Development of a Function-Blocking Antibody Against ... · Cancer Therapy: Preclinical Development...

Cancer Therapy: Preclinical

Development of a Function-Blocking AntibodyAgainst Fibulin-3 as a Targeted Reagent forGlioblastomaMohan S. Nandhu1,2, Prajna Behera1,2, Vivek Bhaskaran1, Sharon L. Longo3,Lina M. Barrera-Arenas2, Sadhak Sengupta4, Diego J. Rodriguez-Gil5,E. Antonio Chiocca1, and Mariano S. Viapiano1,2,3

Abstract

Purpose: We sought a novel approach against glioblastomas(GBM) focused on targeting signaling molecules localized in thetumor extracellular matrix (ECM). We investigated fibulin-3, aglycoprotein that forms the ECM scaffold of GBMs and promotestumor progression by driving Notch and NFkB signaling.

ExperimentalDesign:Weused deletion constructs to identify akey signaling motif of fibulin-3. An mAb (mAb428.2) was gen-erated against this epitope and extensively validated for specificdetection of human fibulin-3. mAb428.2 was tested in cultures tomeasure its inhibitory effect on fibulin-3 signaling. Nude micecarrying subcutaneous and intracranial GBM xenografts weretreated with the maximum achievable dose of mAb428.2 tomeasure target engagement and antitumor efficacy.

Results: We identified a critical 23-amino acid sequence offibulin-3 that activates its signalingmechanisms.mAb428.2 binds

to that epitope with nanomolar affinity and blocks the ability offibulin-3 to activate ADAM17,Notch, andNFkB signaling inGBMcells. mAb428.2 treatment of subcutaneous GBM xenograftsinhibited fibulin-3, increased tumor cell apoptosis, and enhancedthe infiltration of inflammatory macrophages. The antibodyreduced tumor growth and extended survival of mice carryingGBMs as well as other fibulin-3–expressing tumors. Locallyinfused mAb428.2 showed efficacy against intracranial GBMs,increasing tumor apoptosis and reducing tumor invasion andvascularization, which are enhanced by fibulin-3.

Conclusions: To our knowledge, this is the first rationallydeveloped, function-blocking antibody against an ECM target inGBM. Our results offer a proof of principle for using "anti-ECM"strategies toward more efficient targeted therapies for malignantglioma. Clin Cancer Res; 24(4); 821–33. �2017 AACR.

IntroductionGlioblastomas (GBM) are the most common malignant

tumors originating in the CNS (1) and remain one of the deadliestform of cancer despite continuous advances for their treatment(2). Therapeutic strategies for GBMs are stymied by the hetero-geneity of these tumors and their invasive behavior, which makethem highly resistant to therapy and facilitate recurrence (3–5).There is a dire need for strategies capable of overcoming GBMdispersion and heterogeneity, to increase efficacy against tumorcells that may reside in different niches and express differentmolecular signatures.

The extracellular matrix (ECM) that fills the parenchyma ofmalignant gliomas has unique composition and structure com-pared with other solid tumors (6, 7). It contains remnants ofthe original neural ECM rich in hyaluronic acid and proteogly-cans (8, 9) but also includes collagens and other fibrillarproteins produced de novo by tumor cells, resulting in a uniquescaffold that supports GBM cell adhesion and dispersion (10).Prior work describing ECM molecules in malignant gliomas(6, 7, 11–13) and clonal and regional profiling of GBMs(refs. 14, 15; and http://glioblastoma.alleninstitute.org), sug-gest that there may be considerable similarity of ECM compo-nents across GBM molecular subtypes and between tumorregions. Structural ECM molecules could therefore be usefulmolecular targets localized all over the tumor parenchyma,adjacent to GBM cells with different phenotypes and genotypes.Accordingly, ECM disruption could be a feasible approach tostrike multiple populations of tumor cells surrounded by acommon matrix scaffold. This idea has been successfully testedin experimental models, targeting for example GBM-enrichedpolysaccharides and proteoglycans to increase therapeuticdelivery (16, 17) and to disrupt tumor growth and invasion(18, 19). An antibody against the ECM scaffolding proteintenascin-C has even advanced to the clinical stage and com-pleted phases I and II clinical trials (20, 21). However, twomajor limitations for these strategies have been the difficulty toidentify functional motifs underlying the protumoral functionsof ECM proteins, and the absence of reagents to disrupt sig-naling initiated or regulated by these ECM molecules.

1Department of Neurosurgery, Brigham and Women's Hospital and HarvardMedical School, Boston, Massachusetts. 2Department of Neuroscience andPhysiology, SUNYUpstateMedical University, Syracuse, NewYork. 3Departmentof Neurosurgery, SUNY Upstate Medical University, Syracuse, New York. 4BrainTumor Laboratory, Roger Williams Medical Center, Providence, Rhode Island.5Department of Biomedical Sciences, East Tennessee State University, JohnsonCity, Tennessee.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Mariano S. Viapiano, SUNY Upstate Medical University,505 Irving Avenue, IHP Building, Rm 4604, Syracuse, NY 13210. Phone: 315-464-7738; Fax: 315-464-7712; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-1628

�2017 American Association for Cancer Research.

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Fibulin-3 is an ECMglycoprotein normally found in connectivetissues, forming fibrils associated to elastin and collagen (22).This protein is sparsely detected in the body and is essentiallyabsent in adult brain (23). However, fibulin-3 is highly expressedin GBMs (24), where it gains novel functions as autocrine/para-crine activator of Notch andNFkB signaling, which have not beendescribed in normal tissues (25–27). Fibulin-3 enhances GBMinvasion, vascularization, and survival of the tumor-initiatingpopulation, correlating with poor patient survival and acting asa marker for regions of active tumor progression in GBM (26, 28)andother invasive cancers (29–31). The low expression offibulin-3 in normal tissues, high enrichment in GBMs, and nonstructuralfunctions in these tumorsmake it an appealing target to test "anti-ECM" strategies. Approaches focused on blocking the novelfunctions of fibulin-3 in GBM should have limited off-targeteffects because this protein is not expressed in normal brain orknown to act as a soluble signaling factor in other tissues.

We report here the development and preclinical characteriza-tion of a novel antibody that blocks a unique functional motif infibulin-3, resulting in complete inhibition of its signaling func-tions in GBM cells. This antibody has antitumor efficacy againstGBMs and other fibulin-3–secreting solid tumors, being the firstexample of a rationally developed, function-blocking antibodyagainst an ECM protein and capable of inhibiting cancer signal-ing. We propose this antibody as a proof-of-concept of ECM-targeting approaches to potentiate current therapies against GBM.

Materials and MethodsDNA and protein reagents

A full-length clone of human fibulin-3 (1,479 bp) was clonedin pcDNA3.1(þ) as described previously (24). Deletion con-structs lacking N-terminal sequences were generated by PCR.Reporter plasmids carrying firefly luciferase under control ofNotch-dependent (pGL2Pro-CBF1-Luc) or NFkB-dependent(pGL4.32/luc2P/NF-RE) promoters have been described else-where (25, 26). Purified fibulin-3 was from R&D Systems. Puri-fied, endotoxin-free, nonimmunemouse IgGwas fromMolecularInnovations. Fibulin-3 peptides, both free and conjugated to BSAor Keyhole limpet hemocyanin (KLH) were synthesized and

purified at Yenzym Antibodies. Amino acids in the peptides werenumbered to follow their corresponding position in full-lengthhuman fibulin-3. Antibodies and primer sequences used in thiswork are listed in Supplementary Tables SI and SII.

Cells and tissue specimensGBM cell lines, GBM stem-like cells (GSC), and HEK293

cells were cultured following described standard methods(24, 25, 28). Renal cell carcinoma SN12C and colon adenocar-cinoma COLO201 cells were cultured in high-glucose DMEMwith 10% FBS and standard antibiotics, whereas mesotheliomaH226 cells were cultured in RPMI1640 medium with the samesupplements. Endogenous fibulin-3 was detected by qRT-PCRand Western blot analysis (24) in all cells except COLO201 andHEK293. Cells were authenticated and confirmed free of con-taminants at the IDEXX-Research Animal Diagnostic Laboratory.Frozen specimens of GBM and pathologically normal brain wereprocured from NCI's Cooperative Human Tissue Network andSUNY Upstate University Hospital, with patient consent andinstitutional review board approval. Paraffin-embedded tissuesections were from US Biomax.

Antibody production and validationThe sequence N-T25YTQCTDGYEWDPVRQQCK43DIDE47-C

("DSL-like" sequence) from human fibulin-3 was chosen asimmunogen to produce mAbs. To retain the internal disulfidebridge Cys29-Cys42 (23), the peptide was not conjugated withmaleimide to KLH; instead, an N-terminal lysine was added forconjugation using N-hydroxysuccinimide ester and the internalLys43 was changed to arginine (see Fig. 1E). Mouse mAbs wereproduced at the Dana Farber Cancer Institute Monoclonal Anti-body Core Service (Boston, MA). Hybridoma clones were chosenthrough multiple rounds of subcloning as indicated in Supple-mentary Fig. S1. The final anti-fibulin-3 clone (mAb428.2.3C11.H11.G3) was nicknamed mAb428.2.

Binding of mAb428.2 to fibulin-3 and BSA-conjugatedDSL-like peptide was validated using indirect ELISA. Briefly,microtiter plates were coated with fibulin-3 (200 ng/mL) orDSL-like peptide (1,000 ng/mL), blocked with bovine serumalbumin, and probed with mAb428.2 and anti-mouse secondaryantibodies. Antibody binding was quantified by a colorimetricreaction using standard ELISA procedures. The antibody was alsotested by Western blot (Fig. 2) and dot blot (not shown) analysesto confirm binding to purified fibulin-3. mAb428.2 was purifiedin low-endotoxin conditions (< 1 EU/mg) and equilibrated inPBS, pH 6.5. The affinity of mAb428.2 for purified fibulin-3 wasmeasured by surface plasmon resonance (Biacore 3000 biosen-sor) at room temperature. Standard biophysical analysis ofmAb428.2 was performed at Wolfe Laboratories and includedsize-exclusion chromatography to determine polydispersity, iso-electrofocusing to determine PI, and differential scanning calo-rimetry to determine aggregation/solubility.

In vitro assays and histologyCells and tissues were lysed and processed for Western

blotting or semiquantitative real-time PCR (qRT-PCR) usingstandard protocols (24, 25). Reporter cells carrying stablytransfected Notch- or NFkB-reporter plasmids were transfectedwith a Renilla luciferase plasmid (pGL4.75-Rluc/CMV, Pro-mega) as loading control. Cells were stimulated with purifiedfibulin-3 (300 ng/mL) for 6 hours or transfected with fibulin-3

Translational Relevance

The molecular heterogeneity and invasive ability of glio-blastoma (GBM) cells are two major obstacles for successfultherapy of these malignant brain cancers. Targeting the tumorextracellularmatrix (ECM)may help overcome these obstaclesbecause ECM molecules secreted by tumor cells are necessaryfor invasion and relatively conserved across the tumor paren-chyma. New strategies against the ECM must first identifyfunctional domains in ECM targets and develop reagents toblock those domains and disrupt signaling initiated andregulated by ECM molecules. We have developed a func-tion-blocking antibody against a GBM-enriched ECM protein(fibulin-3) that is a "first-in-class" reagent able to inhibitprotumoral signaling and reduce GBMprogression. Anti-ECMreagents may exploit a niche that is currently underexplored,leading toward more efficient combination treatments forGBM and potentially other solid tumors.

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Figure 1.

Fibulin-3 signaling depends on a critical N-terminal sequence. A, Representation of fibulin-3 structure and N-terminal localization of the DSL-like epitope (Thr25-Glu47,underlined) deleted in fibulin-3DDSL (a, Caþþ-binding EGF-like domain; b, EGF-like repeats; c, fibulin consensus domain). B, The activity of a Notch-dependentluciferase reporter inU251MGcellswas increasedbyfibulin-3 transiently transfected for 24hours but not byfibulin-3DDSL (control: emptyexpressionvector).Noneof thefibulin-3 constructs affected Notch reporter activity driven by a constitutive control (NICD). C, Internal transfection controls (10 mg total protein/lane) show thatfibulin-3 and fibulin-3DDSL were expressed at similar levels in the conditioned medium of U251MG cells; total BSA in the medium was used as loading control (IOD:integratedoptical density, relative to the control transfection).D,Transfectedfibulin-3, but notfibulin-3DDSL, also increased theactivity of aNFkB–dependent reporter inU251MG cells (same experimental design as inB). For a positive control of NFkB-driven reporter activity the cellswere treatedwith TNFa (10 ng/mL, 6 hours).E,U251MGcells transfected with fibulin-3 cDNA and processed after 24 hours showed increased mRNA expression of Notch-regulated genes (MMP2 and HES5); transfection offibulin-3DDSL had a much smaller or absent effect. Analyses in (B–E) by one-way ANOVA: �� , P < 0.001; �� , P < 0.01; � , P < 0.05 for each reporter or gene. F,Overexpression of fibulin-3 in U251MG cells significantly increased the proteolytic activity of ADAM17 in cell lysates (RFU: relative fluorescence units); in contrast,transfectionoffibulin-3DDSLwasundistinguishable fromcontrol cells (all cellsprocessed24hourspost-transfection).G,Alignmentof theN-terminal sequenceoffibulin-3against the canonical DSL-motif of Notch ligands; this sequence is very well conserved across species. Amino acids in red indicate conservation of DSL between fibulin-3and Notch ligands and those in green show a major divergence from the consensus DSL motif (b, basic amino acid; a, acidic amino acid). The peptide chosen forimmunization corresponds to Thr25-Glu47 of human fibulin-3 but has Lys43 replaced with Arg as indicated in the Materials and Methods.

Anti-Fibulin-3 Antibody for Glioblastoma Therapy

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cDNA and used 24 hours after transfection. For a positivecontrol of Notch activation, cells were transfected with theplasmid pSG5-FLAG-NICD carrying the constitutively activeNotch1 Intracellular Domain (NICD; ref. 25). For a control ofNFkB activation, cells were treated with TNFa (10 ng/mL, 6hours) as described previously (26). To measure ADAM17proteolytic activity, cells were transfected with fibulin-3 con-structs, lysed after 24 hours, and incubated with a fluorogenicADAM17 substrate peptide (R&D Systems) as described previ-ously (28). All transfections were performed in cultures adjust-ed to a density of 1 � 106 cells/mL.

Tomeasure cell invasion, GSC tumorspheres were labeled witha fluorescent dye (PKH26, Sigma-Aldrich), seeded on freshlyprepared brain slices, and cultured for up to five days followingour established protocols (24). The dispersion of the cells intobrain tissue was imaged daily by fluorescence microscopy (24). Amigration index was calculated as the ratio of area covered by thedispersed cells to the original area of the spheroids. Antibodies(200 mg/mL) were added to the organotypic cultures every 48hours together with fresh culture medium.

Fibulin-3; 5-bromo-20-deoxyuridine (BrdUrd), coexpressedmacrophage markers (Iba1; Arginase-1), and the DNA-damagemarker phospho-histone H2A.X were detected in tissue sectionsfollowing previously described IHC protocols (28, 32, 33). Bloodvessels were stained with an antibody against mouse CD31, andvessel length and density per tumor area were calculated usingparticle image analysis as described previously (28).

In vivo proceduresAll animal experiments were performed in athymic mice

(FoxN1nu/nu, Envigo) following institutional approval at theBrigham andWomen's Hospital (Boston,MA) and SUNYUpstateMedical University (Syracuse, NY). mAb428.2 was prepared inlactated Ringers solution (pH 6.5) and confirmed free of endo-toxins, mycoplasma, and rodent pathogens (Yale University Sec-tion for Comparative Medicine, New Haven, CT). Preliminarytoxicity assays were performed in C57Bl/6 mice and Lewis rats,injected intravenously with mAb428.2 for eight consecutive daysand monitored for up to 7 days after the final injection.

For subcutaneous tumor implantation, animals (N¼ 8/group)received bilateral injections of 1 � 106 tumor cells (in 100 mL)without Matrigel adjuvant. Tumors were measured every otherday with calipers and volumes were calculated as (length �width2)/2. The larger of the two tumors per animal was used todecide treatment initiation and endpoint.When tumors reached a

threshold of 100 mm3 mAb428.2 was injected directly in thetumormass (3� 30mg/kg q48h) or intravenously (8� 30mg/kgq24h). Animals were euthanized at a fixed endpoint 3 days afterthe last antibody injection, or monitored for overall survival andterminated when tumor volumes reached 1,000 mm3. For intra-cranial tumor implantation, animals (N¼ 8/group) were injectedwith 10,000 GBM stem-like cells in the right striatum (2 mL)and one week later intracranial cannulas were implanted todeliver mAb428.2 from a subcutaneous osmotic pump (Alzet#2001; 1.5 mg/mL antibody released at 1.0 mL/hour for 8 days).Pumps were removed after the antibody was fully delivered andthe animals were monitored for overall survival. All euthanizedmice were perfused with PBS and tissues were recovered forbiochemical or IHC analyses. Subclass-specific secondary anti-bodieswere used to detect intratumoral retention ofmAb428.2 byWestern blot of IHC.

To detect antibody distribution in na€�ve and tumor-carryingmice, mAb428.2 was fluorescently labeled with DyLight-755following the manufacturer's protocol (Thermo Fisher antibodylabeling kit #84538), injected intravenously at 5 mg/kg, andvisualized using an in vivo imaging system (Spectral InstrumentsAmi-X, Tucson AZ; Ex/Em ¼ 745/790 nm).

Statistical analysisIn vitro experiments were repeated at least twice with 3–5

independent replicates each time; all results were represented asmean� SD. Animal studies were performedwithN¼ 5/group forfixed-endpoint and N ¼ 8/group for overall survival studies (todetect differences at least 50% larger than the SD of the groups, atpower 80% and P < 0.05). Bilateral tumors were averaged tocalculate a mean tumor volume per animal. Blinding and ran-domization for animal studies followed the ARRIVE guidelinesfor animal research (34).

ResultsAn exposed N-terminal motif of fibulin-3 is critical for itssignaling

Fibulin-3 increases canonical Notch (25, 27) and NFkB(26, 35) signaling in tumor cells. The mechanism is not fullyelucidated but includes activation of the cell-surface metallopro-tease ADAM17, which cleaves (and activates) Notch receptors(28) and can also release TNFa to initiateNFkB signaling (26).Wetherefore sought to identify a functional motif(s) of fibulin-3critical for these signaling mechanisms.

Figure 2.Characterization of anti-fibulin-3 mAb428.2. A,Western blots showing detection of purified fibulin-3 by mAb428.2 in reduced and nonreduced conditions (200 ngprotein/lane). The arrows indicate the position of the 55-kDamonomer, 110-kDa dimer, and high-Mwmultimer (fibrillar) forms of fibulin-3.B,Western blots of serum-free conditionedmedium from HEK293 cells (10 mg total protein/lane) expressing V5-tagged fibulins -3, -4, or -5. Blots were probedwith an antibody against the V5epitope or with the anti-fibulin-3 antibodies mAb428.2 and mAb3-5. The control lane contains medium from untransfected cells. C, Western blots ofserum-free conditionedmedium fromHEK293 cells expressing V5-tagged full-length fibulin-3 (fib3) and fibulin-3DDSL that lacks the target epitope ofmAb428.2.D,Detection of purified fibulin-3 (200 ng/lane) by mAb428.2 was inhibited by the DSL-like peptide from fibulin-3 (Thr25-Glu47) but not by a scrambled version of thispeptide. E, Microtiter plates were coated with BSA-conjugated DSL-like peptide (1,000 ng/mL) and different concentrations of mAb428.2 were used to detect theepitope by indirect-ELISA. mAb428.2 binding to BSA-conjugated peptide was displaced by incubation with free DSL-like peptide following a simple competitivemodel (analyzed in Supplementary Fig. S2A). F,Microtiter plates were coated with purified fibulin-3 (200 ng/mL) and mAb428.2 (1 mg/mL) was used to detect theprotein by indirect-ELISA. Binding of the antibody was inhibited by the DSL-like peptide from human fibulin-3 (pept human) and the modified peptide used forimmunization (pept immun); �� , P <0.001, one-wayANOVA. However, theDSL-like peptide frommouse fibulin-3 (peptmouse, containing Ile38 instead of Val38)wasunable to displace mAb428.2 binding. All peptides were tested at a maximum concentration of 50 mg/mL (18 mmol/L). G, IHC of paraffin-processed tissuesections using mAb428.2 or mAb3-5 showed similar detection of fibulin-3, which increased with tumor grade. The sparse nuclear staining observed with bothantibodies is non-specific. Control staining was performed with non-immune mouse IgG. H, IHC of frozen GBM sections using mAb428.2 or mAb3-5 showed acharacteristic perivascular fibrillar pattern described previously (28). mAb428.2 did not detect perivascular fibulin-3 in blood vessels from normal brain adjacent tothe tumor (brain), as expected. Vessels were stained with an antibody against endothelial CD31.






Fibulin-3 has a unique N-terminal, EGF-like domain(Met1-Ser106) that includes a sequence (Thr25-Cys70) named"DSL-like" for its high homology to the canonical Delta-Serrate-Lag (DSL) motif present in Notch ligands (25). We have shownthat this N-terminal domain is necessary and sufficient to activateNotch signaling (25), suggesting that disrupting this domaincould affect molecular pathways regulated by fibulin-3.

To test this hypothesis, we analyzed short sequences within theDSL-like motif and found that fibulin-3 lacking the sequenceThr25-Glu47 ("fibulin-3DDSL," Fig. 1A) was unable to activateNotch and NFkB reporters in U251MG GBM cells (Fig. 1B–D).The deletion did not affect fibulin-3 secretion (Fig. 1C) or theactivation of the reporters by their respective canonical signals(Fig. 1B and D), suggesting a specific loss of fibulin-3 signalingfunction. In agreement, transfection of fibulin-3DDSL inU251MG cells failed to increase the expression of the Notch-dependent genes HES5 andMMP2 (Fig. 1E), which are regulatedby fibulin-3 in GBM (25, 26). Moreover, fibulin-3DDSL lackedany enhancing effect on ADAM17 catalytic activity (Fig. 1F). Insum, deletion of the Thr25-Glu47 sequence was sufficient tocompletely abolish the signaling functionality of fibulin-3.

Analysis of the sequence Thr25-Glu47 of human fibulin-3 usingthe PHYRE2 Protein Fold Recognition Server (www.sbg.bio.ic.ac.uk/~phyre2; ref. 36) suggested that it is highly exposed on theglobular head of the protein (22) and is well conserved amongspecies (Fig. 1G). We then set out to generate an antibody capableof binding and blocking this exposed functional motif. Becausethe sequence Thr25-Glu47 contains two cysteines thatmay form anintramolecular bridge (23) we modified one amino acid in thesequence (Lys43 ! Arg) to retain this feature in the peptide usedfor immunization (Fig. 1E).

Characterization of mAb428.2, a novel anti–fibulin-3 antibodyThe antibody mAb428.2 was confirmed to be a mouse IgG1

kappa. It has average pI¼ 7.48, which limited its solubility in PBSto approximately 5–6 mg/mL before noticeable aggregation. Atconcentration of 1 mg/mL in this buffer, mAb428.2 remained as>99% monomeric at room temperature.

mAb428.2 detected denatured fibulin-3 in reducing and non-reducing conditions (Fig. 2A), suggesting that the presence of thedisulfide bond in the epitope was not critical for recognition. Theantibody did not cross-react with fibulin-4 and -5 (Fig. 2B), orwith fibulin-3DDSL lacking the target epitope (Fig. 2C). Bindingof mAb428.2 to BSA-conjugated DSL-like peptide or to purifiedfibulin-3 was competitively inhibited by free DSL-like peptide(Fig. 2D–E; and Supplementary Fig. S2A), suggesting that thisepitope is the only part of fibulin-3 recognized by the antibody.Binding of mAb428.2 to fibulin-3 was inhibited by the endoge-nous DSL-like sequence from human fibulin-3 (with Lys43) andby the modified sequence used for immunization (withLys43!Arg; Fig. 2F). Interestingly, a DSL-like peptide derivedfrom mouse fibulin-3, which contains Ile38 instead of Val38, wasunable to displace the binding of mAb428.2, suggesting that ourantibody may show poor to negligible recognition of mousefibulin-3 (Fig. 2F).Measurement of binding kinetics ofmAb428.2to purified human fibulin-3 revealed a high-affinity binding withKd ¼ 5 � 1 nmol/L (Supplementary Fig. S2B).

mAb428.2 also recognized native fibulin-3 in GBM tissuesin a manner comparable to the antibody mAb3-5, which is awell-characterized antibody raised against human fibulin-3for histology (37). mAb428.2 detected pericellular and diffuse-

ly localized fibulin-3 in low-grade astrocytoma and GBM,matching the staining of mAb3-5 that increases with tumorgrade (ref. 25; Fig. 2G). None of these antibodies showedspecific staining of normal adult brain. In frozen (nonparaffi-nized) tissues, both mAb3-5 and mAb428.2 detected a char-acteristic perivascular fibrillar pattern of fibulin-3 that we haveobserved restricted to human GBM blood vessels (Fig. 2H;ref. 28). Further staining of live, dissociated GBM cells andfixed GBM tumorspheres (Supplementary Fig. S3) confirmedthat mAb428.2 detects fibulin-3 that accumulates in the inter-cellular ECM as well as peripherally associated to the mem-brane of the tumor cells.

Taken together, our results validatemAb428.2as anovel antibodythat recognizes human fibulin-3 with high affinity and specificity,under a variety of conditions and specimen preparations.

mAb428.2 is a function-blocking anti–fibulin-3 antibodyWe first tested mAb428.2 on U251MG cells overexpressing

fibulin-3. Transfected fibulin-3 cDNA increased the activity of aNotch-dependent reporter in these tumor cells; however, thiseffect was completely abolished by mAb428.2 in a time- andconcentration-dependentmanner (Fig. 3A andB).mAb428.2 alsoinhibited the enhancing effect of fibulin-3 on a NFkB–dependentreporter (Fig. 3C) and ADAM17 activity (Fig. 3D). The antibodydid not affect the direct activation of the Notch reporter by NICDor the NFkB reporter by TNFa (Fig. 3E), indicating that its effectswere specific against fibulin-3. A preimmune control IgG had noeffects in any of these assays.

As additional confirmation we tested the effect of mAb428.2against endogenous fibulin-3 secreted by GBM cells. mAb428.2added to cell cultures for 24 hours reduced the expression ofseveral Notch andNFkB–regulated genes inU251MG cells as wellas inmesenchymal-type GSCs that have high endogenous expres-sion of fibulin-3 (ref. 25; Fig. 3F). Taken together, our resultsindicate that mAb428.2 is a function-blocking antibody againstfibulin-3.

mAb428.2 inhibits fibulin-3 signaling in vivo, reduces tumorgrowth, and induces antitumor inflammation

For in vivo experiments mAb428.2 was injected intravenouslyonce a day for 8 days (to match the time needed to empty thereservoir of an osmotic pump for local delivery). We did notobserve toxic effects in na€�ve mice treated with mAb428.2 upto `30 mg/kg or in Lewis rats treated with an adjusted dose of20 mg/kg (38). The animals did not show changes in weight orany signs of acute damage in normal tissues that containfibulin-3,such as kidneys' glomeruli, connective tissue in articular joints, orretinal epithelium (Supplementary Fig. S4).

To assess antitumor efficacy, mAb428.2 was tested against thehighly aggressive GSC GBM34 (25). Tumor cells were implantedsubcutaneously and treated with mAb428.2 or nonimmune con-trol IgGonce the tumors reached a threshold volume of 100mm3.A preliminary test with direct intratumoral injections ofmAb428.2 (3 � 30 mg/kg q48h) showed significant reductionof tumor volume and final weight measured at a fixed endpoint(Supplementary Fig. S5A–S5B). Similar reduction in tumorgrowth was observed when the antibody was delivered intrave-nously (8 � 30 mg/kg q24h), suggesting that peripherally deliv-ered mAb428.2 reaches its target in the tumor mass (Fig. 4A).Tumor tissues were subsequently processed to detect indicators ofNotch and NFkB signaling, including expression of Notch1

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Figure 3.

mAb428.2 inhibits fibulin-3 signaling in glioblastoma cells. A, U251MG stably expressing a Notch-dependent reporter (CBF1:Luc) were transfected with fibulin-3 orcontrol cDNAs and after 24 hours treated with 50 mg/mL of mAb428.2. The antibody inhibited the enhancing effect of fibulin-3 on Notch activity within 6 hours. Theinset shows the level of fibulin-3 overexpression in the cells, quantified as shown in Fig. 1C. B, Same experimental design as in A but mAb428.2 was added atdifferent concentrations for 6 hours. Fibulin-3-enhanced Notch activity was completely blocked by mAb428.2 at concentrations above 30 mg/mL. C, Sameexperimental design as in A, using cells stably expressing a NFkB-dependent reporter (NKRE:Luc). mAb428.2 inhibited the enhancing effect of fibulin-3 on NFkBactivity within 6 hours. Firefly luciferase activity of the reporters was normalized to a transfected Renilla Luciferase control as indicated in Materials and Methods.Significant differences in A and C: �� , P < 0.001 (two-way ANOVA). D, U251MG cells were transfected with fibulin-3 or control cDNAs and lysed after 24 hours; thelysateswere incubatedwith anADAM17 fluorogenic substrate tomeasure enzymatic activity in presenceor absence ofmAb428.2. Fibulin-3 overexpression increasedADAM17 activity but this effect was completely abolished when mAb428.2 (50 mg/mL) was present in the reaction mixture. Treatment of cells or lysates withnonimmune IgG in experiments (A–D) had no effects on reporters or enzymatic activity. E, U251MG cells transfected with NICD cDNA (for 24 hours) to activate theNotch reporter, or treated with TNFa (10 ng/mL, 6 hours) to activate the NFkB reporter, showed no changes in reporter activity when they were cotransfected withfibulin-3 cDNA or incubated with antibodies (50 mg/mL). F, U251MG cells and two GSC cultures (GBM09 and GBM34) were treated with mAb428.2 (50 mg/mL) or acontrol IgG for 24 hours and then processed to measure mRNA expression of Notch- and NFkB-dependent genes. mAb428.2 reduced the expression of the fourtested genes compared with control IgG treatment (�P < 0.05; þ P < 0.01; # P < 0.001, by two-way ANOVA for each cell type).






Figure 4.

mAb428.2 inhibits tumor growth and increases inflammatory macrophage infiltration in the tumor. A, Mice carrying bilateral subcutaneous GBM34 tumors(N ¼ 5/group) were treated with daily injections of mAb428.2 or control IgG for eight days (8 � 30 mg/kg, i.v., horizontal bar). Tumor growth was significantlyinhibited inmAb428.2-treatedmice (� , P <0.05; �� , P <0.001; repeatedmeasures ANOVA). Animalswere euthanized three days after the last injection (dashed line)and their tumors were processed for the rest of the experiments in B and C. Expression of Notch1 Intracellular Domain (NICD) and the transcription factorsHes5 and RelA/p65 (and phospho-p65) was probed by Western blot and quantified by densitometry (IOD: integrated optical density). mAb428.2 treatmentsignificantly reduced expression of NICD, Hes5, and phospho-p65, suggesting inhibition of fibulin-3 effects on Notch and NF-kB signaling (� , P < 0.05;�� , P < 0.01; �� , P < 0.001; Student t test for each marker). (Continued on the following page.)

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Intracellular Domain (active form of Notch1); Notch-regulatedtranscription factor Hes5; and phosphorylation of the NFkBtranscription factor RelA/p65. We observed significant downre-gulation of all these proteins in tumors treated with mAb428.2,both by direct intratumoral injection (Supplementary Fig. S5C)and intravenous delivery (Fig. 4B and C).

Further analysis of the GBM34 tumors treated with mAb428.2revealed increased expression of cleaved caspase-3 in the tumormass (Fig. 4D and F) and significant reduction of BrdUrd uptake(Fig. 4E and G), suggesting a cytostatic and possibly cytotoxic

effect of mAb428.2 treatment. Indeed, postmortem examinationrevealed considerable necrosis in the core of mAb428.2-treatedtumors (Supplementary Fig. S5D).

The observation of widespread tumor apoptosis and necrosisin vivo (see also Fig. 6C; comments in Supplementary Fig. S6)was somewhat surprising because highly purified mAb428.2 didnot reduce GBM cell viability significantly when added to culturesfor 24–72 hours (not shown). Therefore, we explored whetherthe antibody had caused any effects on immune cells that couldhelp explain tumor cell death. Indeed, mAb428.2-treated tumors

Figure 5.

mAb428.2 extends survival of mice carrying fibulin-3–secreting tumors. A, Comparative expression of fibulin-3 mRNA in a conventional GBM cell line (U251MG),GSC cultures (GBM34 and GBM09), and three cell lines from the NCI-60 collection (COLO201 colon carcinoma; H226 mesothelioma; and SN12C renal cellcarcinoma). B–F, Mice carrying subcutaneous tumors derived from the cells compared in A were treated with mAb428.2 (8 � 30 mg/kg q24h i.v.) or nonimmunecontrol IgG when the tumors reached a threshold volume of 100 mm3 (black bars indicate the treatment period, N ¼ 8/group). mAb428.2 extended the mediansurvival of mice carrying fibulin-3–expressing tumors (analysis by log-rank test for each tumor model) but failed to improve the survival of mice carryingCOLO201 tumors that have negligible fibulin-3 expression.

(Continued.)D,Expression of full-length and cleaved caspase-3 (casp-3)was probed in three representative tumors per treatment.E,Uptake of BrdUrdwas detectedby IHC in five tumors per treatment; the images show representative staining results. F, Quantitative analysis of caspase-3 expression from D shows increasedcaspase cleavage in mAb428.2-treated tumors (�� , P < 0.01; Student t test). G, Quantitative analysis of BrdUrd staining from (E); each dot represents a tissuesection. mAb428.2 significantly reduced BrdUrd uptake (analysis by Mann–Whitney U test). H, Increased macrophage infiltration in mAb428.2-treated tumors,detected by Iba1-positive staining. I, Comparison of tissue sections with similar number of macrophages in both treatments revealed coexpression of Arginase-1with Iba1-positive cells in control-treated tumors but very lowor absent Arginase-1 inmacrophages ofmAb428.2-treated tumors. J, Tumor treatmentwithmAb428.2correlated with increased mRNA expression of the host's inflammatory cytokines and decreased expression of M2-macrophagemarkers (CD163, CD206, and ARG1);all genes were detected by qRT-PCR with mouse-specific primers. K, Tumor treatment with mAb428.2 also correlated with increased mRNA expression ofinflammatory cytokines in the tumor cells, detected by qRT-PCR with human-specific primers. Results in J and K were analyzed by Student t test corrected formultiple comparisons (� , P < 0.05; �� , P < 0.01; �� , P < 0.001). Scale bars in all the histologic images, 200 mm.






showed increased infiltration of macrophages (Iba1-positivecells; Fig. 4H) and lower or absent expression of the M2-pheno-typemarker Arginase-1 in thosemacrophages (Fig. 4I), comparedwith the control IgG treatment. In agreementwith these histologicfindings, tumors treated with mAb428.2 and processed to mea-

sure mouse-specific mRNAs showed increased expression ofinflammatory cytokines (IL1b, IL10, and IFNg) and reducedexpression of macrophage markers associated with the M2 phe-notype (39) such as Arginase-1 (ARG1), CD163, and CD206(Fig. 4J). Moreover, mAb428.2 increased the mRNA expression

Figure 6.

mAb428.2 has antitumor effects in intracranial GBMs.A,Mice carrying intracranial (IC) xenografts of GBM09 cellswere treatedwithmAb428.2 injected intravenouslyas in Fig. 5, or locally infused with an osmotic pump (OP) as indicated in Materials and Methods (black bars indicate the treatment period, N ¼ 8/group). Onlyanimals that received local mAb428.2 infusion showed improved survival (curves analyzed by log-rank test). B, Tumor spheres of GBM09 cells were seeded oncultured brain slices and their invasion through brain tissue was monitored by microscopy as described in Materials and Methods (24). Addition of mAb428.2to the culturemedium (200 mg/mL, arrows) significantly inhibited cell invasion (�� , P < 0.01; �� , P < 0.001; two-way ANOVA for repeatedmeasures). C andD, Tissuesections from intracranial tumors (GBM09) treatedwith locally delivered antibody were processed for IHC to detect phospho-Histone H2A.X (pH2AX, N¼ 5/group).Quantitative analysis showed a significant increase of pH2AX-positive nuclei in mAb428.2-treated tumors (Mann–Whitney U test). E and F, Intracranialtumors (GBM09-eGFP)were also processed to detect and quantify blood vessels as described (ref. 28; CD31 staining,N¼ 5/group). mAb428.2-treated tumors had asignificant reduction ofmicrovascular density, caused by a lower number of small vessels (� , P <0.05; �� , P <0.01; �� , P <0.001; two-wayANOVA. Scale bars in all thehistologic images, 50 mm.

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of human-specific proinflammatory cytokines, derived from thetumor cells (Fig. 4K). Taken together, the results suggested thatmAb428.2 induces a marked antitumor reaction driven, in part,by inflammation and activation of innate immunity.

mAb428.2 extends survival of mice carrying fibulin-3–expressing tumors and disrupts GBM invasion andvascularization

We next evaluated the efficacy of mAb428.2 to extend survivalof mice carrying fibulin-3–expressing subcutaneous tumors,including our two GSC-derived GBM models as well as cell lines(H226 and SN12C) chosen from the NCI-60 collection for theirhigh expression of fibulin-3 (ref. 40; Fig. 5A). One additional cellline (COLO201) was chosen as negative control for its negligibleendogenous expression of fibulin-3. Intravenously injectedmAb428.2 reduced tumor volume (Supplementary Fig. S6) andsignificantly improved survival in all the fibulin-3–expressingmodels (Fig. 5B–F), extending median survival by 28% (GBM09)to64%(GBM34) in theGBMxenografts.However,mAb428.2didnot prolong the survival of mice carrying fibulin-3–negativeCOLO201 tumor cells (Fig. 5D).

We also tested mAb428.2 against both GSC modelsimplanted intracranially, but were unable to detect a survivalimprovement when the antibody was delivered peripherally(Fig. 6A; Supplementary Fig. S7A). However, when a compa-rable dose of antibody was infused into the intracranial tumormass using an osmotic pump we observed again a significantincrease in median survival (32%, Fig. 6A), suggesting thatmAb428.2 has antitumor efficacy when it is able to accumulatein the tumor parenchyma. Accordingly, a small biodistributionstudy using intravenously injected, fluorescently labeledmAb428.2 suggested that the antibody may be rapidly clearedfrom circulation but nevertheless accumulates in subcutaneoustumors and remains at peak level in the tumor for at least 24hours after injection. In contrast, mAb428.2 fails to accumulatein intracranial tumors unless it has been locally delivered(Supplementary Fig. S8A–S8D).

Further analysis of mAb428.2-treated intracranial tumorsfocused on tumor invasion, apoptotic resistance, and vascu-larization, all of which are enhanced by fibulin-3 (25, 28).We were unable to assess the effect of mAb428.2 on intra-cranial tumor dispersion in vivo due to the modest invasiveprofile and the large size of GBM09 and GBM34 tumors bythe time that the animals could be euthanized. However,mAb428.2 caused significant inhibition of GSC invasion incultured brain slices (Fig. 6B), which is an accurate surrogateof in vivo invasion (24).

Local infusion of mAb428.2 in intracranial tumors (GBM09cells) increased the number of tumor cells expressing the DNAdamage marker phospho-H2A.X (Fig. 6C and D), matching theproapoptotic effect of this antibody in subcutaneous tumors.mAb428.2-treated tumors also showed decreased microvasculardensity, with significant reduction in the number of small vesselsthat are increased byfibulin-3 inGBM(28). Themagnitude of thiseffect was comparable with a previously observed antiangiogeniceffect achieved by fibulin-3 knockdown in the same tumormodel(28). Finally,mAb428.2 increased the infiltration ofmacrophagessurrounding the tumor mass (Supplementary Fig. S7B), suggest-ing an inflammatory effect similar to the one observed in subcu-taneous tumors. Taken together, the results suggest that theantitumor effects of mAb428.2 in intracranial GBMs match a

generalized inhibition of fibulin-3 mechanisms, together withincreased antitumor inflammation.

DiscussionMost antitumor strategies with a focus on tumor ECM have

concentrated on ECM-associated proteins rather than the struc-tural components of the ECM. Successful strategies that have beentranslated to the clinical setting include inhibition of matrix-degrading metalloproteases (41, 42) and integrins (reviewedin ref. 43). In contrast, direct targeting of ECMmolecules in GBM,such as hyaluronic acid, proteoglycans, and glycoproteins,has rarely advanced beyond experimental models. A notableexception is the monoclonal antibody 81C6 that binds the ECMprotein tenascin-C enriched in GBMs (44, 45). 131I-labeled 81C6(Neuradiab) was successfully tested for local radiotherapyof intracranial gliomas (46) and completed a phase II clinicaltrial (NCT00003478) before being discontinued in phase III(NCT00615186) for reasons unrelated to its efficacy. However,neither this antibody nor other approaches against ECM mole-cules have focused on identifying and blocking the signalingmechanisms that are triggered or regulated by these moleculesin GBM.

In this study, we have identified a key motif exposed by theECM protein fibulin-3 and demonstrated that this epitope iscritical for the signaling functions of this protein in GBM. More-over, we have developed and validated an antibody against thisepitope and shown that it blocks the signaling functions offibulin-3 in vitro and its tumor-promoting mechanisms in vivo.To our knowledge, this is the first function-blocking antibodydeveloped to inhibit protumoral signaling mechanisms triggeredby an ECM protein.

mAb428.2 showed significant efficacy against subcutaneoustumor xenografts, reducing tumor cell proliferationand increasingapoptosis. Moreover, the antibody promoted macrophage infil-tration in the tumor, togetherwith increased expression of inflam-matory cytokines and decreased expression of M2/"tumor-pro-moting"macrophagemarkers in the tumor parenchyma (39). Thereasons for the remarkable differences in the infiltrated macro-phages of control- and mAb428.2-treated tumors are currentlyunknown because the effects of fibulin-3 on tumor-associatedmacrophages have not been studied.We can only speculate aboutthe possibility that mAb428.2 disrupted an immunomodulatoryeffect of fibulin-3 on these immune cells. Alternatively, accumu-lation of mAb428.2 in the tumor could have been sufficient toinitiate an inflammatory reaction that contributed to the observedantitumor effects. The increased necrosis in the core ofmAb428.2-treated tumors (Supplementary Fig. S5D) suggests that this anti-tumor reaction was highly efficient and tumor progression likelycontinued only after the treatment was discontinued.

mAb428.2 also showed efficacy against intracranial GBMs,increasing tumor cell apoptosis and decreasing vascularization.In addition, the antibody reduced GBM invasion in organo-typic cultures of brain tissue, suggesting that it probably has asimilar (not assessed) effect in vivo. Fibulin-3 is known toincrease the invasion, apoptotic resistance, and vascularizationof intracranial GBMs via Notch and ADAM17/NFkB activation(25, 26, 28), therefore the effects of mAb428.2 match whatwould be expected from a widespread inhibition of fibulin-3signaling in the tumor. We hypothesize that mAb428.2 pre-vents fibulin-3 from promoting tumor escape and progression






mechanisms (e.g., dispersion and vascularization). Combinedwith antitumor innate immunity, these effects result in tumorcell death and extended survival.

The effects of our antibody may have been restricted in partby its rapid clearance and the requirement of repeated injectionsto "build up" in the tumors. This may have prevented intrave-nously delivered mAb428.2 from reaching sufficient intracranialconcentration to elicit antitumor effects. Another limitation wasthe limited solubility of mAb428.2 in phosphate- or lactate-buffered solutions, which could have also contributed to limitedintratumoral accumulation. Nevertheless, our results suggestthat mAb428.2 has significant antitumor effects that last for aslong as the antibody is delivered and able to accumulate in thetumor mass. These encouraging results warrant further optimi-zation of mAb428.2 to continue improving these effects. Becausethe hybridoma mAb428.2 has been cloned and sequenced(sequences deposited in USPTO application #15/124,826;2016), future work could proceed directly with recombinantvariants of the antibody.

This study illustrates the feasibility of rationally developingbiological agents to target the tumor ECM, which has beenpreviously perceived as a passive barrier for drug efficacy (47)rather than a source of "druggable" targets (48). Targeting theECM of malignant gliomas provides unique advantages such asthe excellent accessibility of the targets; the ability to inhibitoutside-in signalingmechanisms required for tumor progression;and the restricted expression of some ECM proteins to the tumorparenchyma (7, 49). Anti-ECM strategies could take advantage ofa sizable group of novel targets that have been largely overlookedin GBMs and other solid tumors. We propose anti-fibulin-3targeting as a strategy to disrupt signaling mechanisms in GBM

and increase the efficacy of combination therapies for thesemalignant tumors.

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

Authors' ContributionsConception and design: M.S. Nandhu, S. Sengupta, M.S. ViapianoDevelopment of methodology: M.S. Nandhu, P. Behera, V. Bhaskaran,S.L. Longo, L.M. Barrera-Arenas, D.J. Rodriguez-Gil, E.A. ChioccaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.S. Nandhu, P. Behera, V. Bhaskaran, S.L. Longo,L.M. Barrera-Arenas, D.J. Rodriguez-GilAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.S. Nandhu, S. Sengupta, E.A. Chiocca,M.S. ViapianoWriting, review, and/or revision of the manuscript: M.S. Nandhu,E.A. Chiocca, M.S. ViapianoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.S. Nandhu, S.L. LongoStudy supervision: M.S. Nandhu, M.S. Viapiano

AcknowledgmentsThis work was supported by research grants from the NIH (R01CA152065

and R21NS091436) and the B�Cured Foundation (to, M.S. Viapiano).

The costs of publication of this articlewere defrayed inpart 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 June 7, 2017; revisedOctober 10, 2017; acceptedNovember 8, 2017;published OnlineFirst November 16, 2017.

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2018;24:821-833. Published OnlineFirst November 16, 2017.Clin Cancer Res Mohan S. Nandhu, Prajna Behera, Vivek Bhaskaran, et al. a Targeted Reagent for GlioblastomaDevelopment of a Function-Blocking Antibody Against Fibulin-3 as

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