Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

Abstract

Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.

The use of monoclonal antibodies (mAbs) in the clinic has been growing rapidly in the last decade. Fully human antibodies can be selected either from transgenic animals or from large phage-displayed antibody libraries (1). There is, however, a shortage of disease-specific targets for therapeutic antibodies. In fact, just 5 targets constitute one-third of the 55 human mAb anticancer candidates against known targets (1). Unbiased functional identification of clinically relevant antibodies and their targets is an important goal. Developing an mAb without prior knowledge of the target, following a functional screening, has a high potential for innovation (2). In this approach, mAbs are selected based on their ability to bind to complex targets or to elicit a biological response, and their corresponding targets are characterized afterward, usually by a proteomics strategy. Using functional screens in vitro, however, some of the antibodies selected fail to fulfill their intended role in the clinic due to the differences with the in vivo environment, where they might exert their therapeutic function. One of the aspects that need to be considered when selecting antibodies for therapy is the importance of ensuring their ability to reach the target. Several strategies are used to identify ligands that are accessible from bloodstream, including functional genomics analysis (3), subtractive proteomics (4), the in vivo biotinylation of vascular proteins (5), and an in vivo phage display screening for peptides that home to specific targets in the vasculature (6–10).

In vivo peptide phage display has been particularly effective in the identification of markers that distinguish the vessels of diseased tissues from normal vessels (11). Homing moieties—mainly peptides and recombinant antibodies—have been used in targeted delivery of therapeutic compounds to diseased organs and represent a promising area of pharmaceutical intervention (12–14). Whereas antibodies have certain advantages over peptides as targeting agents (e.g., higher affinity and longer circulation time) (15), in vivo antibody screening in tumor-bearing mice has not been accomplished due, among others, to technical limitations such as unspecific binding of phage clones. Here, we overcome some of the limitations and report the isolation of a prostate tumor-homing antibody (011H12) from a human V_H domain antibody library (DAb library), the identification of its receptor, and the subsequent validation in primary and metastatic human prostate cancer samples.

Results

Repertoire Enrichment Strategy.

In preparation for in vivo screening, we hypothesized that the ideal repertoire should be moderately enriched against the target organ, leaving enough diversity for a variety of antigens in the target tissue. We compared enrichment of the phage antibody library in phage that bind to cultured tumor cells (in vitro strategy) or cell suspensions from freshly excised tumors (ex vivo strategy). To avoid overselection for antibody clones to tumor antigens inaccessible from the circulation, we monitored not only the increase in the recovery (Fig. 1A) but also the diversity loss during the rounds of enrichment (Fig. 1B). Sequencing 96 clones from the input and output of each selection round confirmed the expectation that in vitro selection resulted in a bias toward a few dominant sequences whereas ex vivo selection of the phage repertoire enriched a more diverse repertoire of phage clones.

Fig. 1.

Enrichment, diversity loss, and homing of monoclonal phage. The recovery of phage increases as the ex vivo selections progress. The recovery is greater if the repertoire was previously enriched in vitro (A). However, this preenrichment greatly reduces the available diversity (B). (C) Influence of the circulation time. Using an ex vivo enriched repertoire as input, the total recovery of infective particles is maximum at 15 min and drops gradually until 24 h (Upper). This recovery is observed for all of the organs analyzed. However, as the time of circulation increases, there is a higher specific recovery from the tumor (Lower). T, tumor; K, kidney; L, liver; H, heart. (D) Competition experiments using mixtures of fresh monoclonal phage. After 24 h of circulation, the phage were recovered from the organs or the tumor, and the percentage of each was compared with that present in the input (Upper) or with the average of the organs (Lower). (E) Normalized values showing the differential accumulation of phage clones (n = 5 mice per group). (i) α–βGal phage used as a reference: the expected retention for a phage whose target is not present in the tissue. Different clones that show retention in the tumor (iii–v), and retention in all of the organs (ii). Significant differences (P value) U Mann–Whitney.

Optimizing Circulation Time and Selection of Candidates.

A circulation time of 15 min is considered to be sufficient to address most of the targets exposed in the endothelium in nonmalignant tissues but may be too short for dysfunctional tumor vessels (16). A circulation time of 1 h has been proposed for a filamentous phage peptide library (17) based on the half-life of the phage although there is evidence that the displayed exogenous protein or peptide can make the circulation half-life as short as 1.5 min and as long as 4.5 h (18, 19). We administered i.v. a phage pool that had been previously enriched ex vivo and compared the recovery of phage from different organs at different time points after injection [ranging from 15 min to 24 h, using 4 h half-life of wild-type phage as a reference (20)]. The number of phage recovered from the tumor at 15 min after the injection was ∼40 times higher than at 24 h; in the nonmalignant organs, this difference was ∼100-fold (Fig. 1C, Upper). This difference is more easily visualized using a value normalized to control phage (Fig. 1C, Lower). These results suggest that specific accumulation in the target organ is nearly four times greater at 24 h than at 15 min.

Having established a 24-h circulation time as well suited for the in vivo selections, a sample of clones was sequenced from the input pool, from phage recovered from tumor tissue and from reference organs. Candidate tumor-homing phage clones were appraised based on several criteria: abundance in the target output, similarity between self and consensus complementarity determining regions (CDR) sequences obtained from hierarchical clustering, presence and composition of the amber stop codons, and presence and composition of “common known homing motifs” [such as integrin-binding RGD, neuropilin-1 binding tissue penetrating C-end Rule, CD13 binding NGR, etc. (21–23) (SI Appendix, Tables S1 and S2 and Fig. S1)]. We also explored a computer-based approach (24) by analyzing the frequency of all possible combinations of three amino acids among the CDRs for the in vivo output of a single preenriched repertoire. Despite the fact that the number of sequences analyzed in this way was limited, we found (SI Appendix, Fig. S2) significant differences in the sequences obtained from the tumor (TCs) and from the organs (OCs), suggesting that deep sequencing (25) would be helpful in identifying new candidate binders and consensus sequences and in validating previously selected candidates. The similarity heat map for the selected sequences and this computed group of consensus sequences (SI Appendix, Fig. S2, Bottom) show that TCs have more triplets of amino acids also present in other TCs than in OCs. OCs have a similar number of triplets also present in TCs and OCs, and the selected candidates mostly resemble the distribution pattern of TCs. The selected candidate clones were finally evaluated in an in vivo play-off (competitive homing) experiment and compared with a reference irrelevant clone that harbors the anti-β-galactosidase (α−βGal) specificity. Comparison of the recovery to the phage representation in the input pool (Fig. 1D, Upper) or to the average from the normal organs (Fig. 1D, Lower) yielded four promising phage clones (Fig. 1D, arrows) for further analysis.

Homing Assays.

The ability of the four selected clones to home to the tumor was compared with the reference α−βGal phage. A fresh preparation of each phage clone was i.v. injected into a group of mice (n = 5 per group), with the circulation time set at 24 h (Fig. 2). To validate the reproducibility of the normalized value used, two additional mice were injected with a 1:100 dilution of one of the selected clones to compare the total amount of phage recovered and the normalized value for a given specificity and different dosage (SI Appendix, Fig. S3). The reference α−βGal phage (Fig. 1E, i) showed a baseline accumulation in the tumor, similar to the accumulation in the kidney, and a lower accumulation in both heart and liver. Three of the four selected clones showed a higher uptake in the tumor (010H01, 011H12, and 010D6; Fig. 1E, iii–v) whereas the remaining clone, which has one of the lowest scores in the heat map (SI Appendix, Fig. S2, Bottom), accumulated in all of the organs (011A10; Fig. 1E, ii). We focused on the clone designated as 011H12 for further characterization based on the distribution observed.

Fig. 2.

Tumor-specific homing of 011H12 phage. Sections from tumors that have received the phage (either the reference α–βGal or 011H12) were analyzed by immunofluorescence, and the areas positive for CD31 (red) and the phage (green) were quantified. No difference was found in the total area positive for endothelial markers (A). There was a higher phage staining in the 011H12 clone (B). The V parameter (C, total phage/total endothelium) was six times higher in the 011H12 clone (D). **Significant differences (P < 0.0001) U Mann–Whitney. (E) Heterogeneous distribution of the phage. The 011H12 phage accumulates in the interior of the tumors, compared with the reference phage. In both cases, there is unspecific retention of the phage in the rim of the tumor. (F) The distribution of the reference phage (α–βGal) is restricted to the inner side of some vessels, as well as the edge of the tumor (Top). The distribution of the 011H12 phage is heterogeneous: there are small deposits in wide stromal areas (Middle) as well as major deposits around some vessels identified as such by CD31 staining (Bottom). (Scale bar: 50 µm.)

011H12 Phage Accumulates in Tumors.

Phage may be prone to unspecific aggregation (26, 27) and/or interaction with tissue components accessible from the bloodstream. Therefore, we decided to investigate whether the retention observed for the different phage was specific to the antibody harbored, or merely the result of entrapment of phage aggregates in the tumor vascular tree. If the latter were true, (i) a colocalization of endothelial markers with the i.v. administered phage should be expected; and (ii) the differences in the amount of phage recovered should be related to differences in the number of vessels.

We compared the endothelial staining of the tumors from mice dosed with the reference phage, with tumors of mice that had received the 011H12 clone. Analysis of equally vascularized tumors from mice injected with the 011H12 phage or the reference phage (Fig. 2A) showed greater accumulation of the 011H12 phage in the tumors both when the total area positive for the phage staining (Fig. 2B), or the amount of phage over endothelium area (Fig. 2C), was compared (P < 0.0001, Fig. 2D). The high dispersion of the values reflects the heterogeneous accumulation of the phage (Fig. 2E); whereas the reference phage was confined to the lumen of the vessels (Fig. 2F, Top), the 011H12 phage was also found spreading from the vessels into wide areas of surrounding perivascular stroma (Fig. 2F, Middle and Bottom). This specific accumulation pattern was not observed in the reference organs where both phage clones were confined to the vessels (SI Appendix, Fig. S4). We suggest that the progressive accumulation of the 011H12 phage in the tumor is due to the combination of the interaction with a tumor-associated antigen with leaky tumor vessels and long phage circulation time.

PA28 Is the Target of the 011H12 DAb.

To identify the target of the 011H12 DAb, we subjected tumor extracts to affinity chromatography. We used columns coupled with the anti−βGal DAb (SI Appendix, Fig. S5) or the 011H12 DAb in tandem for the separation. Affinity-purified proteins were digested by using the filter-aided sample preparation (FASP) method and then identified by nano liquid chromatography–mass spectrometry (LC-MS). Eluted proteins from the anti−βGal and 011H12 columns were compared, and those present in both eluates (more than one peptide in the control) were disregarded (SI Appendix, Table S3). The remaining proteins were ranked according to the number of peptides found in the eluate from the 011H12 column (SI Appendix, Table S4). To prioritize the potential candidates, the number of spectra associated with each protein [peptide spectrum match (PSM) number] was also considered. Notably, two of the selected proteins are subunits of the 11S proteasome activator complex [PA28 (28, 29)]: subunit α [proteasome activator complex subunit 1 (PSME1)] and subunit β (PSME2). SDS/PAGE revealed two closely spaced bands at about 29 kDa in the 011H12 DAb eluate that were not present in the control DAb eluate (Fig. 3A). These two bands were further identified as PSME1 (Q06323; 28.723 kDa) and PSME2 (Q9UL46; 27.402 kDa) by mass spectrometry (SI Appendix, Table S5), in agreement with the results obtained by the FASP method (SI Appendix, Table S4). The identity of the antigen recognized by the 011H12 DAb was confirmed by ELISA. Soluble purified 011H12 DAb showed reactivity with the active PA28αβ complex (30) and to a lesser extent with individual PA28 subunits (Fig. 3B). No reactivity was observed with two lower score candidates (HPRT and NDKB) or βGal. Furthermore, 011H12 bound specifically to immobilized PA28αβ in a concentration-dependent manner (Fig. 3 C and D).

Fig. 3.

Specific interaction of 011H12 DAb with PA28 subunits. (A) Fresh tumor lysates were purified using affinity chromatography with columns coupled with the α–βGal and 011H12 DAbs. The eluates were subjected to SDS/PAGE and stained using SYPRO dye, and the bands (arrows) were analyzed by mass spectrometry. (B) Purified soluble α–βGal or 011H12 DAbs (20 μg/mL) were tested against a panel of plastic-immobilized candidates (1 μg per well). The interaction between 011H12 and the PA28αβ complex in vitro is dose-dependent. Microtiter plates were coated (1 μg per well) with βGal (C) or PA28αβ complex (D) and incubated with increasing concentration of anti-βGal or 011H12 DAb. Data shown are means ± SD from triplicates and are representative of three independent experiments.

Localization of PSME1 Expression in PPC-1 Human Prostate Cancer Xenografts.

To investigate the expression and subcellular localization of PA28 subunits in tumors, we focused on PSME1 as a surrogate for the complex. The localization of PSME1 was compared with CD31 in sections of PPC-1 tumor xenografts. PSME1 was heterogeneously distributed: most of the blood vessels showed a positive staining, and there were also broad areas that were negative for CD31, but PSME1 positive (SI Appendix, Fig. S6). Tumors from mice injected i.v. with 011H12 phage showed areas of colocalization of the antibody with PSME1 (Fig. 4). The partial colocalization of the antibody phage and PSME1 is compatible with PSME1 being the target of the 011H12 DAb. Control phage remained confined to the blood vessels.

Fig. 4.

Partial colocalization of in vivo administered 011H12 phage with PSME1 in PPC-1 tumor xenografts. PSME1 is heterogeneously distributed: most of the blood vessels seem to be positive, and there are wide areas that seem to be unrelated to CD31 structures (SI Appendix, Fig. S6). The reference phage (α−βGal, first row; third row, magnification of the boxed area) primarily localizes inside PSME1-positive vessel structures but is not seen extravascularly. The 011H12 phage (second row; fourth row, magnification of the boxed area) localizes inside PSME1-positive vessels (white arrowheads) but is also present in extravascular tumor area where 011H12 colocalizes with PSME1 (white arrows). (Scale bar: 50 µm.) Red, PSME1; green, phage.

To study the accessibility of PSME1 expressed in prostate tumors, a rabbit anti-PSME1 antibody and a rabbit control IgG antibody were labeled with Cy7 and i.v. injected into PPC-1 tumor-bearing mice (n = 3 per group). Both antibodies showed a very early bladder signal spike upon injection, which could be attributed to a small percentage of trapped free Cy7 with rapid disappearance (no detectable bladder signal at 24 h postinjection). In both cases, a predominant liver signal was observed (SI Appendix, Fig. S7A), a pattern of distribution expected when IgG rabbit antibodies are used for in vivo imaging (31). The anti-PSME1 antibody localized in the tumors whereas the control IgG was not detected in the tumors (SI Appendix, Fig. S7B). Maximum resolution was achieved at ∼24 h. Ex vivo imaging of the organs further confirmed the specific accumulation of the anti-PSME1 antibody in the tumor. Whereas the uptake by the kidney was similar for both antibodies, the liver accumulated some more anti-PSME1 antibody than control IgG (SI Appendix, Fig. S7 C and D). This accumulation is likely due to differences in the IgG composition and the degree of labeling, between anti-PSME1 and control IgG (31). In fact, the expression of PSME1 in liver was confined to Kupffer cells (nuclear pattern), absent in hepatocytes, and sporadically expressed in ductal and endothelial cells (SI Appendix, Fig. S8), showing that the liver uptake of anti-PSME1 was not antigen-specific.

Validation of PSME1 as Tumor Marker in Primary and Metastatic Human Prostate Cancer.

Validation of a candidate protein biomarker in human normal and tumor tissue samples is an essential step in moving from the initial discovery to possible applications. Therefore, we studied PSME1 expression in human prostate sections by immunohistochemical staining on two tissue microarrays. In normal prostate, the anti-PSME1 antibody staining was restricted to basal cells, with faint cytoplasmic expression in the luminal cells. Most blood vessels were positive, with occasional reactivity in the surrounding stromal cells (Fig. 5 A and B). In contrast, prostate cancer samples showed much higher PSME1 expression levels than the corresponding normal counterparts. Remarkably, a strong staining was observed in cancerous epithelium and in the stromal compartment, where extracellular PSME1-positive deposits were evident. Blood vessels remained positive for PSME1 staining (Fig. 5 B and C). We next investigated PSME1 expression in advanced prostate cancer with metastases to bone and surrounding soft-tissue (Fig. 5E). Most of the tumor cells expressed PSME1. Endothelial cells and some stromal cells were also highly positive for PSME1. The prostatic origin of metastases was confirmed by PSA immunostaining (Fig. 5F).

Fig. 5.

Immunohistochemical detection of PSME1 in normal and tumor human prostate tissues. (A) Panoramic view (15×) of normal prostate showing moderate staining for PSME1. (B) Higher magnification (63×) of the indicated region from A illustrating the staining of basal epithelial cells, blood vessels, and surrounding stroma. (C) Low-magnification (15×) of prostate carcinoma exhibiting a strong widespread PSME1 immunoreactivity. (D) Higher magnification (63×) of the indicated region from C showing strong staining in tumor cells, blood vessels, and stroma. (E) PSME1 and (F) PSA immunostainings (magnification, 15×) in bone metastases with extension to surrounding soft tissues. Insets show higher magnifications (×63) of the boxed areas. (Scale bars: A, C, D, and E, 100 µm; B, D, and Insets, 20 µm.)

Discussion

Phage display is one of the preferred methods to obtain candidate therapeutic human antibodies with one of the highest transition rates between phase I and phase III studies (>65%) and with good perspectives to raise the transition rate between phase III and approval from 12.5% to near 30% (1). However, despite the wide use of the phage display technology to obtain antibodies against known and available targets (32), we believe that the potential of this technology goes further and that it can also be used to isolate antibodies against unknown but relevant targets. This procedure could, at the same time, provide an antibody and unveil a novel target (that could be either a truly unknown target or an epitope not previously identified).

We hypothesized that using a repertoire to select antibodies directly in vivo in a tumor mouse model could provide antibodies able to effectively target the tumor in vivo. Because cells cultivated in vitro can modify the pattern of markers expressed on their surface and because not all these markers would be accessible from the blood vessels, antibodies selected against a purified protein or an in vitro cultured cell may fail to access to the tumor core effectively. The in vivo strategy, which is unbiased by any prior knowledge of overexpressed markers, circumvents these limitations as the only antibodies that can be selected are those that effectively target the tumor. An ex vivo enrichment strategy, before the in vivo selection, facilitates the selection for highly diverse repertoires as the number of mice is drastically reduced. The bias toward the more abundant tumor cells is compensated in the ensuing in vivo selection that will target only the systemically accessible antigens. This procedure has already been carried out successfully using peptide phage display in tumor-bearing mice and in other disease models (9, 33, 34); however, there are very few examples of human antibodies selected with a similar procedure, and always against vascular targets: thymic endothelium (35) and atherosclerotic endothelial and subendothelial tissues (36).

In vivo peptide phage display was originally designed to reveal features specific for the blood vessels of the tissue of interest (6). This design was partially a consequence of the mechanism of selection as the repertoire contacts mainly with the vascular wall and the short circulation time intended not to allow time for the extravasation of the phage particles. However, although targeting of the endothelium has repeatedly been reported as a tumor-targeting strategy (4, 37–39), there is growing evidence that other antigens (40–42) not necessarily restricted to the endothelium may also be valuable targets. Nonetheless, in vivo phage display still faces technical challenges such as the unspecific binding and lack of enrichment. We have studied the dramatic effect of the dosage and the circulation time in both challenges. The circulation time is one of the key parameters in the in vivo selection, which, combined with the size of the phage particles used and the fenestration size [highly heterogeneous in tumor endothelium, ranging from 200 to 780 nm (43)], may influence the outcome of the selection with a greater probability of discovering nonvascular targets in tumors. Increasing the circulation time, although it may compromise the infectivity of the bound phage if they are internalized or exposed to proteases, may allow identification of antigens present in the extravascular compartment of solid tumors.

We explored several combinations of in vitro and ex vivo enrichment before the in vivo selection (SI Appendix, Fig. S9) and, based on extensive thorough sequencing, contrasted the performance of a preenriched repertoire at different circulation times. The results generally agree with previous work (35) for reference organs although we focused only on viable phage (whereas in previous work the quantification has been done by phage staining, which does not differentiate between infective and noninfective particles; therefore the greater accumulation found in others may be a consequence of the mononuclear phagocytic system). However, by using a previously enriched library for the sole purpose of establishing the appropriate circulation time, we were able to detect differences in the target organ that favored the circulation time of 24 h that would have been too subtle if using a naïve library. The differences in half-life due to different fusion proteins displayed (17–19) also suggest that the circulation time is a parameter that should be carefully evaluated for every experimental model.

The selection procedure outlined here facilitates the identification of ligands not restricted to the endothelium. The tumor distribution pattern of 011H12 clone is distinct from the vascular staining pattern. Furthermore, a staining for an endothelial marker and the phage not only can distinguish between an unspecific retention of the phage inside the vessels and a specific retention of the phage (that could show a different staining pattern), but also is useful to quantify these differences. The total positive area of tissue sections positive for CD31 immunoreactivity is similar for the mice that received the α−βGal reference phage and for those that received the 011H12 phage. The six times higher accumulation of 011H12 phage (compared with α−βGal phage) is therefore not a consequence of differences in tumor vascularization.

We believe that the strategy described here addresses several issues considered as crucial for the developing of new therapies (44). It allows for the identification and validation of new targets, which could be related to the host–host microenvironment interactions. Furthermore, the antibodies selected from this library share a high thermal stability and are less prone to aggregation than scFv selected from other commonly used libraries (45, 46). Moreover, they belong to the class of minimal binding proteins, which are the preferred targeting elements for some investigational drugs.

The 011H12 DAb was found to recognize the human PA28αβ complex. We are still investigating the precise epitope. We hypothesize that it recognizes a composite conformational epitope formed by at least two PA28 subunits (PSME1 and PSME2) based on the following considerations: the inability of 011H12 to detect the denatured antigens in Western blotting, the ability to bind to PSME 1, the ability to bind—albeit less strongly—to PSME 2 despite low sequence homology between these two proteins, and the fact that the ELISA signal increased when the PA28αβ complex was used as the target. PA28 is a ring-shaped multimeric complex that binds to the two ends of the standard (20S) proteasome and stimulates its capacity to hydrolyze small peptides. In mammals, PA28 expression is modulated by IFNγ (47), and it is a potent stimulator of MHC class I antigen presentation (48). However, it has been proposed that the involvement of PA28 in antigen presentation may be only a secondary effect of a physiological function (49). PSME1 has recently been proposed as a potential tumor marker in human esophageal squamous cell carcinoma, based on studies using proteomics approaches (50). Moreover, the C terminus of the protein has been identified (51) as a cytoplasmic marker (52). Our data indicate that PA28 can be a tumor marker for human prostate cancer and that it is accessible to targeting with i.v. injected anti-PSME1 antibodies, providing an opportunity to specifically deliver therapeutic agents into primary and metastatic prostate carcinoma. Antibody staining of tumor xenograft sections outlined two main PSME1-positive areas within the tumor (blood vessels and some areas in the perivascular region) that could correspond to tissue disorganized as a result of cellular activation and movement, or intermittent hypoxia and reperfusion. We hypothesize that, in those areas, PA28 subunits may be localized in compartments different from those they usually occupy, becoming available for antibody targeting independently of variations in the level of expression.

The PA28 complex has been observed in the nucleus (mainly PA28γ) and the cytoplasm [mainly PA28αβ (30, 53)]. How PA28 subunits might be targeted to the cell surface is unclear; however, several other intracellular proteins are aberrantly expressed at the cell surface in malignant tumors of various types (4, 42). It is also possible that a fraction of the PA28 subunits are expressed at the cell surface even in normal cells and that the overexpression we demonstrated in prostate cancers makes this protein a selective target in tumors. These possibilities remain to be fully explored, but the results we report here strongly suggest the possibility that PA28 subunits are useful biomarkers and potential drug targets for prostate carcinoma.

Materials and Methods

DAb repertoires were purified by poly(ethylene glycol) precipitation (32). The repertoire was circulated in vivo in tumor-bearing mice, and phage were recovered by trypsin release and infection (32). The target of the DAb was purified in column from a tumor lysate and identified by mass spectrometry. A description of reagents, procedures, and protocols can be found in SI Appendix.