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Microenvironment and Immunology

Vaccination for Invasive Canine Meningioma Induces In SituProduction of Antibodies Capable of Antibody-DependentCell-Mediated Cytotoxicity

Brian M. Andersen1, G. Elizabeth Pluhar5, Charles E. Seiler5, Michelle R. Goulart5, Karen S. SantaCruz2,Melissa M. Schutten4,6, Joyce P. Meints2, M. Gerard O'Sullivan6,4, R. Timothy Bentley7, Rebecca A. Packer7,8,Stephanie A. Thomovsky9, Annie V. Chen9, Dominik Faissler10,Wei Chen1,MatthewA. Hunt3, Michael R. Olin1,and John R. Ohlfest1,3,†

AbstractMalignant and atypical meningiomas are resistant to standard therapies and associated with poor

prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach hasnot been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinicallymeaningful but underutilized model for preclinical testing of novel strategies for aggressive humanmeningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consistingof autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and onlyone dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-g–elaborating Tcells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responsesdeveloped in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surfaceantigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneiccanine and human meningiomas, showing common antigens across breed and species. Histologic analysisrevealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatmentcompared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependentcell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility andimmunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrantfurther development toward human trials. Cancer Res; 73(10); 2987–97. �2013 AACR.

IntroductionMeningioma is the most common primary brain neoplasm,

withmore than 100,000 patients diagnosed in theUnited States

between 2004 and 2008 (1). Newly diagnosed tumors aremanaged by surgical resection alone. Roughly 6,000 patientswill need additional treatment in the United States every yeardue to recurrence (2), which often occurs with invasive ormalignant disease (3, 4). Current salvage approaches includereoperation, radiation, radiosurgery, and chemotherapy; thereis controversy regarding the perceived clinical benefit of theseinterventions (5–8). The 3-year recurrence rate in reoperatedWorld Health Organization (WHO) grade 1meningioma is 50%(9), with risk of recurrence even greater in grade 2 and 3 tumors(10–12). Radiation, although modestly effective in benigndisease, has been associated with cognitive deficits, secondarymalignancies, and the transformation of the tumor to a higher-grade neoplasm (2).

One challenge in treating aggressive meningiomas is thepaucity of animal models with which to test combinations ofsurgery and systemic therapy in a meaningful manner. Spon-taneous meningiomas in dogs make up 45% of primary caninebrain tumors (13) and are an underutilized resource forpreclinical studies. Canine and human meningiomas sharemany features, including histologic resemblance, overexpres-sion of growth factor receptors, deletion of chromosomalsegments, and losses of function in tumor suppressor genes(14–16). More than 40% of canine meningiomas were atypical

Authors' Affiliations: Departments of 1Pediatrics, 2Laboratory Medicineand Pathology, and 3Neurosurgery; 4Comparative Pathology SharedResource, Masonic Cancer Center, University of Minnesota, Minneapolis,Minnesota; Departments of 5Veterinary Clinical Sciences and 6VeterinaryPopulation Medicine, University of Minnesota, St. Paul, Minnesota;7Departments of VeterinaryClinical Sciences and 8BasicMedical Sciences,PurdueUniversity, College of VeterinaryMedicine,West Lafayette, Indiana;9Department of Veterinary Clinical Sciences, College of Veterinary Medi-cine, Washington State University, Pullman, Washington; and 10Depart-ment of Clinical Sciences, Neurology/Neurosurgery, Cummings School ofVeterinary Medicine, Tufts University, North Grafton, Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

†J.R. Ohlfest: Deceased January 21, 2013.

Current address forK.S. SantaCruz:Department ofPathology,University ofNew Mexico, Albuquerque, NM 87131; and current address for M.M.Schutten, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.

Corresponding Author: Brian M. Andersen, 420 Delaware St SE, Minnea-polis, MN 55405. Phone: 612-624-1195; Fax: 612-624-2490; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-3366

�2013 American Association for Cancer Research.

CancerResearch

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on June 29, 2020. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 7, 2013; DOI: 10.1158/0008-5472.CAN-12-3366

http://cancerres.aacrjournals.org/

or malignant in one study (17). Dogs develop many othercancers that are prevalent in humans, and tumors progress5- to 7-fold faster in dogs relative to humans (18). We hypoth-esized that preclinical studies in canine meningioma wouldenable accurate and rapid testing of immunotherapy foraggressive meningioma.

Cancer vaccines have been tested in multiple malignan-cies, including gliomas, with evidence of clinical activity (19,20). A meta-analysis covering more than 100 clinical trialsrevealed that response rates to vaccination with peptidescontaining defined T-cell epitopes were less than half ofthat achieved with whole-cell–based vaccines (21). Thehigher response rate achieved with whole-cell vaccines (asused in this study) could be due to greater antigeniccoverage or the potential to induce tumor-binding antibo-dies. Meningiomas are not subject to the same principles ofimmune privilege as cells within the brain parenchyma suchas gliomas (22). Relative to gliomas, meningiomas may bebetter candidates for immunotherapy because they: (1) arenot insulated by the endothelial tight junctions that limitlarge-molecule (e.g., antibody) diffusion; (2) are in directcontact with cerebrospinal fluid that drains to the venouscirculation and cervical lymph nodes for antigen presenta-tion to T and B lymphocytes; (3) lack the T-cell traffickingcheckpoints present in the Virchow–Robin space (e.g., glialimitans); and (4) are relatively slow growing tumors thatmay be more susceptible to adaptive immune responses.However, invasive, atypical, and malignant meningiomascan infiltrate the brain parenchyma, requiring penetrationof antibodies and/or lymphocytes for control of postsurgi-cal, microscopic disease.

Tumor cell lysates mixed with synthetic toll-like receptor(TLR) ligands function as effective vaccines to induce anti-tumor immune responses in glioma-bearing animals (23, 24).The mechanisms of lysate/TLR ligand vaccines have beenthoroughly characterized and tested in many patients withcancer; however, the activity of this type of vaccine againstmeningioma is unknown. TLR activation on antigen-pre-senting cells facilitates the induction of adaptive immuneresponses by enhancing antigen presentation, expression ofcostimulatory molecules, cytokine production, and homingto secondary lymphoid organs. The TLR9 ligand CpG olid-godeoxynucleotide induced clinical responses in patientswith select melanoma and renal cell carcinoma (25, 26).The U.S. Food and Drug Administration-approved TLR7ligand imiquimod exhibited adjuvant activity in cancerclinical trials (20, 27), with excellent efficacy as a singletopical agent against skin cancers (28).

We conducted a vaccine immunotherapy trial for petdogs with symptomatic, spontaneous meningiomas. Dogsunderwent surgery followed by vaccination with autologoustumor cell lysate that was combined with imiquimod orCpG. Herein, we report safety, robust extension of survival,homology among antibody epitopes between dogs andhumans, and vaccine-induced, local antibody productionin the brain. Vaccination for canine meningioma revealspromising avenues for further development toward humantrials.

Materials and MethodsSurgery, vaccination production, and administration

Dogs were enrolled after obtaining owner consent accord-ing to an approved protocol from the University of Minnesota(Minneapolis, MN) Institutional Animal Care and Use Com-mittee. A presumptive diagnosis of meningioma was based onthe MRI characteristics of a solitary extra-axial mass in thebrain, with heterogeneous T1W signal, usually isointense,homogenous T2W signal, sharply defined borders, homoge-nous contrast enhancement, evidence of a dural tail, that mayhave associated cysts, peritumoral edema, and falx-shift.Surgical resection was conducted under general anesthesiausing the appropriate approach based on tumor location.Dogs were hospitalized with supportive care for 1 to 2 daysafter surgery. Corticosteroids used to minimize peritumoraledema were discontinued by 48 hours before vaccination.Part of the tumor specimen was used for histologic diagnosis,and the remainder of the tumor was cultured for vaccineproduction.

Cultures were established by mincing specimens with scal-pels and digestion at 37�C for 15 minutes in suspension withTrypLE Express (Invitrogen/Life Technologies). Cell suspen-sions were filtered through a 100 mm filter, washed with PBS,and placed in culture in 10 cm culture plates precoated with1:10 Matrigel (BD Biosciences), in serum-free neural stem cellmedia consisting of DMEM:F12 (1:1), with L-glutamine, sodiumbicarbonate, penicillin/streptomycin (100 U/mL), B27 and N2supplements (Gibco), and 0.1 mg/mL Normocin (InVivoGen).Semi-weekly, cells were given 20 ng/mL recombinant humanEGF and recombinant human FGF (R&D Systems) and werecultured at 5%O2 and 5%CO2. Harvest for vaccination involvedscraping cells from one 10 cm dish. Cells were washed thrice inPBS, and underwent 5 freeze-thaw alternations by transferfrom warm water bath to liquid N2 followed by radiation (200Gy). Protein was measured by Bradford assay with standardCoomassie reagent (Pierce), and lysates were stored at�80�C.GMP-grade CpG 685ODNwas provided by SBI Biotech Co., Ltd(Tokyo, Japan), and imiquimod cream was acquired throughthe University of Minnesota Boynton Health Services Phar-macy. In 5 dogs, CpG (2.0 mg) was mixed with thawed lysatesimmediately before intradermal injection at 2 sites in the backof the neck. Six other dogs received imiquimod cream (5%; 0.5g) at 2 intradermal injection sites in the back of the neck 15minutes before lysate injection. The maximally achievablelysate protein concentration was given to each dog, whichvaried by tumor volume and the ability of the tumor toproliferate in culture. Lysate doses ranged from 200 to 1,500mg protein (average of 595 mg), and average dose did not varysignificantly between CpG and imiquimod-treated dogs.

Tumor volume measurementsTwenty consecutive surgical human cases (of M.A. Hunt;

Department of Neurosurgery, University of Minnesota, Min-neapolis, MN) were analyzed for volume by MRI, as calculatedby (length � width � height)/2 for tumor and intracranialvolume. The same procedure was carried out for the 11 dogs inthis study, and the results were compared as described inSupplementary Materials and Methods.

Andersen et al.

Cancer Res; 73(10) May 15, 2013 Cancer Research2988




Western immunoblot analysisCultured tumor cells or homogenized flash-frozen tumors

were used for blotting. Cells were lysed, protease andphosphatase inhibitors (Calbiochem) were added, and forSDS-PAGE, lysates were diluted in Laemmli-reducing sam-ple buffer, heated, and centrifuged. Protein standards (Bio-Rad) were loaded next to each 40 mg lysate and resolved onNuPAGE 4% to 12% Bis/Tris gels (Invitrogen). Proteins weretransferred to nitrocellulose (Amersham) at 5 V constantvoltage using semidry transfer (BioRad). The membraneswere blocked in 5% nonfat dry milk (NFDM)/Tris-bufferedsaline with 0.1% Tween-20 (TTBS) at room temperature for1 hour and cut appropriately into identical blots, each witha molecular weight standard (BioRad) run adjacent tolysate. Each membrane was incubated at room temperaturefor 1 hour in normal, pre or postvaccination sera diluted1:1000 in 5% NFDM/TTBS, washed 6 times for 10 minutes

each in TTBS, followed by room temperature for 1 hour inrabbit anti-canine immunoglobulin G (IgG) horseradishperoxidase (HRP)-conjugated secondary antibody (JacksonImmunoResearch) at 1:50,000 in 5% NFDM/TTBS. Bandswere detected using ECL Western Blotting Detection Sys-tem (Amersham) and HyBlot CL autoradiography film(Denville Scientific). Densitometry was conducted by theGel Analysis tool in ImageJ 1.45s software (NIH), and valueswere normalized by dividing by heavy- and light-chaindensities (areas under the curve) from prevaccination lanes.

Detection of tumor cell surface-reactive antibodiesA Becton Dickinson Custom Canto three-laser flow cyt-

ometer was used for data acquisition. Tumor cells wereremoved from Matrigel-coated 10 cm culture plates byscraping or BD Dispase (BD Biosciences), washed with PBSthrice, and incubated with heat-inactivated normal dog,

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Figure 1. Canine meningiomas model aggressive human disease. A, meningioma surgery specimens stained with H&E, left to right, from cases 3, 4, and1. B, necropsy specimens from 2 dogs with tumors that, in humans, are categorized as WHO grade II on biopsy. C, tumor volume and brain volume of the11 dogs treated with surgery plus immunotherapy and 20 consecutive intrainstitutional patients with human meningioma. �, P < 0.05.

Immunotherapy for Spontaneous Canine Meningioma

www.aacrjournals.org Cancer Res; 73(10) May 15, 2013 2989




prevaccination, or 3-month postvaccination serum at 1:100dilutions at 4�C for 30 minutes. Cells were then washed thricein PBS and incubated with 1 mg anti-canine IgG (HþL)—fluorescein isothiocyanate (American Qualex) at 4�C for anadditional 30 minutes before washing and analyzing.

Immunohistochemistry staining and quantification oflymphocyte infiltration

For lymphocyte analysis, 5 mm tissue sections were cut,prepared with standard procedures, and the following anti-bodies were used: CD3 (AbD Serotec) at 1:2,000, CD20 (ThermoScientific) at 1:2,000, and IgG (HþL; Jackson ImmunoResearch)at 1:2,000. Tissue sections were incubated with the primaryantibodies for 1 hour, rinsed, and a biotinylated secondaryantibody was applied for 30 minutes. CD20 and IgG antibodieswere followed with undiluted Rabbit Link (Covance), and abiotin-conjugated donkey anti-rat IgG (HþL; Jackson Immu-noResearch) at 1:500 was used with CD3. Sections were rinsed,incubated in hydrogen peroxidase, and a tertiary streptavidinHRP link (Covance) for 30 minutes. The immune complex wasvisualized using 3,30-diaminobenzidine as the chromogen.Sections were lightly counterstained with hematoxylin, dehy-drated, coverslipped, and scanned using the Aperio ScanscopeXT.

All surgical resection specimens and all necropsy blockscontaining tumor or inflammation as seen by hematoxylin andeosin stain (H&E) staining were included in counting of CD3þ

and CD20þ cells. Samples were blinded, and all 10� fields inslides were captured and saved as image files. Automated cellquantification was conducted with a customized macro andthe particle analysis tool in Fiji software (ImageJ 1.46j, NIH).Counts underwent statistical analysis as described in Supple-mentary Materials and Methods before unblinding.

Antibody-dependent cell-mediated cytotoxicityDog blood from healthy donors was collected in anticoag-

ulant tubes, lysed osmotically, and peripheral blood leukocytes(PBL) were washed thrice with PBS. PBLs were resuspended incomplete RPMI-1640 (supplemented with 10% heat-inacti-vated FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and0.1 mg/mL Normocin from InVivoGen) in a 96-well plate at 2.5� 106 cells/mL, and stimulated 14 hours with 30 mmol/L of theTLR7/8 ligand resiquimodbefore beingwashed and coculturedwith antibody-coated tumor cells. Primary meningioma cul-tures were coated with antibody as described in the cellsurface-binding procedure. Two washes were conductedbefore addition to PBLs at an effector:target ratio of 25:1.Tumor cell lysis was determined by measurement of lactatedehydrogenase (LDH) activity as indicated by the manufac-turer's protocol after 7 hours of coculture (Roche AppliedScience). Percentage-specific lysis of tumor cells was calculat-ed by: [Sample�(tumor onlyþ PBL only)]/(lysed tumorþ PBLonly) � 100.

Statistical analysisSamples were analyzed by unpaired t test (tumor/brain

volume, flow cytometry, LDH activity). Histologic cell countswere analyzed by an unpaired t test withWelch's correction for

unequal variances with 95% confidence intervals. Survival wasanalyzed using a log-rank test with 95% confidence intervals.All statistics were conducted using GraphPad Prism version4.0c for OS X (GraphPad Software www.graphpad.com).

ResultsCanine meningiomas model aggressive human disease

Meningiomas from dogs treated in our study share his-tologic features with human tumors of the same subtype(Fig. 1A), in addition to features that signify poor survival inhumans. Brain invasion is an independent negative prog-nostic factor that prompted the reclassification of otherwiseWHO grade I meningiomas as grade II (29, 30). Two canine

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cases exhibited gross tumor invasion into the brain atresection, and 3 additional cases showed brain invasionupon postmortem analysis (Fig. 1B, left and SupplementaryTable S1). Tumor invasion into the skull was also present in2 cases (Fig. 1B, right). In addition, canine meningiomasoccupied more than twice the volume of the brain relative tohuman tumors (Fig. 1C). All dogs were symptomatic at thetime of diagnosis, and in 10 dogs, the presenting clinical signwas seizures. The rapid recurrence and progression of thisdisease in canines is consistent with the common appear-ance of invasion into brain, which, in humans, predicts pooroutcomes.

Extension of survival following vaccine immunotherapyEleven dogs underwent craniotomy for tumor removal after

radiographic diagnosis of an intracranial neoplasm consistentwith meningioma. Following histologic confirmation, dogswere administered vaccinations biweekly with lysate derivedfrom their tumors in combinationwithCpGor imiquimod (Fig.2A). Mean follow-up timewas 585 days, with 36% (4/11) of dogsalive (Supplementary Table S1; Fig. 2B and C). Relative tohistoric controls, median survival is extended in the immuno-therapy cohort (645 vs. 222 days, P < 0.05), with no censoring ofdogs that died from other causes (Fig. 2B and SupplementaryTable S2). Neither vaccination cohort contained a case of frank

Figure 3. Vaccination inducesantibody responses to meningiomasurface antigens. A, Westernimmunoblot analysis of autologoustumor cells from culture (cases 10and 3) or snap-frozen tumorspecimen (case 5). Normal dogserum (ND) was used as a control. B,live autologous tumor cells werestained with serum fromprevaccination or postvaccination (at82 days). C, aggregate data of 3 dogsfrom B. �, P < 0.05; ��, P < 0.005.

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tumor progression, althoughmore deaths occurred in the CpGcohort (Fig. 2C). One CpG-treated dog developed 2 meningi-omas, of which the second was not identified before vaccina-tion due to small size. The second tumor was subsequentlyremoved after its growth caused breakthrough seizures, and 4other vaccines were given using tumor lysate of the secondtumor.

Tumor-reactive antibodies bind cell surface antigensand cross-react with human meningiomas

Immune responses before and after vaccination weremeasured in peripheral blood mononuclear cells (PBMC)and sera. IFN-g–elaborating tumor-reactive T cells were

significantly increased in postvaccination PBMCs in 2 of 9dogs tested, suggesting a low frequency of circulating T cells3 months after surgery (Supplementary Table S3). In con-trast, polyclonal tumor-reactive antibody responses towhole-tumor lysate (Fig. 3A) and cell surface antigens (Fig.3B and C) were detected in all 11 dogs (Supplementary TableS3). Probing of allogeneic tumor cells and lysates indicatedrecognition of common tumor antigens among dogs, someof which were present on the cell surface (Fig. 4A–C).Postvaccination antibodies recognized recombinant humanHSP60 and HSP60 from autologous and allogeneic tumor(Fig. 4A), showing the common expression and recognitionof this antigen. Remarkably, postvaccination canine serum

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Figure 4. Antibodies recognizeallogeneic dog and humanmeningiomas. A, Westernimmunoblot analysis of tumor cellsfrom 3 cases and recombinanthuman HSP60. B, representativehistograms of cell surface bindingof primary tumor cells from case 7.C, quantification of serum antibodysurface binding to an allogeneicpapillary (nonstudy dog—recognized by case 3) and ameningothelial meningioma(case 7—recognized by cases 4, 6,7, and 9). D, Western immunoblotanalysis of a human meningiomaprobed with postvaccinationserum from case 3. �, P < 0.05;��, P < 0.005.

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recognized human meningioma tissue, indicating antigenconservation between species (Fig. 4D).

Vaccination induces T-, B-, and plasma cell infiltrationinto peritumoral brainWe conducted analyses of B- and T-cell infiltration in

surgical and necropsy tissue in the 4 cases in which they wereavailable. Three CpG-treated dogs died within 3 weeks of thefinal vaccination and exhibited robust, focal increases in B-(with relatively less T)-cell infiltration into peritumoral brain(Fig. 5A and B; cases 1, 3, and 5). One imiquimod-treated dog

developed acute lymphoblastic lymphoma andwas euthanized25 weeks after the final vaccination. This dog showed mildB-cell infiltration (and no T-cell infiltration) into the brain(Fig. 5B, case 8). As mentioned above, case 3 presented with asecond meningioma in the contralateral hemisphere betweenthe fifth and sixth vaccinations, which was subsequentlyresected. Western blot analysis revealed a profound increasein IgG penetration into the second tumor relative to the first,prevaccination tumor (Fig. 5C and D). Moreover, staining forcanine IgG exposed plasma cells in the brains of 3 CpG-treateddogs (Fig. 5E). Although previous studies have reported plasma

Figure 5. Vaccination inducesB- and plasma cell infiltrates inperitumoral brain. A, representativeimages from CD3 and CD20immunohistochemistry of biopsiesand necropsies from case 1.B, quantification of CD3 andCD20 stains from cases 1, 3, 5,and 8. C, IgG blotted on case3 primary tumor (prevaccination)and contralateral tumor(postvaccination). D, densitometry ofheavy and light chain bands from C.E, immunohistochemical stain ofcanine IgG (HþL), indicating plasmacell infiltrates in normal brain areas ofcases 3 and 5. �, P < 0.005.

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cell entry into the brain in antibody-mediated autoimmune(31) and antipathogen (32) responses, this is thefirst account ofinduced plasma cell homing into brain tumors. In situ antibodyproduction was further suggested by focal areas of extracel-lular IgG staining seen in plasma cell-containing areas of braintissue but not others (data not shown).

Recognition of nonneoplastic brain and meningealantigens by postvaccination sera

Two dogs (cases 3 and 5) were euthanized 7 and 20 daysafter the most recent vaccination. Both animals presentedwith uncontrollable seizures and tumor recurrence wasassumed. At necropsy, case 3 had a microscopic focus ofresidual tumor and case 5 had no evidence of tumor. Toevaluate vaccine-induced autoantibody production, sera fromthese and 3 other dogs was probed against normal dog brain,arachnoid/pia mater, and dura mater. Secondary antibodyrevealed heavy and light chain IgG and IgM deposited inmeninges, but not brain parenchyma (Fig. 6A, left). Theseresults are consistent with the physiologic permeability ofimmunoglobulin into these tissues. Serum from cases 3 and 5reacted to arachnoid/pia and brain parenchyma, respectively.Consistent with the autoreactivity of case 5 sera, analysis ofnecropsy brain tissue from this dog revealed IgG accumula-tion on or in neurons distal to the site of resection (Fig. 6B).The binding of the sera to normal brain antigens in these dogssets them apart from 2 CpG-treated dogs and one imiquimod-treated dog that remained healthy and had nonreactive sera(Fig. 6A).

Postvaccination sera are capable of antibody-dependentcell-mediated cytotoxicity

Antitumor effector activities of antibodies encompass mul-tiple mechanisms, including antibody-dependent cell-mediat-ed cytotoxicity (ADCC). Because antibodies reacted with cellsurface antigens (Figs. 3B and C, 4B and C, and 7A and B), andantibody production in situ could enable opsonization ofinvasivemeningioma cells behind an intact blood brain barrier,we tested whether tumor-reactive antibodies could triggerADCC. PBLs killed few tumor cells when cocultured withmeningioma cells, or when tumor cells were preincubatedwith prevaccination serum; however, ADCC occurred whentumor cells were incubated with postvaccination serum (Fig.7C). Postvaccination serum also triggered ADCC against allo-geneic meningioma cells (Fig. 7D), showing that allogeneicvaccination may be an effective strategy in canines withmeningioma.

DiscussionAs many as 57,000 dogs a year develop meningiomas in the

United States (13, 33), and these dogs are an underutilizedresource for preclinical study. Because the prognosis for caninemeningioma is dismal (34), both dogs and humans couldbenefit from these studies. Our data show the canine modelresembles many histologic subtypes observed in humans andhas features associated with poor prognosis in humans. Thelarge size of the canine brain allows for surgery as a componentof therapy, enabling a more realistic clinical interpretation ofsystemic interventions.

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Figure 6. Reactivity with normalbrain correlates with neurologicsymptoms in CpG-treated dogs.A, brain, arachnoid/pia mater, anddura mater were probed withsecondary anti-canine IgG (HþL)alone, with serum from anallogeneic healthy dog, orpostvaccination sera from 5 cases.Arrows indicate reactivity in dogswith neurologic symptomsfollowing vaccination. B, necropsyspecimen from case 5 stained forcanine IgG (HþL).

Andersen et al.





The survival outcomes of this study call for further inves-tigation into the predictive potential of the canine model,especially with regard to tumor and immune biology in the2 species. Different responses to vaccination may occur due todifferential antigen expression, TLR expression in leukocytecell types, tumor growth kinetics, the relationship betweenhistologic subtype, biologic behavior, and clinical outcome.Addition of lysate/adjuvant vaccination to surgery resulted

in favorable survival compared with historical controls, but aprospectively designed, randomized study is required to makefirm conclusions on therapeutic efficacy. Nonetheless, onlycase 3 experienced tumor growth despite treatment. Thesurvival data may underestimate the benefit of vaccinationbecause censoring was not used (Fig. 2B and C). Two dogsincluded in the analysis died of other malignancies, and 2 dogslacked postmortem analysis but were assumed to die of age-related causes (Supplementary Table S1). The 3 other deathswere due to euthanasia after dogs presented with uncontrol-lable seizures assumed to be caused by tumor recurrence.Postmortemanalysis in these CpG-treated dogs foundminimalor no tumor burden, but immunoreactivity to normal brainstructuresmay have been treatment related and contributed tothe acute onset of neurologic symptoms (Fig. 6A andB). Similarseizure activity in glioma-bearing dogs in clinical trials at ourinstitution has been controlled using additional anticonvul-sants, corticosteroids, or induction of general anesthesia (datanot shown). It is therefore possible that the seizures in menin-gioma-bearing dogs could have been controlled. Whether CpGor imiquimod is more efficacious is still unresolved from thecurrent study due to the small number of dogs and many non-

tumor-related deaths. Given the high level of tumor controland the superior safety profile of imiquimod, however, aprospective randomized trial to compare surgery alone andin combination with vaccines of tumor lysate and a novel TLR7/8 ligand was initiated.

Our study is a starting point for investigation of immuno-therapy for meningioma. The data indicate that B-cell activa-tion and antibody production is the predominant mechanismof immunity induced by vaccination. Tumor-reactive antibo-dies were detected in the serum of all dogs regardless of lysatedose, showing feasibility of autologous lysate/TLR vaccineproduction. Antibodies exhibited considerable intercase and-species cross-reactivity (Fig. 4A and D and SupplementaryTable S3). B-cell infiltration outnumbered T-cell infiltration inpostmortem brain tissue adjacent to the resection cavity (Fig.5B). In contrast, only 2 dogs had increased tumor-reactive T-cell responses as measured by IFN-g ELISpot (SupplementaryTable S3). However, it is likely that reactive CD4 T cells wereprimed following vaccination because antibody responsesoften require CD4 T-cell help. Tumor-reactive T cells in theblood may not represent what occurs in the tumor-draininglymph nodes or tumor site. A limitation of our study was thatELISpot was carried out only in prevaccination and 3-monthpostvaccine blood samples. Nevertheless, infiltrating CD3þ

cells were observed in dogs that died within 2 weeks of thefinal vaccination (Fig. 5A and B), suggesting some T-cellactivation. Future studies will examine T-cell responses ingreater detail.

Tumor antigens in lysate vaccines could include overex-pressed normal antigens, mutated (neo) antigens, oncofetal

Figure 7. Postvaccination serumenables ADCC. A and B, case 3postvaccination serum antibodiesbound autologous tumor (A) andallogeneic tumor (B) of papillaryhistology from a nonstudy dog.Postvaccination serum also enabledkilling of autologous (C) andallogeneic (D) tumors whencombined with allogeneic PBL.�, P < 0.05; ��, P < 0.005.

A Allogeneic tumor

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antigens (expressed during development), or tumor-specificcarbohydrates, glycoproteins, or lipoproteins. That 2 dogs withsevere postvaccine seizure activity had autoreactive sera tobrain and meninges suggests that severe autoreactivity ispossible but infrequent (Fig. 6A and B). Lysates may bedepleted of these autoantigens to yield greater tumor speci-ficity and less risk. Because most dogs had no autoreactivity orrefractory seizures, risk of autoimmunity must be weighedagainst the threat to life posed by aggressive meningiomas.

Identification of meningioma antigens recognized bypostvaccination sera will enable the discovery of crucialepitopes for fully synthetic vaccine strategies in more wide-spread application. We identified HSP60 as one antigenrecognized by sera following vaccination. HSP60 can trans-locate to the cell surface upon stress or malignant trans-formation (35), but it was not expressed on the tumor cellsurface of case 3 (data not shown), arguing against cellsurface protein as the functional target of this antibody.Sera that recognized HSP60 also reacted with a humanmeningioma, and reactivity was observed at 60 kDa; how-ever, more study is needed to determine whether HSP60 is arelevant target in human meningioma.

Antibody-mediated autoimmunity is well characterized,with autoimmune conditions in the central nervous system(CNS) such as multiple sclerosis being re-evaluated in light ofclinical benefit seen from B-cell depleting therapies (36). Braintumor immunotherapy, once focused on T-cell–dependentmechanisms, is similarly expanding its breadth of effectormechanisms. Orthotopic mouse models of glioma indicatethat survival benefit from immunotherapy is absent in B-celldeficient, mMT tumor-bearing mice (ref. 37; unpublishedresults). The importance of B cells and antibodies in braintumor immunity are relatively unstudied. Our study docu-ments for the first time the induction of plasma cells homing tobrain tumors as a consequence of therapeutic intervention. Bcell and plasma cell infiltration into the brain also indicatepromise for immunotherapy to act against invasive cells thatexist behind an intact blood brain barrier. ADCC due to in situantibody production in brain is a novel immune effectormechanism relevant to any brain tumor. Our data suggest

that vaccination can induce a "Trojan horse"-like infiltration ofplasma cells that can in turn trigger ADCC, and thus createexcitement for translation of this approach to human menin-gioma and other CNS cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B.M. Andersen, G.E. Pluhar, W. Chen, M.A. Hunt, M.R.Olin, J.R. OhlfestDevelopment of methodology: B.M. Andersen, G.E. Pluhar, C. Seiler, M.M.Schutten, W. Chen, J.R. OhlfestAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.M. Andersen, G.E. Pluhar, C. Seiler, K.S. SantaCruz,M.G. O'Sullivan, R.T. Bentley, R.A. Packer, S.A. Thomovsky, D. Faissler, M.A. HuntAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B.M. Andersen, G.E. Pluhar, C. Seiler, M.R. Goulart, K.S. SantaCruz, M.M. Schutten, M.G. O'Sullivan, W. Chen, J.R. OhlfestWriting, review, and/or revision of the manuscript: B.M. Andersen, G.E.Pluhar,M.R. Goulart,M.M. Schutten, R.T. Bentley, R.A. Packer, S.A. Thomovsky, A.V. Chen, W. Chen, M.A. Hunt, M.R. Olin, J.R. OhlfestAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B.M. Andersen, G.E. Pluhar, C. Seiler,M.R. Goulart, A.V. Chen, W. Chen, M.A. Hunt, M.R. Olin, J.R. OhlfestStudy supervision: B.M. Andersen, G.E. Pluhar, W. Chen, M.A. Hunt, J.R. OhlfestOther: Histologic cutting and staining of brain tissue and write up of methodsemployed in this task, J.P. Meints

AcknowledgmentsThe authors thank Dr. Robert Schmidt (Washington University, St. Louis MO)

for the micrograph of the human papillary meningioma, GuillermoMarques andJohn Oja (UIC, University of Minnesota) for assistance in quantification andmicrograph capture, Jose L. Gallardo and Patrick T. Grogan for vaccine pro-duction, and Nick J. Erickson (University of Minnesota) for assistance inimmunohistochemistry quantification.

Grant SupportThis work was supported by funding to B.M. Andersen from Torske Klubben

Fellowship for Minnesota Residents, Medical Scientist Training Program GrantT32 GM008244, and the Cancer Biology Training Grant T32 CA009138—36; toM.A. Hunt from the American Brain Tumor Association Discovery Grantsupported by the Anonymous Family Foundation; to J.R. Ohlfest from1R21NS070955-01, R01 CA154345, R01 CA160782, the American Cancer Societygrant RSG-09-189-01-LIB, Minnesota Medical Foundation, the Hedberg FamilyFoundation, and the Children's Cancer Research Fund.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 24, 2012; revised January 4, 2013; accepted January 18, 2013;published OnlineFirst March 7, 2013.

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2013;73:2987-2997. Published OnlineFirst March 7, 2013.Cancer Res Brian M. Andersen, G. Elizabeth Pluhar, Charles E. Seiler, et al. Cell-Mediated CytotoxicityProduction of Antibodies Capable of Antibody-Dependent

In SituVaccination for Invasive Canine Meningioma Induces

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