Abridged from Cancer Research 59, 3192-3198, July 1, 1999.
Five Different Anti-PSMA Antibodies Confirm Prostate-Specific Membrane Antigen (PSMA)[1] Expression in Tumor-Associated Neovasculature[2] Sam S. Chang, Victor E. Reuter, W. D. W. Heston, Neil H. Bander, Lana S. Grauer, and Paul B. Gaudin[3]
Urology Service, Department of Surgery [S.S.C.], George M. O’Brien Urology Research Center [W.D.W.H.], and Department of Pathology [V.E.R., P.B.G.], Memorial Sloan-Kettering Cancer Center, New York, New York, 10021; Hybritech Incorporated, a subsidiary of Beckman-Coulter, Inc., San Diego, CA 92196 [L.S.G.]; Department of Urology, New York Presbyterian Hospital-Weill Medical College of Cornell University and Ludwig Institute for Cancer Research, New York, New York, 10021 [N.H.B.]
ABSTRACT
Prostate-specific membrane antigen (PSMA) is a type II integral membrane glycoprotein initially characterized by the monoclonal antibody (mAb) 7E11. PSMA is highly expressed in prostate secretory-acinar epithelium and prostate cancer as well as in several extra-prostatic tissues. Recent evidence suggests that PSMA is also expressed in tumor-associated neovasculature. We examined the immunohistochemical characteristics of 7E11 and those of four recently developed anti-PSMA mAbs (J591, J415, and Hybritech Incorporated PEQ226.5 and PM2J004.5), each of which binds a distinct epitope of PSMA. We evaluated these mAbs in viable prostate cancer cell lines and various fresh-frozen benign and malignant tissue specimens. In the latter, we compared the localization of the anti-PSMA mAbs to that of the anti-endothelial cell mAb CD34. With rare exceptions, all five anti-PSMA mAbs strongly reacted with the neovasculature of a wide spectrum of malignant neoplasms: conventional (clear cell) renal carcinoma (11/11 cases), transitional cell carcinoma of the urinary bladder (6/6 cases), testicular embryonal carcinoma (1/1 case), colonic adenocarcinoma (5/5 cases), neuroendocrine carcinoma (5/5 cases), glioblastoma multiforme (1/1 cases), malignant melanoma (5/5 cases), pancreatic duct carcinoma (4/4 cases), non-small cell lung carcinoma (5/5 cases), soft tissue sarcoma (5/6 cases), breast carcinoma (5/6 cases), and prostatic adenocarcinoma (2/12 cases). Localization of the anti-PSMA mAbs to tumor-associated neovasculature was confirmed by CD34 immunohistochemistry in sequential tissue sections. Normal vascular endothelium in non-cancer bearing tissue was consistently PSMA-negative. The anti-PSMA mAbs reacted with the neoplastic cells of prostatic adenocarcinoma (12/12 cases) but not with the neoplastic cells of any other tumor type including those of benign and malignant vascular tumors (0/3 hemangiomas; 0/1 hemangioendothelioma; 0/1 angiosarcoma). The mAbs to the extracellular PSMA domain (J591, J415, Hybritech PEQ226.5) bound viable prostate cancer cells (LNCaP and PC3-PIP) while the mAbs to the intracellular domain (7E11 and Hybritech PM2J004.5) did not. All five anti-PSMA mAbs reacted with fresh-frozen benign prostate secretory-acinar epithelium (28/28 cases), duodenal columnar (brush border) epithelium (11/11 cases), proximal renal tubular epithelium (5/5 cases), colonic ganglion cells (1/12 cases), and benign breast epithelium (8/8 cases). A subset of skeletal muscle cells was positive with 7E11 (7/7 cases) and negative with the other four anti-PSMA mAbs. PSMA is consistently expressed in the neovasculature of a wide variety of malignant neoplasms and may be an effective target for mAb-based anti-neovasculature therapy.
INTRODUCTION
Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein of approximately 100 kDa that was initially characterized by the monoclonal antibody (mAb) 7E11 (1, 2). Recent studies have confirmed the location of the PSMA gene on chromosome 11p and have demonstrated the existence of a related PSMA-like gene on 11q (3-5). Two variant forms of PSMA, initially predicted to exist as PSMA and a spliced form PSM¢, have been subsequently confirmed. PSMA is highly expressed in benign prostate secretory-acinar epithelium, prostatic intraepithelial neoplasia, and prostatic adenocarcinoma (2, 6-8), and evidence suggests that PSMA expression is greatest in high-grade and hormone-insensitive cancers (2, 9-11). A shorter, alternatively spliced and presumably cytosolic form of PSMA, named PSM¢, is the predominant form expressed in benign prostate epithelium (12, 13). Several studies have shown that anti-PSMA mAbs bind to several non-prostate tissues, including duodenum and kidney (6, 14, 15) and to the vasculature associated with solid malignant tumors (15, 16).
The function of PSMA is currently under investigation. Pinto et al demonstrated that PSMA has a folate hydrolase-type of activity as LNCaP cells were shown to hydrolyze gamma-glutamyl linkages in MTXGlu3 (17). Others have demonstrated that PSMA has a neuropeptidase-type function (18, 19). Based on these enzymatic characteristics, the nomenclature committee of the International Union of Biochemistry and Molecular Biology has recommended for PSMA the formal name of glutamate carboxypeptidase, #EC 3.4.17.21 (20).
The 7E11 antibody is a specific murine IgG mAb that was derived after immunization of mice with preparations from the LNCaP human prostate cancer cell line (1). 7E11 has been well characterized and is known to bind an intracellular epitope of PSMA not present on PSM¢. As a result, 7E11 does not bind viable prostate cancer cells (1, 16, 21). Modified by the addition of 111In, 7E11 is used currently at some centers as an imaging agent in vivo. Clinical trials have demonstrated that this radioimmuno-conjugate of 7E11, known as 111In-capromab pendetide, may be a useful adjunct in identifying and localizing metastatic or recurrent prostate cancer (22-25).
A number of other anti-PSMA mAbs have been recently developed which bind epitopes distinct from that recognized by 7E11 (13, 16). For example, the mAbs J591, J415, J533, and E99, bind to the extracellular PSMA domain (16). Investigators at Hybritech, Inc. have identified and purified the mAb PEQ226.5 that binds the peptide backbone of the PSMA extracellular domain. In addition, investigators at Hybritech, Inc. have identified PM2J004.5 which binds an epitope of the intracellular PSMA domain distinct from that bound by 7E11 (13).
The purpose of this study was to compare the immunohistochemical profiles of four recently developed anti-PSMA mAb to that of 7E11. Specifically, we evaluated these mAbs in prostate cancer cell lines, benign and malignant prostate tissue, benign non-prostate tissue, and a variety of malignant tissues. In the latter, we sought further to confirm PSMA expression in tumor-associated neovasculature.
RESULTS
Tumor-associated neovasculature. With rare exceptions, all five anti-PSMA mAbs bound tumor-associated neovasculature of non-prostatic tumors (Table 1, Fig. 1). The neovasculature of one breast carcinoma and one soft tissue sarcoma (myxofibrosarcoma) showed no immunoreactivity, however, both contained CD34-positive vasculature. The four cases of breast carcinoma with PSMA-positive neovasculature were ductal carcinomas while the one PSMA-negative case was lobular carcinoma. Interestingly, only a small subset of prostate cancer specimens showed PSMA-positive neovasculature (2/12 cases). In these cases, we found the CD34-stained sections useful in localizing so-called “hot-spots” of neovasculature that we then compared to the anti-PSMA mAb-stained sections. This helped us confirm the location of vessels amidst strongly PSMA-positive tumor cells. We noted no significant histologic differences between prostate cancers with PSMA-positive neovasculature and those with PSMA-negative neovasculature. In all the tumors, 7E11, J591, J415, PEQ226.5, and PM2J004.5 mAbs bound neovasculature in a like manner (Fig. 2). The results of CD34 immunohistochemistry in sequential tissue sections confirmed localization of the anti-PSMA mAbs to neovasculature endothelium (Fig. 2). In contrast to tumor-associated neovasculature, none of the anti-PSM mAbs reacted with vasculature in the non-cancer-bearing tissue sections. The staining intensity of the external domain binding mAbs (J591, J415, and PEQ226.5) in tumor-associated neovasculature was greater than that of the internal domain binding mAbs (7E11 and PM2J004.5).
Prostate cancer cell lines. The external domain binding mAbs (J591, J415, and PEQ226.5) bound viable LNCaP and PC3-PIP cells known to express PSMA. In contrast, the internal domain binding mAbs (7E11 and PM2J004.5) did not bind viable LNCaP and PC3-PIP cells (Fig. 3). After formalin fixation, all anti-PSMA mAbs, including 7E11 and PM2J004.5, reacted with LNCaP and PC3-PIP cells. None of the mAbs bound viable or formalin-fixed PC3 cells that are known to lack PSMA expression.
Benign tissues. Although benign prostatic secretory-acinar epithelium displayed heterogeneous staining with the five mAbs, all 28 benign prostate cases were PSMA-positive. Immunoreactivity was typically concentrated at the luminal aspect of the cytoplasmic membrane. Basal epithelium and stromal cells were PSMA-negative. The immunoreactivity of the benign secretory-acinar epithelium was less intense than that of prostatic adenocarcinoma, and the staining intensity of the external domain binding mAbs, J591, J415, and PEQ226.5 was greater than that of the internal domain binding mAbs, 7E11 and PM2J004.5 (data not shown).
The anti-PSMA mAbs reacted with several of the 19 benign non-prostate tissues (Table 2). All five mAbs reacted with duodenal columnar (brush border) epithelium (11/11 cases), renal proximal tubular epithelium (5/5 cases), benign breast epithelium (8/8 cases), and colonic ganglion cells (1/12 cases). In skeletal muscle, a subset of muscle fibers were positive only with 7E11 and negative with the other four mAbs (Fig. 4). The vasculature in all benign tissues was uniformly PSMA-negative. The staining intensity of these PSMA-positive benign tissues was less than that of prostate cancer and tumor-associated neovasculature.
DISCUSSION
Our study confirms PSMA expression in the neovasculature of a wide spectrum of malignant neoplasms. Specifically, we found PSMA expression in various epithelial (carcinomas), neuroendocrine, and mesenchymal tumors (soft tissue sarcomas), and malignant melanoma and glioma. In contrast to previous studies, we utilized five anti-PSMA mAbs, each of which binds a different epitope of the intracellular or extracellular PSMA domain. Thus, our results provide further evidence that PSMA, rather than a PSMA-like molecule, is expressed in tumor-associated neovasculature. Also in contrast to previous studies, we confirmed localization of PSMA to endothelial cells with the mAb CD34, an anti-endothelial cell marker used to study angiogenesis and determine microvessel density (26-30).
Our findings are consistent with previous studies showing PSMA expression in tumor-associated neovasculature. For example, Silver et al demonstrated 7E11 binding and “neoexpression of PSMA in endothelial cells” in a subset of tumors including renal cell carcinoma (unspecified type), transitional cell carcinoma of the urinary bladder, and colonic adenocarcinoma (15). More recently, Liu et al studied four external domain-binding anti-PSMA mAbs (J591, J415, J533, and E99) and showed that each bound the tumor-associated neovasculature in several non-prostatic carcinomas (16). While it is unclear if PSMA is produced by endothelial cells of tumor-associated neovasculature or if it is produced in other tissues and sequestered from the serum, we favor the former since PSMA is expressed only in a limited number of benign tissues and in prostate cancer but is not expressed in other malignant cell type. In addition, circulating PSMA has not been demonstrated in serum (Liu H. and Bander N.H., unpublished observations). Additional studies, however, are necessary to confirm this hypothesis.
We found that endothelial cell expression of PSMA was restricted to the neovasculature of malignant neoplasms. In fact, neither the vascular endothelial cells of benign tissues nor the neoplastic cells of vascular tumors expressed PSMA. These results suggest that endothelial cell-PSMA expression may be stimulated by one or more tumor-secreted angiogenic factors. The fact that all of the vascular neoplasms we studied, including the one example of angiosarcoma, were PSMA-negative is not surprising given that in these tumors the endothelium itself is neoplastic and presumably not stimulated by angiogenic factors. The presence or absence of PSMA-expression in benign neovasculature (e.g. granulation tissue, endometrium, etc. . .) remains to be established.
The neovasculature associated with organ-confined (OC) prostatic adenocarcinoma only rarely expressed PSMA. Others also have found no detectable PSMA expression in OC prostate cancer-associated neovasculature (9, 15). These observations are remarkable given the ubiquity of PSMA expression in tumor-associated neovasculature of other cancer types. They are, however, not altogether surprising given the histologic features of OC prostate cancer. For example, in contrast to many other epithelial tumors such as ductal carcinoma of the breast or pancreas, OC prostate cancer typically is not associated with an exuberant host-stromal reaction. Lobular carcinoma of the breast, like prostatic adenocarcinoma, typically does not induce a marked desmoplastic stromal response. Interestingly, the one breast cancer specimen in our series with PSMA-negative neovasculature was an example of lobular carcinoma. These results suggest that PSMA expression in tumor-associated neovasculature may be related to the degree and nature of neo-angiogenesis. The relationship between primary tumor stage in different malignancies and PSMA expression in neovasculature is unknown.
Consistent with most previous studies, we found that mAbs to the intracellular PSMA domain (7E11 and PM2J004.5) do not bind viable prostate cancer cells, whereas mAbs to the external domain (J591, J415, PEQ226.5) do bind live cells (16, 21). Only one study has reported 7E11 binding with viable prostate cancer cells (31). It is postulated that 7E11 binds predominantly to apoptotic cells within prostate cancer sites in vivo. Apoptotic cells, unfortunately, comprise only a minority of the total prostate tumor-cell population. This, no doubt, has contributed to the relatively low sensitivity of 111indium-capromab pendetide as an imaging agent for prostate cancer. In this regard, targeting the extracellular PSMA domain with radioimmuno-conjugates may enhance prostate cancer cell labeling in vivo.
The results of several, but not all, immunohistochemical studies using the 7E11 mAb have shown that PSMA is expressed in a limited number of non-prostatic tissues (1, 6, 15). Our findings support the results of other studies showing PSMA expression in duodenal (brush border) epithelium and renal proximal tubular epithelium but suggest that PSMA expression in these tissues is less than it is in prostate cancer and tumor-associated neovasculature (15, 16). Duodenal brush-border epithelium has high levels of folate hydrolase activity that is essential for folate absorption (17). This folate hydrolase activity is localized to the luminal membrane and is consistent with the staining pattern of the anti-PSMA mAbs. Proximal renal tubular epithelium also actively reabsorbs folate through the luminal membrane (32). Halsted et al found significant sequence homology between pig intestinal folate hydrolase (folypoly-gamma-glutamate carboxypeptidase) and human PSMA, suggesting that human duodenal membrane folate hydrolase may represent PSMA (33). Alternatively, it may represent a closely related enzyme that cross-reacts with anti-PSMA mAbs. In contrast to previous studies, we found consistent PSMA expression in mammary ductal epithelium. The reasons for our conflicting results are unclear, however, previous studies showing no PSMA expression in breast may have included specimens with inadequate amounts of ductal epithelium. One of our 12 colon specimens displayed PSMA expression in ganglion cells. The relatively sparse immunoreactivity observed in colonic ganglia may be indicative of peripheral neuronal PSMA expression previously described in non-myelinating, perisynaptic Schwann cells near motoneuron terminal endplates (34).
The staining profile of skeletal muscle is unique in that a subset of cells is positive with only 7E11. Liu et al also showed a subset of skeletal muscle cells bind 7E11 and not other anti-PSMA mAbs (16). Of note is the fact that the other internal domain binding anti-PSMA mAb, PM2J004.5, did not bind skeletal muscle. Thus, it is likely that in skeletal muscle 7E11 uniquely cross-reacts with either a yet to be defined PSMA-like or PSMA-unrelated molecule. The patchy distribution suggests that expression of this molecule may be restricted to either fast-twitch or slow-twitch muscle fibers.
Novel PSMA-based prostate cancer therapies, including anti-PSMA mAb-based therapies, are currently under investigation (35-37). The results of our study indicate that anti-PSMA mAb-based diagnostic and therapeutic modalities may be expanded to include anti-neovasculature targeting for a wide variety of malignant neoplasms. The importance of angiogenesis in neoplasia is well-documented (38-40), and endothelial cell expression of PSMA appears highly restricted to tumor-associated neovasculature and may represent a novel target for anti-neovasculature based therapy. Recent in vivo localization by the 111indium-labeled 7E11 mAb to a conventional (clear cell) renal cell carcinoma demonstrates the potential clinical utility of anti-PSMA mAbs in a non-prostate cancer (41). Enthusiasm for mAb-based therapy, however, must be tempered by the fact that PSMA is expressed in several benign tissue types; the potential side effects of anti-PSMA mAbs on these tissues in vivo is unknown. Other mAbs, however, currently in clinical trials or FDA-approved for clinical use, also are not tumor-specific and bind antigens expressed in benign tissues (42, 43).
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[1] The abbreviations used are: PSMA, prostate-specific membrane antigen; mAb, monoclonal antibody; MSKCC, Memorial Sloan-Kettering Cancer Center; OC, organ confined.
[2] This work was supported by grants from NIH, NIDDKD/NCI 47650, and CaP CURE. L.S.G. is a senior research scientist at Hybritech Inc, a subsidiary of Beckman-Coulter, Inc.
[3] To whom requests for reprints should be addressed at: Dept. of Pathology
Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021,
Fax #: (212) 717-3203, email: gaudinp@mskcc.org
This work was supported by grants from the NIH, NIDDKD/NCI 47650, and CaP CURE.
