Download Target Antigens for Prostate Cancer Immunotherapy

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
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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
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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
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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
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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
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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.
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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
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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. Since antigens such as PSCA and
PSMA appear to be up-regulated in advanced disease,
perhaps a combination of both immune- based and antiandrogen therapies might be most effective in treating
those CaP patients.
Acknowledgements
We would like to thank Lianna Doan, Jayne Bennett,
and Rene Hubert for their excellent assistance in the
preparation of this manuscript.
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