**2. The Prostate Specific Membrane Antigen (PSMA)**

### **2.1 Structure, function and expression**

PSMA, also known as Glutamate Carboxypeptidase II (GCPII, EC 3.4.17.21), N-acetyl-α-linked acidic dipeptidase I (NAALADase) or folate hydrolase, is a type II transmembrane protein, which is anchored in the cell membrane of prostate epithelial cells (Carter et al., 1996; Pinto et al., 1996). In 1998, the gene encoding PSMA was mapped to chromosome 11p11-p12, where it encompasses 19 exons spanning about 60 kb of genomic DNA (O'Keefe et al., 1998). The cDNA of PSMA codes for a glycoprotein of 750 amino acids (aa) with a molecular mass of about 100 kDa. The protein is partitioned into a small intracellular domain of 19 aa, a transmembrane domain of 21 amino acids, and a large extracellular domain of 707 aa. Crystallization data revealed that the extracellular domain of PSMA folds into three distinct structural and functional domains: a protease domain (aa 56-116), an apical domain (aa 117- 351), and a C-terminal domain (aa 592-750). Furthermore, it was shown that PSMA is expressed as a compact homodimer, which is highly glycosylated with oligosaccharides accounting up to 25% of the molecular weight (Davis et al., 2005; Mesters et al., 2006).

PSMA contains a binuclear zinc site and can act as glutamate carboxypeptidase or folate hydrolase, catalyzing the hydrolytic cleavage of glutamate from poly-γ-glutamated folates (Ghosh & Heston, 2003). Therefore, PSMA is thought to play a role in the folate metabolism of the prostate. This hypothesis is supported by recent studies, where PSMA expression correlated with proliferation and folate uptake of PSMA transfected cells (Yao & Bacich, 2006; Yao et al., 2009).

In contrast to other prostate-related antigens, like prostate specific antigen (PSA), prostate acidic phosphatase (PAP) or prostate secretory protein (PSP), PSMA is not secreted into

Prostate Specific Membrane Antigen as Biomarker and Therapeutic Target for Prostate Cancer 83

Recently, a new splice variant (PSM-E) was described, which is specifically overexpressed in prostate carcinomas and which correlates with the Gleason score (Cao et al., 2007). PSM-E, which is expressed in the cytoplasm, could account for the lack of correlation between histological positive staining of anti-PSMA antibodies with clinical grade (stage)

Despite of such controversies, it is apparent that the enhanced expression and enzymatic activity of PSMA in aggressive prostate tumors is indicative of a selective advantage on the

PSMA was found to associate with the anaphase-promoting complex and to induce chromosomal instability (Rajasekaran et al., 2008). Moreover, PSMA favoured prostate cancer development in a permissive folate environment (Yao & Bacich, 2006; Yao et al., 2009). One mechanism by which PSMA contributes to prostate tumor growth is its ability to activate IL-6 and CCL5 synthesis. These cytokines acted synergistically to enhance the

Taken together, assessment of PSMA levels, either alone or in combination with PSA status, might prove useful in future for the diagnosis of metastatic prostate cancer, risk assessment,

Specific characteristics of PSMA concerning its structure, function and expression make it an ideal candidate as a target antigen for the treatment of advanced prostate cancer. (1) Its high and specific expression on the prostate cancer cell surface and the fact that it is not shed into the circulation allows an effective systemic delivery of PSMA targeting therapeutics. (2) Its high organ specificity leads to a minimal binding of anti-PSMA drugs to normal organs and therefore to a maximal reduction of potential side effects. (3) Its expression at all tumor stages enables a therapeutic intervention at any time of the disease. (4) Its internalization after ligand binding can be used for the targeted delivery of intracellular acting drugs. (5) Its enzymatic activity allows the cleavage of prodrugs to active molecules on the surface of

Many preclinical and clinical studies were performed in the last years, which used PSMA as target antigen. They include radioimmunotherapy, the use of immunotoxins, targeted virotherapy, retargeting of immune cells, PSMA vaccination, prodrug activation,

Radioimmunoconjugates generally consist of an antibody moiety as target domain coupled to a therapeutic radionuclide (alpha- or beta-particle) with the biologic effect of high linear energy transfer (LET) radiation. Compared to conventional radiotherapy, radioimmunoconjugates allow the targeted delivery of reduced radiation doses to the tumor, which ideally leads to a reduction of side effects. Moreover, the radiation of a radioimmunoconjugate is not restricted to cells presenting the target antigen. It also affects neighboring cells with a heterogeneous antigen expression or insufficient vascularization,

An initial immunoscintigraphic approach targeting PSMA was done with the 111Indium (111In) labeled anti-PSMA monoclonal antibody 7E11 (Capromab Pendetide (Prosta Scint®), Cytogen, Philadelphia, PA) (Kahn et al., 1994; Sodee et al., 1996; Kahn et al., 1998). With this

photodynamic therapy, and PSMA targeting nanoparticles.

which is so called "bystander effect" (Rzeszowska-Wolny et al., 2009).

**2.3.1 Anti-PSMA radioimmunotherapy** 

part of cells expressing it and that it contributes to prostate carcinogenesis.

growth of LNCaP cells by activating the MAPK pathway (Colombatti et al., 2009).

(Mannweiler et al., 2009).

and the prognosis of disease outcome.

**2.3 PSMA as therapeutic target** 

prostate cancer cells.

circulation. Instead of that, PSMA undergoes constitutive internalization, which is about threefold enhanced after antibody binding (Liu et al., 1998). It is therefore suggested that PSMA has transport function and that anti-PSMA antibodies might act as surrogates for a yet unknown ligand. The endocytic pathways of PSMA after antibody binding were specified in a recent study and comprise clathrin-mediated endocytosis, macropinocytosis, and clathrin-, calveolae-independent endocytosis (Liu et al., 2009b).

To examine the PSMA expression in the prostate, immunohistochemical analyses were performed. In a study with prostate tissue specimen from 184 patients with prostate cancer, the percentage of PSMA positive stained cells averaged about 69.5% (range 20%-90%) in the benign epithelium, 77.9% (range 30-100%) in high grade prostatic intraepithelial neoplasia (PIN) and was highest in adenocarcinomas with a mean of 80.2% (range 30-100%). In contrast, tumor stroma, urothelium, normal vasculature and, with rare exceptions, basal cells were PSMA negative (Bostwick et al., 1998). Other immunohistochemical studies demonstrated a heterogeneous, weak to moderate staining of normal prostate epithelial cells, and a homogeneous, extensive staining of prostate adenocarcinomas and metastases (Silver et al., 1997; Wolf et al., 2010a).

PSMA expression is highly organ specific. An extraprostatic expression was only detected in secretory cells of the salivary glands (Israeli et al., 1994; Troyer et al., 1995; Wolf et al., 2010a), in cryptic cells of the duodenal brush border (Chang et al., 1999; Wolf et al., 2010a), and in a subset of proximal renal tubules (Liu et al., 1997; Silver et al., 1997; Chang et al., 1999). In some studies, an additional expression was found in the brain and in the colon, but these results are controversially discussed (Troyer et al., 1995; Silver et al., 1997; Chang et al., 1999; Sacha et al., 2007). Nonetheless, potential side effects of anti-PSMA therapeutics against PSMA expressing normal organs were not described until today.

Interestingly, PSMA is also discussed as an unique anti-angiogenetic target, since it is expressed in the neovascularization of numerous solid tumors (bladder, kidney, breast, pancreas, lung, melanoma), but not in normal blood vessels (Liu et al., 1997; Chang et al., 1999; Chang et al., 2001; Baccala et al., 2007). In this respect, it was found that PSMA regulates cell invasion and tumor angiogenesis by modulating integrin signal transduction in endothelial cells (Conway et al., 2006).

### **2.2 PSMA as prognostic and diagnostic biomarker**

Generally, prostate carcinoma tissues show a higher PSMA expression and an increased enzymatic activity of PSMA compared with normal prostate and benign prostate hyperplasia (BPH) tissues (Lapidus et al., 2000; Burger et al., 2002). Therefore, the question was raised, if PSMA might serve as a valuable biomarker for the management of prostate cancer.

Indeed, in different studies a direct correlation between PSMA expression and the Gleason score, which is used for the staging of prostate cancer, was determined for adenocarcinomas (Su et al., 1995; Kawakami & Nakayama, 1997; Burger et al., 2002). Moreover, an upregulation of PSMA was shown in tumor cells of patients with hormonerefractory prostate cancer (Wright et al., 1996; Kawakami & Nakayama, 1997). In a study with tissue specimen from 136 patients it was demonstrated that PSMA can serve as a prognostic biomarker, because it significantly correlates with adverse prognostic factors, like tumor grade, pathological stage, aneuploidy, and biochemical recurrence, and therefore independently predicts disease outcome (Ross et al., 2003).

Recently, a new splice variant (PSM-E) was described, which is specifically overexpressed in prostate carcinomas and which correlates with the Gleason score (Cao et al., 2007). PSM-E, which is expressed in the cytoplasm, could account for the lack of correlation between histological positive staining of anti-PSMA antibodies with clinical grade (stage) (Mannweiler et al., 2009).

Despite of such controversies, it is apparent that the enhanced expression and enzymatic activity of PSMA in aggressive prostate tumors is indicative of a selective advantage on the part of cells expressing it and that it contributes to prostate carcinogenesis.

PSMA was found to associate with the anaphase-promoting complex and to induce chromosomal instability (Rajasekaran et al., 2008). Moreover, PSMA favoured prostate cancer development in a permissive folate environment (Yao & Bacich, 2006; Yao et al., 2009). One mechanism by which PSMA contributes to prostate tumor growth is its ability to activate IL-6 and CCL5 synthesis. These cytokines acted synergistically to enhance the growth of LNCaP cells by activating the MAPK pathway (Colombatti et al., 2009).

Taken together, assessment of PSMA levels, either alone or in combination with PSA status, might prove useful in future for the diagnosis of metastatic prostate cancer, risk assessment, and the prognosis of disease outcome.
