Proteasome Activator Complex PA28 Identified As an Accessible Target In

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

David Sánchez-Martína,1, Jorge Martínez-Torrecuadradab, Tambet Teesaluc,d,e, Kazuki N. Sugaharac,d, Ana Alvarez-Cienfuegosa, Pilar Ximénez-Embúnb, Rodrigo Fernández-Periáñeza, M. Teresa Martínf, Irene Molina-Privadoa, Isabel Ruppen-Cañásb, Ana Blanco-Toribioa, Marta Cañamerog, Ángel M. Cuestaa,2, Marta Comptea, Leonor Kremerf, Carmen Bellash, Vanesa Alonso-Caminoa,3, Irene Guijarro-Muñoza, Laura Sanza, Erkki Ruoslahtic,d, and Luis Alvarez-Vallinaa,4

aMolecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, 28222 Majadahonda, Madrid, Spain; bProteomics Unit, Centro Nacional de Investigaciones Oncológicas, 28029 Madrid, Spain; cCancer Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; dCenter for Nanomedicine, University of California, Santa Barbara, CA 93106; eLaboratory of Cancer Biology, Institute of Biomedicine, Centre of Excellence for Translational Medicine, University of Tartu, 50090 Tartu, Estonia; fProtein Tools Unit, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientíﬁcas, 28049 Madrid, Spain; gHistopathology Unit, Centro Nacional de Investigaciones Oncológicas, 28029 Madrid, Spain; and hPathology Department, Hospital Universitario Puerta de Hierro Majadahonda, 28222 Majadahonda, Madrid, Spain

Edited by Richard A. Lerner, The Scripps Research Institute, La Jolla, CA, and approved July 9, 2013 (received for review January 3, 2013)

Antibody cancer therapies rely on systemically accessible targets function. One of the aspects that need to be considered when and suitable antibodies that exert a functional activity or deliver selecting antibodies for therapy is the importance of ensuring their a payload to the tumor site. Here, we present proof-of-principle of ability to reach the target. Several strategies are used to identify in vivo selection of human antibodies in tumor-bearing mice that ligands that are accessible from bloodstream, including functional identified a tumor-specific antibody able to deliver a payload and genomics analysis (3), subtractive proteomics (4), the in vivo biotinylation of vascular proteins (5), and an in vivo phage display unveils the target antigen. By using an ex vivo enrichment process SCIENCES fi against freshly disaggregated tumors to purge the repertoire, in screening for peptides that home to speci c targets in the vascu-

– APPLIED BIOLOGICAL combination with in vivo biopanning at optimized phage circula- lature (6 10). fi In vivo peptide phage display has been particularly effective tion time, we have identi ed a human domain antibody capable fi of mediating selective localization of phage to human prostate in the identi cation of markers that distinguish the vessels of diseased tissues from normal vessels (11). Homing moieties— cancer xenografts. Affinity chromatography followed by mass mainly peptides and recombinant antibodies—have been used in spectrometry analysis showed that the antibody recognizes the fi targeted delivery of therapeutic compounds to diseased organs proteasome activator complex PA28. The speci city of soluble an- and represent a promising area of pharmaceutical intervention tibody was confirmed by demonstrating its binding to the active – αβ (12 14). Whereas antibodies have certain advantages over pep- human PA28 complex. Whereas systemically administered con- tides as targeting agents (e.g., higher affinity and longer circu- fi trol phage was con ned in the lumen of blood vessels of both lation time) (15), in vivo antibody screening in tumor-bearing normal tissues and tumors, the selected phage spread from tumor mice has not been accomplished due, among others, to technical vessels into the perivascular tumor parenchyma. In these areas, limitations such as unspecific binding of phage clones. Here, we the selected phage partially colocalized with PA28 complex. Fur- overcome some of the limitations and report the isolation of thermore, we found that the expression of the α subunit of PA28 a prostate tumor-homing antibody (011H12) from a human VH [proteasome activator complex subunit 1 (PSME1)] is elevated in domain antibody library (DAb library), the identification of its primary and metastatic human prostate cancer and used anti- receptor, and the subsequent validation in primary and meta- PSME1 antibodies to show that PSME1 is an accessible marker in static human prostate cancer samples. mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention Results in prostate cancer. Repertoire Enrichment Strategy. In preparation for in vivo screening, we hypothesized that the ideal repertoire should be moder- phage library | phage display | single domain antibody | ately enriched against the target organ, leaving enough diversity physiological selection | tumor-associated antigen for a variety of antigens in the target tissue. We compared enrichment of the phage antibody library in phage that bind to he use of monoclonal antibodies (mAbs) in the clinic has Tbeen growing rapidly in the last decade. Fully human antibodies can be selected either from transgenic animals or from Author contributions: D.S.-M., J.M.-T., T.T., K.N.S., L.K., E.R., and L.A.-V. designed research; D.S.-M., J.M.-T., T.T., K.N.S., A.A.-C., P.X.-E., R.F.-P., M.T.M., I.M.-P., I.R.-C., A.B.-T., Á.M.C., large phage-displayed antibody libraries (1). There is, however, M. Compte, V.A.-C., and I.G.-M. performed research; D.S.-M., J.M.-T., T.T., K.N.S., P.X.-E., a shortage of disease-specific targets for therapeutic antibodies. M.T.M., I.R.-C., M. Cañamero, L.K., C.B., L.S., E.R., and L.A.-V. analyzed data; and D.S.-M., In fact, just 5 targets constitute one-third of the 55 human mAb J.M.-T., T.T., K.N.S., E.R., and L.A.-V. wrote the paper. anticancer candidates against known targets (1). Unbiased func- The authors declare no conflict of interest. tional identification of clinically relevant antibodies and their tar- This article is a PNAS Direct Submission. gets is an important goal. Developing an mAb without prior 1Present address: Laboratory of Cellular Oncology, Center for Cancer Research, National knowledge of the target, following a functional screening, has Cancer Institute, National Institutes of Health, Bethesda, MD 20892. a high potential for innovation (2). In this approach, mAbs are 2Present address: Buchmann Institute of Molecular Life Science, Goethe University Frank- selectedbasedontheirabilitytobindtocomplextargetsortoelicit furt, 60438 Frankfurt, Germany. a biological response, and their corresponding targets are charac- 3Present address: Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905. terized afterward, usually by a proteomics strategy. Using func- 4To whom correspondence should be addressed. E-mail: [email protected]. tional screens in vitro, however, some of the antibodies selected fail org. fi to ful ll their intended role in the clinic due to the differences with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the in vivo environment, where they might exert their therapeutic 1073/pnas.1300013110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1300013110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 cultured tumor cells (in vitro strategy) or cell suspensions from that had been previously enriched ex vivo and compared the freshly excised tumors (ex vivo strategy). To avoid overselection recovery of phage from different organs at different time points for antibody clones to tumor antigens inaccessible from the cir- after injection [ranging from 15 min to 24 h, using 4 h half-life of culation, we monitored not only the increase in the recovery (Fig. wild-type phage as a reference (20)]. The number of phage re- 1A) but also the diversity loss during the rounds of enrichment covered from the tumor at 15 min after the injection was ∼40 (Fig. 1B). Sequencing 96 clones from the input and output of each times higher than at 24 h; in the nonmalignant organs, this dif- selection round confirmed the expectation that in vitro selection ference was ∼100-fold (Fig. 1C, Upper). This difference is more resulted in a bias toward a few dominant sequences whereas ex easily visualized using a value normalized to control phage (Fig. vivo selection of the phage repertoire enriched a more diverse 1C, Lower). These results suggest that specific accumulation in the repertoire of phage clones. 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 Optimizing Circulation Time and Selection of Candidates. A circula- the in vivo selections, a sample of clones was sequenced from the tion time of 15 min is considered to be sufficient to address most input pool, from phage recovered from tumor tissue and from of the targets exposed in the endothelium in nonmalignant tis- reference organs. Candidate tumor-homing phage clones were sues but may be too short for dysfunctional tumor vessels (16). A appraised based on several criteria: abundance in the target out- circulation time of 1 h has been proposed for a filamentous put, similarity between self and consensus complementarity de- phage peptide library (17) based on the half-life of the phage termining regions (CDR) sequences obtained from hierarchical although there is evidence that the displayed exogenous protein clustering, presence and composition of the amber stop codons, or peptide can make the circulation half-life as short as 1.5 min and presence and composition of “common known homing and as long as 4.5 h (18, 19). We administered i.v. a phage pool motifs” [such as integrin-binding RGD, neuropilin-1 binding

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 = 5mice 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 re- tentioninthetumor(iii–v), and retention in all of the organs (ii). Significant differences (P value) U Mann– Whitney.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1300013110 Sánchez-Martín et al. Downloaded by guest on September 24, 2021 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.

Fig. 2. Tumor-specific homing of 011H12 phage. Sections from tumors that Homing Assays. The ability of the four selected clones to home to α–β the tumor was compared with the reference α−βGal phage. A have received the phage (either the reference Gal or 011H12) were analyzed by immunofluorescence, and the areas positive for CD31 (red) and the fresh preparation of each phage clone was i.v. injected into n = 5 phage (green) were quantified. No difference was found in the total area a group of mice ( per group), with the circulation time set at positive for endothelial markers (A). There was a higher phage staining in the SCIENCES 24 h (Fig. 2). To validate the reproducibility of the normalized 011H12 clone (B). The V parameter (C, total phage/total endothelium) was six value used, two additional mice were injected with a 1:100 di- fi <

times higher in the 011H12 clone (D). **Signi cant differences (P 0.0001) U APPLIED BIOLOGICAL lution of one of the selected clones to compare the total amount Mann–Whitney. (E) Heterogeneous distribution of the phage. The 011H12 of phage recovered and the normalized value for a given speci- phage accumulates in the interior of the tumors, compared with the refer- ficity and different dosage (SI Appendix, Fig. S3). The reference ence phage. In both cases, there is unspecific retention of the phage in the α−βGal phage (Fig. 1E, i) showed a baseline accumulation in the rim of the tumor. (F) The distribution of the reference phage (α–βGal) is re- tumor, similar to the accumulation in the kidney, and a lower stricted to the inner side of some vessels, as well as the edge of the tumor accumulation in both heart and liver. Three of the four selected (Top). The distribution of the 011H12 phage is heterogeneous: there are small clones showed a higher uptake in the tumor (010H01, 011H12, deposits in wide stromal areas (Middle) as well as major deposits around some and 010D6; Fig. 1E, iii–v) whereas the remaining clone, which vessels identified as such by CD31 staining (Bottom). (Scale bar: 50 μm.) 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 PA28 Is the Target of the 011H12 DAb. To identify the target of the characterization based on the distribution observed. 011H12 DAb, we subjected tumor extracts to affinity chromatography. We used columns coupled with the anti−βGal DAb (SI 011H12 Phage Accumulates in Tumors. Phage may be prone to un- Appendix, Fig. S5) or the 011H12 DAb in tandem for the sepa- specific aggregation (26, 27) and/or interaction with tissue com- ration. Affinity-purified proteins were digested by using the filter- ponents accessible from the bloodstream. Therefore, we decided aided sample preparation (FASP) method and then identified by to investigate whether the retention observed for the different nano liquid chromatography–mass spectrometry (LC-MS). phage was specific to the antibody harbored, or merely the result Eluted proteins from the anti−βGal and 011H12 columns were of entrapment of phage aggregates in the tumor vascular tree. If compared, and those present in both eluates (more than one the latter were true, (i) a colocalization of endothelial markers peptide in the control) were disregarded (SI Appendix, Table with the i.v. administered phage should be expected; and (ii)the S3). The remaining proteins were ranked according to the differences in the amount of phage recovered should be related to number of peptides found in the eluate from the 011H12 col- differences in the number of vessels. umn (SI Appendix,TableS4). To prioritize the potential can- We compared the endothelial staining of the tumors from didates, the number of spectra associated with each protein mice dosed with the reference phage, with tumors of mice that [peptide spectrum match (PSM) number] was also considered. had received the 011H12 clone. Analysis of equally vascularized Notably, two of the selected proteins are subunits of the 11S tumors from mice injected with the 011H12 phage or the ref- proteasome activator complex [PA28 (28, 29)]: subunit α [pro- erence phage (Fig. 2A) showed greater accumulation of the teasome activator complex subunit 1 (PSME1)] and subunit β 011H12 phage in the tumors both when the total area positive for (PSME2). SDS/PAGE revealed two closely spaced bands at the phage staining (Fig. 2B), or the amount of phage over en- about 29 kDa in the 011H12 DAb eluate that were not present dothelium area (Fig. 2C), was compared (P < 0.0001, Fig. 2D). in the control DAb eluate (Fig. 3A). These two bands were The high dispersion of the values reflects the heterogeneous ac- further identified as PSME1 (Q06323; 28.723 kDa) and PSME2 cumulation of the phage (Fig. 2E); whereas the reference phage (Q9UL46; 27.402 kDa) by mass spectrometry (SI Appendix, was confined to the lumen of the vessels (Fig. 2F, Top), the 011H12 Table S5), in agreement with the results obtained by the FASP phage was also found spreading from the vessels into wide areas of method (SI Appendix, Table S4). The identity of the antigen surrounding perivascular stroma (Fig. 2F, Middle and Bottom). recognized by the 011H12 DAb was confirmed by ELISA. Sol- This specific accumulation pattern was not observed in the refer- uble purified 011H12 DAb showed reactivity with the active ence organs where both phage clones were confined to the vessels PA28αβ complex (30) and to a lesser extent with individual (SI Appendix,Fig.S4). We suggest that the progressive accumu- PA28 subunits (Fig. 3B). No reactivity was observed with two lation of the 011H12 phage in the tumor is due to the combination lower score candidates (HPRT and NDKB) or βGal. Further- of the interaction with a tumor-associated antigen with leaky tumor more, 011H12 bound specifically to immobilized PA28αβ in vessels and long phage circulation time. a concentration-dependent manner (Fig. 3 C and D).

Sánchez-Martín et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 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 micro- arrays. 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 occa- sional 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 epithe- lium 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. 3. Specific interaction of 011H12 DAb with PA28 subunits. (A) Fresh Discussion tumor lysates were purified using affinity chromatography with columns Phage display is one of the preferred methods to obtain candidate coupled with the α–βGal and 011H12 DAbs. The eluates were subjected to therapeutic human antibodies with one of the highest transition SDS/PAGE and stained using SYPRO dye, and the bands (arrows) were ana- rates between phase I and phase III studies (>65%) and with lyzed by mass spectrometry. (B) Purified soluble α–βGal or 011H12 DAbs (20 good perspectives to raise the transition rate between phase III μg/mL) were tested against a panel of plastic-immobilized candidates (1 μg and approval from 12.5% to near 30% (1). However, despite the per well). The interaction between 011H12 and the PA28αβ complex in vitro wide use of the phage display technology to obtain antibodies is dose-dependent. Microtiter plates were coated (1 μg per well) with βGal against known and available targets (32), we believe that the (C) or PA28αβ complex (D) and incubated with increasing concentration of potential of this technology goes further and that it can also be anti-βGal or 011H12 DAb. Data shown are means ± SD from triplicates and used to isolate antibodies against unknown but relevant targets. are representative of three independent experiments. 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). Localization of PSME1 Expression inPPC-1HumanProstateCancer We hypothesized that using a repertoire to select antibodies Xenografts. To investigate the expression and subcellular localiza- directly in vivo in a tumor mouse model could provide antibodies tion of PA28 subunits in tumors, we focused on PSME1 as a sur- able to effectively target the tumor in vivo. Because cells culti- rogate for the complex. The localization of PSME1 was compared vated in vitro can modify the pattern of markers expressed on with CD31 in sections of PPC-1 tumor xenografts. PSME1 was their surface and because not all these markers would be acces- heterogeneously distributed: most of the blood vessels showed sible from the blood vessels, antibodies selected against a purified 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. 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 attrib- uted to a small percentage of trapped free Cy7 with rapid disap- pearance (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 resolu- tion 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.S7C 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 Fig. 4. Partial colocalization of in vivo administered 011H12 phage with expression of PSME1 in liver was confined to Kupffer cells (nuclear 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 pattern), absent in hepatocytes, and sporadically expressed in ductal seem to be unrelated to CD31 structures (SI Appendix, Fig. S6). The reference and endothelial cells (SI Appendix,Fig.S8), showing that the liver α−β fi fi fi phage ( Gal, rst row; third row, magni cation of the boxed area) pri- uptake of anti-PSME1 was not antigen-speci c. marily localizes inside PSME1-positive vessel structures but is not seen extravascularly. The 011H12 phage (second row; fourth row, magnification Validation of PSME1 as Tumor Marker in Primary and Metastatic of the boxed area) localizes inside PSME1-positive vessels (white arrowheads) Human Prostate Cancer. Validation of a candidate protein bio- but is also present in extravascular tumor area where 011H12 colocalizes with marker in human normal and tumor tissue samples is an essential PSME1 (white arrows). (Scale bar: 50 μm.) Red, PSME1; green, phage.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1300013110 Sánchez-Martín et al. Downloaded by guest on September 24, 2021 protein or an in vitro cultured cell may fail to access to the tumor lack of enrichment. We have studied the dramatic effect of the core effectively. The in vivo strategy, which is unbiased by any dosage and the circulation time in both challenges. The circulation prior knowledge of overexpressed markers, circumvents these time is one of the key parameters in the in vivo selection, which, limitations as the only antibodies that can be selected are those combined with the size of the phage particles used and the fen- that effectively target the tumor. An ex vivo enrichment strategy, estration size [highly heterogeneous in tumor endothelium, rang- before the in vivo selection, facilitates the selection for highly ing from 200 to 780 nm (43)], may influence the outcome of the diverse repertoires as the number of mice is drastically reduced. selection with a greater probability of discovering nonvascular The bias toward the more abundant tumor cells is compensated in targets in tumors. Increasing the circulation time, although it may the ensuing in vivo selection that will target only the systemically compromise the infectivity of the bound phage if they are inaccessible antigens. This procedure has already been carried ternalized or exposed to proteases, may allow identification of out successfully using peptide phage display in tumor-bearing antigens present in the extravascular compartment of solid tumors. mice and in other disease models (9, 33, 34); however, there are We explored several combinations of in vitro and ex vivo en- SI Appendix very few examples of human antibodies selected with a similar richment before the in vivo selection ( , Fig. S9) and, procedure, and always against vascular targets: thymic endothe- based on extensive thorough sequencing, contrasted the perfor- lium (35) and atherosclerotic endothelial and subendothelial mance of a preenriched repertoire at different circulation times. tissues (36). The results generally agree with previous work (35) for reference organs although we focused only on viable phage (whereas in In vivo peptide phage display was originally designed to reveal fi features specific for the blood vessels of the tissue of interest (6). previous work the quanti cation has been done by phage stain- This design was partially a consequence of the mechanism of ing, which does not differentiate between infective and non- selection as the repertoire contacts mainly with the vascular wall infective particles; therefore the greater accumulation found in and the short circulation time intended not to allow time for the others may be a consequence of the mononuclear phagocytic extravasation of the phage particles. However, although target- system). However, by using a previously enriched library for the ing of the endothelium has repeatedly been reported as a tumor- sole purpose of establishing the appropriate circulation time, we targeting strategy (4, 37–39), there is growing evidence that other were able to detect differences in the target organ that favored the circulation time of 24 h that would have been too subtle if antigens (40–42) not necessarily restricted to the endothelium using a naïve library. The differences in half-life due to different may also be valuable targets. Nonetheless, in vivo phage display – fi fusion proteins displayed (17 19) also suggest that the circula- still faces technical challenges such as the unspeci c binding and tion time is a parameter that should be carefully evaluated for

every experimental model. SCIENCES The selection procedure outlined here facilitates the identiﬁ-

cation of ligands not restricted to the endothelium. The tumor APPLIED BIOLOGICAL 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 unspecificre- tention 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 αβ Fig. 5. Immunohistochemical detection of PSME1 in normal and tumor PA28 complex was used as the target. PA28 is a ring-shaped human prostate tissues. (A) Panoramic view (15×) of normal prostate multimeric complex that binds to the two ends of the standard showing moderate staining for PSME1. (B) Higher magnification (63×)ofthe (20S) proteasome and stimulates its capacity to hydrolyze small indicated region from A illustrating the staining of basal epithelial cells, peptides. In mammals, PA28 expression is modulated by IFNγ blood vessels, and surrounding stroma. (C) Low-magnification (15×)ofpros- (47), and it is a potent stimulator of MHC class I antigen pre- tate carcinoma exhibiting a strong widespread PSME1 immunoreactivity. (D) sentation (48). However, it has been proposed that the involvement Higher magnification (63×) of the indicated region from C showing strong of PA28 in antigen presentation may be only a secondary effect of staining in tumor cells, blood vessels, and stroma. (E)PSME1and(F)PSA a physiological function (49). PSME1 has recently been proposed as immunostainings (magnification, 15×) in bone metastases with extension to a potential tumor marker in human esophageal squamous cell surrounding soft tissues. Insets show higher magnifications (×63) of the boxed carcinoma, based on studies using proteomics approaches (50). areas. (Scale bars: A, C, D,andE,100μm; B, D,andInsets,20μm.) Moreover, the C terminus of the protein has been identified (51) as

Sánchez-Martín et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 a cytoplasmic marker (52). Our data indicate that PA28 can be PA28 subunits are useful biomarkers and potential drug targets a tumor marker for human prostate cancer and that it is accessible for prostate carcinoma. to targeting with i.v. injected anti-PSME1 antibodies, providing an opportunity to specifically deliver therapeutic agents into primary Materials and Methods and metastatic prostate carcinoma. Antibody staining of tumor DAb repertoires were purified by poly(ethylene glycol) precipitation (32). The xenograft sections outlined two main PSME1-positive areas within 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 the tumor (blood vessels and some areas in the perivascular region) fi that could correspond to tissue disorganized as a result of cellular in column from a tumor lysate and identi ed by mass spectrometry. A de- activation and movement, or intermittent hypoxia and reperfusion. scription of reagents, procedures, and protocols can be found in SI Appendix. We hypothesize that, in those areas, PA28 subunits may be local- ACKNOWLEDGMENTS. We thank A. Gómez-Robles for advice, discussion, ized in compartments different from those they usually occupy, and statistical guidance; M. J. Coronado for help with the confocal micro- becoming available for antibody targeting independently of varia- scope; and O. Coux (Centre de Recherche en Biochimie Macromoléculaire- tions in the level of expression. Centre National de la Recherche Scientifique, Universités Montpellier, The PA28 complex has been observed in the nucleus (mainly France) for providing the PA28αβ complexes. We thank the Centro Nacional PA28γ) and the cytoplasm [mainly PA28αβ (30, 53)]. How PA28 de Investigaciones Oncológicas Tumor Bank and the H. U. Marqués de Val- decilla Tumor Bank (Santander, Spain) for kindly providing the cases in- subunits might be targeted to the cell surface is unclear; however, cluded in this study. This study was supported by Ministerio de Economía y several other intracellular proteins are aberrantly expressed at Competitividad Grant BIO2011-22738 and Comunidad de Madrid Grant the cell surface in malignant tumors of various types (4, 42). It is S2010/BMD-2312 (to L.A.-V.), and by US Department of Defense Prostate also possible that a fraction of the PA28 subunits are expressed Cancer Program Grant W81XWH-10-1-0199 (to E.R.). D.S.-M. was supported at the cell surface even in normal cells and that the overexpression by Comunidad de Madrid/Fondo Social Europeo Training Grant FPI-000531, by a Fundación de Investigación del H. U. Puerta de Hierro travel grant, and by we demonstrated in prostate cancers makes this protein a selective a Boehringer Ingelheim Fonds travel grant. P.X.-E. was partially supported target in tumors. These possibilities remain to be fully explored, by Instituto de Salud Carlos III Grant CA10/01231. V.A.-C. was supported by but the results we report here strongly suggest the possibility that Predoctoral Fellowship BFI07.132 from the Gobierno Vasco.

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