MOLECULAR CANCER RESEARCH |

A Bispecific Antibody Targeting the av and a5b1Integrins Induces Integrin Degradation in ProstateCancer Cells and Is Superior to Monospecific AntibodiesRaghav Joshi, Wenying Ren, and Paul Mathew

ABSTRACT◥

Fibronectin-binding integrins a5b1 and av collaborate inprostate cancer–bone stromal interactions relevant to the colo-nization of the bone marrow microenvironment. Combinatorialinactivation of these integrins on prostate cancer cells wasassessed. Monospecific antibodies to a5b1and av integrins alone(MAb) and in combination (cMAb), and a bispecific antibodythat simultaneously targets a5b1and av integrins (BsAba5b1/av) were compared in assays of chemotaxis, clonogenic survival,and induction of endothelial migration. Cellular expression ofintegrins, their transcription, translation, and degradation fatewas compared. The BsAba5b1/av was superior to MAbs andcMAbs in abrogating adhesion, migration, clonogenic survival,and induction of endothelial migration responses by prostatecancer cells. Integrin upregulation observed with MAbs orcMAbs was abrogated with the BsAba5b1/av. Loss of integrinexpression was uniquely induced by the BsAba5b1/av andblocked by lysosomal inhibition.

Implications: A novel and effective combinatorial strategyto target a5b1and av integrins is defined for translationalstudies.

Visual Overview: http://mcr.aacrjournals.org/content/molcanres/18/1/27/F1.large.jpg.

IntroductionThe preferential colonization of the bone marrow microenviron-

ment by disseminated prostate cancer cells underpins its lethal met-astatic phenotype. Deconvolution of the molecular mechanisms thatmediate this behavior can define novel therapeutic strategies toimprove mortality and morbidity from the disease. Bone marrow–

derived mesenchymal stromal cells have been implicated as architectsof both the hematopoietic (1) and bone metastatic niche (2). Earlierstudies indicated that human bone marrow–derived mesenchymalstromal cells (hBM-MSC) induce a strong chemotactic and adhesiveresponse in prostate cancer cells. The relevant bioactive fraction of thehBM-MSC secretome was purified and proteolytic fragments offibronectin (FNFr) signaling via the a5b1 integrin in prostate cancercells identified as the basis of the chemotactic response (3). Geneticinactivation of the a5 integrin induced programmed cell death inprostate cancer cells indicating a role in adhesion-mediated surviv-al (4). Accordingly, the FNFr–a5b1 integrin interaction was proposedas a seed-and-soil mechanism of bone colonization by prostatecancer (3).

We hypothesized that the av integrin, an alternative fibronec-tin-binding integrin (5) cooperates with the a5b1 integrin inmediating the metastatic niche interactions of prostate cancer cells.By comparing the impact of monospecific neutralization of a5b1and av integrins alone (MAb) and in combination (cMAb) to afirst-in-class integrin-targeting bispecific antibody, BsAba5b1/av,

Division of Hematology-Oncology, TuftsMedical Center, Boston,Massachusetts.

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

Corrected online January 6, 2020.

Corresponding Author: Paul Mathew, Division of Hematology-Oncology, TuftsMedical Center, 800 Washington St, #245, Boston, MA 02111. Phone: 617-636-8483; Fax: 617-636-8538; E-mail: pmathew@tuftsmedicalcenter.org

Mol Cancer Res 2020;18:27–32

doi: 10.1158/1541-7786.MCR-19-0442

�2019 American Association for Cancer Research.

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we find that the BsAba5b1/av optimally neutralizes tumor–stromalinteractions with a novel mechanism of action.

Materials and MethodsProstate cancer, stromal and endothelial cell lines, and culture

Prostate cancer cell lines PC-3, DU-145, LNCaP, VCaP, and C4-2B and human umbilical vein endothelial cells (HUVEC) wereobtained from ATCC and the Characterized Cell Line Core Facility(MD Anderson Cancer Center, Huston, TX). Plastic adherenthBM-MSCs were generated from human bone marrow aspirates(Lonza) as described previously. Cell lines represent genomicallyand phenotypically diverse prostate cancer cell lines: integrin a5/av coexpressing lines include bone-derived PTEN-null androgenreceptor (AR)-negative PC-3 cells, bone-derived PTEN-null AR-positive hormone-resistant C4-2B cells, lymph node–derivedPTEN-null AR-positive hormone-sensitive LnCAP cells, and PTENwild-type AR-negative DU-145 cells. TMPRSS2-ERG–positive,PTEN wild-type AR-positive VCAP cells lack membrane a5expression.

Generation and validation of a bispecific antibody to integrinsa5b1 and av

A genetic construct was designed to express a bispecific antibodythat targetsav (6) anda5b1 integrins (7). The construct was expressedin 293T cells, and the resulting supernatant was purified by protein Achromatography (Creative Biolabs). A similar approach was used togenerate a5b1 and av IgG control antibodies with the same antigen-binding sequences as BsAba5b1/av. Further description of structure,purity, and binding of BsAba5b1/av is provided in SupplementaryFig. S1.

Generation of conditioned mediahBM-MSC conditioned media (CM) was generated as described

previously (3). Briefly, confluent hBM-MSCs were cultured in serum-free media with CM harvested after 24 hours and gently centrifugedbefore being stored at 4�C.

Cell migration and adhesion assaysProstate cancer adhesion and migration assays were performed as

described previously (3). Briefly, migration of cancer cells to hBM-MSCCMin aBoyden chamberwas resolved after 24 hourswith calceinAM or crystal violet staining with average counts of five representativefields reported. For endothelial migration, 50,000 prostate cancer cellswere layered onto confluent hBM-MSC in the bottom chamber. After24 hours of coculture, migration of 10,000 HUVEC was assessed asabove. Adhesion of cancer cells to hBM-MSC CM-coated wells wasassessed after 1 hour (PC-3 and DU-145) or 6 hours (C4-2b) usingMTS Reagent (Promega) and was reported directly as optical densityor relative fluorescent units.

Cell clonogenic potential assayCancer cells (20,000) were treated and then plated in a 24-well plate

in full culturemedia. Forty-eight hours later, cells were trypsinized andreseeded in a series of limiting dilutions (1,000, 500, 250, and 100 cellsper well) in a 24-well format. After 14 days, cells were fixed and stainedwith crystal violet and colonies of greater than 50 cells were counted.Plating efficiencies are reported as a ratio to the number of inoculatedcells in respective wells. Only one limiting dilution was used forcalculations for each experiment.

Quantitative real-time reverse-transcriptase PCRRNA extractions, cDNA syntheses, and quantitative real-time

reverse-transcriptase PCR reactions were performed as describedpreviously (4). Primer sequences are provided in SupplementaryMaterials and Methods.

Flow cytometryCells were labeled with conjugated primary antibodies or with

unconjugated primary antibodies followed by incubation with fluo-rescently labeled secondary antibodies. All incubations were for 1 hourat room temperature. For intracellular detection, cells were stained inthe presence of 0.2% saponin. Median fluorescence intensity of stainedpopulations was detected using a CyAn ADP Analyzer (BeckmanCoulter).

Western blot analysisWestern blot analysis was conducted as described previously (4).

See Supplementary Materials and Methods for the list of antibodiesused.

ResultsCombinatorial integrin a5 and av blockade is superior toblockade of either integrin in inhibiting prostate cancer cellchemotaxis and induced endothelial migration

A strong chemotactic and adhesive response of prostate cancercells to hBM-MSC CM was identified previously (3). Furthermore,coculture of prostate cancer cells with hBM-MSCs strongly inducesthe migration of human endothelial cells. Using these in vitrotumor–niche interaction models, we tested the hypothesis thatcombinatorial blockade of integrins a5 and av would be superiorto single integrin blockade alone across a panel of genomicallydiverse, integrin a5/av coexpressing prostate cancer cells. Ashypothesized, combined a5 and av blockade with dual MAbs wassuperior to individual single agents in inhibiting prostate cancer cellmigration, adhesion, and induced endothelial migration in each ofthese cell lines (Fig. 1A).

Generation of a bispecific antibody to integrins a5b1 and avOn the basis of the hypothesis that a bispecific antibody targeting

these two integrins would be superior to a combination of MAbs, wedesigned and generated a bispecific antibody. The molecule is com-posed of an IgG that targets the av integrin (6) and a single-chainvariable fragment (scFv) that targets the a5b1 integrin (ref. 7; Sup-plementary Fig. S1A). By including a pan-av integrin–targetingsequence, the problem of diverse av heterodimers with overlappingfunctions was skirted whereas a5b1 is the obligate heterodimer of a5integrin. BsAba5b1/av purity was demonstrated by gel electropho-resis (Supplementary Fig. S1B). BsAba5b1/av binding to targetintegrin receptors was correlated with commercially available a5 andav antibodies across a panel of variably expressing integrin a5 and avcell lines (Supplementary Fig. S1C). High affinity, specific binding ofBsAba5b1/av to target integrins was demonstrated using the Biacoreplatform (Supplementary Fig. S2).

BsAba5b1/av is superior to monospecific combination ininhibiting prostate cancer cell chemotaxis and inducedendothelial migration

Wehypothesized that BsAba5b1/avwould be superior to cMAbs inassays of tumor–hBM-MSC interaction. Bsa5/avAb was superior tothe combination of sequence-matched monospecific IgG controls

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(MAba5b1 or MAbav) in inhibiting prostate cancer cell migration,adhesion, and induced endothelial migration (Fig. 1B). A scFv controlwas not considered to be an optimal control as its half-life in vivo isexpected to be low given its low molecular weight and susceptibility torapid renal clearance.

BsAba5b1/av treatment uniquely induces loss of integrinexpression and/or blocks integrin upregulation in prostatecancer cells

In assessment of the pharmacodynamic effects of the BsAba5b1/av, a marked loss of total cellular integrin av and a5 in PC-3 cells at48 hours following treatment was observed (Fig. 2A). This reductionin expression was not observed withMAbs or cMAbs. In DU-145, C4-2B, and VCAP cells, treatment with MAbs and/or cMAbs, variablyresulted in upregulation of either or both integrins. However, in eachcase this upregulation was either abrogated or markedly inhibited byBsAba5b1/av treatment (Fig. 2A). In contrast, preferential impact ofthe BsAba5b1/av on focal adhesion kinase, Akt, or Erk signaling wasinconsistent across cell lines (Supplementary Fig. S3). MMP-14, amaster regulator of matrix degradation, MMP-2 and TGF-beta acti-vation, was strongly downregulated with cMAbs and the BsAba5b1/av but not MAbs (Supplementary Fig. S4).

Loss of integrin expression induced by BsAba5b1/av treatmentis correlated with inhibition of prostate cancer chemotaxis andclonogenic survival

We hypothesized that the persistent reduction of integrin expres-sion observed with BsAba5b1/av treatment at the 48-hour timepointwould correlate with a differential functional impact on chemotaxisand clonogenic survival of prostate cancer cells compared with MAbs

or cMAbs. Accordingly, in PC-3 and DU-145 cells recovered 48 hoursafter antibody exposure, chemotaxis was most significantly impairedin cells treated with the BsAba5b1/av (Fig. 2B; SupplementaryFig. S5) compared with MAbs and cMAbs. Similarly, clonogenicsurvival of both PC-3 and DU-145 cells was maximally impaired byBsAba5b1/av compared with cMAbs (Fig. 2B). In AR-positive linesC4-2B,VCaP, and LNCaP,av-directed treatments universally resultedin a marked loss in replating and migratory capacity, prolongedproliferative arrest, and reduced numbers of filopodia-like projections(Fig. 2B and C).

Regulation of integrin expression dynamics by BsAba5b1/av ispost-translational and related to altered trafficking andlysosomal degradation of target integrins

Using PC-3 prostate cancer cells as a model line, we investigatedthe mechanism by which the BsAba5b1/av induced a markeddecrease in cellular integrin expression. We initially hypothesizedthat these dynamic changes might be explained by rapid endocy-tosis and degradation of antibody-bound receptor as seen previ-ously with other antibodies. However, BsAba5b1/av treatmentresulted in slow progressive loss of av membrane expressionobserved over 24–48 hours post-treatment (Fig. 3A). This loss ofmembrane expression correlated with diminished total cellularexpression of av (data not shown).

To assess the fate of these integrins more closely, we assessedwhether integrin synthesis or degradation were specifically impact-ed by BsAba5b1/av treatment. Quantitative reverse-transcriptionPCR studies showed no significant change in ITGA5 or ITGAVtranscript expression following either MAbs, cMAbs, or BsAba5b1/av treatment (Fig. 3B). Treatment with cycloheximide, an inhibitor

Figure 1.

Combinatorial integrin a5 and av blockade is superior to monospecific blockade and BsAba5b1/av is superior to combinatorial integrin blockade ininhibiting prostate cancer cell migration, adhesion, and induction of endothelial migration. PC-3, DU-145, or C4-2B cells were harvested and treated with50 mg/mL of MAbs or cMAbs (A) or 10 mg/mL of BsAba5b1/av or cMAbs (20mg/mL total; B) for 20 minutes on ice before use in functional assays. � , P < 0.05;�� , P < 0.01; ��� , P < 0.001; ���� , P < 0.0001.

Bispecific Antibody Targeting of av and a5b1 Integrins

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of protein translation, failed to abrogate the differential impact ofBsAba5b1/av on target expression (Fig. 3C), indicating the reg-ulation was post-translational. Because the major post-translationalregulator of integrin expression is transition from recycling path-ways to lysosomal/endosomal degradation (8), we hypothesized thatBsAba5b1/av induced preferential lysosomal degradation of boundtarget receptors. Treatment with chloroquine, a disruptor of lyso-somal integrity and function, blocked the downregulation of integ-rin expression observed with BsAba5b1/av therapy (Fig. 3D;Supplementary Fig. S6).

DiscussionOur studies have implicated fibronectin-binding a5 and av integ-

rins in the key interactions of prostate cancer cells with hBM-MSCs,the putative metastatic niche-regulating cell in the bone microenvi-ronment.We show that combined antibody blockade of these integrinsis superior to single-integrin blockade and that a bispecific antibodystrategy optimally abrogates chemotaxis, clonogenic tumor survival,and tumor-induced endothelial migration in prostate cancer cells. Adistinct mechanism of action for the BsAba5b1/av is demonstrable.Membrane depletion, blocked upregulation, and induced lysosomal

degradation of target integrins by the BsAba5b1/av contrasts stronglywith upregulated integrin expression following single or combinedmonospecific antibody therapy. The data presented in this articleprovide foundational data to advance in vivo and translational studiesof the novel BsAba5b1/av strategy.

From a historical perspective in targeting integrins in prostatecancer, the a5b1 mAb volociximab was abandoned in the absence ofdiscernible clinical efficacy in a limited set of solid tumor studies (9).Only a handful of patients with prostate cancer were included, and theefficacy data are insufficient to draw conclusions. Synthetic peptideapproaches to block the a5b1 integrin (10) did not advance in clinicaltrials and it is unclear if peptide-based competitive binding strategiesare capable of interdicting both ligand-dependent and ligand-independent integrin functions, both of which are implicated in tumorsurvival and dissemination (11). From other lines of investigation, theav integrin has been shown to be expressed in primary tumors,mediate bone homing in experimental models, mediate interactionwith endothelial cells and bone matrix components to drive tumorprogression, and endow prostate cancer cells with stem-repopulatingcapacity (12). Integrin av was targeted with the cyclic peptide cilengi-tide but without significant clinical impact (13). Contrasted to a5b1,the av integrin has multiple heterodimeric forms implicated in the

Figure 2.

Loss of integrin expression induced by BsAba5b1/av treatment is correlated with inhibition of prostate cancer chemotaxis and clonogenic survival. A, In PC-3 cells,BsAba5b1/av treatment results in loss of integrin a5 andav expression from baseline, compared with MAbs and cMAbs. In other lines, upregulation ofa5 (C4-2B) orbotha5 andav (DU-145 andVCAP) integrins is observed variablywithMsAbs and/or their combination, but in each case this upregulation ismitigated by BsAba5b1/av treatment. Prostate cancer cells were treated for 20 minutes on ice with MAbs (10 mg/mL), cMAbs (20 mg/mL total), or BsAba5b1/av (10 mg/mL) before beingplated for culture. After 48 hours, cell extracts were harvested for Western blot assessment of integrin expression. B, The migration and clonogenic survival ofAR-negative prostate cancer cells assessed 48 hours after treatment is more significantly impaired by BsAba5b1/av compared with cMAbs (PC-3 and DU-145). InAR-positive lines, for example, C4-2B, av-directed treatments universally resulted in a marked loss in migratory and clonogenic capacity. C, Photomicrograph ofLNCaP cells 48 hours posttreatment exemplifies markedly reduced numbers of filopodia-like projections with BsAba5b1/av. � , P < 0.05; �� , P < 0.01; ���, P < 0.001.

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progression of prostate cancer including avb1 and avb6 (14, 15). Therestricted avb3 and avb5 heterodimeric targeting by celingitide, itsshort half-life administered by intermittent intravenous infusion, andrapid adaptive resistance by membrane recycling of a5b1 (16) likelycontribute to treatment failure. A pan-av mAb, abituzumab, demon-strated intriguing biological activity by delaying progression of bonemetastases in men with metastatic castration–resistant disease. How-ever, overall survival differences were not demonstrable (17). Thesedata suggest that the av integrin is probably implicated in the biologyof prostate cancer progression in bone but that adaptive resistance tomonotherapy likely dictates the lack of survival advantage. Rapidmembrane recycling of a5b1 in response to av blockade with celingi-tide offers evidence of cross-regulation of these two integrins toaccount for drug resistance. The enriched expression of a5 and avintegrins in disseminated tumor cells recovered from bone marrowaspirates and in pathologic specimens of bone metastases in prostatecancer (18, 19) lends additional rationale to the importance andnecessity of targeting both integrins.

The potential advantages of a bispecific antibody targeting a5b1and av over a combination of MAbs include harmonized pharma-cokinetic and pharmacodynamic considerations within one mole-cule, lack of steric hindrance between two large molecules targetingproximate targets in the cell membrane, cross-linking of targetsleading to increased binding affinity and superior on-target time,and the induction of novel biology such as induced degradation oftarget proteins. More than one of these mechanisms of action likelyaccount for the markedly improved in vitro activity of theBsAba5b1/av over MAbs.

In addition, upregulated cellular integrin a5 and/or av expressionnoted with either MAbs or cMAbs may represent an autoregulatorycellular adaptive response that could generate resistance to therapy. InVCAP cells, which are a5 membrane-negative, cellular integrin a5expression is robustly upregulated by av blockade demonstrating thatupregulation of integrins emerges despite lack of detectable baselineexpression. We have not noted an increase in membrane-specificexpression of integrins that correlates with this cellular upregulation,suggesting that intracellular pooling of these integrins in endosomesfrom impaired recycling may occur primarily as a result of MAbtherapy. Persistent low-level recycling or intracellular signaling fromendosomal integrins (20) may account for resistance toMAb or cMAbtherapy. Such resistance pathways are probably mitigated by theBsAba5b1/av, which triggers lysosomal degradation, loss of intracel-lular integrins, and membrane recycling.

The precise mechanism by which BsAba5b1/av induces degrada-tion is unknown, but we speculate that the steric consequences ofBsAba5b1/av binding may destabilize the Rab family–regulatedmachinery required for integrin recycling (8). Exceptionally, com-bined MAbs may also trigger lysosomal degradation of integrins, asdemonstrated in C4-2B cells. The BsAba5b1/av effect on targetintegrin expression is slower to develop, that is, over 48 hours butalso long-lasting, reversing after 7 days. These remarkably slowpharmacodynamic changes after a single brief exposure of tumor cellsto the BsAba5b1/av have challenged the accurate tracking of integrinfates with traditional immunofluorescent tagging. When our data aretaken together, the BsAba5b1/avmay be seen to impose an early effecton chemotaxis, clonogenic survival, and induction of endothelial

Figure 3.

Regulation of integrin expression dynamics by BsAba5b1/av is post-translational and related to altered trafficking and lysosomal degradation of target integrins.A,In PC-3 cells, BsAba5b1/av treatment results in progressive loss (over 24 hoursþ) of membrane av expression. B, ITGA5 and ITGAV transcript expression is notsignificantly altered (fold change > 1.5 or <0.5) in PC-3 cells following MAb, cMAb, or BsAba5b1/av treatment. Blockade of protein translation with cycloheximidefailed to abrogate the differential impact of MsAbs or BsAbav/a5b1 on integrin expression in PC-3 cells (C). D, Lysosomal inhibition with chloroquine blocked thedownregulation of integrin expression with BsAba5b1/av therapy in PC-3 cells. C, 10 mg/mL cycloheximide; D, 50 mmol/L chloroquine.

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migration secondary to highly effective dual blockade of themembraneintegrins on tumor cells followed by a persistent late effect driven bydegradation and loss of the tumor integrins.

Integrin depletion offers a method for pharmacodynamicmonitoring of tumor cells in vivo. There is lack of consistency inthe impact of BsAba5b1/av on downstream signaling intermedi-ates which may reflect FAK-dependent and FAK-independentintegrin signaling (11) and the varied genetic backgrounds ofthese prostate cancer cells. Notwithstanding these signaling andpharmacodynamic variations, the functional consequences of theBsAba5b1/av have been consistent and justify further transla-tional study of this first-in-class strategy to target integrins inprostate cancer.

The bispecific antibody strategy may also prove relevant as amolecular therapeutics strategy to target other components of theenabling tumor microenvironment regulated by av and a5b1integrins including immunosuppressive cancer-associated fibro-blasts, bone remodeling cells, and blood vessels. Other neoplasticand nonneoplastic disease states including pathologic fibrosis andangiogenesis in which these two integrins have been implicated mayalso be susceptible.

Disclosure of Potential Conflicts of InterestP.Mathew is an inventor on a pending patent assigned to Tufts Medical Center on

the bispecific antibody strategy. No potential conflicts of interest were disclosed by theother authors.

Authors’ ContributionsConception and design: P. MathewDevelopment of methodology: R. Joshi, W. Ren, P. MathewAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): R. Joshi, W. Ren, P. MathewAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Joshi, W. Ren, P. MathewWriting, review, and/or revision of the manuscript: R. Joshi, W. Ren, P. MathewAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): R. JoshiStudy supervision: P. Mathew

AcknowledgmentsFunding support from theWill and Julie Person Prostate Cancer Research Fund is

gratefully acknowledged. Technical advice and assistance from Dr. Edi Goihberg andDr. Albert Tai with the Biacore system is appreciated.

Received April 25, 2019; revised September 3, 2019; accepted October 16, 2019;published first October 21, 2019.

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