Survivin Monoclonal Antibodies Detect Survivin Cell Surface Expression and Inhibit Tumor Growth in Vivo Robert A

Published OnlineFirst March 14, 2018; DOI: 10.1158/1078-0432.CCR-17-2778

Cancer Therapy: Preclinical Clinical Cancer Research Survivin Monoclonal Antibodies Detect Survivin Cell Surface Expression and Inhibit Tumor Growth In Vivo Robert A. Fenstermaker1,2,3,4, Sheila A. Figel1, Jingxin Qiu5, Tara A. Barone1, Sanam S. Dharma1, Evan K. Winograd4, Phillip M. Galbo1, Laura M. Wiltsie6, and Michael J. Ciesielski1,2,4

Abstract

Purpose: Survivin is an inhibitor of apoptosis protein (IAP) that Results: Here we demonstrate that survivin is present on the is highly expressed in many cancers and represents an attractive outer cell membrane of a wide variety of cancer cell types, molecule for targeted cancer therapy. Although primarily regarded including both murine and human glioma cells. In addition, as an intracellular protein with diverse actions, survivin has also antibodies to survivin that are derived from the immunogen been identified in association with circulating tumor exosomes. display antitumor activity against murine GL261 gliomas in both Experimental Design: We have reported that active, specific flank and intracranial tumor models and against B16 melanoma vaccination with a long peptide survivin immunogen leads to the as well. development of survivin-specific CD8-mediated tumor cell lysis Conclusion: In addition to immunogen-induced, CD8-medi- and prolongation of survival in tumor-bearing mice. In addition ated tumor cell lysis, antibodies to the survivin immunogen have to cellular antitumor responses, circulating anti-survivin antibo- antitumor activity in vivo. Cell-surface survivin could provide a dies are detected in the serum of mice and human glioblastoma specific target for antibody-mediated tumor immunotherapeutic patients following vaccination with the survivin immunogen. approaches. Clin Cancer Res; 1–11. 2018 AACR.

Introduction therapy (8). Epitopes of survivin are presented by MHC I com- plexes on the surface of tumor cells making them immunolog- Survivin (BIRC5, API4) is a protein that belongs to a family of þ ically targetable by specific cytotoxic CD8 T cells. Patients with apoptosis inhibitors and acts in concert with the mitotic spindle cancer have anti-survivin antibodies (9) and survivin-specificT apparatus to regulate cell division and chromosomal segregation cells (10) in their peripheral blood. Therefore, survivin is immu- (1). It is expressed during the G –M phase of the cell cycle in 2 nogenic and its immunogenicity may be enhanced with the aid of association with the spindle microtubule organizing center (2). survivin mimics (11, 12). Several studies have looked at active, Survivin functions in critical roles at other cellular loci to regulate specific vaccination against survivin to treat various cancers (13– the cell cycle and to inhibit apoptotic cell death. It is frequently 15), including malignant gliomas (16). expressed by cancer cells of many different types, but its expres- Although it was initially defined as an intracellular molecule, sion is uncommon in normal adult fully differentiated tissues (3). more recently, survivin has been identified in association with Expression of survivin in gliomas and other cancers is associ- exosomes produced by cervical and prostatic carcinoma cells (17, ated with a poor prognosis (4–7) and refractoriness to chemo- 18). While the intracellular functions of survivin have been characterized extensively, less well appreciated is its presence and 1Department of Neurosurgery, Roswell Park Comprehensive Cancer Center, function on the outer membrane of tumor cells where it is Buffalo, New York. 2Department of Immunology, Roswell Park Comprehensive potentially targetable using antibody-mediated immunothera- Cancer Center, Buffalo, New York. 3Center for Immunotherapy, Roswell Park peutic strategies. Here we identify survivin expression on the Comprehensive Cancer Center, Buffalo, New York. 4Department of Neurosur- surface of a wide range of cancer cell types using complementary gery, Jacobs School of Medicine and Biomedical Sciences, State University of techniques and demonstrate that mAbs derived from immuniza- New York at Buffalo, Buffalo, New York. 5Department of Pathology, Roswell Park tion with an engineered survivin peptide have antitumor prop- Comprehensive Cancer Center, Buffalo, New York. 6Department of Pediatrics, Jacobs School of Medicine and Biomedical Sciences, State University of New erties in different mouse tumor models. York at Buffalo, Buffalo, New York. R.A. Fenstermaker and M.J. Ciesielski are co-inventors listed on patents regard- Methods ing the survivin vaccine and are co-founders of MimiVax, LLC, which has licensed Cell lines and culture conditions such patents from Roswell Park Comprehensive Cancer Center. GL261, U87, HT-29, HeLa, A2780, A1207, B16f1, Jurkat, Raji, Corresponding Author: Michael J. Ciesielski, Roswell Park Comprehensive MCF7, PC-3, and HEK293T cells were grown in complete DMEM Cancer Center, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845- containing 10% FCS, 5,000 units of penicillin/streptomycin, 50 8850; Fax: 716-845-8980; E-mail: [email protected] mmol/L 2-mercaptoethanol, 25 nmol/L HEPES, and 1 nones- doi: 10.1158/1078-0432.CCR-17-2778 sential amino acids at 37 Cin5%CO2 with media changes two to 2018 American Association for Cancer Research. three times per week. For animal studies, GL261 and B16f1 were

www.aacrjournals.org OF1

Fenstermaker et al.

(SVN53-67) and engineered (SVN53-67/M57) peptides. Two Translational Relevance stable subclonal cell lines of each primary clone were chosen for Survivin is one of the most specific and ubiquitous cancer cryopreservation based on confirmed antigen-recognition and molecules identified to date. Attention has been directed to normal doubling time. Isotype identification for all the subcloned targeting this protein via small-molecule inhibitors and spe- cell lines was performed. Subclone cultures were scaled up for cific immunologic approaches. To date, vaccine therapy has production of purified IgG. been the primary immunotherapeutic strategy studied, based on extensive research, which has focused on survivin as an DNA constructs intracellular molecule. Thus, proteasomal processing of the To generate hSurvivin-Myc/FLAG, human survivin fused to molecule leads to presentation of survivin epitopes on the Myc and FLAG tags at its C-terminus was first excised from a surface of tumor cells where they are subject to immune commercial cDNA clone (OriGene). The survivin-Myc/FLAG – recognition via T-cell receptor mediated processes. Our data insert was then used as the template in a PCR reaction with fi show for the rst time that the survivin protein is present on primers hSVN-EcoRI-F (50-GCGATCGAATTCGTCGACTGG-30) the surface of a wide variety of tumor cell types and that anti- and hSVN-XbaI-R (50-ACTCCTCTAGAAAACCTTATCGTCGTC- fi survivin mAbs have signi cant therapeutic antitumor effects 30). Digested insert was then ligated into pcDNA3.1 (Thermo in vivo. Thus, antibody-mediated, immunotherapeutic strate- Fisher Scientific), and the construct was validated by sequenc- gies that target survivin could offer promising treatment ing. To generate the GST-hSurvivin plasmid used for protein approaches in the future. purification, the survivin ORF (but not Myc or FLAG tag sequence) was excised from a commercial cDNA clone (Ori- Gene) using EcoRI and NotI, and ligated into pGEX-5X-2 (GE Healthcare Life Sciences). obtained from the Division of Cancer Treatment and Diagnosis (DCTD) repository at the National Cancer Institute, NIH Purification of GST-hSurvivin (Bethesda, MD). Additional cell lines were obtained through Overnight cultures of BL21-CodonPlus (DE3)-RIL (Agilent) ATCC. No further authentication studies were done. containing GST-hSurvivin or the empty pGEX-5X-2 vector (500 mL) were used to inoculate 10 mL of Terrific Broth containing Antibodies ampicillin and 80 mmol/L ZnCl2, which was then grown to mid- Anti-c-Myc Tag (9E10) was purchased from Thermo Fisher log phase at 37 C with shaking. Expression was induced with 0.2 Scientific. Anti-Caveolin-1 and anti-CD71 (D7G9X) were pur- nmol/L IPTG for 5 hours at 30 C, after which bacteria were chased from Cell Signaling Technology. Anti-Flotillin-1 (18/Flo- pelleted, resuspended in buffer containing 50 nmol/L Tris-Cl, pH tillin-1) was purchased from BD Transduction Laboratories. Anti- 7.4, 150 nmol/L NaCl, 5 nmol/L MgCl2,80mmol/L ZnCl2,1 GST was purchased from ABM labs. Anti-survivin (60.11) was nmol/L DTT plus protease inhibitors, and lysed by freeze/thaw purchased from Novus Biologicals. Anti-Ga q/11/14 (G7), anti- and sonication. Supernatants were incubated with Glutathione EGFR (R-1) and anti-GAPDH (FL-335) were purchased from Agarose (Thermo Fisher Scientific) for 4 hours at 4 C, and then Santa Cruz Biotechnology. beads were collected and washed. GST and GST-Survivin were eluted in buffer containing 50 nmol/L Tris-Cl, pH 8.0, 20 nmol/L Peptide synthesis and antibody production reduced L-Glutathione and 0.1% Triton-X by incubation for 10 Survivin peptide synthesis was performed using Fmoc chem- minutes at room temperature, then elutions were concentrated istry and solid support resin and followed by conjugation to using 10 KD MWCO concentrator columns (Thermo Fisher fi keyhole limpet hemocyanin (KLH) for immunization (Genscript Scienti c). Protein concentrations were measured by bicincho- USA). Additional survivin peptides, including N-terminal bioti- ninic acid (BCA) assay and adjusted by adding buffer without nylated survivin, were synthesized by CS Bio. Hybridomas were glutathione or Triton-X. generated under contract with Genscript USA. Ten C57BL/6 mice were prebled at 4 days and followed with immunization sub- Measurement of antibody affinity cutaneously with SVN53-67/M57-KLH on day 0. Mice were Antibody binding to survivin peptides was measured using boosted on days 14, 28, and 56 and test bled at 21 and 35 days. the AlphaScreen reagent Mouse IgG Detection Kit (Perkin- Cells were fused to create hybridomas on day 60. Immune Elmer). Reactions were carried out in volumes of 50 mLin responses were tested by ELISA and Western blot analysis with wells of an opaque, half-area 96-well plate, in buffer consisting target and irrelevant tagged protein 7 days after each boost of PBS pH 7.4, 0.1% BSA, and 0.01% Tween-20. Each well immunization. On the basis of the test bleed result, two animals contained a final concentration of 20 mg/mL Acceptor Beads, were selected for electrofusion. A fusion efficiency of one hybrid- 20 mg/mL Donor Beads, 0.5 nmol/L antibody (2C2 or 30H3) oma per 2,500 splenocytes was expected. An average of 1 108 B and 0.02–20 nmol/L biotinylated survivin peptide. Briefly, cells from each spleen of immunized mouse provides an antic- antibody was incubated with anti-mouse IgG Acceptor Beads ipated recovery of 2 104 hybridoma clones. All fused cells from plus dilutions of biotinylated survivin peptide for 30 minutes each cell fusion were plated into 15 96-well plates. Supernatants at room temperature, after which Streptavidin Donor Beads were screened by indirect ELISA with antigen peptide. Twenty were added. The reactions were incubated another 30 minutes positive clones were seeded into 24-well plates, and five positive in the dark at room temperature and read on an EnVision primary clones were subcloned by limiting dilution. The clones Excite Multilabel Reader (Perkin-Elmer). Data was fitusing were carried for a maximum of three generations. Subcloned cells GraphPad Prism 7, and Kd values were extrapolated from were again screened by indirect ELISA with both wild-type saturation curves.

OF2 Clin Cancer Res; 2018 Clinical Cancer Research

Survivin mAbs Inhibit Tumor Growth

Immunofluorescence microscopy plus protease inhibitors. In the case of HEK293T, cells in 15-cm Cultured cells were seeded on sterile glass coverslips in com- dishes were transfected with the hSVN-Myc/FLAG construct using plete media overnight. Cells were washed with PBS, fixed for 15 Lipofectamine 2000 (Thermo Fisher Scientific) 24 hours prior to minutes at room temperature in 4% paraformaldehyde and (if collection. After 15 minutes on ice, cells in Base Buffer were noted) permeabilized for 10 minutes using 0.05% Triton-X. homogenized through a 22-gauge needle 20 times and centri- Coverslips were incubated with the indicated survivin antibodies fuged at 1,000 g for 10 minutes. The post-nuclear supernatant for 2 hours at room temperature, washed with PBS, incubated was collected on ice, and the nuclear pellet was subjected to with secondary antibodies for 1 hour at room temperature, and homogenization and extraction again in 1 mL Base Buffer. Both then washed in PBS overnight at 4C before mounting on glass post-nuclear supernatants were pooled and stored at 20C. slides and analyzing by fluorescence microscopy. Upon thawing, the post-nuclear supernatant was mixed with 2 mL of 50% Optiprep (Stemcell Technologies) and dispensed into Immunohistochemistry a 12-mL tube. Two milliliters each of 20%, 15%, and 10% Formalin-fixed paraffin-embedded sections from a multi- Optiprep solutions were then layered above the post-nuclear tumor tissue microarray and one multi-organ normal tissue array supernatant, followed by 2-mL base buffer. Gradients were cen- (FDA999n; Biomax; 0.6 mm per core) were cut at 4 mm, placed on trifuged at 52,000 g for either 2 (U87) or 3 hours (A1207 and charged slides, and dried at 60C for 1 hour. Slides were depar- HEK293T) in a Beckman Coulter L9-55M Ultracentrifuge affinized at room temperature through a series of xylene and equipped with SW28 rotor. After centrifugation, fractions were graded ethanol baths. Slides were pretreated in citrate buffer collected as 0.67 mL, and 25 mL of each was loaded onto 12% SDS- (BioCare Medical CB910) for 60 minutes in a steamer and then PAGE for Western blot analysis. cooled for 20 minutes. Slides were quenched in 0.3% H2O2 for 10 minutes, followed by protein block (X909, Dako) for 5 minutes. Lipid raft visualization Slides were incubated with 2C2 antibody (1:100; 2 mg/mL) in a Lipid rafts in GL261 cells were labeled using the Vybrant Lipid humidity chamber for 16 hours. Slides were loaded onto the Dako Raft Labeling Kit (Molecular Probes) as recommended by the Autostainer PLUS and mouse Envision HRP was applied for 30 manufacturer. Briefly, cells were detached from culture dishes and minutes (Dako K4007). Diaminobenzidine (Dako) was applied resuspended in cold complete media containing 1 mg/mL Alexa for 10 minutes. Slides were counterstained with hematoxylin for 2 Fluor 594 dye-labeled CT-B, incubated for 10 minutes at 4C, minutes, dehydrated, cleared, and coverslipped. washed in cold PBS, and then resuspended in anti-CT-B antibody solution (anti-CT-B diluted 1:200 in compete media) for 15 Flow cytometry staining of tumor cell lines minutes at 4 C. Cells were washed twice with PBS, then resus- Aliquots of anti-survivin 2C2 (0.2 mg each) were preincubated pended in Cell Staining Buffer (Biolegend) and stained with 0.2 with 100 mg of immunizing survivin peptide or scrambled peptide mg of 2C2 for 45 minutes at room temperature, washed with PBS, for 30 minutes at room temperature. Cells were trypsinized, incubated with FITC goat anti-mouse antibody, and analyzed by washed, and suspended in staining buffer. Preincubated antibody imaging flow cytometry on an ImageStreamX Mark II Imaging was added, and cells were incubated at room temperature for 1 Flow Cytometer (AMNIS/Millipore). hour. Cells were washed with PBS and stained with anti-mouse- FITC antibody for 30 minutes at room temperature, washed again, Western blotting and data acquired with a BD Fortessa flow cytometer running Cells were lysed in RIPA buffer and protein concentration was FACSDiva software with final analysis using FCS Express v4.0. measured using Pierce BCA Assay Reagent. Aliquots of 20 mg total protein were mixed with Laemmli buffer, boiled, and separated Protease treatment of cells on 12% SDS-PAGE gels, and transferred to polyvinylidene Cultured cells were collected either by gently dislodging with difluoride membranes, and then blocked with 5% nonfat milk PBS or by limited trypsinization, then washed with PBS and in TBST for 1 hour. Antibodies were applied in blocker overnight resuspended in DMEM to a concentration of 106/mL. Cells were at dilutions of 1:1,000 (60.11) or 1:100 (2C2 and 30H3), after treated with either trypsin (1 mg/mL) or proteinase K (0.5 mg/ which the membranes were washed, incubated with HRP-conju- mL) for 20 minutes at 37C, then proteases were inactivated by the gated secondary antibody, washed again, incubated with ECL addition of either 1 mg/mL soybean trypsin inhibitor (for trypsin) reagent, and exposed to X-ray film. For Western blotting of or protease inhibitor diluted to 1 and 1 mmol/L phenylmethyl- purified protein, 0.2 mg of GST or GST-Survivin were loaded in sulfonylfluoride. Cells were washed twice with PBS, then resus- triplicate onto a 12% SDS-PAGE gel, transferred, and blots were pended in Cell Staining Buffer (Biolegend) or Cell Staining Buffer processed as above using anti-GST at 1:1,000 and 2C2/30H3 at plus Fixation Buffer (Biolegend) plus 0.1% Triton-X. Cells were 1:500. ECL Chemiluminescence was captured using the Bio-Rad then stained with 0.2 mg of 2C2 for 1 hour at room temperature, ChemiDoc Touch Imaging System. washed once with PBS, incubated with FITC goat anti-mouse antibody, and analyzed by flow cytometry. Cell growth GL261 cells were plated on sterile 96-well plates (2 103 cells Isolation of lipid raft fractions per well). Cells were treated with 2C2 and 30H3 antibodies in Lipid rafts were isolated following the detergent-free method of DMEM at 10 mg/mL in quadruplicate. Daily, cells were washed Macdonald and Pike, 2005 (19). Briefly, cells were scraped from with PBS and fixed with 100% methanol at 4 C for 10 minutes. three to four 15-cm culture plates into PBS, collected, and resus- Fixed cells were stained with crystal violet for 10 minutes, washed, pended in 1 mL of Base Buffer containing 20 nmol/L Tris-Cl, pH and allowed to dry. Crystal violet was solubilized in 10% acetic 7.8, 250 nmol/L sucrose, 1 nmol/L CaCl2, and 1 nmol/L MgCl2, acid and absorbance measured on a Synergy (TM) HTX

www.aacrjournals.org Clin Cancer Res; 2018 OF3

Fenstermaker et al.

multi-mode microplate reader at 595 nm. Data were analyzed CO2 incubator for 5 to 6 hours, after which luminescence was with GraphPad PRISM. measured using Bio-Glo Reagent (Promega).

GL261 and B16 flank tumor models Results C57BL6 and nude (nu/nu) mice were implanted with murine Mouse mAbs detect wild-type and engineered survivin peptides GL261 glioma cells or B16f1 melanoma cells. For flank tumors, a A panel of 19 murine hybridoma cell lines was developed suspension of 2 107 Gl261 glioma cells or 1 106 B16f1 against the engineered survivin peptide (SVN53-67/M57). Super- melanoma cells in 100 mL of PBS was injected subcutaneously in natants were screened, and high-affinity IgG mAbs were produced. male C57Bl/6 (immunocompetent) mice (Charles River) and in Purified antibodies were characterized for target recognition and nude (immunocompromised) NCr-nu/nu mice (Charles River). specificity. Cross-reactivity of individual hybridoma culture After 3 days, mice were randomly assigned to groups and were supernatants to wild-type survivin peptide SVN53-67 from 13 given intraperitoneal (i.p.) injections of 10 mg of anti-survivin representative cell lines are shown in Fig. 1A. We chose to focus on antibody 2C2 or 10 mg of IgG control antibody. Antibodies were two of these clones (2C2 and 30H3) with different affinities and administered every 5 days for a total of four doses. Tumor growth isotype. Antibodies were compared with a commercially available was measured daily using calipers and volumes were calculated survivin antibody (60.11; Novus Biologicals) in Western blots of according to the formula V ¼ {a b2}/2, where V is volume and a total protein derived from human embryonic kidney 293T cells and b are perpendicular diameters of the tumor. Subcutaneous (Fig. 1B). All three antibodies detected a 16.5 kDa survivin species tumor volume significance was measured using Wilcoxon with limited minor bands (Fig. 1C). To measure antibody affinity, matched pairs signed rank test. All mouse flank tumor model we utilized the fluorescence proximity-based AlphaLISA assay in experiments were approved by the Roswell Park Institutional which binding K was measured from the saturation of antibodies Animal Care and Use Committee (IACUC). d by biotinylated wild-type survivin peptide SVN53-67, (Fig. 1F). Anti-survivin antibody 2C2 (IgG2a) displayed approximately 4- Intracerebral GL261 tumor model fold greater binding affinity (Kd ¼ 0.56 nmol/L) than mAb 30H3 Male C57BL/6 mice (Charles River) were anesthetized with (IgG1; Kd ¼ 2.3 nmol/L) for wild-type survivin (Fig. 1F). isoflurane and fixed in a stereotactic head frame (David Kopf Instruments). A midline scalp incision was made and the bregma Antibody specificity and detection of survivin in human cancers was identified. Stereotactic coordinates were measured (2.0 mm Both antibodies 2C2 and 30H3 detected filter-bound survivin lateral and 1.2 mm anterior to the bregma). A burr hole was drilled peptides SVN53-67 and SVN 53-67/M57 with little reactivity to 5 at this point and 1 10 GL261 cells suspended in 2.5 mLof scrambled peptide or KLH carrier protein (Fig. 1D). The addition DMEM was injected through a Hamilton syringe with a fixed, 25- of wild-type survivin peptide to the antibodies during immuno- gauge needle at a depth of 3.0 mm relative to the dura mater. blotting effectively blocked their detection of survivin peptide, Injections were performed at 1 mL/minute. After 3 days, mice were demonstrating high antibody specificity (Fig. 1E). Antibody 2C2 randomized into groups and injected with 10 mg anti-survivin detected strong survivin expression in a wide range of human antibody or 10 mg IgG for control. Antibodies were administered cancers, including glioblastoma, invasive ductal breast carcino- subcutaneously every 5 days for a total of five doses. SurVaxM ma, lobular breast carcinoma, carcinoid tumor, clear cell renal (100 mg) was administered in Montanide ISA 51 with 100 ng carcinoma, hepatocellular carcinoma, medullary thyroid carcino- mGM-CSF. Mice were followed for signs of neurologic deficits as ma and pheochromocytoma in a tissue microarray (Fig. 4). As an indicator of tumor growth and were sacrificed according to determined by the pathology coinvestigator (J. Qiu), patterns of established criteria. Significance was assessed by Kaplan–Meier survivin expression included nuclear, cytoplasmic, apical, and methodology and Mantel–Cox test for all groups. All mouse membranous. Normal human brain, breast, and liver revealed no intracranial implantation experiments were approved by the specific survivin expression, although modest nuclear staining Roswell Park IACUC. was seen in renal tubular epithelium (Fig. 4H).

Antibody-dependent cell-mediated cytotoxicity Survivin expression on the surface of tumor cells Antibody-dependent cell-mediated cytotoxicity (ADCC) was Analysis of nonpermeabilized U87 and A1207 human glioma assessed using a mFcgRIV ADCC Reporter Bioassay Kit (Pro- cells by flow cytometry confirmed that cell-surface binding was mega). For experiments using prebound target cells, U87 cells present and was competed out by wild-type survivin peptide, but were trypsinized, washed with complete media, resuspended at not scrambled peptide (Fig. 2A). Survivin was not detected on the a concentration of 106 cells/mL, and recovered at 37Cfor2 surface of nonpermeabilized cells that had been treated with hours. Cells were collected and resuspended in assay buffer and either trypsin or proteinase K (Fig. 2B). Treatment with proteases incubated with nonspecific mouse IgG or mAb 2C2 for 30 did not impair the ability of antibody to detect intracellular minutes at 37C. Cells were washed to remove unbound survivin following subsequent permeabilization. Flow cytometry antibody, diluted, and used as directed for the assay. Effector analysis using anti-survivin 2C2 identified survivin expression on cells were diluted in assay buffer according to the manufac- the surface of a diverse set of human and murine cancer cell lines, turer's instructions. In 96-well opaque culture plates, 25 mL including those derived from gliomas (U87, A1207, and GL261), effectors were added to either 50 mL of prebound U87 cells, ovarian (A2780), breast (MCF7), prostate (PC3), colon (HT29), 50 mL of assay buffer containing increasing concentrations of skin (B16 melanoma) and cervical (HeLa) cancers, leukemia 2C2, or to 25 mL of peptides at increasing concentrations, after (Raji), and lymphoma (Jurkat; Fig. 2A and 2C). Survivin was also which 25 mL2C2wasaddedtoafinal concentration of 4 mg/mL. detected in permeabilized tumor cell lines with 2C2 and 30H3 by Plates were covered and incubated at 37Cinahumidified, 5% immunofluorescence microscopy (Fig. 2D), and similar results

OF4 Clin Cancer Res; 2018 Clinical Cancer Research

Survivin mAbs Inhibit Tumor Growth

Figure 1. A, Antibody panel raised following vaccination of mice with survivin peptide (SVN53-67/M57) present in SurVaxM. Relative binding of different hybridoma clones to immobilized survivin peptide (ELISA) is displayed with indicated amount of supernatant volume (MFI ¼ mean fluorescence index). B, Western blot analysis of whole-cell lysates from human embryonic kidney 293T cells with indicated antibodies demonstrating the dominant 16.5 kDa survivin species. C, Western blot analysis of purified recombinant GST or GST-survivin proteins detected by the indicated antibodies (GST, glutathione-S-transferase). D, Slot blots detecting filter- immobilized KLH carrier protein and scrambled, mutant, and wild-type survivin peptides with indicated survivin antibodies. E, Competition blocking of mAbs preincubated with peptides (left) and then used to detect filter-immobilized, wild-type SVN53-67 peptide. F, AlphaLISA assay performed to determine relative affinity of purified 2C2 and 30H3 antibodies forSVN53-67 peptide.

were obtained with a commercially available survivin antibody growth in immunodeficient (nude) mice as well (Fig. 5C and D). (60.11) to an undefined survivin sequence. Nonpermeabilized Vaccination of immunocompetent (C57BL/6) mice with SVN53- cells showed significant surface binding of survivin antibodies 67/M57-KLH (SurVaxM) produced effective suppression of with focal concentrations of fluorescence on the surface of the cell tumor growth (Fig. 5F). In comparison, treatment of tumor- membranes of both murine and human cancer cell lines. bearing mice with pooled serum from vaccinated mice was also effective at suppressing tumor growth in na€ve GL261 tumor- Cell membrane and lipid raft localization of survivin bearing mice, although less so than vaccine (Fig. 5F; P < 0.05). To test whether survivin is contained within lipid rafts of the cell Survivin antibody (2C2) also significantly prolonged the survival membrane, we performed cell fractionation. Western blotting of of C57BL/6 mice with orthotopic (intracerebral) GL261 gliomas fractionated lysates from U87 (Fig. 3B) and A1207 (Fig. 3C) compared with either nonspecific PBS or IgG control (Fig. 5E; P ¼ human glioblastoma cells revealed the presence of survivin in 0.004). There was no significant difference between nonspecific the low-density lipid raft fraction (Fig. 3A) along with known lipid IgG and PBS controls (P ¼ 0.089). In these experiments, treat- raft markers flotillin-1, EGFR, and caveolin-1. Cells transfected ments with vaccine and antibodies began 4 days after tumor cell with an expression vector encoding a c-Myc/FLAG-tagged survivin implantation. construct also revealed detectable recombinant myc-survivin within the lipid raft fractions (Fig. 3D). Similarly, imaging flow Antibody-dependent cytotoxicity and survivin expression on cytometry showed colocalization of survivin and bound cholera effector cells toxin B (CT-B) (a lipid raft marker) in some, but not all, lipid rafts To assess one potential mechanism by which anti-survivin (Fig. 3E). antibodies could mediate antitumor effects, we measured for ADCC in vitro. Preliminary experiments using splenocytes did Effects of survivin antibodies on tumor growth in vivo not demonstrate measurable ADCC. To improve sensitivity, Both 2C2 and 30H3 anti-survivin antibodies inhibited growth ADCC was assessed using a reporter assay that measures biolu- of GL261 gliomas and B16 melanomas in flank tumor models minescent signal from engineered Jurkat effector cells expressing a compared with irrelevant IgG control antibody (Fig. 5A–D). There FcgRIV-luciferase construct. We also found that Jurkat cells express was no significant difference between 2C2 and 30H3 with respect survivin on their cell surface (Fig. 2C). Consequently, 2C2 anti- to tumor inhibition, despite their difference in affinity for the body triggers FcgRIV-mediated luciferase expression, through target antigen. Although the inhibition of tumor growth by 2C2 binding Jurkat effector cells even in the absence of target cells and 30H3 was more effective in immunocompetent mice (Fig. 5A (Fig. 6A). In addition, competition using free wild-type survivin and B), both survivin antibodies inhibited GL261 and B16 tumor peptide disrupts the 2C2-mediated, autoactivation of effector

www.aacrjournals.org Clin Cancer Res; 2018 OF5

Fenstermaker et al.

Figure 2. A, Flow cytometric detection of cell-surface survivin in unfixed (i.e., nonpermeabilized) human U87 and A1207 glioma cells. Purified anti-survivin 2C2 mAb was used to stain cells with or without preincubation with SVN53-67 peptide, or a scrambled peptide to assess specificity through blocking. B, Loss of cell surface survivin binding by 2C2 in nonpermeabilized A1207 cells treated with proteinase K or trypsin (left) and detection of intracellular survivin in protease-stripped and permeabilized cells (right). C, Flow cytometric measurement of cell-surface survivin (red) staining compared to IgG control (black) in nonpermeabilized cancer cells lines, including MCF7 (human breast carcinoma), HeLa (human cervical carcinoma), Jurkat (human T-cell leukemia), B16f1 (mouse melanoma), HT-29 (human colon carcinoma), Raji (human Burkitt's lymphoma), PC3 (human prostate carcinoma), A2780 (human ovarian carcinoma) cells, and GL261 (murine glioma cells). D, Immunofluorescence microscopic detection of total (permeabilized) and cell-surface (nonpermeabilized) survivin in human (U87) and murine (GL261) glioma cells with 2C2, 30H3, and commercially available 60.11 anti-survivin mAbs.

cells; whereas, scrambled peptide does not (Fig. 6B). Because of unbound antibody (Fig. 6C). Target U87 cells preincubated with antibody recognition of survivin by effector (Jurkat) cells, subse- normal mouse IgG did not trigger effector cell luminescence, quent assays were performed using U87 target cells that had been whereas target cells preincubated with 2C2 stimulated mFcgRIV preincubated with 2C2 antibody, followed by washing to remove engagement and luciferase expression. In contrast to the results

Figure 3. A, Photograph of gradient separation in U87 cells with arrow indicating low- density lipid raft band corresponding to survivin-containing fraction #4. Western blot analysis detection of lipid raft–associated proteins and survivin in subcellular fractions derived from U87 (B) and A1207 (C) glioblastoma cells. D, Transiently expressed recombinant c-Myc– tagged human survivin in association with lipid raft protein fractions in HEK293T cells detected with an anti-c-Myc-tag antibody (9E10). E, Imagestream imaging ﬂow cytometric visualization of colocalized bound cholera toxin B (CT-B) and survivin in lipid rafts.

OF6 Clin Cancer Res; 2018 Clinical Cancer Research

Survivin mAbs Inhibit Tumor Growth

Figure 4. Survivin IHC with 2C2 mAb in human cancers and normal tissues with indicated staining patterns, including glioblastoma (nuclear; A), normal brain (negative; B), invasive ductal breast carcinoma (nuclear; C), lobular breast carcinoma (nuclear; D), normal breast tissue (negative; E), typical carcinoid tumor (apical; F), clear cell renal cell carcinoma (membranous and cytoplasmic; G), normal kidney (nuclear; H), hepatocellular carcinoma (membranous and cytoplasmic; I), normal liver (negative; J), medullary thyroid carcinoma (cytoplasmic; K), and pheochromocytoma (cytoplasmic; L). Images are shown at 400. with 2C2, mAb 30H3 did not increase luciferase activity due to the targeting of survivin associated with the outer surface of the tumor fact that mouse IgG1 does not bind mFcgRIV (20, 21). To rule out cell membrane. Our work shows, for the first time, that antibodies any direct nonimmunologic effect upon cell viability, we assayed generated through survivin vaccination can also have antitumor cell growth in the presence of antibody. Incubation of GL261 cells effects. Thus, the finding of surface expression of survivin on with 2C2 and 30H3 antibodies had no detectable effect upon in tumor cells changes the paradigm for survivin-targeted immuno- vitro cell viability (Fig. 6D). therapy to include antibody-mediated approaches as well. Although the origin of circulating anti-survivin antibodies in patients with cancer is unclear, survivin protein has been identi- Discussion fied in patient serum (26–30) and in association with tumor- Survivin is expressed by many different types of cancer cells. derived exosomes (18). We have recently reported that the survi- Patients with cancer have been shown to have anti-survivin vin protein is detectable by imaging flow cytometry on the outer antibodies in their circulation, even without active, specific surface of glioma-derived exosomes present in the circulation of vaccination (9). The presence of anti-survivin antibodies and patients with malignant gliomas, but not in normal noncancer survivin-specificTcells(9,10)inpatientswithcancerindicates controls (31). Moreover, vaccination of glioma patients using the that survivin is released systemically by tumor cells and that it is SVN53-67/M57-KLH peptide vaccine may affect levels of survi- immunogenic to some degree. Survivin peptide sequences, vin-bearing exosomes in the bloodstream (31). This study sug- including those derived from the wild-type survivin molecule, gests that antibodies could arise from an immunologic response and proteins encoded by various survivin splice variants, have to tumor membrane survivin expression as well. It is also possible been used to develop different cancer vaccine strategies (16, 22, that exosomal survivin is derived from lipid raft survivin during 23–25). In these studies, the assumption that survivin is largely the process of exosome generation and release (31). an intracellular protein target has led to a focus on T-cell– Treatment of mice with purified anti-survivin antibodies or mediated antitumor mechanisms in these vaccine-based with pooled antisera from survivin-vaccinated mice inhibited approaches. As survivin has been regarded as an intracellular tumor growth. Vaccination was more effective in preventing molecule, its presentation as an immunologic target is postu- tumor progression than injection of antibody alone, and tumor lated to be via processed survivin epitopes displayed by MHC-I growth inhibition was greater in syngeneic, immunocompetent, molecules for TCR recognition (10, 11, 15). C57BL/6 mice than in immunodeficient (nu/nu) mice, suggesting In this study, we found that the survivin protein is present on that cellular immunologic elements may be necessary for anti- the outer surface of a wide range of human and murine tumor cell body-mediated tumor inhibition. While some ADCC activity is lines. In addition, we developed mouse mAbs that inhibited observed in highly sensitive luciferase reporter systems, it is tumor growth in several murine tumor models, perhaps via unclear whether ADCC is a major component of the antitumor

www.aacrjournals.org Clin Cancer Res; 2018 OF7

Fenstermaker et al.

Figure 5. Subcutaneous (A–D and F) and intracranial (E) tumor models in C57BL/6 (A, B, E,andF) and nude (C and D) mice with GL261 glioma (A, C, E,andF), and B16f1 melanoma (B and D) cells. Mice were given the indicated treatments once every 5 days beginning 3 days following tumor cell implantation (A, C; n ¼ 8–10 per group; B, D; n ¼ 5 per group). Subcutaneous tumors (A–D and F) were measured and tumor volumes were calculated as described. E, Mice with GL261 intracranial tumors were treated with PBS (control), nonspecific IgG, mAb 2C2 at a dose of 10 or 20 mg, or SurVaxM vaccine (n ¼ 11–17 per group) and survival was determined by the Kaplan–Meier method. Median survival time and range; control ¼ 22 days (range, 17–31 days), nonspecificIgG¼ 19 days (range, 17–27 days), mAb 2C2 ¼ 28 days (range, 21–58þ days), SurVaxM ¼ 45 days (range, 21–120þ days). F, Mice with GL261 flank tumors were treated with conjugated survivin vaccine (SVN53-67/M57-KLH; SurVaxM), pooled antiserum derived from nontumor-bearing mice that had been vaccinated with SurVaxM, or nonspeci fic IgG control antibody (n ¼ 4 per group). Statistical significance in A–D and F was assessed using the Wilcoxon matched pairs test (, P < 0.05; , P < 0.0014). Statistical significance in E was assessed using the log-rank Mantel–Cox test (, P ¼ 0.0041; , P < 0.0001).

effect of survivin antibodies in vivo. Nevertheless, both antibodies As previously shown with SurVaxM vaccine (11) and replicated were similarly effective suggesting that the biological actions of here, antibody 2C2 prolongs the survival of immunocompetent anti-survivin antibodies are more complex in vivo than in cell mice with orthotopic (cerebral) GL261 gliomas indicating that culture and that ADCC probably does not account solely for the the blood–brain barrier does not fully prevent survivin-specific antitumor effects of anti-survivin antibodies. antibodies and activated effector cells from gaining access to For this study, we specifically selected antibodies generated in cerebral tumors. Moreover, it has been shown that the GL261 response to vaccination that were cross-reactive to the wild-type glioma model produces some disruption of the BBB as measured survivin sequence so that high affinity for the target antigen on by high molecular weight tracers such as Evans Blue (32). Thus, tumor cells would be assured. In addition to activity in immu- cellular and antibody-mediated, survivin immunotherapies may nocompetent mice, these antibodies have efficacy against GL261 have utility in treating cerebral tumors. and B16 tumors in immunocompromised mice. In fully immu- Although our work highlights the potential clinical importance nocompetent mice, vaccine was more effective than mAbs in of membrane-bound survivin, the cellular mechanics of surface inhibiting tumor growth, consistent with the fact that cytotoxic expression and the biological role of survivin on the cell surface T cells and helper support are important components of vaccine both remain to be defined. In one study, the protein encoded by action against tumors (11). As nude mice retain B cells, NK cells, solute carrier gene family 5a, member 8 (SLC5A8) was shown to macrophages and dendritic cells, which can all potentially medi- translocate survivin to the plasma membrane in cancer cells by ate ADCC and other immunologic processes, additional studies way of direct protein–protein interaction (33). Beyond that, will be required to define the mechanism underlying antibody- survivin lacks a transmembrane domain and it is not known to mediated antitumor effects. For example, we cannot exclude the interact with specific cell membrane components; however, it possibility that antitumor effects of survivin antibodies in vivo are does possess amphipathic alpha helical structure (34). The focal either fully, or in part, the result of opsonization, cell-mediated pattern of expression observed by immunofluorescence micros- tumor killing, or interference with survivin-containing exosomes copy suggests the likelihood that cell-surface survivin may be in the tumor microenvironment (29). specifically associated with membranous lipid rafts. Packaging of

OF8 Clin Cancer Res; 2018 Clinical Cancer Research

Survivin mAbs Inhibit Tumor Growth

Figure 6. mAb 2C2 produces FcgRIV activation in luciferase reporter assays A, Jurkat lymphoma cells as both effector cells and reporter targets. Jurkat cells have cell-surface survivin expression (Fig. 2C) leading to antibody (2C2) dose-dependent activation of luciferase reporter via mFcgRIV. Dilutions of murine anti-survivin mAb (2C2) or anti-IgG were added to effector cells and luminescence was measured. B, Blocking of Fc-mediated activation of luciferase in Jurkat effector cells by SVN53-67 peptide binding to 2C2 compared with scrambled peptide. C, U87 human glioma cells engage mAb 2C2 with activation of FcgRIV in Jurkat effector cells (RLU ¼ relative luminescence units). D, Growth curve of Gl261 cells, in vitro, exposed to 10 mg/mL of the indicated antibody over 11 days.

exosomal survivin has previously been reported to be dependent nature of survivin biology and raise the possibility that the on lipid rafts and to be associated with Hsp70, Hsp90, and PRDX1 unique survivin epitopes present in these isoforms may be proteins (35). If, as several studies suggest, exosomal survivin has differentially targetable via specific antibodies. It is possible tumor-promoting properties in the microenvironment, antibo- that a surface-survivin isoform or conformational variant of dies to survivin could also interfere with the action of these survivin on the cell membrane is being detected by 2C2 and nanobodies, thus inhibiting tumor growth. 30H3. In our study, the survivin epitope from which 2C2 and There are at least six survivin isoforms that appear to be self- 30H3 antibodies are derived is conserved in all survivin iso- regulating and may have distinct functions (36, 37). Survivin forms described to date. Therefore, we are unable to make firm splice variants are generated through combinations of exons 1– conclusions about which specificisoformsarepresentonthe 4 with two alternate exons 2B and 3B (36–38). Survivin-2B is outer surface of tumor cells. Such information could help guide formed by the addition of an alternate exon 2, whereas, the development of highly specific antibody-mediated surviv- survivin-DEx3 contains a deletion of exon 3 that results in a ing-targeting tumor therapies. truncated protein due to a frame shifted stop codon (36). A high ratio of survivin-DEx3 to wild-type survivin has been identified in aggressive cancers. In contrast, when survivin-2B Conclusion is predominant over wild-type survivin, a reduced antiapopto- While cancer vaccines provide one method for anti-survivin tic potential is observed (39–46). High expression of survivin- cancer immunotherapy, there are several potential advantages DEx3 is also associated with high proliferative activity and to antibody mediated approaches. Unlike with peptide vac- tumor recurrence, while survivin-2B is associated with a lower cines, the effectiveness of mAbs is not HLA-dependent. Anti- rate of tumor recurrence (47–49). body therapy could potentially also have more immediate Survivin variants can differentially traffic within the cell and action against cancer cells than vaccines, which may take weeks can have variable subcellular localization (50, 51). For exam- to generate active immune responses and which may not work ple, survivin-2B has been reported to preferentially localize in well in patients who are immunocompromised by their tumors the cytoplasm, whereas, survivin-DEx3 preferentially accumu- or other treatments. Finally, with the presence of survivin lates in the nucleus (50, 52). Wild-type survivin and survivin- protein on the surface of cancer cells, surface-survivin anti- DEx3 have also been observed to interact within the mitochon- body-armed, CAR T-cells could have broad applicability for dria (40). Such observations demonstrate the multifaceted treating survivin-expressing cancers.

www.aacrjournals.org Clin Cancer Res; 2018 OF9

Fenstermaker et al.

Disclosure of Potential Conflicts of Interest Administrative, technical, or material support (i.e., reporting or organizing R.A. Fenstermaker is an employee of and has ownership interests (including data, constructing databases): R.A. Fenstermaker, S.A. Figel, M.J. Ciesielski patents) at MimiVax, LLC. J. Qiu reports receiving speakers bureau honoraria Study supervision: M.J. Ciesielski from and is a consultant/advisory board member for Merck. M.J. Ciesielski is an employee of and has ownership interests (including patents) at and reports Acknowledgments receiving commercial research grants from MimiVax, LLC. No potential conflicts of interest were disclosed by the other authors. This project was funded by research grants from American Cancer Socie- tyRSG-11-153-01-LIB (to M.J. Ciesielski), the Roswell Park Alliance Founda- Authors' Contributions tion, Mr. Philip H. Hubbell, and the Linda Scime Endowment. This work was supported by Roswell Park Comprehensive Cancer Center and National Cancer Conception and design: R.A. Fenstermaker, S.A. Figel, M.J. Ciesielski Institute (NCI) grant P30CA016056. Development of methodology: R.A. Fenstermaker, S.A. Figel, J.-X. Qiu, P.M. Galbo, M.J. Ciesielski Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the provided facilities, etc.): S.A. Figel, J.-X. Qiu, T.A. Barone, S.S. Dharma, payment of page charges. This article must therefore be hereby marked E.K. Winograd, P.M. Galbo, L.M. Wiltsie, M.J. Ciesielski advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Analysis and interpretation of data (e.g., statistical analysis, biostatistics, this fact. computational analysis): R.A. Fenstermaker, S.A. Figel, J.-X. Qiu, E.K. Wino- grad, P.M. Galbo, M.J. Ciesielski Writing, review, and/or revision of the manuscript: R.A. Fenstermaker, Received September 22, 2017; revised January 25, 2018; accepted March 6, S.A. Figel, J.-X. Qiu, T.A. Barone, E.K. Winograd, L.M. Wiltsie, M.J. Ciesielski 2018; published first March 14, 2018.

References 1. Kallio MJ, Nieminen M, Eriksson JE. Human inhibitor of apoptosis protein 14. Berinstein NL, Karkada M, Oza AM, Odunsi K, Villella JA, Nemunaitis JJ, (IAP) survivin participates in regulation of chromosome segregation and et al. Survivin-targeted immunotherapy drives robust polyfunctional T cell mitotic exit. FASEB J 2001;15:2721–3. generation and differentiation in advanced ovarian cancer patients. 2. Rosa J, Canovas P, Islam A, Altieri DC, Doxsey SJ. Survivin modulates Oncoimmunology 2015;4:e1026529. microtubule dynamics and nucleation throughout the cell cycle. Mol Biol 15. Becker JC, Andersen MH, Hofmeister-Muller V, Wobser M, Frey L, Cell 2006;17:1483–93. Sandig C, et al. Survivin-specific T-cell reactivity correlates with 3. Adida C, Crotty PL, McGrath J, Berrebi D, Diebold J, Altieri DC. Develop- tumor response and patient survival: a phase-II peptide vaccination mentally regulated expression of the novel cancer anti-apoptosis gene trial in metastatic melanoma. Cancer Immunol Immunother 2012; survivin in human and mouse differentiation. Am J Pathol 1998;152: 61:2091–103. 43–9. 16. Fenstermaker RA, Ciesielski MJ, Qiu J, Yang N, Frank CL, Lee KP, et al. 4. Chakravarti A, Noll E, Black PM, Finkelstein DF, Finkelstein DM, Clinical study of a survivin long peptide vaccine (SurVaxM) in patients with Dyson NJ, et al. Quantitatively determined survivin expression levels recurrent malignant glioma. Cancer Immunol Immunother 2016;65: are of prognostic value in human gliomas. J Clin Oncol 2002;20: 1339–52. 1063–8. 17. Khan S, Jutzy JM, Aspe JR, McGregor DW, Neidigh JW, Wall NR. Survivin is 5. Kajiwara Y, Yamasaki F, Hama S, Yahara K, Yoshioka H, Sugiyama K, et al. released from cancer cells via exosomes. Apoptosis 2011;16:1–12. Expression of survivin in astrocytic tumors: correlation with malignant 18. Khan S, Jutzy JM, Valenzuela MM, Turay D, Aspe JR, Ashok A, et al. Plasma- grade and prognosis. Cancer 2003;97:1077–83. derived exosomal survivin, a plausible biomarker for early detection of 6. Uematsu M, Ohsawa I, Aokage T, Nishimaki K, Matsumoto K, Takahashi H, prostate cancer. PLoS One 2012;7:e46737. et al. Prognostic significance of the immunohistochemical index of survivin 19. Macdonald JL, Pike LJ. A simplified method for the preparation of deter- in glioma: a comparative study with the MIB-1 index. J Neurooncol 2005; gent-free lipid rafts. J Lipid Res 2005;46:1061–7. 72:231–8. 20. Michaelsen TE, Kolberg J, Aase A, Herstad TK, Hoiby EA. The four 7. Lv S, Dai C, Liu Y, Shi R, Tang Z, Han M, et al. The impact of survivin on mouse IgG isotypes differ extensively in bactericidal and opsonopha- prognosis and clinicopathology of glioma patients: a systematic meta- gocytic activity when reacting with the P1.16 epitope on the outer analysis. Mol Neurobiol 2015;51:1462–7. membrane PorA protein of Neisseria meningitidis. Scand J Immunol 8. Virrey JJ, Guan S, Li W, Schonthal AH, Chen TC, Hofman FM. Increased 2004;59:34–9. survivin expression confers chemoresistance to tumor-associated endothe- 21. Bruhns P. Properties of mouse and human IgG receptors and their con- lial cells. Am J Pathol 2008;173:575–85. tribution to disease models. Blood 2012;119:5640–9. 9. Soling A, Plugge EM, Schmitz M, Weigle B, Jacob R, Illert J, et al. Auto- 22. Tsuruma T, Iwayama Y, Ohmura T, Katsuramaki T, Hata F, Furuhata T, antibodies to the inhibitor of apoptosis protein survivin in patients with et al. Clinical and immunological evaluation of anti-apoptosis pro- brain tumors. Int J Oncol 2007;30:123–8. tein, survivin-derived peptide vaccine in phase I clinical study for 10. Andersen MH, Pedersen LO, Capeller B, Brocker EB, Becker JC, thor Straten patients with advanced or recurrent breast cancer. J Transl Med 2008; P. Spontaneous cytotoxic T-cell responses against survivin-derived MHC 6:24. class I-restricted T-cell epitopes in situ as well as ex vivo in cancer patients. 23. Miyazaki A, Kobayashi J, Torigoe T, Hirohashi Y, Yamamoto T, Yamaguchi Cancer Res 2001;61:5964–8. A, et al. Phase I clinical trial of survivin-derived peptide vaccine therapy for 11. Ciesielski MJ, Ahluwalia MS, Munich SA, Orton M, Barone T, Chanan-Khan patients with advanced or recurrent oral cancer. Cancer Sci 2011;102: A, et al. Antitumor cytotoxic T-cell response induced by a survivin peptide 324–9. mimic. Cancer Immunol Immunother 2010;59:1211–21. 24. Nitschke NJ, Bjoern J, Met O, Svane IM, Andersen MH. Therapeutic 12. NoeDominguez-Romero A, Zamora-Alvarado R, Servin-Blanco R, Perez- vaccination against A modified minimal survivin epitope induces func- Hernandez EG, Castrillon-Rivera LE, Munguia ME, et al. Variable epitope tional CD4 T cells that recognize survivin-expressing cells. Scand J Immu- library carrying heavily mutated survivin-derived CTL epitope variants as a nol 2016;84:191–3. new class of efficient vaccine immunogen tested in a mouse model of breast 25. Gross S, Lennerz V, Gallerani E, Mach N, Bohm S, Hess D, et al. Short cancer. Hum Vaccin Immunother 2014;10:3201–13. peptide vaccine induces CD4þ T helper cells in patients with different solid 13. GrossS,LennerzV,GalleraniE,SessaC,MachN,BoehmS,etal.First- cancers. Cancer Immunol Res 2016;4:18–25. in-human trial focusing on the immunologic effects of the survivin- 26. Guney N, Soydine HO, Derin D, Tas F, Camlica H, Duranyildiz D, et al. derived multiepitope vaccine EMD640744. J Clin Oncol 2011; Serum and urine survivin levels in breast cancer. Med Oncol 2006;23: 29:2515–. 335–9.

OF10 Clin Cancer Res; 2018 Clinical Cancer Research

Survivin mAbs Inhibit Tumor Growth

27. Shehata HH, Abou Ghalia AH, Elsayed EK, Ziko OO, Mohamed SS. 40. Caldas H, Jiang Y, Holloway MP, Fangusaro J, Mahotka C, Conway EM, Detection of survivin protein in aqueous humor and serum of retino- et al. Survivin splice variants regulate the balance between proliferation and blastoma patients and its clinical significance. Clin Biochem 2010; cell death. Oncogene 2005;24:1994–2007. 43:362–6. 41. Vivas-Mejia PE, Rodriguez-Aguayo C, Han HD, Shahzad MM, Valiyeva F, 28. Hong JY, Ryu KJ, Park C, Hong M, Ko YH, Kim WS, et al. Clinical impact of Shibayama M, et al. Silencing survivin splice variant 2B leads to antitumor serum survivin positivity and tissue expression of EBV-encoded RNA in activity in taxane–resistant ovarian cancer. Clin Cancer Res 2011;17: diffuse large B-cell lymphoma patients treated with rituximab-CHOP. 3716–26. Oncotarget 2017;8:13782–91. 42. Ling X, Cheng Q, Black JD, Li F. Forced expression of survivin-2B abrogates 29. Ren YQ, Zhang HY, Su T, Wang XH, Zhang L. Clinical significance of serum mitotic cells and induces mitochondria-dependent apoptosis by blockade survivin in patients with pancreatic ductal adenocarcinoma. Eur Rev Med of tubulin polymerization and modulation of Bcl-2, Bax, and survivin. Pharmacol Sci 2014;18:3063–8. J Biol Chem 2007;282:27204–14. 30. Lancaster GI, Febbraio MA. Exosome-dependent trafficking of HSP70: a 43. Atlasi Y, Mowla SJ, Ziaee SA. Differential expression of survivin and its novel secretory pathway for cellular stress proteins. J Biol Chem 2005; splice variants, survivin-DeltaEx3 and survivin-2B, in bladder cancer. 280:23349–55. Cancer Detect Prev 2009;32:308–13. 31. Galbo PM Jr, Ciesielski MJ, Figel S, Maguire O, Qiu J, Wiltsie L, 44. Cheng Z, Hu L, Fu W, Zhang Q, Liao X. Expression of survivin and its splice et al. Circulating CD9þ/GFAPþ/survivinþ exosomes in malignant variants in gastric cancer. J Huazhong Univ Sci Technolog Med Sci 2007; glioma patients following survivin vaccination. Oncotarget 2017;8: 27:393–8. 114722–35. 45. Mahotka C, Wenzel M, Springer E, Gabbert HE, Gerharz CD. Survivin- 32. Leten C, Struys T, Dresselaers T, Himmelreich U. In vivo and ex vivo deltaEx3 and survivin-2B: two novel splice variants of the apoptosis assessment of the blood brain barrier integrity in different glioblastoma inhibitor survivin with different antiapoptotic properties. Cancer Res 1999; animal models. J Neurooncol 2014;119:297–306. 59:6097–102. 33. Coothankandaswamy V, Elangovan S, Singh N, Prasad PD, Thangaraju M, 46. Li F. Role of survivin and its splice variants in tumorigenesis. Br J Cancer Ganapathy V. The plasma membrane transporter SLC5A8 suppresses 2005;92:212–6. tumour progression through depletion of survivin without involving its 47. Takashima H, Nakajima T, Moriguchi M, Sekoguchi S, Nishikawa T, transport function. Biochem J 2013;450:169–78. Watanabe T, et al. In vivo expression patterns of survivin and its splicing 34. Verdecia MA, Huang H, Dutil E, Kaiser DA, Hunter T, Noel JP. Structure of variants in chronic liver disease and hepatocellular carcinoma. Liver Int the human anti-apoptotic protein survivin reveals a dimeric arrangement. 2005;25:77–84. Nat Struct Biol 2000;7:602–8. 48. Moore AS, Alonzo TA, Gerbing RB, Lange BJ, Heerema NA, Franklin J, 35. Valenzuela MMA, Bennit HRF, Osterman CJD, Khan S, Casiano C, Wall NR. et al. BIRC5 (survivin) splice variant expression correlates with refrac- Survivin packaging into exosomes is lipid raft-dependent [abstract]. In: tory disease and poor outcome in pediatric acute myeloid leukemia: a Proceedings of the 105th Annual Meeting of the American Association for report from the Children's Oncology Group. Pediatr Blood Cancer Cancer Research; 2014 Apr 5–9; San Diego, CA. Philadelphia (PA): AACR; 2014;61:647–52. 2014. Abstract nr 4856. 49. Cho GS, Ahn TS, Jeong D, Kim JJ, Kim CJ, Cho HD, et al. Expression of the 36. Wesierska-Gadek J, Schmid G. Transcriptional repression of anti-apoptotic survivin-2B splice variant related to the progression of colorectal carcino- proteins mediated by the tumor suppressor protein p53. Cancer Ther 2007; ma. J Korean Surg Soc 2011;80:404–11. 5:203–12. 50. Mahotka C, Liebmann J, Wenzel M, Suschek CV, Schmitt M, Gabbert HE, 37. UniProt Consortium. Activities at the Universal Protein Resource (Uni- et al. Differential subcellular localization of functionally divergent survivin Prot). Nucleic Acids Res 2014;42:D191–D8. splice variants. Cell Death Differ 2002;9:1334–42. 38. Zheng W, Ma X, Wei D, Wang T, Ma Y, Yang S. Molecular cloning and 51. Mahotka C, Krieg T, Krieg A, Wenzel M, Suschek CV, Heydthausen M, et al. bioinformatics analysis of a novel spliced variant of survivin from human Distinct in vivo expression patterns of survivin splice variants in renal cell breast cancer cells. DNA Seq 2005;16:321–8. carcinomas. Int J Cancer 2002;100:30–6. 39. Krieg A, Mahotka C, Krieg T, Grabsch H, Muller W, Takeno S, et al. 52. Mull AN, Klar A, Navara CS. Differential localization and high expression Expression of different survivin variants in gastric carcinomas: first clues of SURVIVIN splice variants in human embryonic stem cells but not in to a role of survivin-2B in tumour progression. Br J Cancer 2002;86: differentiated cells implicate a role for SURVIVIN in pluripotency. Stem 737–43. Cell Res 2014;12:539–49.

www.aacrjournals.org Clin Cancer Res; 2018 OF11

Survivin Monoclonal Antibodies Detect Survivin Cell Surface Expression and Inhibit Tumor Growth In Vivo

Robert A. Fenstermaker, Sheila A. Figel, Jingxin Qiu, et al.

Clin Cancer Res Published OnlineFirst March 14, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-2778

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2018/04/17/1078-0432.CCR-17-2778. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.