2/neu has been observed in breast cancer patients, including presence of HER-2/neu specific CTL (reviewed in [34]). In a variety of model systems, HER-2/neu has been shown to induce specific T-cell immunity, suggesting its potential as a cancer vaccine candidate [34]. In the early 1990’s MAb against HER-2/neu were developed which could inhibit growth of breast cancer cell lines both in vitro and in vivo (reviewed in [44]). Further pre-clinical studies demonstrated that a combination of anti-HER-2/neu MAbs and chemotherapy were most effective in eradicating established breast cancer xenografts [45–47]. Based on successful clinical trials, the anti-HER-2 MAb (Herceptin) has been approved in combination with chemotherapy by the FDA for treatment of advanced breast cancer [48–50]. The evaluation of Herceptin in pre-clinical models of CaP, especially AI xenografts, is warranted. HER-2/neu is a member of the EGFR family, and the EGFR has been considered as a target in CaP [51]. Using the chimeric C225 MAb to the EGFR, Prewett et al. demonstrated significant inhibition of growth of established EGFR-positive prostate tumors PC-3 and DU145 in vivo [52]. There was no difference in tumor growth inhibition using the C225 MAb alone or in combination with the chemotherapeutic drug doxorubicin. It remains to be seen whether Herceptin would be most effective as either a monotherapy or in combination with chemotherapy. B. Secreted proteins Prostate-Specific Antigen PSA, originally identified from human seminal plasma, is a 34 kD serine protease and a member of the human kallikrein gene family [53–55]. PSA is produced exclusively in normal and malignant prostate epithelial cells and is normally found at high concentrations in the seminal fluid where it is believed to play a role in liquefaction of the semen [56,57]. Circulating 442 PSA levels are absent or present at low concentrations (0–4 ng/ml) in the serum of normal males, but in patients with benign prostatic hypertrophy (BPH) or CaP, PSA levels rise dramatically making it a useful serum marker for diagnosis [56,58]. Currently PSA is the best available biomarker to diagnose CaP and to follow disease progression after treatment [59]. In addition to its prostate-specificity, PSA is also regulated by androgen and contains distinct androgen-responsive regulatory elements in the promoter [60]. However, in AI CaP, both AR and PSA expression is retained, suggesting a dysregulation of gene expression in hormone-independent CaP possibly via HER-2/neu. The fact that PSA expression is retained in both AD and metastatic AI disease makes it a good target for immune intervention. The ability of PSA to induce specific T-cell mediated immune responses has been evaluated in both murine and human in vitro and in vivo model systems. In one approach, human PSA cDNA was constructed into plasmid DNA for nucleic acid immunization of mice [61]. A murine homologue of human PSA has not yet been identified. Immunized mice demonstrated strong antibody and cell-mediated responses against PSA that lasted for a minimum of three months after immunization. In a different approach, Frelinger and colleagues overexpressed human PSA in murine tumor cells to address its immunogenicity. Immunization of mice with the syngeneic P815 mastocytoma cell line transfected with human PSA cDNA resulted in induction of PSAspecific CTL clones [62]. Additionally, the immunized mice were protected from tumor challenge with a syngeneic, aggressive lung carcinoma cell line, Line 1, also engineered to overexpress human PSA cDNA. This demonstrated the ability of PSA to act as a target antigen for CTL generation in mice. They extended this model system further by creating strains of human PSA transgenic mice [63]. In these mice, PSA expression was mainly confined to ductal epithelial cells in mouse prostate tissue. Challenge of PSA transgenic mice with Line 1/PSA tumor cells resulted in generation of PSA-specific tumor infiltrating lymphocytes (TIL) which had cytotoxic activity against PSA expressing targets in vitro. This demonstrated that in spite of transgene expression, an immune response could still be generated against human PSA. This human PSA transgenic mouse model should be useful in the future for studying mechanisms to overcome tolerance to self antigens, especially if crossed with human CD8/HLA-A2 transgenic mice to examine immune responses in the context of human MHC molecules [64]. The induction of human CTL specific for PSA has been achieved using peptides targeted to the MHC Class I allele HLA-A2 [65–67]. HLA-A2 MHC molecules have been shown to bind 9-mer peptides with specificity that is determined by aa residues at positions 2 and 9 of the peptide [68]. In vitro stimulation of PBL from normal HLA-A2 individuals with the PSA peptide encompassing aa residues 146–154 resulted in induction of PSA-specific CTL [65]. In a separate set of studies, Correale et al. [66,67] demonstrated that PSAspecific CTL could be generated from PBL of normals or a CaP patient using two distinct PSA peptides (aa residues l41–150 and 154–163). The PSA-specific CTL had the ability to lyse either peptide-pulsed target cells or the HLA-A2 positive CaP cell line LNCaP, which endogenously produces PSA. They have also demonstrated that a 30-mer peptide comprising multiple PSA peptide epitopes could be processed and stimulate CTL against the distinct epitopes. These studies demonstrate that human CTL can be generated against PSA, even in a CaP patient, and that multiple epitopes can induce a significant immune response. Recently two clinical trials have been initiated to evaluate the efficacy of PSA as a cancer vaccine in CaP patients. A recombinant vaccinia virus expressing PSA, called PROSTVAC, has been evaluated in a Phase I clinical trial in patients with recurrent CaP after radical prostatectomy [69]. PROSTVAC had previously been shown to induce long-lived PSA-specific T-cell responses after immunization of rhesus monkeys in a preclinical study [70]. In humans, PROSTVAC was well tolerated and at least one patient out of six showed a clinical response (no detectable serum PSA) as a result of the vaccine. In a second Phase I trial, a liposomal preparation of PSA, called OncoVax-P, was evaluated in patients with surgically incurable CaP [71]. OncoVax-P is comprised of baculovirus produced human PSA protein mixed with liposomes and the adjuvant lipid A. Administration of OncoVax-P caused no serious side effects and a significant proportion of patients demonstrated successful immunization against PSA as measured by circulating antibodies and a DTH response. Immunity to the vaccine was only effective in the presence of immunoenhancing agents such as cytokines, BCG, or light mineral oil, but not when administered alone. Both studies demonstrate the potential utility of PSA as a cancer vaccine candidate for CaP. 443 Antibodies against PSA have also been investigated for their ability to target drugs to and inhibit growth of CaP cells and tissues [72,73]. A polyclonal rabbit anti-PSA IgG Ab, conjugated with a labeled derivative of 5-FU was used to stain prostate tissue sections from BPH or CaP patients after radical prostatectomy. The immunoconjugate was specific for prostate epithelial cells in both cases. The anti-PSA antibody was also tested in vivo for its ability to target and affect growth of either PSA-positive LNCaP or PSA-negative DU145 tumors [73]. The conjugated antibodies localized to and were cytotoxic for LNCaP but not DU145 tumors as determined by immunohistochemical analysis of the tumors in treated mice. These studies demonstrated that although PSA is a secreted protein, antibodies against it can localize to prostate epithelial cells and effectively deliver therapeutic drugs. Although the mechanism of uptake of the conjugated antibodies was not determined, it was proposed to be via endocytosis at the cell membrane. This suggests that the antibodies recognized cell-associated PSA and that there was no inhibition of uptake of the immunoconjugate by circulating PSA in LNCaP-tumor bearing mice. Recently, two novel serine proteases similar to PSA were identified, both which are androgen-regulated and overexpressed in CaP. The first, called prostase, was identified from a prostate cDNA library by suppression subtractive hybridization and independently by a positional cloning approach [74,75]. The gene is localized on the same region of chromosome 19 (19q13) as several other serine proteases including PSA (hK3), human glandular kallikrein 2 (hK2), pancreatic/renal kallikrein (hK1), and protease M. Prostase mRNA is highly expressed in normal and malignant prostate tissue and expressed at lower levels in testis, mammary gland, adrenals, uterus, thyroid, and salivary glands [74,75]. The prostase gene encodes a putative secreted 254 aa protein that has 78% aa identity with porcine enamel matrix serine proteinase I and 35% aa identity with PSA [74]. A second serine protease was identified by Nelson and colleagues using androgen-stimulated LNCaP derived mRNA to probe cDNA microarrays [76]. This gene, called TMPRSS2, had previously been identified by exon trapping on chromosome 21 [77]. The TMPRSS2 gene encodes a 492 aa type II integral membrane protein with a serine protease domain of the S1 family, a transmembrane domain, a scavenger receptor cysteine-rich domain, an LDL receptor class A domain, and a cytoplasmic domain. In situ hybridization of clinical samples demonstrated that TMPRSS2 is expressed in the basal cells of normal prostate and in epithelial cells in prostate adenocarcinoma. TMPRSS2 is most closely related to hepsin, a cell surface protease overexpressed in ovarian cancer [78]. Since both prostase and TMPRSS2 are potentially overexpressed in CaP, antibody therapy or vaccine approaches might be considered for these targets. Further expression analysis of prostase and TMPRSS2 on a larger panel of CaP specimens will be required to confirm the relevance of these proteins as target antigens for immunotherapy. Prostatic Acid Phosphatase PAP was originally identified in 1936 as a phosphatase activity associated with osteoblastic metastasis of CaP [79]. The gene was cloned and found to encode a secreted protein of 386 aa in length [80]. Analysis of PAP expression at both the nucleic acid and protein level has demonstrated that PAP is very prostate-specific, with expression in normal or cancerous epithelial cells, but not in any other tissues investigated [81–83]. Circulating PAP levels in the serum of CaP patients increased progressively with the stage of disease, and elevated levels in advanced disease was associated with a poor prognosis [84]. Although PAP is a secreted protein, like PSA, it represents a potential cancer vaccine target due to its prostate specificity and overexpression in all stages of CaP. Immunogenicity of PAP has been evaluated in the Copenhagen rat in a model of tissue-specific autoimmune prostatitis [85]. Immunization of rats with either rat or human PAP protein in CFA led to a PAP-specific Ab response, but no CTL response or prostatitis. Alternatively, immunization with vaccinia virus expressing PAP was also examined. Interestingly, human PAP, but not rat PAP, induced a CTL response and prostatitis in this context. Three points can be made from this study: (i) CTL and not Ab were responsible for autoimmune prostatitis; (ii) vaccinia virus delivery was efficient at inducing an antigen-specific CTL response; and (iii) xenogeneic immunization might be an efficient mechanism of breaking immunological tolerance. Experimental autoimmune prostatitis has also been induced by immunization of rats with syngeneic prostate homogenates, resulting in induction of strong antibody and T-cell responses against rat prostatic steroid-binding protein, PSBP [86]. This 444 suggests that syngeneic immunization can also be effective and perhaps the form of antigen or mode of delivery governs the immune response that is generated. In a human in vitro stimulation model, a peptide of human PAP has been used to induce Ag-specific CTL responses [87]. Dendritic cells, derived from normal peripheral blood, were pulsed with a PAP peptide to generate CD8+ T-cells that recognized and lysed peptide-pulsed HLA-A2+ target cells. The PAPspecific CD8+ CTL also lysed PAP expressing LNCaP cells, demonstrating that endogenous, processed PAP was also recognized. This data suggests that PAP is a potential cancer vaccine candidate, and confirms that in vitro stimulation with peptide-pulsed DC may be an effective way to generate Ag-specific T-cell responses in CaP. Antibodies to PAP have been investigated for their ability to target drugs to CaP cells and tissues [83,88,89]. Deguchi and colleagues conjugated either methotrexate or adriamycin to the same antiPAP MAb and tested its ability to target and inhibit growth of LNCaP cells [88,89]. The conjugated antibodies targeted to and inhibited growth of LNCaP cells in vitro, but never with the same efficacy as the free drug. In vivo, the conjugated antibodies could specifically localize to the tumors and inhibit, but not prevent, growth of established LNCaP tumors compared with conjugated control antibodies. In a more recent study, a rabbit anti-PAP polyclonal Ab, conjugated with a labeled derivative of 5-FU was used to stain either benign or malignant prostate sections [83]. The immunoconjugate was specific for prostate epithelial cells in both cases, but did not recognize epithelial cells from normal colon or kidney tissue sections. Additionally, the anti-PAP immunoconjugate also localized to epithelial cells derived from organ cultures of human prostate tissue. These studies demonstrate that antibodies against PAP can localize to prostate epithelial cells and deliver therapeutic drugs to specific sites. The mechanism of uptake of the conjugated antibodies was proposed to be via endocytosis at the cell membrane, similar to anti-PSA antibody conjugates. However in the study using anti-PAP conjugated to adriamycin, the authors caution that diminished effectiveness of the conjugate, especially when treating larger tumors, might be a result of binding to circulating PAP resulting in clearance of the antigen– antibody conjugate [89]. C. Intracellular (nuclear) proteins PAGE antigens Cancer testis antigens represent genes that are expressed in normal testes and that are also activated in many cancers, the most notable being malignant melanoma. In melanoma the MAGE genes, which are expressed at high levels, are currently being evaluated as targets for vaccine-based approaches to treat the disease [90]. Homologues of MAGE have also been identified which include the GAGE and BAGE genes, both expressed in melanoma and other tumors [91,92]. Recently, a new family of prostate-specific cancer testis antigens homologous to the MAGE/GAGE families, appropriately named PAGE (‘prostate-associated gene’), have been independently identified by two groups. In the first case, sublines of LNCaP were used to define genes differentially expressed between AD, non-metastatic cells and AI, metastatic cells [93]. Using differential display, two novel transcripts were identified which were homologous to GAGE-family genes. One gene, termed PAGE-1, was expressed in CaP, testes, and placenta, and had 45% homology to the GAGE family. PAGE-1 mRNA levels were found to be elevated 5-fold in the LNCaP AI, metastatic versus the parental AD, non-metastatic LNCaP cells. A second gene was found to be a new GAGE family member and was termed GAGE-7. This gene was also expressed in testes and placenta, but in contrast to PAGE-1, GAGE-7 mRNA levels were the same in both the parental and metastatic LNCaP sublines. In a second approach, publicly available databases were used to identify genes up-regulated in normal prostate and CaP. This technique, referred to as ‘database mining’, has resulted in the identification of several novel PAGE-family genes [94,95]. PAGE-1, located on the human X chromosome, was expressed in prostate, testicular, and uterine cancer as well as normal male and female reproductive tissues [95]. The name of this gene was later changed to PAGE-4, since it was different from PAGE-1 originally described by Chen et al., but was still a PAGE family member [96]. Expression of PAGE-4 was verified at the RNA level by dot blot, Northern, and RT-PCR analysis and was consistent with the pattern observed by EST representation in the specific tissues. PAGE-4 has homology to the GAGE family, but is more homologous to the PAGE family members PAGE-2 and PAGE-3 that are both expressed in testis [95]. Expression of the PAGE family 445 and GAGE-7 genes needs to be examined in a larger panel of clinical samples, but nonetheless PAGE-4 is currently being evaluated as a target for vaccine therapy of CaP [96]. Future perspectives There is a continued need to find additional markers for improved diagnosis, prognosis, and treatment of CaP, especially with respect to advanced disease. With the current explosion in genomics-based research, new markers are being identified at a rapid pace, providing an ever-increasing number of attractive candidates for immunotherapy. Perhaps the real challenge is the development of new and improved technologies relating to immune-based therapies. MAb therapy has recently made a re-emergence as a viable method to treat cancer, such as anti-CD20 (Rituxan) in B-cell lymphoma and anti-HER-2/neu (Herceptin) in breast cancer, mainly due to the ability to create chimeric or humanized antibodies that target specific antigens [97]. Furthermore, the development of transgenic mice that produce high affinity human antibodies after immunization will provide a source of fully human MAbs that can be used for diagnostic or therapeutic purposes [98,99]. Phage display technologies have recently begun to show promise in recognizing antigens with high affinity and specificity and might provide an alternative to deriving engineered MAbs [100]. For the generation of specific T-cell mediated immunity, breaking immunological tolerance is a consideration since the antigens generally expressed in cancers, including prostate cancer, are self antigens. New technologies are currently being developed to overcome tolerance, including antigen delivery, development of novel adjuvants, and combinations of antigen and specific cytokines to elicit the desired humoral or cellmediated effects. With respect to antigens, ‘functional antigenics’ has recently emerged as a new field to identify peptide sequences from a given protein that will serve as the best immunogens [101]. The determination of specific peptide sequences that might serve as the best antigen has recently been demonstrated using MHC-tetrameric complexes to identify patientreactive T-cells against specific antigens in melanoma [102]. Evasion of tumor immunity is an issue to contend with since there is evidence demonstrating that prostate tumors may down-regulate MHC Class I expression [103]. In those cases, MAb therapy might be a preferable alternative. Ultimately, understanding the expression pattern of the target antigen at specific disease stages in CaP may help to guide the appropriate immunotherapy strategies to consider. This is especially critical in advanced CaP, where currently there are no effective treatment options available for those patients. 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