the role of anti-drug antibodies in the pharmacokinetics,...

1521-0103/356/3/574–586$25.00 http://dx.doi.org/10.1124/jpet.115.229864THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 356:574–586, March 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

The Role of Anti-Drug Antibodies in the Pharmacokinetics,Disposition, Target Engagement, and Efficacy of a GITR AgonistMonoclonal Antibody in Mice

Nicholas D. Brunn, Smita Mauze, Danling Gu, Derek Wiswell, Roanna Ueda,Douglas Hodges, Amy M. Beebe, Shuli Zhang, and Enrique EscandónBiologics DMPK and Disposition (N.D.B., D.W., D.H., E.E.), Immuno-oncology (S.M., D.G., R.U., A.M.B.), and BioanalyticsDepartment (S.Z.), Merck Research Laboratories, Merck & Co. Inc., Palo Alto, California

Received October 7, 2015; accepted December 14, 2015

ABSTRACTAdministration of biologics to enhance T-cell function is part of arapidly growing field of cancer immunotherapy demonstrated bythe unprecedented clinical success of several immunoregulatoryreceptor targeting antibodies. While these biologic agents confersignificant anti-tumor activity through targeted immune re-sponsemodulation, they can also elicit broad immune responsespotentially including the production of anti-drug antibodies(ADAs). DTA-1, an agonist monoclonal antibody against GITR,is a highly effective anti-tumor treatment in preclinical models.We demonstrate that repeated dosing with murinized DTA-1(mDTA-1) generates ADAs with corresponding reductions in drugexposure and engagement of GITR on circulating CD31 CD41

T cells, due to rapid hepatic drug uptake and catabolism. Miceimplanted with tumors after induction of preexisting mDTA-1ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1,

an efficacious dose in naive mice. Nonetheless, increasingmDTA-1 treatment to 30 mg/kg in ADA-positive mice restoresmDTA-1 exposure and GITR engagement on circulating CD31

CD41 T cells, thereby partially restoring anti-tumor efficacy.Formation of anti-mDTA-1 antibodies and changes in drugexposure and disposition does not occur in GITR2/2 mice,consistent with a role for GITR agonism in humoral immunity.Finally, the administration of muDX400, a murinized monoclonalantibody against the checkpoint inhibitor PD-1, dosed alone orcombined with mDTA-1 did not result in reduced muDX400exposure, nor did it change the nature of the anti-mDTA-1response. This indicates that anti-GITR immunogenicity may notnecessarily impact the pharmacology of coadministered mono-clonal antibodies, supporting combination immunomodulatorystrategies.

IntroductionDue to their long half-life, high specificity, and excellent

therapeutic window, antibodies and antibody-based biologicsconstitute one of the more successful classes of recently de-veloped products in the biotechnology industry (Beck et al.,2010; Reichert 2011). Significant focus has recently been di-rected toward immune systemmodulating biologics, withmono-clonal antibodies directed against immune checkpoint receptorsincludingPD-1 andCTLA-4 showing favorable pharmacokinetic(PK) and significant survival benefit in mouse models andclinical studies (Melero et al., 2007; Peggs et al., 2009; Hodiet al., 2010; Robert et al., 2011; Topalian et al., 2012; Hamidet al. 2013; Wolchok et al., 2013; Page et al., 2014).In addition to targeting immune checkpoint inhibitors,

biologics targeting members of the tumor necrosis factorreceptor superfamily are emerging as an additional avenue

of cancer immunotherapy (Sugamura et al., 2004; Murataet al. 2006; Croft et al., 2013). One of these targets is the GITR,a T-cell costimulatory receptor (Ronchetti et al., 2004; Schaeret al., 2012). It has been shown that GITR can regulateimmunity by potentiating CD41 and CD81 effector T-cellfunctionwhilemodulating regulatory T cells (Tone et al., 2003;Kanamaru et al., 2004; Kohm et al., 2004; Nocentini andRiccardi, 2005; Nishikawa et al., 2008). In tumor-bearingmice, treatment with the GITR agonist monoclonal antibodymurinized DTA-1 (mDTA-1) has been shown to increaseintratumoral effector T-cell function and decrease regulatoryT-cell stability, culminating in effective tumor regression(Cohen et al., 2006, 2010; Zhou et al., 2007; Coe et al., 2010).While modulation of costimulatory pathways have shown

great preclinical potential as immunotherapies, immune-related adverse events ranging from hyper-costimulation tocytokine dysregulation and immunogenicity have been observed(Chirino et al., 2004; Reuben et al., 2006; Suntharalingamet al., 2006; Brennan et al., 2010; Clarke 2010; Murphy et al.,2014). Ultimately, the administration of any biologic has thepotential to generate anti-drug antibodies (ADAs) (Pepinskyet al., 2001; Shankar et al., 2006, 2007; Badylak and Gilbert

This work was supported by Merck Research Laboratories (MRL), Merck &Co. Inc. The MRL Postdoctoral Research Program provided financial supportto N.D.B.

dx.doi.org/10.1124/jpet.115.229864.

ABBREVIATIONS: ADA, anti-drug antibody; FACS, fluorescence-activated cell sorting; HMW, high molecular weight; HPLC, high-performanceliquid chromatography; IC, immune complex; KC, Kupffer cell; mDTA-1, murinized DTA-1; MFI, median fluorescence intensity; MSD, mesoscale discovery;PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PK, pharmacokinetic; Q7D, every 7 days; SEC, size exclusion chromatography.

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2008). Although the degree of immunogenicity against aparticular therapeutic is typically based on inherent proper-ties of the molecule being administered (Chirino et al., 2004;De Groot and Moise 2007; De Groot and Martin 2009;Badylak and Gilbert 2008), other factors such a dose, doseregimen, route of administration, and disease status of thehost could also play a significant role. The presence of ADAscan dramatically alter the PK and pharmacodynamic proper-ties of administered biologics due to immune-complex formationchanges in disposition and in the case of neutralizing antibodiesby modulating biologic activity. Furthermore, due to theirunique mechanism of action, biologics targeting immunoreg-ulatory receptors may enhance not only specific anti-tumorimmune responses, but also lead to nonspecific immunologicactivation and the possible emergence of immune disorders(Kohm et al., 2004; Brennan et al., 2010). Therefore, acomplete characterization of the biologic responses to potentimmunomodulatory drugs following extended exposure iscritical to better predicting their therapeutic window. Here,we describe for the first time the dramatic GITR-mediatedPK and pharmacological changes that follow repeatedtreatment with a murinized IgG2a version of the anti-GITR agonist monoclonal antibody DTA-1 (mDTA-1) whendosed alone or in combination with the PD-1 antagonistantibody muDX400.

Materials and MethodsAll mouse procedures were performed in accordance with the Institu-

tional Animal Care and Use Committee at Merck, and housed in a facilityapproved by theAmericanAssociation forAssessment andAccreditation ofLaboratory Animal Care, and maintained under a protocol in accordancewith the guidelines of the American Association for Assessment andAccreditation of Laboratory Animal Care (http://www.aaalac.org/).

Mice, Tumor Cell Lines, and Anti-Tumor Efficacy Experi-ments. Unless otherwise stated, all dosing was conducted as a s.c.bolus dose given in the upper scruff on the back of the neck. In tumorgrowth experiments, care was given to dose as far away from thetumor as possible. C57BL/6J mice were obtained from the JacksonLaboratory (Bar Harbor, ME), and BALB/c mice were obtained fromTaconic (Hudson, NY). Mice were approximately 8–14 weeks of agewith body weights ranging from 20 to 25 g. In the anti-tumor efficacyexperiments, BALB/c mice were inoculated s.c. with 3.0 � 105 CT26murine colon carcinoma cells (ATCC, Manassas, VA) and C57BL/6Jmicewere inoculated s.c. with 1.0� 106MC38murine colon carcinomacells (National Cancer Institute, Bethesda,MD). Cells were injected in100 ml volume. For anti-tumor efficacy experiments, upon achieving atumor burden of approximately 100 mm3, as gauged by electroniccalipers, mice were given a single s.c. injection of 3 or 30 mg/kg mDTA-1or IgG2a isotype control. Additional cohorts of naive andADA-positivemice were given three separate s.c. doses of 3 mg/kg mDTA-1 everyseven days (Q7D). Tumor growth was monitored twice per week forapproximately 3 weeks, at which point the animals were euthanized.Tumor length and width were measured using the electronic caliperand tumor volume determined using the following formula:

Volume�mm3�50:5� length

�width2; where length is the longer dimension

Monoclonal Antibodies. mDTA-1 IgG2a (anti-GITR), muDX400IgG1 D265A (anti-PD-1), and mouse anti-VP2 IgG2a (isotype control)were produced, purified, and characterized by the Protein SciencesGroup at Merck & Co. Inc. Fluorescein isothiocyanate anti-mouseCD3, eFluor 450 anti-mouse CD4, and Alexa Fluor-488/Anti-F4/80were purchased from BD Biosciences (San Jose, CA).

Fluorescent Labeling. DyLight 650N-hydroxysuccinimide esterlabeling kits (Life Technologies, Carlsbad, CA) were used to conjugatean N-hydroxysuccinimide ester fluorophore (excitation/emission of646/674) to label the experimental proteins of interest. Prior tolabeling, antibodies were dialyzed into 50 mM sodium borate (LifeTechnologies) utilizing a 10-kDa mol. wt. cutoff Slide-A-Lizer dialysiscassette (Life Technologies). Each reaction contained protein at a finalconcentration of approximately 2 mg/ml. The conjugation was initi-ated by combining the protein with the dimethylsulfoxide-dissolveddye and incubating for 1 hour at room temperature. UnreactedDyLight 650 was removed using a Thermo Fisher Scientific DyeRemoval Column (Thermo Fisher Scientific, Waltham, MA) accordingto the manufacturer’s protocols. If needed, protein samples wereconcentratedwith a 30-kDamol. wt. cutoff Amicon ultracentrifugationfilter unit (EMD Millipore Corp., Billerica, MA) and sterile filteredusing a 0.22-mm Millex syringe filter (EMD Millipore). Labeledantibodies were characterized using a NanoDrop UV-visible spectro-photometer (Thermo Fisher Scientific) to determine the proteinconcentration and degree of labeling as dye/protein (mol/mol) ratio.The integrity of the labeled antibodies was additionally assessed bysize exclusion chromatography (SEC) and high-performance liquidchromatography (HPLC), and by extended incubation in phosphate-buffered saline (PBS) and wild-type BALB/c mouse plasma (data notshown). All reagents were analyzed for purity and activity by severalmethods prior to administration (data not shown). All DyLight650–labeled antibodies had an average degree of labeling of 2.1 dyemolecules per antibody.

Pretreatment of BALB/c Mice. For generating ADA-positivemice, either two or three doses of indicated antibody were adminis-tered with 7 or 14 day intervals. Previous experiments have shown nomeasurable differences between ADA titers of animals pretreatedwith 2, 3, or 4 doses of mDTA-1. All antibodies were unlabeled andadministered by s.c. injection in either 100 or 200 ml of sterile PBS orappropriate buffer. In PK experiments, mice were dosed twice, 7 daysapart, with either 3 mg/kg single agent or 1.5 mg/kg of each agent incombination in 100 ml PBS. For tumor model experiments, mice weredosed three times, 14 days apart, with 3mg/kgmDTA-1. In all cohorts,there was a 10–20 day washout period prior to experimentation toallow for the removal of unlabeledmDTA-1 ormuDX400 (confirmed byenzyme-linked immunosorbent assay).

Blood and Tissue Collection. Animals were euthanized withcarbon dioxide followed by terminal cardiac puncture. Blood wascollected into EDTA-K2 CapiJect microcollection tubes (TerumoMedical Corporation, Somerset, NJ) and placed on ice. Plasma wasseparated from whole blood via centrifugation for 6 minutes at 6000gat 4°C, and stored at 280°C. Likewise, whole blood samples werealiquoted into polypropylene vials (Corning Glassworks, Corning, NY)and stored at280°C. For tissue lysates, organ samples were collectedand immediately placed into 2-ml Precellys Lysing Tubes (BertinTechnologies, Rockville, MD) and placed into dry ice. Upon partialthawing, a 1:10 dilution of Dulbecco’s PBS containing 1% Triton X-100(MP Biomedicals, Solon, OH) and 1� Halt Protease Inhibitor single-use cocktail (Thermo Fisher Scientific) was added to the samples.Then, tissue slurries were produced using a Precellys EvolutionHomogenizer. The slurry was centrifuged (10,000g at 4°C for10 minutes) and the tissue lysate supernatants were collected andeither processed immediately or stored at 280°C until analysis.

SEC-HPLC Analysis. Samples were applied onto a BioSep-SEC-S3000 column with an inline SecurityGuard filter (Phenomenex,Torrance, CA) and analyzed using an Agilent 1200 HPLC system(Agilent Technologies, Santa Clara, CA), utilizing an integrated UVand fluorescence detector (Hamamatsu Corperation, Bridgewater,NJ). The size exclusion protocol used a 15-minute isocratic run withDulbecco’s PBS buffer (HyClone, GE Life Sciences, Little Chalfont,United Kingdom) as the mobile phase, and was run at a constant flowrate of 1 ml/min21 and kept at room temperature. The effluent wasmonitored continuously by optic observation at 280 nm and by the netfluorescence intensity at an excitation wavelength of 646 nm and

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emission of 674 nm. For mDTA-1 incubation experiments, plasmafrom naive and mDTA-1–pretreated mice (after a 15-day washoutperiod) was collected as previously described. Defined quantities ofmDTA-1 or an IgG2a isotype control (50, 100, or 250mg/ml) was spikedinto 20 ml of neat plasma, incubated for 15 minutes at 37°C, andapplied to the BioSep-SEC-S 3000 column. The analysis and datacollection were performed using Agilent Technologies’ ChemStationsoftware as well as EXCEL (Microsoft Corporation, Seattle, WA) andGraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA). Thecolumn performance was assessed prior to and following each batch ofsamples by running 20 ml of a standard set of molecular weightmarkers (Bio-Rad Laboratories, Hercules, CA).

Fluorescence Emission–Linked Assay. Net fluorescence in-tensity was quantified utilizing a Promega Glowmax plate reader(Promega, Madison, WI) equipped with a fluorescence optical filterwith excitation/emission wavelengths of 625/660–720. Concentrationsof fluorophore-labeled protein were calculated by generating individ-ual calibration curves in naive BALB/c liver tissue, whole blood, andplasma for each DyLight 650–labeled test article. These calibrationcurves were analyzed using low fluorescence–background black 96-well polystyrene assay plates (Corning Glassworks), and blank tissuelysates, blood, and plasma were used for background corrections.A linear correlation function was fitted to the data using best-fitparameters (EXCEL). Concentrations of DyLight-labeled test articleswere calculated as microgram equivalents per gram of wet tissue.Subsequent tissue/blood ratios were calculated by comparing thequantified concentrations of the test articles in the respective tissuetypes versus the concentration in blood at the indicated time point.Ratios were calculated using EXCEL.

Capillary Fluorescent Electrophoresis Analysis. Tissue ly-sates were analyzed using a Caliper Lifesciences LabChip GX II

microfluidic capillary electrophoresis apparatus (Perkin Elmer,Hopkinton, MA) according to the manufacturer’s protocol. Briefly, thetissue lysates (previously diluted 1:10 in tissue lysate buffer) werediluted an additional 2- to 10-fold in the caliper sample buffer,denatured at 75°C for 5 minutes, centrifuged at 2000g for 2 minutes,and then run. Electropherograms were generated by LabChip GXIITouch software (Perkin Elmer).

Tissue Distribution and PK Analysis. For PK analysis andbiodistribution of mDTA-1 and muDX400 antibodies, mice wereseparately given a single s.c. bolus dose of either 5 or 30 mg/kgDyLight 650–labeled mDTA-1 or muDX400. The animals weresacrificed at the previously indicated time points of 2, 4, 6, 8, 24,and 96 hours (5 mg/kg) or 4, 8, 16, 48, and 96 hours (30 mg/kg). Theconcentrations of test articles in serum, whole blood, and selectedtissues were quantified by a fluorescence emission–linked assay,with lower detection limits of approximately 5 ng/ml for DyLight650–labeled experimental proteins. The PK parameters were calcu-lated in WinNonlin (Pharsight, Mountain View, CA) using non-compartmental methods on the concentration-time data quantified inthe fluorescence emission–linked assay. The concentration-time datafor all antibodies were plotted using GraphPad Prism 6 (GraphPadSoftware, Inc.), and are presented with their corresponding S.D.values.

Fluorescent Analysis of Immune Complex (IC) Uptake inMouse Kupffer Cells (KCs). Naive and mDTA-1–pretreated wild-type BALB/c mice (previously inoculated with 3 mg/kg mDTA-1 Q7D� 2)were injected s.c. with 30 mg/kg DyLight-labeled mDTA-1 and KCswere enriched from liver-associated cells as previously described(Zeng et al., 2013; Li et al., 2014). Briefly, 24 hours postdose micewere euthanized and underwent cardiac perfusion with 3 ml �min21

PBS for 3 minutes. The livers were removed, minced, and collected in

Fig. 1. The concentration time profiles of mDTA-1 in naive BALB/c mice (A) and littermates previously dosed 3 mg/kg mDTA-1 Q7D � 2 (B). Bothcohorts were injected s.c. with 5 or 30 mg/kg of DyLight-labeled mDTA-1 and the concentrations were determined by fluorescence emission–linkedassays. Data are representative of two independent experiments (n = 3 mice per group). Error bars correspond to the S.D. calculated using GraphPadPrism 6.

TABLE 1PK parameters determined by noncompartmental analysis (WinNonlin)

Pretreatment Regimen Dose t1/2 Cmax AUClast CL Vss

mg/kg day mg/ml hr·mg/ml ml/day/kg ml/kgNone 5 3.7 51.1 3536.7 16.7 87.9None 30 4.0 277.8 18510.8 17.7 101.13 mg/kg mDTA-1 Q7D � 2 5 0.4 2.7 147.0 786.7 906.83 mg/kg mDTA-1 Q7D � 2 30 0.8 75.5 2614.3 273.8 188.1

AUClast, last area under the curve; CL, clearance.

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30 ml RPMI media 1 10% fetal bovine serum 1 0.1% collagenase IV,and subsequently incubated at 37°C for 30 minutes. The KCs wereenriched from hepatocytes, nonparenchymal, and liver sinusoidalendothelial cells by gradient centrifugation. Heavy sediments wereremoved by centrifugation at 20g for 2 minutes and the pellet wasdiscarded. Larger cells were removed by centrifugation at 50g for3 minutes and the pellet was discarded. Cells were clarified by twoalternating rounds of centrifugation of 50g for 3 minutes (discarding thepellet) and 300g for 3 minutes (discarding the supernatant), and alwaysresuspended in 5 ml of RPMI media. On the final spin, cells wereresuspended with 500 ml RPMI media and incubated with 5 mg/mlAlexa Fluor 488/anti-F4/F80 antibody for 30 minutes at 4°C. Cells

were then plated directly atopmicroscopy slides and allowed to adherefor 2 hours. Nonadherent cells were removed by three washes withPBS. Cells were then visualized with an EVOS FL Cell ImagingSystem (Life Technologies) containing both the green fluorescentprotein and Cy5 light cubes. Images were acquired and overlaidutilizing the onboard EVOS FL software.

ADA Detection and Free-Drug Detection Assays. Mice werepretreated with the indicated antibody (3 mg/kg Q7D� 3) and plasmasampleswere collected as described previously. Sixmicewere used pertime point. Plasma samples were then run on a mesoscale discovery(MSD) bridging immunogenicity assay. MSD plates were coated with150 ml of Blocker A solution [5 g bovine serum albumin (5%) in 100 mlDulbecco’s PBS] per well and stored at 4°C overnight (or up to 4 days).Amixture of 1mg/ml biotin-DTA-1 and1mg/ml sulfo-DTA-1was preparedin assay diluent buffer (0.5% bovine serum albumin, 0.05% Tween 20,0.25% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate,5 mMEDTA, 0.35 MNaCl, in PBS, pH7.4); 100 ml of the DTA-1 mix fromthe previous step was added to each well of the reaction plate (CorningGlassworks, nonbinding surface); 35 ml of the assay diluent buffer wasadded to each well; and 15 ml of neat sample plasma (or pooled BALB/cmouse serum forbackgroundwells)was added to eachwell andmixedandincubated on a plate shaker at 300 rpm for 2 hours at room temperature.Samples were washed, while MSD plates were blocked 3� with washingbuffer (PBSwith 0.5%Tween 20). Then, 50ml of the samplemixture fromtheprevious stepwasadded toduplicatewells on theMSDreadplate. Theplate was incubated on the shaker at 300 rpm for 1 hour at roomtemperature. Plateswerewashed 3�withwashing buffer. Then, 150ml ofMSD 1� Read Buffer T was added to each well and read on the MSDinstrument (SI-6000 Meso Scale Discovery LLC, Gaithersburg,MD). To determine the concentration of free mDTA-1 or muDX400in circulation, a drug-capture enzyme-linked immunosorbent assayutilizing a recombinant mouse GITR or PD-1 protein as the captureantigen was used, followed by a horseradish peroxidase–conjugatedgoat anti-mouse IgG as the detection antibody, adhering to themanufacturer’s protocols.

Flow Cytometry and Fluorescence-Assisted Cell Sorting.Mouse peripheral blood mononuclear cells (PBMCs) were isolatedfrom naive or mDTA-1–pretreated (ADA-positive) mice 4 hours after abolus dose of either 3 or 30 mg/kg labeled mDTA-1 or DyLight 650/mIgG2a isotype control. Briefly, whole blood was collected by terminal

Fig. 2. Plasma samples from naive (A) and mDTA-1–pretreated (B) BALB/c mice s.c. dosed with 30 mg/kg DyLight-labeled mDTA-1. Specimens werecollected at 4, 8, 16, 24, 48, and 96 hour time points and characterized by SEC-HPLC analysis as described inMaterials andMethods. The retention timeof 8.1 minutes corresponds to a theoretical molecular weight of ∼155,000 kDa on the Phenomenex 3000, indicating that the major peaks correspond tointact monomeric mDTA-1 monoclonal antibody. The scale of the x-axis has been expanded to highlight the HMW complexes present in (B), indicated bythe asterisks. Data are representative of two independent experiments (n = 3 mice per group).

Fig. 3. HMW complex formation in plasma samples from mDTA-1–pretreated mice spiked in vitro with the indicated concentrations ofDyLight 650/mDTA-1. Naive plasma and an IgG2a isotype antibody wereused as controls. Aliquots were incubated for 15 minutes at 37°C prior toSEC-HPLC analysis. The retention time of 8.1 minutes corresponds to atheoretical molecular weight of ∼155,000 kDa. The HMW complexes areindicated by the asterisks. Data are representative of two independentexperiments (n = 3 mice per group).


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cardiac puncture, placed into EDTA-treated collection tubes, andplaced on ice. Ammonium-chloride-potassium red blood cell lysisbuffer was added to whole blood at a 20-fold excess to lyse the RBCsaccording to the ammonium-chloride-potassium lysing buffer kit (LifeTechnologies) protocol. Samples were then centrifuged at 300g for5 minutes at 4°C. The supernatant was aspirated and the isolatedPBMCs were washed three times with ice-cold fluorescence-activatedcell sorting (FACS) buffer (Sigma-Aldrich, St. Louis, MO).

Cells from mice treated with DyLight 650–labeled mDTA-1 orisotype control were divided into two groups; one group was incubatedwith anti-CD3 and anti-CD4 (experimental) and the other group wasincubated with anti-CD3, anti-CD4, and 5 mg/ml DyLight 650/DTA-1[maximum median fluorescence intensity (MFI) control]. Cells werethen washed three times in ice-cold FACS buffer, followed by FACSand analysis.

To establish the relative maximum andminimumMFIs of mDTA-1GITR engagement, negative controls received no ex vivo DTA-1staining (negative control) and (maximum) GITR engagement wasaccomplished through ex vivo incubation with 5 mg/ml DyLight 650/DTA-1 for 30 minutes. For labeled mDTA-1 or IgG2a isotype control,the percent GITR binding was calculated using the following formula:

%Bound5 ðexperimental MFI2negative control MFIÞ4ðmaximum MFI2negative control MFIÞ � 100

ResultsDyLight 650–Labeled mDTA-1 Antibody Plasma Con-

centration Decreases Rapidly over Time in BALB/cMice Previously Treated with mDTA-1. Figure 1 showsthe dramatic changes in the concentration time profiles ofcirculating mDTA-1 antibodies when administered to micepreviously exposed to the drug relative to naive control mice.By 96 hours following s.c. administration the plasma concen-trations in naive mice were 28 and 161 mg/ml (5 and 30 mg/kgdoses) versus 0.1 and 1.5 mg/ml, respectively in mDTA-1–pretreated mice. The PK parameters calculated from theconcentration time profiles of both cohorts (Table 1) indicatedthat previous treatment with mDTA-1 resulted in a nonlinearreduction in exposure [area under curve (AUC)]; 24-fold (5mg/kg)

Fig. 4. Tissue distribution of DyLight 650–labeled mDTA-1 in naive (A, B) and mDTA-1–pretreated (C and D) BALB/c mice s.c. dosed with 30 mg/kgmDTA-1. Drug concentrations were determined by fluorescence emission–linked assay as described inMaterial andMethods. Tissue/blood ratios greaterthan 1 are indicative of tissue uptake (A and C), and mDTA-1 concentration (microgram/milliliter) was measured at 4, 8, 16, 24, 48, and 96 hours afterdosing (B and D). Data are representative of two independent experiments (n = 3 mice per group) and error bars correspond to the S.D. calculated usingGraphPad Prism 6.

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and 7-fold (30mg/kg). Corresponding changes in clearance andhalf-life were consistent with these reductions in the AUC inpretreatedmice, and the PK parameters in naivemice assumethe elimination phase kinetics remain constant after the96-hour collection point.DTA-1 Forms High Molecular Weight (HMW) Com-

plexes in Plasma from mDTA-1–Pretreated Mice. Togain better understanding of the increased mDTA-1 elimina-tion in pretreated versus naive mice, mDTA-1 stability andpotential protein interactions were characterized by fluores-cent SEC-HPLC analysis. In the plasma of naive mice,DyLight 650/mDTA-1 elutes as a single monomeric peak at alltime points (Fig. 2A), consistent with intact IgG. No HMWcomplexes, aggregates, or catabolic products were observed,whereas in pretreated BALB/c mice we observed the presenceof HMW complexes with retention times of approximately 6and 7 minutes corresponding to approximate molecularweights of 1 MDa and 350 kDa, respectively (Fig. 2B).Consistent with the fast elimination of mDTA-1 in pretreatedmice (Fig. 1B), the DyLight 650–labeled mDTA-1 chromato-graphic signal was essentially depleted by 48 hours. Theformation of HMW complexes in vivo is indicative of potentialdrug/anti-drug ICs.

Furthermore, a major shift was observed in the sizeexclusion profile of labeled mDTA-1 when incubated in vitroin plasma from mice previously exposed to mDTA-1 changingfrom monomeric IgG to mostly HMW forms (Fig. 3). This is inagreement with the presence of high ADA levels and the rapidformation of drug/anti-drug ICs. As expected, no changes inthe chromatographic profiles were observed in naive mouseplasma spiked with labeled mDTA-1 or plasma from pre-treated mice spiked with an isotype control DyLight650–labeled antibody. The relatively low level of mDTA-1HMW complexes in circulation versus in spiked pretreatedplasma (Fig. 2B versus Fig. 3) is consistent with a mechanismfor rapid hepatic elimination of ICs (Løvdal et al., 2000;Ganesan et al., 2012).Hepatic Uptake of DyLight 650–Labeled DTA-1 in

Pretreated BALB/c Mice. Total fluorescence profiles (mi-crogram equivalents per milliliter of tissue lysate) of labeledmDTA-1 after s.c. injection of 30 mg/kg in naive and pre-treated BALB/c mice were measured over time in severalorgans and whole blood. A tissue/blood ratio greater than 1 isindicative of tissue uptake and accumulation. In naiveanimals tissue/blood ratios remained below 0.5 (Fig. 4A), withno evidence of drug accumulation in any organ analyzed (Fig.4B). In contrast, mice previously exposed to mDTA-1 experi-enced intense mDTA-1 organ uptake after dosing. Tissue/blood ratios in the liver, and to a lesser degree in the kidneys,reached their highest values (∼150 and 20, respectively) at96 hours for the liver and 48 hours for the kidney (Fig. 4C). Thetotal levels of DyLight 650/DTA-1 (mg/g tissue equivalents)were 10-foldhigher by24hourspostdose in the liver of pretreatedmice relative to naive animals (150 versus 17.5 mg/g) (Fig. 4, Band D). Overall, these results demonstrate that pretreatmentwith mDTA-1 dramatically changes the PK and dispositionof fluorescent mDTA-1 resulting in dramatic liver uptake,atypical of normal IgGs.

Fig. 5. Plasma and liver lysates of naive (A) and pretreated (B) BALB/cmice s.c. dosed with 30 mg/kg labeled mDTA-1 were collected at 4, 8, 16,24, 48, and 96 hours and characterized by capillary fluorescent elec-trophoresis analysis. Arrows indicate the presence of antibody-derivedcatabolites.

Fig. 6. Fluorescent microscopy of KCs from naive and mDTA-1–pretreated BALB/c mice s.c. dosed with 30 mg/kg DyLight 650–labeled mDTA-1. KCs were isolated by gradient centrifugation asdescribed in Materials and Methods. DyLight 488–labeled anti-F4/F80monoclonal antibody (green) was used for KC identification. Thefluorescent and transmitted light signals were overlaid using theEVOS FL Cell Imaging System (Life Technologies). The white barscorrespond to 50 mM. All images are representative of typical cellfluorescence and morphology. The experiment was replicated threetimes with identical results.


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Hepatic Catabolism of DyLight 650/mDTA-1. To bettercharacterize the dramatic changes in antibody exposure andliver uptake in pretreated mice, tissue lysates of naive andpretreated BALB/c mice dosed with 30 mg/kg fluorescentmDTA-1 were analyzed by capillary fluorescent electrophore-sis analysis to determine drug integrity (Fig. 5). Themajor IgGband at approximately 150 kDa corresponds to intact mDTA-1antibody and it is the main component in plasma from naivemice (Fig. 5A). Only trace amounts of degradation productswere found in any organ analyzed, including the liver (Fig. 5A).Conversely, consistent with the increased clearance, only

low levels of intact mDTA-1 were present in plasma fromBALB/cmice previously treated withmDTA-1 (Fig. 5B). In theliver of these mice the majority of the experimental antibodywas catabolized as indicated by the abundance of smallmolecular weight degradation products denoted by the ar-rows. These results support the presence of circulating ADAsin pretreated mice that upon retreatment form drug/anti-drugICs. Then, the ICs likely undergo rapid fragment-crystallizablegamma receptor–mediated hepatic clearance and degradationas previously shown by others (Bogers et al., 1991; Løvdal

et al., 2000; Ganesan et al., 2012). The efficient removal ofpotential ICs is in agreement with the low levels of circulatingDTA-1 containing HMW complexes in pretreated mice asshown in Fig. 2B.KC Uptake of DyLight 650/mDTA-1. KCs are one of the

major cell types involved in removal of ICs (Løvdal et al.,2000). To investigate their role in the increased hepaticcatabolism of mDTA-1 in pretreated mice, KCs were isolatedfrom livers of naive and mDTA-1–pretreated animals andanalyzed 24 hours after DyLight 650/mDTA-1 s.c. adminis-tration (Fig. 6). Intense DyLight 650/mDTA-1 staining wasprevalent in KCs from pretreated mice (Fig. 6, red area). NomDTA-1 fluorescence signal was observed in KCs from naivemice, suggesting that the increased elimination of mDTA-1 inpretreatedmice was due in part to KC uptake of anti-mDTA-1/mDTA-1 ICs. Furthermore, ex vivo staining with an AlexaFluor-488–conjugated anti-F4/80 antibody, used to identifyKCs (Fig. 6, left panels), confirmed that all KCs were alsopositive for DyLight 650/mDTA-1 signal.DTA-1 Treatment Elicits a Target-Mediated ADA

Response. To assess the relative levels of ADAs and thepotential role of the target (GITR) in anti-mDTA-1 immuno-genicity and altered clearance, plasma samples from naive ormDTA-1–pretreated wild-type and GITR2/2 C57BL/6 micewere collected at 3, 30, 60, and 90 days following a s.c. mDTA-1dose of 3mg/kg mDTA-1.Treatment with mDTA-1 in wild-type mice led to a robust

ADA response by day 30 following the most recent mDTA-1administration and then gradually declined but remainedpositive through at least day 120. The ADA signal was over105-fold higher than background levels observed in GITR2/2

knockout mice (Fig. 7A). Likewise, there was no detectablemDTA-1 in plasma from wild-type mice (lower limit of quan-tification of 0.09 mg/ml) after 3 days postdosing, whereas atdays 3, 30, 60, and 90 the GITR2/2 mice had average mDTA-1concentrations of 31, 13, 3, and 0.6 mg/ml, respectively (Fig.7B). These results clearly demonstrate that the agonisticantibodymDTA-1 critically requires the presence of the target(GITR) to induce a specific anti-drug response.Restoration of DTA-1 Anti-Tumor Efficacy in ADA-

Positive Mice. The impact of ADAs on mDTA-1 anti-tumorefficacy was evaluated in naive and ADA-positive BALB/c(Fig. 8, A and B) and C57BL/6J mice (Fig. 8, C and D)implanted with CT26 and MC38 syngeneic tumors, respec-tively. Mice with established tumors (average 100 mm3) weredosed s.c. with single or multiple doses of 3 mg/kg mDTA-1at day 0 or at days 0, 7, and 14, respectively, or with a single30 mg/kg dose. The average tumor volume was recorded every3 to 4 days (Fig. 8, A and C) and the final tumor volumes wereindividually plotted and statistically analyzed (Fig. 8, B andD). In naive BALB/c and C57BL/6Jmice, a 3mg/kg single dosewas sufficient to induce tumor regression in both theCT26 andMC38 tumor models. Efficacy was comparable between co-horts treated with a single dose of 3 or 30 mg/kg mDTA-1 orthree weekly doses of 3 mg/kg. Conversely, in ADA-positivemice, neither a single nor multiple doses of 3 mg/kg mDTA-1showed any anti-tumor activity. However, a single 30 mg/kgdose of mDTA-1 did slow tumor progression in mice with pre-existing ADA, particularly in the MC38 model. The sameresponse, although to a lesser degree, was observed in the CT26model. The presence of ADAs in these mice was confirmed bythe MSD assay and SEC-HPLC (data not shown).

Fig. 7. Wild-type and GITR2/2 BALB/c mice pretreated with mDTA-1were given a s.c. dose of 3 mg/kg unlabeled mDTA-1. Plasma samples weretaken at 3, 30, 60, 90, and 120 days and examined for ADAs using a MSDbridging immunogenicity assay (A), and for mDTA-1 concentrations bydirect enzyme-linked immunosorbent assay (B). Experiments were carriedout in duplicate, n = 6 mice per time point, and error bars correspond to6S.D. calculated using GraphPad Prism 6.

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Efficient mDTA-1 Target Engagement in ADA-PositiveMice Can Be Achieved with a 10-Fold Increase inDose. The presence of ADAs against mDTA-1 dramaticallydecreased tumoricidal activity. This was likely due in part tothe enhanced elimination of the drug upon IC formation. To

determine if the ADAswould also neutralizemDTA-1 binding,we evaluated the effects of ADAs on mDTA-1 GITR engage-ment 4 hours after dosing using flow cytometry and FACS.A s.c. dose of 3 mg/kg of DyLight 650–labeled mDTA-1

engaged approximately 95% of circulating CD31 CD41 GITR

Fig. 8. Dose escalation of mDTA-1 in ADA-positive mice restores anti-tumor efficacy. C57BL/6J and BALB/cAnNmice split into groups that were eithers.c. pretreated with mDTA-1 to establish an ADA (+) response or were not pretreated and remained ADA (2). The BALB/c mice were implanted with CT-26 tumors (A and B) and C57BL/6 mice were implanted with MC38 tumors (C and D). Upon reaching an average of approximately 100 mm3, mice weretreated withmDTA-1 as a single dose on day 0 or asmultiple doses on days 0, 7, and 14 at the indicated dosages. Mean tumor volume (mm3)6 S.E.M., n =10 mice per condition, was plotted with respect to time (A and C) and the individual tumor volumes were compared at day 21 for CT26 (B) or day 18 forMC38 (D). Median tumor volume for the respective cohort plotted as horizontal bars, and *P , 0.05 and ****P , 0.0001 compared with isotype controlbased on the Mann-Whitney U-test calculated in GraphPad Prism 6 (B and D).


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cells in naive mice in comparison with only ∼5% in ADA-positive mice (Fig. 9A). Increasing the mDTA dose to 30mg/kgrestored the mDTA-1 target engagement to 91%. The MFIfrom the negative controls (no antibody) were comparable tothe MFI observed from s.c. dosing of an IgG2a isotype control.Purified PBMCs were also visualized for mDTA-1 binding inall groups using a fluorescent microscope (Fig. 9B). AbundantDyLight 650 cellular staining was observed in both PBMCsfrom naive mice dosed with 3 mg/kg and ADA-positive micedosed with 30 mg/kg. No fluorescence signal was detected inPBMC fromADA-positive mice dosed with 3mg/kg of mDTA-1or with 3 or 30 mg/kg of isotype control antibody (data notshown). Flow cytometry analysis of circulating CD31 CD41

T-cell populations in naive and ADA-positive mice (Fig. 9C)demonstrated strong mDTA-1-GITR engagement in naivemice dosed with 3 mg/kg. However, when ADA-positive micewere given the same 3mg/kg DyLight 650/mDTA dose no MFIchanges were observed, consistent with decreased concentra-tions of available mDTA-1 in the circulation due either to fastIC elimination and/or direct ADA neutralization of mDTA-1binding. Importantly, mDTA-1 target engagement on periph-eral blood CD41 CD31 T cells was almost restored to naivelevels in ADA-positive mice treated with 30 mg/kg.Effect of Pretreatment in the Disposition of PD-1

Antagonist muDX400 in BALB/c Mice. Drug combinato-rial approaches are rapidly becoming a hallmark of cancertreatment. Therefore, better understanding of potential phar-macological drug-drug interactions is critical to improvingrational combination therapy design. To that end, in vivo PK

and disposition were used to assess whether pretreatmentwith mDTA-1 dosed in combination with the PD-1 antagonistantibody muDX400 would further accelerate mDTA-1 dispo-sition and/or immunogenicity (Hirsch et al., 2015; Pinheiroet al., 2015). In addition, we compared the effects of mDTA-1versus muDX400 pretreatment, dosed alone or in combina-tion, in the PK and disposition of muDX400.Figure 10 shows muDX400 concentration time profiles

in naive, muDX400, and muDX400 1 mDTA-1–pretreatedBALB/c mice. Fluorescently labeled muDX400 concentrationtime profiles and PK parameters were indistinguishableirrespective of pretreatment combinations (Table 2). In con-trast, there is a dramatic reduction in drug exposure formDTA-1 in pretreated mDTA-1/muDX400 combo mice. In-terestingly, the changes in mDTA-1 PK parameters in micepretreated with mDTA-1 alone (Table 1) were similar to thosepretreated in combination with muDX400. This suggestslimited cross reactivity between these two immunoregulatorytargets in the generation of an anti-drug response. Likewise,analysis of muDX400 disposition in muDX400-pretreatedmice (Fig. 11, A and B) and muDX400 1 mDTA-1–pretreatedmice (Fig. 11, C and D) revealed that tissue/blood ratios ofmuDX400 remained below 0.5 in both cohorts at all collectedtime points (Fig. 11, A and C). This is indicative of negativetissue uptake or accumulation. Furthermore, distribution ofmuDX400 revealed no evidence of drug accumulation in anyorgan analyzed (Fig. 11, B and D), demonstrating thatmuDX400 is highly stable in circulation with a nonspecificorgan disposition pattern typical of normal IgGs. In addition,

Fig. 9. ADAs impair mDTA-1 engagement of GITR onCD3+ CD4+ T cells. Naive and pretreatedmDTA-1 BALB/cmice were injected s.c. with 3 or 30mg/kgof unlabeled mDTA-1, DyLight 650/mDTA-1, or DyLight 650–labeled IgG2a isotype control antibody. PBMCs were isolated fromwhole blood 4 hours lateras previously described inMaterials andMethods. FACSwas used to select CD3+ CD4+ T cells from PBMCs and quantify the percent of GITR engagement(A). TheMFI of cells incubated ex vivo in the absence of antibody or incubated with 5 mg/ml DyLight 650/mDTA-1 were used as negative and positive GITRbinding controls, respectively (A). CD3+ CD4+ T cells isolated from naive versus ADA-positive mice dosed with fluorescent mDTA-1 (3 and 30 mg/kg,respectively) were also imaged on an EVOS FL fluorescent microscope (B). Images are representative of prevailing cell fluorescence and morphology.Typical FACS-MFI shifts for CD3+ CD4+ T cells in all study groups were plotted using FlowJo (FlowJo, LLC Ashland, OR) (C). All in vivo target engagement andFACS experiments were carried out in duplicate, n = 3 mice per time point, and error bars correspond to 6S.D. calculated using GraphPad Prism 6.

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plasma samples from a separate group of BALB/c micepretreated Q7D � 2 with 3.0 mg/kg mDTA-1 (alone), 1.5 mgmDTA-1 1 muDX400, or 3.0 mg/kg muDX400 (alone) werecollected 21 days after the second dose, spiked with labeledmuDX400, and examined by SEC-HPLC. The resulting chro-matographs showed a single monomeric peak correspondingto intact IgG and no detectible HMW complexes (data notshown).

DiscussionThis study characterized the impact of ADAs on the

pharmacology of the GITR agonist monoclonal antibodymDTA-1. We compared the PK, disposition, target engage-ment, and anti-tumor efficacy of mDTA-1 in naive and ADA-positive mice. This study demonstrated that treatment withmDTA-1 in naive wild-type mice generated anti-mDTA-1antibodies. Subsequent doses of mDTA-1 formed HMW com-plexes in circulation, which led to hepatic deposition and drugelimination, thereby limiting drug exposure and reducingtarget engagement and anti-tumor efficacy of mDTA-1.

Moreover, we showed that this ADA response was GITRmediated, mDTA-1 specific, and did not alter the PK of acoadministered PD-1 antagonist, muDX400.Interestingly, the lack of an ADA response against mDTA-1

in GITR2/2 mice following repeated treatment stronglysupports the hypothesis that direct agonistic engagement ofGITR by mDTA-1 is responsible for the immunogenicity of themDTA-1 molecule in wild-type mice, as opposed to an un-usually strong immunogenicity of the molecule itself. Aninvestigation into this target-mediated ADA mechanismmight clarify whether mDTA-1 engagement of GITR onT or B cells (Zhou et al., 2010) is potentiating mDTA-1immunogenicity.The PK, disposition, and target engagement of biotherapeu-

tics are typically evaluated through the use of either radioactivelylabeled molecules (Rojas et al., 2005; Kelley et al., 2013),fluorescently labeled molecules (Alvarez et al., 2012), or anti-drug assays (Bourdage et al., 2007; Kelley et al., 2013). In ourstudy of mDTA-1 in naive and ADA-positive mice, we usedfluorescent labeling of the mDTA-1 andmuDX400 monoclonalantibodies to follow their fate in vivo at relevant pharmaco-logical doses. Direct quantitation of labeled antibodies cir-cumvents potential assay interference issues when assessingdrug levels in the presence of ADAs. SEC-HPLC analysis ofmDTA-1 fromplasma of ADA-positivemice dosed or incubatedwith fluorescently labeled mDTA-1 revealed the presence ofHMW complexes consistent with the presence of anti-mDTA-1/mDTA-1 ICs (Løvdal et al., 2000; Rojas et al., 2005; Ganesanet al., 2012). The relatively low concentration of ICs detectedin blood by this method in ADA-positive mice treated withDyLight 650–labeled DTA-1 versus the total conversion toHMW complexes formed by spiking DyLight 650/mDTA-1 exvivo in plasma fromADA-positive mice suggests that ICs wereefficiently removed from circulation. The nonlinear reductionin DTA-1 exposure observed in pretreated animals dosed with5 versus 30 mg/kg suggests saturation of IC formation or aplateau in the elimination capacity of DTA-1/anti-DTA-1 ICsby the reticuloendothelial system. To ascertain the clearancemechanisms of mDTA-1 ICs in BALB/c mice, we conductedvarious tissue distribution analyses of DyLight 650/mDTA-1administered to naive and ADA-positive cohorts. Our resultssuggested that, following administration to mice with apreexisting ADA response against mDTA-1, the ADAs boundto and formed ICs with mDTA-1. These ICs were then rapidlycleared through hepatic uptake, at least partially by KCs,followed by catabolism and subsequent elimination. Whilethis is consistent with previous studies (Løvdal et al., 2000;Ganesan et al., 2012) it does not preclude the possibility of ICuptake by other liver-associated cells, such as liver sinusoidalendothelial cells, which in our studies showed sporadicDyLight 650/mDTA-1 staining (data not shown). Additionally,

Fig. 10. Concentration time profiles of PD-1 antagonist muDX400 innaive, muX400, andmuX400 +mDTA-1–pretreated BALB/c mice. Cohortswere dosed s.c. with 30 mg/kg of DyLight 650–labeled muDX400 (A, B, andC) or mDTA-1 (D) and the concentrations were determined by fluorescenceemission–linked assay as previously described; n = 3 mice per group anderror bars correspond to S.D. calculated using GraphPad Prism 6.

TABLE 2Effect of mDTA-1, muDX400, or combination pretreatments in the PK of DyLight 650–labeled muDX400 and mDTA-1

Pretreatment Regimen DyLight 650 Antibody Dose t1/2 Cmax AUClast CL Vss

mpk day mg/ml hr·mg/ml ml/day/kg ml/kgmDTA-1 + muDX400 3mg/kg Q7D � 2 mDTA-1 30 0.5 81.4 3020.6 235.2 178.1Naive muDX400 30 7.3 267.2 20717.6 10.1 106.4muDX400 3mg/kg Q7D � 2 muDX400 30 7.4 290.5 22735.5 9.1 97.8mDTA-1 + muDX400 3mg/kg Q7D � 2 muDX400 30 8.4 281.6 23259.4 8.2 99.3

AUClast, last area under the curve; CL, clearance.


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the renal uptake observed in ADA-positive mice may bepartially due to the excretion of free fluoroprobe in circulationresulting fromADA-related drug processing (Nlend et al., 2014).We demonstrated that while single- and multiple-dose

regimens of 3 mg/kg mDTA-1 provided equivalent anti-tumor efficacy in naive mice, neither was sufficient to achieveanti-tumor efficacy in mice with preexisting ADA. Thisrevealed that mDTA-1 anti-tumor efficacy was ablated bythe anti-mDTA-1 antibody response. Furthermore, we showedthat in ADA-positive mice, a 3 mg/kg mDTA-1 dose wasinsufficient to engage GITR on circulating CD31 CD41

T cells. This lack of target engagement was likely responsiblefor the diminished anti-tumor efficacy of mDTA-1 in ADA-positive mice. When the mDTA-1 dose in ADA-positive micewas increased to 30 mg/kg, GITR engagement on circulatingCD31CD41T cells was comparable to that of naivemice dosedwith 3 mg/kg. Anti-tumor efficacy was observed, but not to the

same degree as in ADA-negative mice dosed with 3 mg/kgmDTA-1. The lack of complete anti-tumor efficacy may be dueto limited intratumoral receptor engagement, compared withreceptor engagement in circulation. Overall, this supports acorrelation between GITR engagement and anti-tumor effi-cacy, and of critical importance, that restoration of anti-tumorefficacy was possible.Recently, the development of novel routes of administration,

such as intratumoral dosing (Marabelle et al., 2013, 2014),have been evaluated as an option to limit systemic T-cellactivationwhilemaintaining intratumoral immune activationand anti-tumor efficacy. Given that the mDTA-1 ADA re-sponse can be overcome in vivo, localized GITR activation inthe tumor may be sufficient to confer anti-tumor efficacy.Therefore, our results warrant the preclinical exploration oflocalized treatments with agonistic therapeutics to explore thedynamics of anti-tumor efficacy versus ADA responses.

Fig. 11. Tissue distribution of DyLight 650–labeled muDX400 (30 mg/kg s.c.) in BALB/c mice pretreated with muDX400 alone (A and B) or muDX400 +mDTA-1 (C and D). Drug concentrations were determined by fluorescence emission–linked assay. Tissue/blood ratios greater than 1 are indicative oftissue uptake (A and C). MuDX400 concentrations (microgram/milliliter) were measured at 4, 8, 16, 24, 48, and 96 hours (B and D); n = 3 mice per timepoint and error bars correspond to S.D. calculated using GraphPad Prism 6.

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Given recent trends of cancer immunotherapy towardemploying combinations of various immunoregulatory recep-tor agonists and antagonists (Blank 2014; Lu et al., 2014;Westin et al., 2014), our study sought to characterize whetherrepeated administration of mDTA-1, an agonist anti-GITRantibody, would enhance ADA to a coadministered anti-PD-1antibody (muDX400). Our results suggest that while mDTA-1elicits a rapid anti-drug response, it is specific to mDTA-1, andmuDX400 PK and disposition are not impacted, at least in theduration of the study. This does not preclude the possibility ofan eventual anti-muDX400 response, but we can infer thattreatment with mDTA-1 will not necessarily induce asimilarly enhanced ADA response to molecules administeredsimultaneously.Of importance in our studies is whether the emergence

of ADA responses against GITR agonists is translatable tohumans. Anaphylaxis induced by ADAs against mDTA-1 andOX-86 have already been characterized in preclinical andclinical studies (Werier et al., 1991; Abramowicz et al., 1992;Baudouin et al., 2003; Murphy et al., 2014), Although, themanifestations of immunogenicity in preclinical models aregenerally not considered predictive to humans, our resultssuggest that close attention to the appearance of ADAresponses should be givenwhen administering T-cell agonists.Anti-GITR agonist antibodies are already in the clinic asmonotherapy and in combination with anti-PD-1. Ultimately,understanding how the presence of ADAs changes the PK,drug exposure, and clearance of mDTA-1 and muDX400 inmouse models could be helpful for anticipating potentialdose adjustments and safety concerns in the clinic, both asmonotherapies and in combination.In conclusion, we showed that exposure to mDTA-1 in mice

results in GITR-mediated mDTA-1 immunogenicity leadingto the generation of neutralizing ADAs, formation of drug/anti-drug ICs, rapid mDTA-1 clearance, hepatic disposition,and catabolism. Likewise, we observed diminished targetengagement and loss of mDTA-1 anti-tumor efficacy in micewith pre-existing ADA. Of critical importance, mDTA-1 anti-tumor efficacy in CT26 and MC38 murine syngeneic tumormodels could be partially restored at pharmacological dosesthat reestablish target engagement on circulating CD31

CD41 T cells in ADA-positive mice. We also demonstratedthat mDTA-1 did not impact the pharmacology of muDX400when dosed in combination. Taken together, our resultsfacilitate better understanding of ADA responses againstimmunomodulatory monoclonal antibodies and the role oftarget engagement in regulating drug pharmacology.

Acknowledgments

The authors acknowledge the contributions of Dr. LaurenceFayadat-Dilman and Dr. Brian Reardon at the Department of ProteinSciences for providing the mDTA-1, muDX400, and VP2 antibodies;Priscilla Lapresca for Animal Facility support; Dr. Wolfgang Seghezziat the Department of Bioanalytics for MSD and enzyme-linkedimmunosorbent assay support; Dr. Terri McClanahan, Dr. FernandoUgarte, and Dr. Doug Wilson at the Department of Profiling andExpression for support of flow cytometry facilities and equipment; andDr. Jennifer Yearley, Dr. Meric Ovacik, and Dr. Mohammad Tabrizifardfor valuable discussions.

Authorship Contributions

Participated in research design: Brunn, Beebe, Escandón.Conducted experiments: Brunn, Mauze, Ueda, Gu, Wiswell.

Contributed new reagents or analytic tools: Brunn, Mauze, Gu,Zhang, Hodges, Escandón.

Performed data analysis: Brunn, Mauze, Gu, Zhang.Wrote or contributed to the writing of the manuscript: Brunn,

Escandón.

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Address correspondence to: Dr. Enrique Escandón, 901 S. CaliforniaAvenue, Merck Research Laboratories, Merck & Co. Inc., Palo Alto, CA 94304-1104. E-mail: [email protected]

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