Improved Tumor Penetration and Single-Cell ... · with T-DM1 improves efﬁcacy (tumor growth reduction and overall survival as measured by tumor volume endpoint) in a trastuzumab-insensitive

Translational Science

Improved Tumor Penetration and Single-CellTargetingofAntibody–DrugConjugates IncreasesAnticancer Efficacy and Host SurvivalCornelius Cilliers1, Bruna Menezes1, Ian Nessler1, Jennifer Linderman1,2, andGreg M. Thurber1,2

Abstract

Current antibody–drug conjugates (ADC) have made advancesin engineering the antibody, linker, conjugation site, small-mole-cule payload, and drug-to-antibody ratio (DAR). However, therelationship between heterogeneous intratumoral distribution andefficacy of ADCs is poorly understood. Here, we compared trastu-zumab and ado-trastuzumab emtansine (T-DM1) to study theimpact ofADC tumordistributiononefficacy. In amouse xenograftmodel insensitive to trastuzumab, coadministration of trastuzu-mab with a fixed dose of T-DM1 at 3:1 and 8:1 ratios dramaticallyimproved ADC tumor penetration and resulted in twice theimprovement in median survival compared with T-DM1 alone. Inthis setting, the effective DAR was lowered, decreasing the amountofpayloaddelivered to each targeted cell but increasing thenumberof cells that receivedpayload. This result is counterintuitive becausetrastuzumab acts as an antagonist in vitro and has no single-agent

efficacy in vivo, yet improves the effectiveness of T-DM1 in vivo.Novel dual-channel fluorescence ratios quantified single-cell ADCuptake andmetabolism and confirmed that the in vivo cellular doseof T-DM1alone exceeded theminimumrequired for efficacy in thismodel. In addition, this technique characterized cellular pharma-cokineticswith heterogeneousdelivery after 1 day, degradation andpayload release by 2 days, and in vitro cell killing and in vivo tumorshrinkage 2 to 3 days later. This work demonstrates that theintratumoral distribution of ADC, independent of payload doseor plasma clearance, plays a major role in ADC efficacy.

Significance: This study shows how lowering the drug-to-antibody ratio during treatment can improve the intratumoraldistribution of a antibody-drug conjugate, with implicationsfor improving the efficacy of this class of cancer drugs. Cancer Res;78(3); 758–68. �2017 AACR.

IntroductionAntibodies and antibody–drug conjugates (ADC) make up the

largest portion of the growing biologics market. Currently, thereare over 50 FDA-approved antibodies and nearly 500 in thevarious stages of the clinical pipeline (1). Although there arecurrently over 70 ADCs in the clinical pipeline (2, 3), only four,Adcetris, Besponsa, Mylotarg, and Kadcyla (T-DM1), are currentlyapproved by the FDA. Although these ADCs have had clinicalsuccess, the factorial optimization of the antibody, linker, con-jugation site, small-molecule payload, drug loading, and targetselection make development of each ADC a unique challenge.Although there havebeen advances in engineering thebiophysicalcharacteristics to improve safety, stability, and develop morehomogeneous products, ADCs continue to be limited by toxicity,which is typically driven by the toxicity of the small-molecule

payload (4, 5). In particular, several of the recent ADC failuresmay have been prevented by marginal gains in tolerability (2).

It iswidely known that antibodies/ADCs exhibit heterogeneousdistribution in solid tumors (6–11); however, it is not wellunderstood how the heterogeneous tissue distribution of ADCsimpacts their overall efficacy. ADC efficacy requires a multistepprocess, which includes the distribution of intact ADC in thetumor, cellular uptake, degradation of the antibody, release of thesmall-molecule payload, induction of apoptosis by the cytotoxin,and potentially bystander effects on neighboring cells (12–14).Therefore, there is a need to understand the ADC's effects from thesubcellular scale (e.g., howmanyADCs are required to achieve celldeath in vivo) to the tissue level (e.g., howmany cells in the tumorare receiving a therapeutic dose) to whole organ biodistribution(e.g., what is the healthy tissue exposure and resulting toxicity) todevelop effective therapeutics.

Previously, we developed a combined tissue and physiologi-cally based pharmacokinetic model to describe both the tumorand systemic distribution of T-DM1 (15). We found that coad-ministration of trastuzumab with T-DM1 (trastuzumab linked tothe payloadDM1,which therefore competes for the same bindingepitope of HER2) dramatically improved tumor penetration, buttotal tumor uptake of ADC was unchanged. Coadministration oftrastuzumab with T-DM1 lowers the effective drug-to-antibodyratio (DAR)while competingwith T-DM1 forHER2 receptors anddriving penetration deeper into the tissue. The higher the trastu-zumab dose, the farther the ADC will penetrate into the tumor;however, the averageDM1payload concentration in each targetedcell will be lower. Several studies in the literature demonstrate that

1Department of Chemical Engineering, University of Michigan, Ann Arbor,Michigan. 2Department of Biomedical Engineering, University of Michigan, AnnArbor, Michigan.

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

Corresponding Author: Greg M. Thurber, University of Michigan, 2800 Ply-mouth Rd., Ann Arbor, MI 48109. Phone: 734-764-8722; Fax: 734-763-0459;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-1638

�2017 American Association for Cancer Research.

CancerResearch

Cancer Res; 78(3) February 1, 2018758

on February 9, 2021. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 7, 2017; DOI: 10.1158/0008-5472.CAN-17-1638

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-1638&domain=pdf&date_stamp=2018-1-18

http://cancerres.aacrjournals.org/

cohorts of mice treated with ADCs having different DAR but thesame overall payload dose (i.e., DAR2 given at 2 mg/kg vs. DAR4given at 1 mg/kg) had better outcomes with the lower DAR (andtherefore higher antibody dose, which correlates with bettertumor penetration; ref. 15). These results are consistent withtumor penetration playing a major role in efficacy independentof the target antigen, antibody, linker, payload, or bystandereffects.However, thesewere not prospective studies, and althoughcertain mechanisms, such as DAR-dependent clearance, did notappear to play a role in the analysis, they could have affected thedata interpretation. Therefore, we wanted to design an experi-mental study to isolate the impact of distribution on efficacy.

Here, we demonstrate that coadministration of trastuzumabwith T-DM1 improves efficacy (tumor growth reduction andoverall survival as measured by tumor volume endpoint) in atrastuzumab-insensitive mouse xenograft model. Against trastu-zumab-insensitive cell lines, the addition of trastuzumab wasantagonistic in vitro (i.e., lowered efficacy by blocking T-DM1uptake), as expected. Counterintuitively, coadministration oftrastuzumab (which acts as an antagonist in vitro and has nosingle-agent efficacy in this animal model in vivo) with T-DM1showed a significant improvement in efficacy in a mouse xeno-graft model despite the same small-molecule dose and tumoruptake as T-DM1 alone. In fact, the combination of trastuzumab,which had no single-agent efficacy, and T-DM1 was synergistic,meaning the net improvement was greater than additive (astrastuzumab alone had no impact on efficacy in the absence ofT-DM1). Histologic imaging showed a significant increase inT-DM1 tumor penetration with the coadministered trastuzumab.In addition, we present a novel near-infrared (NIR) fluorescenceratio technique with dually labeled ADCs to track themetabolismand distribution of ADCs at the single-cell level. Applying thistechnique to single-agent T-DM1 therapy showed the delivery ofADC to cellswithin the targeted population in vivowashigher thanthe threshold required for cell death, while the majority of tumorcells did not receive any ADC. These results demonstrate that theintratumoral distribution of ADCs in tumor tissue plays a majorrole in determining their efficacy independent of the amount oftotal tumor payload delivered. To our knowledge, this is thefirst time that the distribution itself, independent of the otherparameters that affect efficacy and tumor penetration such asdose, plasma clearance, and molecular weight, significantlyimpacted survival.

Materials and MethodsAntibodies and NIR imaging agents for ratio measurements

Herceptin (trastuzumab, Roche) and Kadcyla (T-DM1, Roche)were obtained from the University of Michigan Pharmacy. AlexaFluor 680 NHS Ester (AF680, Thermo Fisher Scientific, A37567),IRDye 800CW NHS Ester (IRDye, LI-COR, 929-70020), andCellTrace Far Red DDAO-SE (DDAO, Thermo Fisher Scientific,C34553) were conjugated to the antibodies following the man-ufacturer's instructions as described previously (15, 16). Anti-body/ADC at 2 mg/mL supplemented with 10% sodium bicar-bonate (v/v) was reacted with dye at molar ratios of 0.5 (AF680,IRDye) and 1.5 (DDAO) for 2 hours at room temperature andpurified using P6 Biogel (1 g gel/10mL PBS), resulting in dye toprotein ratios of approximately 0.3 (AF680, IRDye) and 0.7(DDAO). Our previous work has shown that the distribution ofT-DM1 is unchanged after labeling with AF680 at dye-to-protein

ratio of 0.3 or less (17). Antibody/ADC dye conjugates were runon SDS-PAGE and scanned on theOdyssey CLx Scanner (LI-COR)to ensure free dye was removed. For fluorescence histology, anti-mouse CD31 (BioLegend, 102402) was conjugated with AlexaFluor 555 (Thermo Fisher Scientific, A37571),mouse anti-humanIgG Fc antibody (BioLegend, 409302) was conjugated with AlexaFluor 488 (Thermo Fisher Scientific, A20000), and trastuzumabwas conjugated with Alexa Fluor 750 (Thermo Fisher Scientific,A20011) at dye to protein ratios of 1.5.

Cell lines and in vitro toxicityNCI-N87 and HCC1954 cells were purchased from ATCC in

May 2015 and June 2016, respectively. Cell line authenticationwas performed by ATCC using cytochrome C oxidase testing andshort tandem repeat profiling. Cells were grown at 37�C with 5%CO2 inRPMI1640 growthmediumsupplementedwith 10%(v/v)FBS, 50 U/mL penicillin, and 50 mg/mL streptomycin. Mycoplas-ma testing was performed yearly using the Mycoalert TestingKit (Thermo Fisher Scientific, NC9719283). Cells were cultured2 to 3 times per week up to passage number 50 (approximately 3–4 months). For cell viability assays, 5,000 cells were plated in 96-well plates. Titrations of T-DM1 or T-DM1 and trastuzumab werereplaced daily for 6 days, and viability was measured using thePrestoBlue Cell Viability Reagent (Thermo Fisher Scientific,A13261). Briefly, cells were washed twice with media, and a1:10dilutionofPrestoBlue inmediawas incubated for 25minutesat 37�C. After incubation, the fluorescence (560/590, Ex/Em) ofeach well was measured using a Biotek Synergy plate reader.Background signal from wells without cells was subtracted fromall samples, and then, viability was normalized to untreated cells.

In vitro NIR fluorescence ratio measurements and fluorescencemicroscopy

ADC metabolism was studied by dually labeling T-DM1 withDDAOand IRDye as described above. As the labeledADCbinds tothe cell surface receptor, gets internalized, and subsequentlydegraded, the low molecular weight and more lipophilic DDAOdiffuses out of the cell while the IRDye remains trapped (16).DDAO therefore approximates the intact protein (as it is clearedupon degradation), whereas IRDye approximates the cumulativeuptake in the cell (18). Unlike pH effects (19) or quenching/FRET,this provides an irreversible measurement of both intact proteinand payload delivery without requiring a high degree of labeling(self-quenching approach) or larger dye-quencher conjugates.NCI-N87 and HCC1954 cells were plated in 96-well plates. Cellswere labeled for 30 minutes at 37�C at different times over a48-hour period. After each labeling, cells were washed twiceto remove excess media. After 48 hours, cells were washedthree times and then harvested using Cellstripper (Corning,25-056-CI), a nonenzymatic cell dissociation solution, and fluo-rescence intensity was quantified using an Attune Acoustic Focus-ing Cytometer (Life Technologies). The signal for each dye wasnormalized to the initial time point, and the normalized ratio ofDDAO divided by IRDye was plotted to show the ratio of intactADC to cumulative uptake. Alternatively, cells were imaged usingfluorescence confocalmicroscopy (Olympus) with a 635 nm laserfor DDAO and a 748 nm laser for IRDye.

Tumor growth studiesAll animal studies were conducted in accordance with the

University of Michigan Institutional Animal Care and Use

Improving ADC Efficacy through Increased Tumor Penetration

www.aacrjournals.org Cancer Res; 78(3) February 1, 2018 759




Committee. For all tumor xenografts studies, 5 � 106 NCI-N87cells were inoculated in the rear flanks of 4–8 week old femalenude (Foxn1nu/nu) mice obtained from Jackson Laboratories(one flank for tumor growth studies, both flanks for all others).For tumor growth studies, tumor volume was measured usingcalipers every other day using the formula volume¼ 0.5� length� width2. Trastuzumab, T-DM1, both trastuzumab and T-DM1(all unlabeled), or saline were injected via tail vein once tumorsreached 250 mm3. For tumor growth studies, 10 animals wereused for all treated and untreated cohorts. Kaplan–Meier survivalcurves were generated in PRISM and were analyzed by log-ranktest at significance level of P � 0.05.

In vivo NIR fluorescence ratio measurements and fluorescencehistology

To study the cellular uptake and metabolism kinetics in vivo,tumor xenografts were treated with DDAO and IRDye-labeledT-DM1, and the tumors were resected, digested into a single-cellsuspension and analyzed on flow cytometry (similar to the in vitroassay). Once the longest axis of the tumor reached 9 to 10 mm,100 mg of dually labeled T-DM1 was injected via tail vein, andanimals were sacrificed at 24, 48, and 72 hours. After sacrifice,tumors were resected and sliced before being placed in a colla-genase IV solution (5 mg/mL). The tissue was digested for25 minutes before centrifugation (5 minutes, 300 � g). The cellpellet was resuspended in media, washed twice, and filteredthrough a 40-mm filter. Cells were then analyzed by flow cyto-metry. Uninjected negative control tumor digests were used toestablish gates for DDAO and IRDye fluorescence. To determinethe percent intact (from the DDAO/IRDye ratio), we examinedcells that were targeted with T-DM1 (IRDyeþ). The background

mean fluorescence intensity in RL1 and RL2 fromnegative controltumors was subtracted from the mean fluorescence intensity ofDDAO (RL1) and IRDye (RL2) to get fluorescence per targetedcell. Then, the DDAO signal was divided by IRDye to get theDDAO/IRDye ratio. To get the percent intact, this ratio wasnormalized to the initial intact ratio, which was determined byharvesting in vitro cells, labeling on ice for 25 minutes, washingtwice, and analyzing by flow cytometry. In addition to runningtumor cells on flow cytometry, part of the tumor was used forfluorescence biodistribution. The low autofluorescence in thenear-IR makes IRDye a suitable fluorophore to determine organuptake (17, 20, 21), and fluorescence biodistribution was per-formed as described previously (15, 17, 22). Fluorescence histol-ogy was performed as described previously (15, 17). A detaileddescription is provided in the Supplementary Material.

ResultsCoadministrationof trastuzumabwithT-DM1 improves T-DM1tumor penetration

ADC tumor distribution is dependent on many parameters,including dose, DAR, systemic clearance, antigen expression andinternalization rate in tumor (and healthy tissue), and the surfaceto volume (S/V) ratio of the vasculature. The clinical dose ofT-DM1-AF680 (3.6 mg/kg) shows a heterogeneous, perivasculardistribution in NCI-N87 tumor xenografts (Fig. 1A; Supplemen-tary Fig. S1) consistent with high-affinity antibodies that targethighly expressed receptors and are dosed at subsaturating levels(9, 23, 24). Coadministration of unlabeled trastuzumab at 10.8or28.8mg/kg (3:1 or 8:1, respectively) dramatically increases tumorpenetration of a constant T-DM1 dose (Fig. 1B and C), allowing

Figure 1.

Improving T-DM1 tumor distribution through coadministration of trastuzumab. A, Administration of T-DM1 at 3.6 mg/kg (single agent) results in aheterogeneous, perivascular distribution due to rapid binding relative to transport in the tissue. B and C, The tumor penetration of a constant dose of T-DM1 isimproved when coadministered with a subsaturating (B) or saturating (C) dose of trastuzumab. Trastuzumab competes for binding sites, increasing T-DM1penetration. The middle column shows distribution of AF680-labeled T-DM1 (green) at 3.6 mg/kg, with unlabeled trastuzumab at 0:1, 3:1, and 8:1 trastuzumab:T-DM1 ratios (0, 10.8, and 28.8 mg/kg, respectively). Immunofluorescence staining with CD31-AF488 (red) shows the tumor vasculature. The right columnshows immunofluorescence staining with antihuman IgG Fc-AF555 (gray). The window leveling between images is different as the intensity of the T-DM1 decreaseswith an increasing ratio, while the anti-Fc staining labels both trastuzumab and T-DM1, thereby maintaining a constant intensity while the penetration increases(see Supplementary Material). Scale bar, 200 mm.

Cilliers et al.

Cancer Res; 78(3) February 1, 2018 Cancer Research760




more cells to receive the cytotoxic payload. However, addingtrastuzumab lowers the effective DAR, increasing the number oftargeted cells while reducing the average number of payloadmolecules delivered per cell. Immunofluorescence staining withantihuman IgG Fc-488 shows antibody distribution is morehomogeneous with increasing doses of trastuzumab (Fig. 1A–C).Similarly, increasing the dose of T-DM1 alone also improvedtumor penetration (Supplementary Fig. S2), but this exceeds themaximum tolerated dose (MTD) in humans. HER2 was stainedex vivo with trastuzumab-AF750 to ensure the heterogeneousdistribution is not from lack of available antigen (SupplementaryFig. S3). T-DM1 binding affinity was unchanged by fluorophoreconjugation (Supplementary Fig. S4).

T-DM1 is effective in vitro when occupying only a fraction ofHER2 receptors even under saturating antibody conditions

The increasing doses of trastuzumab improve T-DM1 penetra-tion into tumor tissue but lower the DM1 payload delivery bycompeting with HER2 receptors. To determine whether the lower

payload delivery was still sufficient to kill cells in vitro, wemeasured efficacy with T-DM1 alone or a saturating combinationof total antibody (T-DM1 þ trastuzumab) while varying theT-DM1 to trastuzumab ratio. Toxicity assays with trastuzumabalone showed only slight growth inhibition at the highest con-centrations (Supplementary Fig. S5), consistent with literaturereports that the NCI-N87 cell line is sensitive to T-DM1, whiletrastuzumab has a slight growth inhibitory effect in vitro (25). Wemeasured the toxicity of T-DM1 in vitro with the NCI-N87and HCC1954 cell lines, and we found IC50 values of 82 and33 pmol/L for NCI-N87 and HCC1954 (Fig. 2A and B), respec-tively, consistent with other reports (26, 27). Next, we performedtoxicity assays with fluorescently tagged T-DM1-AF680 and exam-ined cellular uptake of T-DM1 by flow cytometry. The IC50 foreach cell linewasmuch less than the concentrationneeded for halfof the normalized uptake, indicating that complete surface recep-tor saturation is not needed for T-DM1 cytotoxicity in vitro. Inaddition, we found that fluorophore conjugation had no impacton T-DM1 cytotoxicity in vitro (Supplementary Fig. S6). The

Figure 2.

In vitro cytotoxicity.A andB, In vitro cytotoxicity of T-DM1 against NCI-N87 (A) andHCC1954 (B) cell lines. Assayswere performed in triplicate and average IC50swere82 � 10 pmol/L and 33 � 20 pmol/L for NCI-N87 and HCC1954 cell lines, respectively. The number of DM1 molecules per cell (gray) was estimated by measuringT-DM1-680 uptake with flow cytometry and quantitative beads. C and D, In vitro cytotoxicity varying the T-DM1 concentration, while keeping total antibodyconcentration (T-DM1þ trastuzumab) constant at 10 nmol/L. Cellswere incubatedwith T-DM1 and/or trastuzumab inmedia for 6days, andmediawere replaceddailyfor all in vitro cytotoxicity assays. Data, mean � SD.






maximum cellular uptake occurred at concentrations below theKd (1.8 nmol/L; ref. 17), indicating treatment likely impactsuptake over this time scale. Tomimic the effect of coadministeringtrastuzumab in vivo, we performed toxicity assays where theT-DM1 concentration was varied, but the total antibody concen-tration was kept constant at 10 nmol/L (saturating) to see howcompetition for receptors would affect toxicity (Fig. 2C and D).T-DM1 still showed toxicity below the antibody Kd against bothcells lineswith the additionof trastuzumab, although the IC50washigher likely due to competition from the trastuzumab. Using asimple competitive inhibition binding model, we found thatT-DM1 cytotoxicity was similar when adjusting for the fractionof receptors bound by T-DM1 (Supplementary Fig. S7). In addi-tion, a similar number of molecules of T-DM1 were required toachieve 50% cell death in both cases (Supplementary Fig. S8).Because trastuzumab increases the penetration (while loweringthe single-cell delivery) of T-DM1 and T-DM1 remains toxic atsubsaturating conditions, we tested the efficacy of this combina-tion in a trastuzumab-insensitive mouse xenograft model.

Addition of trastuzumab (an in vitro antagonist) to T-DM1therapy improves in vivo efficacy in a trastuzumab-insensitivexenograft model

To examine the impact of ADC tumor distribution on efficacy,we administered a fixed dose (3.6 mg/kg) of T-DM1 with varyingratios of trastuzumab in an NCI-N87 tumor xenograft mousemodel. Nonfluorescently labeled (clinical) T-DM1 and trastuzu-mab were used for in vivo efficacy studies. We selected the NCI-N87 cell line because (i) it was less sensitive than the HCC1954cell line to T-DM1 in vitro (Fig. 2) and in vivo (27), providingmoreroom to detect improvements in efficacy, and (ii) other groupshave shown that at moderate doses, like the ones used in thisstudy, trastuzumab treatment did not significantly alter tumorgrowth from control (25, 28). Trastuzumab treatment results inmodest (but statistically significant) growth inhibition at higherdosages (>60 mg/kg total dose) but does not result in tumorreduction even at highest doses of 280 mg/kg total dose over

several weeks (28–30). In addition, we chose to use large estab-lished tumors that were 250 mm3 or greater. Others have shownantibody-dependent cell-mediated cytotoxicity is reduced in larg-er established tumors, albeit with a different HER2-expressing cellline (31). Expectedly, the clinically approved T-DM1 therapyalone showed significant improvement over control (Fig. 3).Although others have shown complete tumor regression usingT-DM1 (26), the larger tumors and single administration pre-vented consistent cures. Addition of trastuzumab to T-DM1resulted in slower tumor growth than T-DM1 alone for all dosagelevels (Fig. 3A), with 3:1 and 8:1 (T:T-DM1) ratios having astatistically significant effect. The 3:1 and 8:1 dosing levels hadseveral more partial responses and exhibited a statistically signif-icant increase in survival with increasing trastuzumab doses(Fig. 3B). Kaplan–Meier survival plots with 95% confidenceintervals calculated byPRISMand individual tumor growth curvesare shown in the SupplementaryMaterial (Supplementary Figs. S9and S10, respectively). Animals receiving trastuzumab only weresimilar to saline control, demonstrating that trastuzumab has nodirect effect on efficacy at these doses. In addition, mice receivingtreatment were weighed over the course of the study and showedcomparable tolerability (Supplementary Fig. S11), consistentwith the payload and not the antibody dose driving toxicity. Astrastuzumab is tolerated at much higher doses than T-DM1 (32)and the same T-DM1 dose was given, all treatments were consis-tently well tolerated.

Single-cell T-DM1 uptake and metabolism in vivo highlights afractionof cellswithhigher delivery thanneeded for cell killing,while other cells receive negligible T-DM1

The fluorescence histology images (Fig. 1) qualitatively showbetter penetration, but they cannot be used to quantify payloaddelivery. We utilized a novel NIR fluorescence ratio technique todetermine the absolute uptake and payload delivery per cellin vivo. In particular, we applied the technique to single-agentT-DM1 therapy to confirm that targeted tumor cells were receivingmore payload than necessary to achieve cell death, and the lack of

Figure 3.

Coadministration of trastuzumab with T-DM1 results in a significant reduction in tumor growth compared with T-DM1 alone. A, Tumor growth curves for micebearing NCI-N87 tumor xenografts following treatment with a single administration of saline, single-agent trastuzumab (10.8 mg/kg), single-agent T-DM1(3.6 mg/kg), or coadministration of trastuzumab and T-DM1 at 1:1, 3:1, and 8:1 dosage levels (T-DM1 constant at 3.6 mg/kg; trastuzumab varied at 3.6, 10.8,and 28.8 mg/kg). Nonfluorescently labeled T-DM1 and trastuzumab were used for tumor growth experiments. Data, mean � SE. The number of partial responses,complete responses, and durable complete responses is tabulated. B, Kaplan–Meier survival curves of time to progression to 1,000 mm3. Survival curves wereanalyzed by log-rank test (significance level ofP�0.05). All treatments except single-agent trastuzumab resulted in statistically significant improvements in survival(P¼0.0442, 0.0021, 0.0006, 0.001 for 0:1, 1:1, 3:1, 8:1, respectively). The 3:1 and8:1 treatments significantly improved survival over the single-agent T-DM1 (P¼0.0486and 0.0484 for 3:1 and 8:1, respectively). n ¼ 10 for each treatment. T, trastuzumab; PR, partial response; CR, complete response; DCR, durable completeresponse.

Cilliers et al.





tumor penetration was limiting efficacy in vivo. Figure 4A shows agraphic depiction of the technique. Two NIR fluorescent dyes,DDAO and IRDye, were chosen because of their widely differingresidualization rates (16). The nonresidualizing dye rapidly leaksout of the cell upon degradation, thereby approximating intactprotein, while the residualizing dye is trapped in the cell, approx-imating cumulative ADC uptake. This approach is analogous toradiolabeling with 125I as the nonresidualizing probe and 111In asthe residualizing probe (33).

To visualize the change in fluorescence ratio and validate themethod in vitro, cells were imaged using a confocal microscopeafter pulsing with labeled ADC for 30 minutes at different timesover 48 hours. Figure 4B shows separate andmerged channels forDDAO (red) and IRDye (green) for dually labeled T-DM1. Ini-tially, DDAO and IRDye are only seen on the surface. As ADC isinternalized and degraded, however, the DDAO signal graduallydecreases, while IRDye forms punctate spots in lysosomes. Figure4C shows the fraction intact of T-DM1 for HCC1954 (black) andNCI-N87 (gray) cell lines over time. Although both express the

same antigen, the HCC1954 cell line degraded T-DM1 slightlyfaster than the NCI-N87 cell line. Using the residualizing IRDyesignal, we quantified the single-cell uptake of T-DM1 and esti-mated the release of DM1 payload (Supplementary Tables S1 andS2). To quantify the kinetics of cell death following cell targeting,we measured the viability of both cell lines over 6 days (Fig. 4D).Consistent with the faster T-DM1 degradation of HCC1954cells and published link between intracellular payload concen-tration and toxicity (13), their viability decreased more quicklythan NCI-N87 cells. We used flow cytometry to quantitativelymeasure the cellular signal for DDAO and IRDye over time at thesingle-cell level (Fig. 4E). Initially, the ADC is intact and the cellpopulation is positive for DDAO and IRDye. Over time, there is agradual shift to DDAO�/IRDyeþ, indicating that the ADC isdegraded and DDAO has washed out.

After demonstrating the NIR fluorescence ratio techniquein vitro, we applied the technique in vivo. Mice were injected viatail vein with 100 mg (�4 mg/kg) of dually labeled T-DM1 andeuthanized at 24, 48, and 72 hours. Then tumors were resected

Figure 4.

In vitro T-DM1 metabolism and pharmacodynamics. A, A graphic depiction of the NIR fluorescence ratio technique. The dually labeled antibody binds thetarget, is internalized, and degraded. The nonresidualizing DDAO (red star) leaks out of the cell, while the residualizing IRDye800CW (green star) is trappedwithin in the cell. B, Representative confocal images of dually labeled T-DM1. DDAO (red) shows cell surface labeling with a loss of signal over time. IRDye (green)shows initial cell surface labeling, followed by the formation of punctate spots as it is trapped in the lysosomes. Scale bar, 10 mm. C, T-DM1 metabolism. Fractionof intact ADC following pulse of dually labeled T-DM1 for HCC1954 (black) and NCI-N87 (gray) cells. Data, mean � SD. D, Timing of T-DM1 pharmacodynamics.The fraction of viable cells over time for HCC1954 (black) and NCI-N87 (gray) cells when treated with a constant 5 nmol/L T-DM1. Nonfluorescently labeled(clinical) T-DM1 was used for this assay. Data, mean � SD. E, Representative flow cytometry plots of dually labeled T-DM1 gated on cells. Intact dually labeledT-DM1 appears in DDAOþ/IRDyeþ quadrant. Over time as ADC is degraded, there is a gradual shift toward DDAO�/IRDyeþ.






and digested into a single-cell suspension and analyzed by flowcytometry (Fig. 5). Representative flow cytometry plots of single-cell tumor digests are shown in Fig. 5A. Twenty-four hourspostinjection, targeted cells (IRDyeþ) show mostly intact protein(DDAOþ/IRDyeþ; Fig. 5A and B) because there is a constantsupply of intact ADC from the blood. However, by 48 and 72hours, there is a shift from DDAOþ/IRDyeþ to DDAO�/IRDyeþ

for the targeted cells, indicating that much of the surface-boundADC was internalized and degraded. In addition, consistentwith the tumor histology (Fig. 1), only a small fraction of cellsin the tumor (around 10% according to flow cytometry andconsistent with histology and a �10-fold higher dose for satura-tion) is targeted with ADC, despite administering the clinicaldose. Because IRDye is a residualizing dye and approximates thecumulative uptake of ADC (16), we used quantitative beads toconvert the IRDye signal from targeted cells into the number ofADCs per cell. Combining the number of ADCs targeted per cellwith the percent intact, we estimated the number of DM1 payloadmolecules released in targeted cells (Fig. 5C). Consequently, theamount of DM1 released increased dramatically between 24 and48 hours and started to plateau by 72 hours.

In addition to single-cell tumor metabolism from flow cyto-metry, the NIR fluorescence ratio technique was used to measurethe biodistribution of ADC in the same animals (Fig. 5D) to verifythe normal systemic distribution of the antibodies. The reducedautofluorescence in the near infrared window makes IRDye anappropriate dye for biodistribution studies (17, 20, 21). We havepreviously shown that at a dye-to-protein ratio of 0.3 or less, thereis no impact on protein pharmacokinetics over the first 3 to 4 days

(17). The plasma clearance of dually labeled trastuzumab wassimilar to trastuzumab-IRDye, indicating the addition of theDDAO fluorophore did not impact clearance (SupplementaryFig. S12). The biodistribution of dually labeled T-DM1 showsprimarily liver and tumor uptake and is similar to radiolabelingstudies of trastuzumab, albeit with lower tumor uptake in thismodel (34). Consistent with the single-cell flow cytometry data,themaximum tumor uptake was reached 24 hours after injection,and there was a gradual decrease at 48 and 72 hours (Fig. 5D).

Untargeted tumor cells can sustain tumor growth throughnewly functional tumor vessels

To better understand the relationship between the heteroge-neous T-DM1 distribution and efficacy, we imaged the distribu-tion of T-DM1 and trastuzumab at maximum uptake and duringtreatment. The NIR fluorescence ratio technique showed that themaximum uptake was reached 24 hours postinjection, and pay-load release appeared to plateau around 3 days. From the tumorgrowth curves, it appeared that the maximum shrinkage wasoccurring several days after payload release, around 5 days afterinitial injection for T-DM1.Once tumor cells are killed, they are nolonger able to internalize and degrade the drug, potentiallyallowing ADCs to penetrate deeper into the tissue. However,the tumor distribution of T-DM1-680 and trastuzumab-680(3.6 mg/kg) at one and 5 days postinjection remained heteroge-neous and perivascular with a significant fraction of the tumoruntargeted (Fig. 6A). Fifteenminutes prior to sacrifice, we injectedHoechst 33342 to stain functional tumor vasculature and thenstained histology slices with anti-mouse CD31 ex vivo to show all

Figure 5.

In vivo T-DM1 metabolism in tumor. A, Representative flow cytometry plots of single-cell suspension from NCI-N87 tumors at 24, 48, and 72 hourspostinjection of 3.6 mg/kg of dually labeled T-DM1. Intact dually labeled T-DM1 appears in DDAOþ/IRDyeþ quadrant. Over time as ADC is degraded, there is agradual shift toward DDAO�/IRDyeþ. Cells targeted with T-DM1 (IRDyeþ) were used to calculate percent intact as described in Materials and Methods. B, T-DM1degradation in tumor cells. The fraction of intact ADC (for the fraction of cells that is targeted by T-DM1 at this dose). Data, mean� SD. C,Molecules of DM1 payloadreleased per target cell in vivo for the targeted cells calculated using the total cell uptake and fraction intact. Data, mean � SD. D, T-DM1 biodistribution.T-DM1 shows maximum uptake 24 hours postinjection.

Cilliers et al.





(functional and nonfunctional) vasculature. Using an automatedimage analysis algorithm, we calculated the absolute vessel sur-face area to tumor volume ratio (S/V) along with the fraction ofthese vessels that had Hoechst and/or T-DM1 signal around them(see Supplementary Material; Fig. 6B). Twenty-four hours afterinjection, T-DM1 distribution was localized to functional vessels(Fig. 6A, arrows). By 5 days after injection, there were severalregions of the tumor that had functional vessels but no perivas-cular ADC (Fig. 6A, arrowheads), and the image analysis indicateda significant increase (Student t test, P < 0.0001) in the fraction offunctional vessels (Hoechst and CD31) lacking ADC (Fig. 6B),consistent with angiogenesis and/or opening of collapsed vessels.Collapsed vessels (CD31 vessels stained with ADC but notHoechst, indicating they are no longer functional) were alsopresent. These phenomena were present in both T-DM1- andtrastuzumab-treated tumors, indicating that they are not neces-sarily a result of T-DM1 efficacy. However, the formation of newlyfunctional vessels in regions untargeted by T-DM1 could play arole in rescuing the tumor, further supporting the importance ofADC distribution on efficacy.

DiscussionThe efficacy of ADCs is determined by a complex interplay

between tumor uptake, distribution, cellular targeting, internal-ization, antibody degradation, and release of the small-moleculepayload. Here, we show that improving tumor penetration bycoadministering trastuzumab enhances the efficacy of T-DM1 in atrastuzumab-insensitive mouse xenograft model. These resultshave significant implications for the development of ADCs.Although substantial efforts have been made in optimizing thedrug itself (high-affinity antibodies, stable linkers, and highlypotent smallmolecules), these data demonstrate the intratumoraldistribution (independent of the payload dose) plays amajor role

in determining efficacy. Given that the ADC dose is often limitedby the small-molecule payload dose and not the amount ofantibody, matching the potency of the ADC with delivery (ratherthan trying to maximize potency) may provide a way to improvethe efficacy of ADCs while maintaining tolerability (5).

The rapid binding of antibodies relative to their tissue pene-tration results in receptor saturation of perivascular cells (8, 10).To penetrate deeper into the tissue, additional antibody mustenter the tissue, but the toxicity of ADCs generally prevents theadministration of higher ADC doses. Although there are otherstrategies to improve tumor penetration, such as decreasingprotein size (i.e., F(ab) or F(ab')2 fragments) or lowering affinity,increasing the antibody dose has the potential to improve mul-tiple mechanisms of action. The coadministration of unconju-gated antibody improves penetration (Fig. 1) and is generallywell tolerated relative to the cytotoxic payload. For example,trastuzumab is well tolerated even at high doses, such as anintensive loading schedule totaling 18 mg/kg given over 15 days(32). This does not increase (or decrease) the amount of ADCuptake in the tumor; it only changes the distribution as long asthe dose remains subsaturating (15). In the clinic, saturatingdoses can requiremultiple grams of antibody for highly expressedand/or rapidly internalized antigens (35), possibly higher withheavy tumor burdens due to target mediated binding (36). Moreuniformly delivering the ADC could potentially lower efficacyon the perivascular cells that typically receive a high concentrationof ADC. However, ADCs tend to have low IC50s, often overan order of magnitude below the Kd (Fig. 2; refs. 4, 37, 38),indicating subsaturating concentrations can result in cell death.Therefore, the heterogeneous delivery of ADC in the tumor canresult in "overkill" of perivascular cells, where they receive moretherapeutic than necessary, while other cells receive none. Whencoadministering trastuzumab with T-DM1, the perivascular cellsreceive a smaller payload dose; however,more cells overall receive

Figure 6.

Immunofluorescence histology after treatment. A, Tumor distribution of 3.6 mg/kg of T-DM1-680 (top, green) or trastuzumab-680 (bottom, green) at24 hours (left) or 5 days (right) after tail-vein injection. Fifteen minutes prior to sacrifice, 15 mg/kg Hoechst 33342 (blue) was administered via tail vein tolabel functional vasculature. CD31-555 (red) was stained ex vivo to label all vasculature (functional and nonfunctional). Arrows highlight examples of functionalvessels that contain perivascular T-DM1 labeling, which dominate at early times after treatment. By 5 days, a significant fraction of functional vessels (CD31 andHoechst labeled) lacks perivascular T-DM1 (arrowheads). As some vessels still contain perivascular T-DM1 at this time point, presumably these vessels arenewly formed (or became functional) once a significant fraction of T-DM1 cleared. Thewindow leveling is different for each image (qualitative distribution only). Scalebar, 200 mm. B, Image analysis of histologic samples. The fraction of functional vessels (Hoechst and CD31) containing perivascular T-DM1 at 5 days aftertreatment was significantly less than at 1 day (P < 0.0001), indicating angiogenesis and/or opening of collapsed vessels.






therapeutic levels of payload. At supersaturating doses (e.g.,60–120 mg/kg in this high expression model), it is anticipatedefficacy would decrease as there would be no increase in pene-tration for a saturated tumor and payload uptake would decrease.Likewise, administering a saturating antibody dose a day beforethe ADC (e.g., ref. 39) can decrease efficacy.

These data show that the addition of an antagonist (trastuzu-mab, which antagonizes T-DM1 at high concentrationsin vitro; Fig. 2) with no single-agent efficacy (Fig. 3) can improvein vivo efficacy and survival. The converse of this conceptmust alsobe considered. Newer andmore potent payloads may be requiredfor targets with low to moderate receptor expression and/or slowinternalization rates (40). However, an ADC with higher in vitropotency may actually be less efficacious in vivo for some targetsthat are highly expressed. The increased potency (whether fromhigher DAR or a more toxic payload) could lower the MTD. Thislower dose reduces the number of cells that can be targeted,thereby lowering the overall efficacy. When developing newADCs, these results indicate that neither the maximum cellularpotency nor the maximum antibody dose is optimal. Rather, thiswork emphasizes the need to match the single-cell potency withsingle-cell delivery to maximize efficacy.

In previous work (15), we identified studies that used a con-stant small-molecule dosewithdifferentDAR/antibodydoses anddemonstrated the higher antibody dose (and correspondinglylower DAR) exhibited better efficacy. These studies includedmultiple targets, antibodies, linkers, and payloads (with andwithout bystander effects, e.g., refs. 13, 41; see SupplementaryMaterial), indicating that the impact of tissue penetration isimportant across all ADCs studied to date. Since this publication(15), two other studies reported the same result: keeping thesmall-molecule dose the same and increasing the antibody doseimproved efficacy (37, 42). However, a potentially confoundingfactor in these studies was that higher DAR ADCs tend to havefaster clearance (DAR-dependent clearance), although the differ-ence in payload AUC was less than 25% for these cases (42, 43).The current work avoids potential DAR-dependent clearance byonly using T-DM1. Another possible explanation could be thatadding trastuzumab resulted in a dose-dependent slower clear-ance of T-DM1 (44). However, the plasma clearance rates aresimilar with or without trastuzumab (15).

Using the NIR fluorescence ratio technique, the fraction oftumor cells targeted by ADC, the number of ADC moleculesdelivered per cell, and the fraction of intact ADC versus degradedwere measured (Fig. 5). The slowly clearing T-DM1 showedmostly intact protein (�85%) at 24 hours postinjection due tocontinuous delivery from the blood during this period with thehighest plasma concentrations. Conversely, over half of the ADCwas degraded by 24 hours in vitro when the ADC was pulsed(Fig. 4). After 3 days, once the tumor had surpassed maximaluptake and plasma concentrations decreased, the majority of theADC in the tumor was degraded (Fig. 5). Consistent with thehistology images, only approximately 10% of the tumor cells aretargeted by a 3.6 mg/kg dose of T-DM1 at maximum uptake. Thisis also in agreement with the 9-fold higher antibody dose requiredfor saturation of the tumor (Fig. 1). The number of DM1 mole-cules delivered per targeted cell is estimated at 1.7 � 0.3 million.Given the 2- to 3-day residualization half-life of IRDye (16), thismeasurement at 3 days is likely lower than the actual payloaddelivery but significantly higher than the uptake at the IC50 in vitro(Fig. 2). These results are consistent with a rapid targeting of ADC

(�1 day) in perivascular cells at a higher concentration thanneeded for cell death. A large fraction of cells within the tumor(�90%) is not exposed to T-DM1 even after 48 to 72 hours whenmaximum payload delivery is achieved within the tumor. There-fore, a significant fraction of cells receives more drug than neededfor cell killing, while a large fraction of cells completely escapestherapy, lowering the overall tumor efficacy despite efficienttargeting (15% ID/g in these 300–400 mm3 tumors).

The fluorescently tagged ADC was used to image distributionduring tumor response (the nadir in the tumor growth curvesoccurs around 5–7 days; Fig. 3). The tumor distribution of bothtrastuzumab and T-DM1 5 days after injection shows both func-tional (CD31 vessels labeled with intravenous Hoechst) andnonfunctional vessels with signal. In addition, there are function-al vessels that do not have detectable ADC signal, indicating thatafter maximum uptake in the tumor is reached, there may be newvessels that form,whichwill not receive significant payload until ahigh plasma concentration is achieved with the next dose (every3 weeks in the case of T-DM1). The irregular, dynamic vasculaturehas important implications for ADC treatment when a significantfraction of cells is untargeted by ADC (Fig. 6). New vessels deliverboth oxygen/nutrients for survival and drugs for cell killing.However, the ADC requires approximately 1 day for uptake, 1 dayfor complete metabolism, and several days for cell killing, whileoxygen and nutrients can rescue the cells more quickly. Therefore,the dynamic vasculature within the tumor has the potential torepeatedly rescue untreated regions of the tumor even withcontinuous ADC in the plasma, which may stymie attempts tokill cells layer by layer with successive treatments.

Although our results demonstrate that ADC tumor distributionhas a significant role in efficacy, there remain several challenges toclinical implementation. First,matching the single-cell potency tosingle-cell delivery is challenging given that many targets aremeasured using IHC rather than a more quantitative methodcapable of reporting receptors/cell. Second, selecting the opti-mum potency could be challenging given intra- and interpatientvariability where one could perfectly "match" potency and deliv-ery of an ADC to a primary tumor but not metastasis withmuch higher or lower expression. It is unknown whether it isbetter to err on the side of higher potency (targeting fewer cellswith a higher dose thannecessary for cell killing, which could helpavoid mechanisms of drug resistance; ref. 45) or higher delivery(increased tumor penetration at a subtoxic dose). In this modelsystem, higher penetration appears to be more beneficial. Theuse of higher antibody doses to increase ADC penetration hasadditional potential benefits, such as maximizing other mechan-isms of action, including receptor signaling blockade and/orimmune cell interactions (46). Sacituzumab govitecan, an ADCthat has received Breakthrough Therapy designation from theFDA, takes this approach using a lower potency payload withmuch higher antibody doses (8–10 mg/kg, ClinicalTrials.govidentifier: NCT01631552; ref. 47). In addition, our modelingwork shows that healthy tissue with low target expression wouldhave less uptake when ADC and antibody are administeredtogether compared with ADC alone (15). Finally, imaging mayplay a useful role in identifying optimal treatment regimens (48).The ZEPHIR trial looks to combine pretreatment and early met-abolic responsemolecular imaging to select patients that respondbest to T-DM1 therapy (49). Combining molecular imaging andpharmacokinetic models (17) to determine the optimum anti-body/ADC dosage could provide an individualized treatment

Cilliers et al.





with potentially better outcome. Nonetheless, some patients thatare HER2 positive may not have trastuzumab uptake (49), whileother patients that have HER2-negative primary cancers may havemetastases that are HER positive (50), making individualizedtreatment challenging.

In conclusion, we have shown that improving tumor penetra-tion of a constant dose of T-DM1 by coadministration of trastu-zumab (in a trastuzumab-insensitive xenograft model) results insignificantly better efficacy than T-DM1 alone. Maximizing tumorpenetration of ADCs in addition to optimizing the antibody,linker, and payload during development may help improve theefficacy of future ADCs in the clinic.

Disclosure of Potential Conflicts of InterestG.M. Thurber reports receiving a commercial research grant from Eli Lilly.

No potential conflicts of interest were disclosed by the other authors.

DisclaimerThe content is solely the responsibility of the authors and does not neces-

sarily represent the official views of the NIH.

Authors' ContributionsConception and design: C. Cilliers, G.M. ThurberDevelopment of methodology: C. Cilliers, B. Menezes, G.M. Thurber

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Cilliers, I. NesslerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Cilliers, B. Menezes, G.M. ThurberWriting, review, and/or revision of the manuscript: C. Cilliers, B. Menezes,I. Nessler, J. Linderman, G.M. ThurberAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. CilliersStudy supervision: J. Linderman, G.M. Thurber

AcknowledgmentsWe would like to thank John Rhoden for helpful comments and Eli

Lilly for providing funding for the studies (to C. Cilliers, I. Nessler, andG.M. Thurber). Additional support was provided by R01-CA196018(to B. Menezes and J. Linderman) and a National Science FoundationGraduate Research Fellowship (to I. Nessler). Research reported in thispublication was supported by the NCI of the NIH under Award NumberP30CA046592 by the use of the following Cancer Center Shared Resource(s):histology.

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 1, 2017; revised October 18, 2017; accepted November 28,2017; published OnlineFirst December 7, 2017.

References1. Reichert JM. Antibodies to watch in 2016. mAbs 2016;8:197–204.2. Polakis P. Antibody drug conjugates for cancer therapy. Pharmacol Rev

2016;68:3–19.3. Chari RVJ. Expanding the reach of antibody–drug conjugates. ACS Med

Chem Lett 2016;7:974–76.4. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, et al. Site-

specific conjugation of a cytotoxic drug to an antibody improves thetherapeutic index. Nat Biotechnol 2008;26:925–32.

5. Donaghy H. Effects of antibody, drug and linker on the preclinicaland clinical toxicities of antibody-drug conjugates. mAbs 2016;8:659–71.

6. Bhatnagar S, Deschenes E, Liao J, Cilliers C, Thurber GM. Multichannelimaging toquantify four classes of pharmacokinetic distribution in tumors.J Pharm Sci 2014;103:3276–86.

7. Baker JHE, Lindquist KE,HuxhamLA, Kyle AH, Sy JT,MinchintonAI.Directvisualization of heterogeneous extravascular distribution of trastuzumabin human epidermal growth factor receptor type 2 overexpressing xeno-grafts. Clin Cancer Res 2008;14:2171–79.

8. Thurber GM, Schmidt MM, Wittrup KD. Antibody tumor penetration:Transport opposed by systemic and antigen-mediated clearance. Adv DrugDeliv Rev 2008;60:1421–34.

9. Rhoden JJ, Wittrup KD. Dose dependence of intratumoral perivasculardistribution of monoclonal antibodies. J Pharm Sci 2012;101:860–7.

10. Adams GP, Schier R, McCall AM, Simmons HH, Horak EM, Alpaugh RK,et al.High affinity restricts the localization and tumor penetration of single-chain Fv antibody molecules. Cancer Res 2001;61:4750–55.

11. Blumenthal RD, Fand I, Sharkey RM, Boerman OC, Kashi R, GoldenbergDM. The effect of antibody protein dose on the uniformity of tumordistribution of radioantibodies - an autoradiographic study. Cancer Immu-nol Immunother 1991;33:351–58.

12. Singh AP, Sharma S, Shah DK. Quantitative characterization of in vitrobystander effect of antibody-drug conjugates. J Pharmacokinet Pharma-codyn 2016;43:567–82.

13. Li F, Emmerton KK, Jonas M, Zhang X, Miyamoto JB, Setter JR, et al.Intracellular released payload influences potency and bystander-killingeffects of antibody-drug conjugates in preclinical models. Cancer Res2016;76:2710–19.

14. Kovtun YV, Audette CA, Ye Y, Xie H, Ruberti MF, Phinney SJ, et al.Antibody-drug conjugates designed to eradicate tumors with homoge-

neous and heterogeneous expression of the target antigen. Cancer Res2006;66:3214–21.

15. Cilliers C, GuoH, Liao J, ChristodoluN, Thurber GM.Multiscalemodelingof antibody-drug conjugates: connecting tissue and cellular distribution towhole animal pharmacokinetics and potential implications for efficacy.AAPS J 2016;18:1117–30.

16. Cilliers C, Liao J, Atangcho L, Thurber GM. Residualization rates of near-infrared dyes for the rational design of molecular imaging agents. MolImaging Biol 2015;17:757–62.

17. Cilliers C, Nessler I, Christodolu N, Thurber GM. Tracking Antibodydistribution with near-infrared fluorescent dyes: impact of dye struc-ture and degree of labeling on plasma clearance. Mol Pharm 2017;14:1623–33.

18. Williams S-P. Tissue distribution studies of protein therapeutics usingmolecular probes: molecular imaging. AAPS J 2012;14:389–99.

19. Urano Y, Asanuma D, Hama Y, Koyama Y, Barrett T, Kamiya M, et al.Selective molecular imaging of viable cancer cells with pH-activatablefluorescence probes. Nat Med 2009;15:104–9.

20. Cohen R, Vugts DJ, Stigter-van Walsum M, Visser GW, van Dongen GA.Inert coupling of IRDye800CW and zirconium-89 to monoclonal anti-bodies for single- or dual-mode fluorescence and PET imaging. Nat Protoc2013;8:1010–8.

21. Oliveira S, Cohen R,WalsumMS-v, vanDongenGA, Elias SG, vanDiest PJ,et al. A novel method to quantify IRDye800CW fluorescent antibodyprobes ex vivo in tissue distribution studies. EJNMMI Res 2012;2:50.

22. Zhang L, Thurber GM.Quantitative impact of plasma clearance and down-regulation on GLP-1 receptor molecular imaging. Mol Imaging Biol 2016;18:79–89.

23. Thurber GM, Zajic SC, Wittrup KD. Theoretic criteria for antibodypenetration into solid tumors and micrometastases. J Nucl Med2007;48:995–9.

24. Rudnick SI, Lou JL, Shaller CC, Tang Y, Klein-Szanto AJP, Weiner LM, et al.Influence of affinity and antigen internalization on the uptake and pen-etration of anti-HER2 antibodies in solid tumors. Cancer Research2011;71:2250–59.

25. Barok M, Tanner M, K€oninki K, Isola J. Trastuzumab-DM1 is highlyeffective in preclinical models of HER2-positive gastric cancer. Cancer Lett2011;306:171–79.






26. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al.Targeting HER2-positive breast cancer with trastuzumab-DM1, an anti-body-cytotoxic drug conjugate. Cancer Res 2008;68:9280–90.

27. Jackson D, Atkinson J, Guevara CI, Zhang C, Kery V, Moon SJ, et al. In vitroand in vivo evaluation of cysteine and site specific conjugated herceptinantibody-drug conjugates. PLoS One 2014;9:e83865.

28. Fujimoto-Ouchi K, Sekiguchi F, Yasuno H, Moriya Y, Mori K, Tanaka Y.Antitumor activity of trastuzumab in combination with chemotherapy inhuman gastric cancer xenograft models. Cancer Chemother Pharmacol2007;59:795–805.

29. Yamashita-Kashima Y, Iijima S, Yorozu K, Furugaki K, Kurasawa M, OhtaM, et al. Pertuzumab in combinationwith trastuzumab shows significantlyenhanced antitumor activity in HER2-positive human gastric cancer xeno-graft models. Clin Cancer Res 2011;17:5060–70.

30. Singh R, Kim WJ, Kim P-h, Hong HJ. Combined blockade of HER2 andVEGF exerts greater growth inhibition of HER2-overexpressing gastriccancer xenografts than individual blockade. Exp Mol Med 2013;45:e52–e52.

31. BarokM, Isola J, Palyi-KrekkZ,NagyP, Juhasz I, VerebG, et al. Trastuzumabcauses antibody-dependent cellular cytotoxicity-mediated growth inhibi-tion of submacroscopic JIMT-1 breast cancer xenografts despite intrinsicdrug resistance. Mol Cancer Ther 2007;6:2065–72.

32. Leyland-Jones B, Colomer R, Trudeau ME, Wardley A, Latreille J, CameronD, et al. Intensive loading dose of trastuzumab achieves higher-than-steady-state serum concentrations and is well tolerated. J Clin Oncol2010;28:960–6.

33. Ferl GZ, Kenanova V, Wu AM, DiStefano JJ. A two-tiered physiologicallybasedmodel for dually labeled single-chain Fv-Fc antibody fragments.MolCancer Ther 2006;5:1550–58.

34. Chang AJ, DeSilva R, Jain S, Lears K, Rogers B, Lapi S. 89Zr-radiolabeledtrastuzumab imaging in orthotopic and metastatic breast tumors. Phar-maceuticals 2012;5:79–93.

35. Thurber GM, Weissleder R. Quantitating antibody uptake in vivo: condi-tional dependence on antigen expression levels. Mol Imaging Biol 2011;13:623–32.

36. Oude Munnink TH, Dijkers EC, Netters SJ, Lub-de Hooge MN, BrouwersAH, Haasjes JG, et al. Trastuzumab pharmacokinetics influenced by extenthuman epidermal growth factor receptor 2–positive tumor load. J ClinOncol 2010;28:e355–e56.

37. Pabst M, McDowell W, Manin A, Kyle A, Camper N, De Juan E, et al.Modulation of drug-linker design to enhance in vivo potency ofhomogeneous antibody-drug conjugates. J Control Release 2017;253:160–64.

38. Hamblett KJ, Senter PD, Chace DF, Sun MMC, Lenox J, Cerveny CG, et al.Effects of drug loading on the antitumor activity of amonoclonal antibodydrug conjugate. Clin Cancer Res 2004;10:7063–70.

39. Hong EE, Erickson H, Lutz RJ, Whiteman KR, Jones G, Kovtun Y, et al.Design of coltuximab ravtansine, a CD19-targeting antibody-drugconjugate (ADC) for the treatment of B-cell malignancies: struc-ture-activity relationships and preclinical evaluation. Mol Pharm2015;12:1703–16.

40. de Goeij BE, Lambert JM. New developments for antibody-drugconjugate-based therapeutic approaches. Curr Opin Immunol 2016;40:14–23.

41. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, et al.Engineered thio-trastuzumab-DM1 conjugate with an improved therapeu-tic index to target human epidermal growth factor receptor 2-positivebreast cancer. Clin Cancer Res 2010;16:4769–78.

42. Sun X, Ponte JF, Yoder NC, Laleau R, Coccia J, Lanieri L, et al. Effects ofdrug–antibody ratio on pharmacokinetics, biodistribution, efficacy, andtolerability of antibody–maytansinoid conjugates. Bioconjug Chem 2017;28:1371–81.

43. Lyon RP, Bovee TD, Doronina SO, Burke PJ, Hunter JH, Neff-LaFord HD,et al. Reducing hydrophobicity of homogeneous antibody-drug conjugatesimproves pharmacokinetics and therapeutic index. Nat Biotechnol2015;33:733–36.

44. Reddy N, Ong GL, Behr TM, Sharkey RM, Goldenberg DM, Mattes MJ.Rapid blood clearance ofmouse IgG2a andhuman IgG1 inmanynude andnu/þ mouse strains is due to low IgG2a serum concentrations. CancerImmunol Immunother 1998;46:25–33.

45. Loganzo F, Sung M, Gerber H-P. Mechanisms of resistance to antibody–drug conjugates. Mol Cancer Ther 2016;15:2825–34.

46. M€uller P, Kreuzaler M, Khan T, Thommen DS, Martin K, Glatz K, et al.Trastuzumab emtansine (T-DM1) renders HER2þ breast cancerhighly susceptible to CTLA-4/PD-1 blockade. Sci Transl Med 2015;7:315ra188.

47. Starodub AN, Ocean AJ, ShahMA, GuarinoMJ, Picozzi VJ, Vahdat LT, et al.First-in-human trial of a novel anti-trop-2 antibody-SN-38 conjugate,sacituzumab govitecan, for the treatment of diverse metastatic solidtumors. Clin Cancer Res 2015;21:3870–78.

48. Williams S-P, Ogasawara A, Tinianow JN, Flores JE, Kan D, Lau J, et al.ImmunoPET helps predicting the efficacy of antibody-drug conjugatestargeting TENB2 and STEAP1. Oncotarget 2016;7:25103–12.

49. Gebhart G, Lamberts LE, Wimana Z, Garcia C, Emonts P, Ameye L, et al.Molecular imaging as a tool to investigate heterogeneity of advancedHER2-positive breast cancer and to predict patient outcome under trastu-zumab emtansine (T-DM1): The ZEPHIR trial. Ann Oncol 2016;27:619–24.

50. Ulaner GA, Hyman DM, Ross DS, Corben A, Chandarlapaty S, Goldfarb S,et al. Detection of HER2-positive metastases in patients with HER2-negative primary breast cancer using 89Zr-trastuzumab PET/CT. J NuclMed 2016;57:1523–28.


Cilliers et al.




2018;78:758-768. Published OnlineFirst December 7, 2017.Cancer Res Cornelius Cilliers, Bruna Menezes, Ian Nessler, et al. Drug Conjugates Increases Anticancer Efficacy and Host Survival−

Improved Tumor Penetration and Single-Cell Targeting of Antibody

Updated version

10.1158/0008-5472.CAN-17-1638doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2017/12/07/0008-5472.CAN-17-1638.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/78/3/758.full#ref-list-1

This article cites 50 articles, 18 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/78/3/758.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

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

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/3/758To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-1638

http://cancerres.aacrjournals.org/content/suppl/2017/12/07/0008-5472.CAN-17-1638.DC1

http://cancerres.aacrjournals.org/content/78/3/758.full#ref-list-1

http://cancerres.aacrjournals.org/content/78/3/758.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/78/3/758


Documents

Improved Tumor Penetration and Single-Cell ... · with T-DM1 improves efﬁcacy (tumor growth reduction and overall survival as measured by tumor volume endpoint) in a trastuzumab-insensitive