**2. AGR2 as a cancer biomarker**

*Advances in Precision Medicine Oncology*

targetable molecule is an ongoing endeavor.

specific expression of these proteins. PSCA (prostate stem cell antigen), for example, is misidentified as a stem cell marker, and is expressed by the bladder, colon, kidney and stomach as well [5]. Vaccines against PSA (prostate-specific antigen) in PROSTVAC-VF and PAP (prostatic acid phosphatase) in Provenge were used to prime immune cells. However, inconsistent trial results with modest survival benefits were reported [6]. Strategies to target immune checkpoints (CTLA-4, PD-1, PD-L1) with the intention to amplify T cell responses in eradicating tumors have not been particularly successful in prostate cancer, with one of the main side effects being immune-related adverse events with tissue damage caused by overly activated T cells [6]. These potential therapies are beset by response monitoring although a subset of patients with advanced disease did show some benefits. There are even fewer similar types of immunotherapeutic approaches developed for pancreatic and bladder cancer, which is largely due to fewer suitable targets identified. Immunotherapy based on antibodies, on the other hand, would not require tinkering the immune system to achieve an outcome. Tumor-associated antigens (TAA) [7] constitute a pool of candidates for targeted cancer therapies. Antibodies raised against TAA mediate cancer cell killing through antibody-dependent cellular cytotoxicity (ADCC) by recruiting cytotoxic T cells [8] and complement-dependent cytotoxicity (CDC) by assembling complement components into a membrane attack complex [9]. The antibody-bound cancer cells are lysed by T-cell secreted enzymes and water uptake through a perforated cell membrane, respectively. A major complication is the cancer non-specificity of most TAA identified to date, because other normal cell types also express these TAA leading to unintended collateral damage of healthy tissue. Therefore, the quest for a truly cancer-specific

Our work in Urologic Cancer Biomarker Development identified AGR2 as a TAA for prostate cancer. AGR2 is present in prostate cancer cells but absent in the normal luminal cells [10, 11]. Similarly, AGR2 expression is detected in various cancers including pancreatic [12], breast [13], lung [14], colorectal [15], oral [16], subsets of ovarian [17], and bladder [18]. What makes AGR2 attractive for cancer therapy besides its ubiquity in solid tumors is its differential subcellular localization between cancer and normal cells [19] as we demonstrated previously for bladder cancer [18]. Intracellular iAGR2 is localized to the endoplasmic reticulum (ER) of normal cells where, as a protein disulfide isomerase, it functions in protein folding [19]. Extracellular eAGR2 is localized to the cell surface of and secreted by cancer

Antibodies targeting eAGR2 on cancer cells would thus spare normal cells as the iAGR2 antigen is cell interior. We have generated mouse monoclonal antibodies, P1G4 (mIgG1) and P3A5 (mIgG2a), recognizing two epitopes of AGR2 [20]. The use of mouse monoclonal antibodies for therapeutics is problematic. Mouse antibodies, besides being immunogenic in human, do not interact efficiently with human immune system components. To overcome these drawbacks, we have replaced the constant domains of AGR2 antibodies by the analogous human constant domains via recombinant DNA technology [21], generating human:mouse chimeric hIgG1, hIgG2, hIgG4 for both P1G4 and P3A5 for potential therapeutic

With these chimeric human:mouse antibodies, direct antigenic stimulation of T cells via CAR-T cell therapy can potentially also be achieved [23]. We could link via the engineered restriction enzyme sites the antigen-binding VH and VL domains of the AGR2 antibodies to T cell activator molecules for triggering response upon binding of the T cells via eAGR2 on the cancer cell surface. In the future, one might be able to use patient-derived induced pluripotent stem (iPS) cells to differentiate into dendritic cells in vitro (by bone marrow factors and interaction with bone marrow

cells. Thus, eAGR2 is a unique TAA that it is not found on normal cells.

**96**

development [22].

We used comparative transcriptomic analysis between sorted CD26+ prostate cancer cells from a Gleason pattern 3 (well-differentiated glandular adenocarcinoma) tumor focus and CD26+ luminal cells of benign glands to identify differentially expressed genes that encode secreted proteins for use as urine biomarkers [10]. AGR2 was the top candidate with elevated expression in cancer cells, which was verified by immunohistochemistry (**Figure 1**). A similar analysis was carried out between CD9+ urothelial cancer cells and CD9<sup>+</sup> normal urothelial cells [25]. In this case, AGR2 was down-regulated in cancer. AGR2 is expressed by urothelial cells (iAGR2 for normal cells) and was found absent in 75% of bladder cancer cases [18]. Although the AGR2 gene contains a leader signal peptide characteristic of secreted proteins, in normal cells iAGR2 is retained in the ER (via a *C*-terminus ER-retention motif). Over-expression of AGR2 in cancer cells provides a possible explanation for its secretion due to saturation of ER anchorage [26]. For example, urothelial cells were immunostained for AGR2 at moderate intensity *vs*. prostate cancer cells. The immunostaining corroborated a 35-fold difference in expression levels measured

#### **Figure 1.**

*AGR2 in prostate cancer and bladder. The prostate tumor glands (specimen 99-010D, top) are stained positive for AGR2 while benign glands are negative. Faint staining in the cancer-associated stroma (red arrow) suggests AGR2 secretion from the adjacent cancer cells. The urothelium (specimen 03-043B1, bottom) is stained at moderate intensity. Two different parts of the specimen indicate uniform expression throughout the entire urothelium. Black arrow indicates the lamina propria below the urothelium.*

by DNA microarray analysis of CD9+ urothelial cells (signal value = 105.54) [25] and CD26+ prostate cancer cells (signal value = 4168.03) [10]. A previous study estimated that adenocarcinoma cells had 80% iAGR2 and 20% eAGR2 while in all non-tumor cells AGR2 was located intracellularly [19]. Faint AGR2 staining of the stroma surrounding prostate tumor glands could be evidence of AGR2 secretion [27] (**Figure 1**, top panel). No such staining was detectable in the lamina propria next to the iAGR2+ urothelium [18] (**Figure 1**, bottom panel) or benign glands of the prostate. In prostate cancer cells, AGR2 could be activated by ER stress with down-regulation of unfolded protein response genes leading to an accumulation of misfolded proteins like that in pancreatic cancers cells [28].

In summary, three AGR2 expression patterns have been described in solid tumors: (1) iAGR2 positive for normal urothelial cells, eAGR2 positive for 25% primary tumors [18]; (2) no AGR2 for prostate epithelial cells, eAGR2 positive for >95% primary tumors [10], similar to pancreas [12] and breast [13]; (3) iAGR2 positive for normal bronchial epithelial cells, eAGR2 positive for>95% non-small cell lung tumors [14].
