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Therapeutics, Targets, and Chemical Biology

Intracellular Released Payload Influences Potencyand Bystander-Killing Effects of Antibody-DrugConjugates in Preclinical ModelsFu Li1, Kim K. Emmerton1, Mechthild Jonas2, Xinqun Zhang3, Jamie B. Miyamoto1,Jocelyn R. Setter3, Nicole D. Nicholas4, Nicole M. Okeley3, Robert P. Lyon3,Dennis R. Benjamin2, and Che-Leung Law1

Abstract

Antibody-drug conjugates (ADC) comprise targeting anti-bodies armed with potent small-molecule payloads. ADCsdemonstrate specific cell killing in clinic, but the basis of theirantitumor activity is not fully understood. In this study, weinvestigated the degree to which payload release predicts ADCactivity in vitro and in vivo. ADCs were generated to targetdifferent receptors on the anaplastic large cell lymphoma lineL-82, but delivered the same cytotoxic payload (monomethylauristatin E, MMAE), and we found that the intracellularconcentration of released MMAE correlated with in vitroADC-mediated cytotoxicity independent of target expressionor drug:antibody ratios. Intratumoral MMAE concentrationsconsistently correlated with the extent of tumor growth inhi-bition in tumor xenograft models. In addition, we developed a

robust admixed tumor model consisting of CD30þ and CD30�

cancer cells to study how heterogeneity of target antigenexpression, a phenomenon often observed in cancer specimens,affects the treatment response. CD30-targeting ADC deliveringmembrane permeable MMAE or pyrrolobenzodiazepinedimers demonstrated potent bystander killing of neighboringCD30� cells. In contrast, a less membrane permeable payload,MMAF, failed to mediate bystander killing in vivo, suggestinglocal diffusion and distribution of released payloads representsa potential mechanism of ADC-mediated bystander killing.Collectively, our findings establish that the biophysical prop-erties and amount of released payloads are chief factors deter-mining the overall ADC potency and bystander killing. CancerRes; 76(9); 2710–9. �2016 AACR.

IntroductionAntibody-drug conjugates (ADC) are targeted therapies con-

sisting of three components: an antibody targeting a specifictumor antigen, a linker, and a potent payload. Recent advancesin these ADC technologies have resulted in two FDA-approveddrugs (ADCETRIS and KADCYLA) and more than 30 ADCs inclinical trials (1). The remarkable clinical response to ADCETRISvalidates the potential of ADCs as cancer drugs (2, 3).

Currently, payloads utilized in ADCs primarily include micro-tubule-disrupting agents [e.g., monomethyl auristatin E (MMAE)and maytansinoid-derived DM1 and DM4] and DNA-crosslink-ing agents [e.g., calichaemicin and pyrrolobenzodiazepines(PBD) dimers]. Although ADCs have demonstrated clinical andpreclinical activity, it has been unclear what factors determinesuch potency in addition to antigen expression on targeted tumorcells. For example, drug:antibody ratio (DAR), ADC-binding

affinity, potency of the payload, receptor expression level, inter-nalization rate, trafficking,multiple drug resistance (MDR) status,and other factors have all been implicated to influence theoutcome of ADC treatment in vitro (4–7). Thus, it is difficult tocompare activity of different ADCs across cell lines, due tomultiple variable parameters. Indeed, two studies found nocorrelation between cell surface CD22 expression and the activityof ADCs targeting CD22 (using either MMAE or DM1; refs. 8, 9),demonstrating the complexity of the multiple mechanisms con-tributing to ADC activity.

In addition to the direct killing of antigen-positive tumorcells, ADCs also have the capacity to kill adjacent antigen-negative tumor cells: the so-called "bystander killing" effect(10). In vitro, this capacity has been observed in colony assaysand coculture systems. For example, huC242-DM1 ADCs werefound to mediate bystander killing using a colony cocultureassay in vitro and an admixed tumor model in vivo (11).Similarly, intracellular released MMAE was reported to mediatebystander killing in a coculture system (12). Recently, Breij andcolleagues suggested such bystander activity is relevant inpatient-derived xenograft models (13). Although these studiesprovide examples of bystander killing, the underlying factorscontrolling this property remain to be defined. Here, we studiedtwo key parameters of ADCs: (i) the relationship between theconcentration of the released payload and ADC potency in vitroand in vivo and (ii) the relationship between the membranepermeability of the released payload and the extent of ADCbystander killing. By targeting different receptors capable ofinternalization and lysosomal trafficking on L-82 cancer cells

1Preclinical Science, Seattle Genetics, Inc., Bothell, Washington.2Translational Research, Seattle Genetics, Inc., Bothell, Washington.3Chemistry, Seattle Genetics, Inc., Bothell,Washington. 4ExperimentalPharmacology, Seattle Genetics, Inc., Bothell,Washington.

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

CorrespondingAuthor: Fu Li, Seattle Genetics, Inc., 21823 30th Drive Southeast,Bothell, WA 98021. Phone: 425-527-4626; Fax: 425-527-4609; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-1795

�2016 American Association for Cancer Research.

CancerResearch

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using ADCs with various drug:antibody ratios (DAR), we deter-mined how the concentration of the released MMAE correlatedwith in vitro cytotoxic response and in vivo antitumor activity.We found that intratumoral MMAE concentration correlatedwith antigen-specific antitumor activity in vivo. Furthermore, weestablished a robust admixed tumor model consisting ofCD30þ Karpas 299 cells and CD30�, cAC10-vcMMAE–resistantKarpas-35R cells, in which we observed potent bystander killingof CD30� cells with ADCs containing MMAE or PBD dimers.The roles of linker chemistry and the biophysical nature of thepayload in this bystander killing were also studied.

Materials and MethodsCell culture

Karpas 299 and L-82 cells were obtained from Dr. AbrahamKarpas of the University of Cambridge and from the GermanCollection of Microorganisms and cultures (DSMZ). The Karpas-35R cell linewasderived fromKarpas 299by continuous exposureto cAC10-vcMMAE, and they were confirmed as derivative ofKarpas 299 using SNP fingerprint analysis by the ATCC. Cellswere grown inRPMI1640media (Invitrogen) containing 10%FBSand 100 U/mL Penicillin and Streptomycin in a 37�C humidifiedincubator with 5% CO2. For cytotoxicity assays, cells were platedat an average density of 7,000 to 10,000 per well in 96-well tissueculture plates and incubated with ADCs for 72 hours or 96 hours.Each assay was performed in triplicate. Cell viability was deter-mined using CellTiter-Glo (Promega), and IC50 values werecalculated using GraphPad Prism.

In vivo antitumor activity studyAll animal studies were conducted following Institutional

Animal Care andUseCommittee protocols. For xenograft studies,5 million Karpas 299 or Karpas-35R cells were implanted subcu-taneously into SCID mice (Harlan). The admixed tumor modelwas implanted with a mixture containing 2.5 million Karpas 299and 2.5 million Karpas-35R cells. To titrate CD30þ cells inadmixed tumor model, a total of 5 million cells were implantedin each mouse, whereas the number of Karpas 299 and Karpas-35R cellswas adjusted to achieve the ratios of 10%, 25%, and 50%CD30þ Karpas 299 cells. One million L-82 cells were implantedsubcutaneously to NOD SCID IL2Rgammanull (NSG) mice forxenograft studies (Jackson Laboratory). Treatment was initiatedwhen the average tumor size reached at least 100 mm3 for tumorefficacy studies. Tomeasure releasedpayload in tumors, treatmentwas initiated at 250 to 400 mm3. Tumors were measured twice aweek, and the volume was calculated with the formula volume¼1/2 � length � width � width.

Flow cytometryReceptor expression on Karpas 299, Karpas-35R, and L-82 cells

was quantified using QIFIKit following the manufacturer'sinstructions (Dako). Themonoclonal antibodies used tomeasureCD30, CD70, and CD71 surface expression were BerH2 (BDBiosciences), h1F6 (14), and chimeric OKT9. Chimeric OKT9was developed from mouse hybridoma cell line OKT9 (ATCC).Flow cytometry ofCD30onKarpas-35R cells and admixed tumorswas performed using mouse monoclonal antibody BerH2 con-jugatedwith R-phycoerythrin or isotype control (BDBiosciences),and CD45 conjugated with allophycocyanin (BD Biosciences).Stained cells were analyzed on FACS Calibur (BD).

ImmunohistochemistryTumors were fixed in 10% neutral buffered formalin, embed-

ded in paraffin, and sections were cut at 4-mm thickness. Stainingincluding de-paraffinization and heat-induced target retrieval inthe presence of EDTA was done on a Bond autostainer (LeicaBiosystems). Bond autostainer reagents were used for antibodydetection, nuclear counterstain, and buffer rinses. The followingprimary antibodies were applied: rabbit anti-CD71 polyclonalantibody (Proteintech Group) and mouse monoclonal anti-CD30 Clone BerH2 (Dako). For detection, horseradish peroxi-dase-3,30-diaminobenzidine (HRP-DAB) was used for CD30 IHC(Bond Polymer Refine DAB Kit), and alkaline phosphatase-fastred was used for CD71 (Bond Polymer Refine Red Kit). Hema-toxylin was used to counter stain nuclei. Images were acquired onan Olympus BX41 microscope equipped with a Nikon DS-Fi1camera.

Antibody-drug conjugationThe syntheses of MMAE, MMAF, and PBD dimers have been

described previously (6, 15, 16). ADCs were prepared usingvaline-citrulline (vc)–PAB linkers with an average drug loadingof 4 (15), unless specified otherwise. The glucuronide linker andPBD linker conjugation has been described elsewhere (16, 17).

Intracellular and intratumoral drug measurement by LC-MSCell pellets were collected 24 hours after ADC treatment. Cell

count, diameter, and circularity were determined on Vi-CellCounter (BeckmanCoulter).MMAEextraction andquantificationmethod was identical to the protocol described in Okeley andcolleagues (12). Briefly, tumors or cell pellets were homogenizedwith methanol and acetonitrile containing internal standard (d8-MMAE forMMAEdetection and 13C-MMAF forMMAFdetection).The homogenates were centrifuged at 10,000 rpm to precipitateprotein and protein-bound payloads. The supernatant was thensubjected to solid phase extraction, and signals of MMAE andMMAF were detected by LC-MS.

Measurement of ADCs in plasmaMicro-titer plates were coated overnight with a murine IgG1

mAbwith specificity to human light chain kappa (Jackson Immu-noResearch), or an anti-idiotype mAb that binds to cAC10.Samples were diluted into the dynamic range of the assay withna€�ve mouse plasma. After a 1:40 dilution into assay diluent,standards, controls, and samples were incubated on the coatedand blocked plates. Bound ADCs were detected with HRP-con-jugated goat polyclonal antibody reagent with specificity to the Fcregion of human IgG (Jackson ImmunoResearch). After incuba-tion and subsequent washing, 3,30,5,50 tetramethylbenzidine wasapplied to the wells and the signal measured by absorbance atOD450 nm–OD630 nm.

ResultsThe intracellular concentration of released MMAE drives ADCcytotoxicity and potency in vitro

It has been shown that ADCs bind to tumor cells in vitro andrelease payload inside cancer cells upon trafficking to the appro-priate subcellular organelles, e.g., lysosomes (12, 18, 19). Sincethe released payload is the active component of the ADC, wesought to evaluate whether the amount of released payload iscorrelated with ADC activity by targeting the anaplastic large cell

Released Payload Impacts ADC Activity and Bystander Killing

www.aacrjournals.org Cancer Res; 76(9) May 1, 2016 2711




lymphoma (ALCL) cell line L-82, which expresses CD30, CD70,and CD71 (transferrin receptor) simultaneously (Fig. 1A;ref. 20). An in vitro cytotoxicity assay of ADCs targeting CD30(cAC10-vcMMAE, with a DAR of 2, 4, or 8), CD70 (h1F6-vcMMAE with a DAR of 4), or CD71 (cOKT9-vcMMAE with aDAR of 4) was used to determine the in vitro potency of theseADCs. Their IC50 values varied from 2 ng/mL to 55 ng/mL,which may reflect the variance of antigen expression level,internalization rate, and DAR (Fig. 1B), consistent with ourprevious report (21).

We then assessed how much MMAE was released inside L-82cells after the treatment with these five ADCs at their respectiveIC50 concentrations. Interestingly, the intracellular MMAE con-centrations from these ADCs were similar, ranging between 98nmol/L to 150 nmol/L, a variance of only approximately 1.5-foldin contrast with the >20-fold difference in their IC50 values (Fig.1C). These results indicate that the concentration of released

MMAE determines the extent of cell killing in vitro, independentof antigen expression level or DAR.

The intratumoral concentration of MMAE correlates with ADCantitumor activity in vivo

We then evaluated whether released MMAE correlates withADC potency in vivo. To minimize the effects of drug loading onADCpharmacokinetics in vivo (21),weonly testedADCswith fourMMAE molecules per antibody, conjugated to cAC10, cOKT9, orh1F6. After a single dose of cAC10-vcMMAE treatment, L-82tumors either had no response (0.5 mg/kg), growth delay (1mg/kg), or complete remission (3 mg/kg). Similarly, cOKT9-vcMMAE and h1F6-vcMMAE demonstrated dose-dependent anti-tumor response in the L-82 xenografts (Fig. 2A). Interestingly,despite their different in vitro potencies (Fig. 1A and B), L-82xenografts responded similarly to cAC10-vcMMAE, h1F6-vcMMAE, and cOKT9-vcMMAE (Fig. 2A).

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Figure 1.Intracellular MMAE correlates withADC potency in L-82 cells in vitro. A,receptor expression of CD30, CD70,and CD71 on the surface of L-82 cells,as determined by quantitative flowcytometry. cAC10, h1F6, and cOKT9are monoclonal antibodies used toconjugate ADCs. B, in vitrocytotoxicity assay of ADCs withvarying antibody or DAR in L-82cells. Samples were analyzed inquadruplicates 72 hours after ADCtreatment. Numbers in parenthesisstand for drug loading ratio. C,intracellular MMAE concentrationmeasurement in L-82 cells 24 hoursafter treatment with ADCs at theirrespective IC50 concentrations (n¼ 2).Data represent mean and SD. mAb,monoclonal antibody.

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Cancer Res; 76(9) May 1, 2016 Cancer Research2712




We also used LC-MS tomeasure the concentration of MMAE ina parallel cohort of L-82 tumors with an identical treatmentregimen. Although tumor volume was not different among treat-ment groups 3 days after dose, the intratumoral MMAE measure-ment revealed two patterns (Fig. 2B). First, intratumoral MMAEconcentration increased proportionally to the ADC dose, whichcorresponded to stronger antitumor activity. Second, the intratu-moral MMAE concentration obtained from treatment with bothcOKT9-vcMMAE and cAC10-vcMMAE was similar at each dose,consistent with the observation that tumor responded similarly tothese two ADCs (Fig. 2A). These results indicate that the intratu-moral concentration of MMAE determines the antitumor activityof ADCs in vivo, independent of antigen expression level, as wasthe case for in vitro treatments.

Intratumoral MMAE correlates with antigen-specific antitumoractivity in Karpas 299 xenograft

To directly determine how released MMAE correlates withADC potency in vivo, we treated CD30þ, ALCL cell line Karpas

299 tumor-bearing SCID mice with cAC10-vcMMAE, or non-binding IgG-vcMMAE, followed by measurement of plasmaADC, plasma MMAE, as well as intratumoral released MMAEconcentration. A single dose of 2 mg/kg cAC10-vcMMAE treat-ment resulted in tumor regression within 10 days, whereas IgG-vcMMAE had no impact on tumor growth (Fig. 3A). Thisobservation is consistent with our previous report of cAC10-vcMMAE–specific antitumor activity in Karpas 299 cell model(22). Concordantly, intratumoral MMAE concentration mea-surement revealed a clear separation between cAC10-vcMMAEand IgG-vcMMAE–treated animals (Fig. 3B). Approximately300 nmol/L intratumoral MMAE was detected in tumors 3days after cAC10-vcMMAE treatment. In contrast, the highestconcentration was only at 50 nmol/L in the group received IgG-vcMMAE treatment. Overall, cAC10-vcMMAE yielded 5-foldgreater MMAE exposure than IgG-vcMMAE in tumors (areaunder curve: 1,628 vs. 328 day.nmol/L).

Neither the pharmacokinetics of ADCs or small-moleculeMMAE in plasma correlated with antigen-specific antitumor

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Figure 2.Intratumoral MMAE concentrationcorrelates with ADC antitumoractivity in L-82 xenograft model.A, tumor growth after treatment ofcAC10-vcMMAE, cOKT-vcMMAE, orh1F6-vcMMAE (0.5, 1, or 3 mg/kg;once i.p.). ADCswere conjugatedwithaverage DAR of 4. n¼ 5 per group. B,intratumoral MMAE concentrationmeasurement by LC/MS in tumorstreated with cAC10-vcMMAE, cOKT-vcMMAE, or h1F6-vcMMAE. Tumorswere collected 72 hours aftertreatment. n ¼ 3 tumor-bearingmice per group. Data representmean þ SEM.






activity. The profiles of cAC10-vcMMAE and IgG-vcMMAE weresimilar in plasma, with an average half-life of 6.7 days for cAC10-vcMMAE (Supplementary Fig. S1A). The plasma concentration ofMMAE in both treated groups was around 2 nmol/L at 4 hoursafter dose, but cleared quickly in circulation (Supplementary Fig.S1B). These data collectively show: (i) targeted ADCs deliver ahigh concentration of MMAE to tumor, (ii) the amount ofintratumoral released MMAE correlates with ADC antitumoractivity, and (iii) the amounts of released MMAE determined theextent of cytotoxicity in vitro and in vivo.

Development of admixed tumor model with heterogeneousCD30 expression

We have recently developed Karpas-35R cells (resistant tocAC10-vcMMAE treatment) from the Karpas 299 cell line throughcontinuous exposure to SGN-35 (23). This derivative cell line haslost surface CD30 expression but has retained CD71 expression asconfirmed by flow cytometric analysis (Supplementary Fig. S2A).The absence of CD30 prevented cell killing by cAC10-vcMMAE orcAC10-vcMMAF (IC50 > 5 mg/mL) in vitro. However, Karpas-35Rcells remain sensitive to CD71 binding ADCs such as cOKT9-vcMMAE and cOKT9-vcMMAF (IC50 1�4 ng/mL; Fig. 4A). In vivo,Karpas-35R tumors and Karpas 299 tumors grew at similar rateswhen implanted subcutaneously in SCID mice. Karpas-35R cellsremained CD30 negative and hence were insensitive to cAC10-vcMMAE treatment in vivo, while they were eradicated by cOKT9-vcMMAE (Supplementary Fig. S2B).

These results prompted us to mix Karpas 299 and Karpas-35Rcells to develop a tumor model consisting of both CD30þ andCD30� cells. A 1:1 ratio mixture of Karpas 299 and Karpas-35Rcells grew together at a similar rate to those tumors from indi-vidual cell lines (Fig. 4B). IHC staining revealed a heterogeneousCD30 expression pattern in the admixed tumors, unlike thehomogeneous CD30 expression in Karpas 299 tumors or absenceof CD30 in Karpas-35R tumors (Fig. 4C). Importantly, theCD30þ

and CD30� cells formed an interspersed pattern, resembling the

distribution seen in analyses of human tumors (24). IHC analysisalso confirmed comparable CD71 expression in Karpas 299,Karpas-35R, and the admixed tumors (Fig. 4C). FACS analysisalso demonstrated coexistence of CD30þ and CD30� cells inadmixed tumors (Supplementary Fig. S3A)

MMAE, but not MMAF, mediates bystander killing in vivoWe then studied the in vivo bystander killing potential of two

potent auristatin payloads, MMAE and MMAF, in this admixedtumor model. MMAE and MMAF are structurally similar, andcAC10-vcMMAE and cAC10-vcMMAF were both potently cyto-toxic to Karpas 299 cells (Fig. 4A). However, MMAF is a morehydrophilic molecule and lessmembrane permeable thanMMAE(6). In general, MMAF has a higher IC50 than MMAE in cell lines(Fig. 5A), consistent with its lower membrane permeability.

After cAC10-vcMMAE (3 mg/kg, once i.p.) treatment, theadmixed tumors underwent complete remission within 1 weekafter dose. Three of the five tumors remained in remission for 4weeks. In contrast, cAC10-vcMMAF caused only a moderategrowth delay but no complete remissions (Fig. 5B). Nontargetingcontrols IgG-vcMMAE or IgG-vcMMAF did not affect tumorgrowth of the admixed tumors, whereas positive controls(cOKT9-vcMMAE and cOKT9-vcMMAF) targeting CD71 led tocomplete remissions (Fig. 5B). This experiment indicated thatcomplete tumor remission could result from bystander killing ofMMAE.

IHC analysis revealed that nonbinding control-treated tumorsconsist of bothCD30þ andCD30� cells, presumably because theydo not kill either CD30þ or CD30�Karpas 299 cells. Only CD30�

cells were found in cAC10-vcMMAF–treated tumors, illustratingthat cAC10-vcMMAF eliminated most CD30þ cells (Fig. 5C).Interestingly, the two tumors that relapsed from cAC10-vcMMAEtreatment were also found to be CD30� by the end of study,indicating a small fraction of CD30� cells might have escapedfrom bystander killing in these two remaining tumors (Fig. 5C).We also performed LC-MS analysis of admixed tumors treated

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Figure 3.Intratumoral MMAE correlates with cAC10-vcMMAE–specific antitumor activity in Karpas 299 xenograft model. A, Karpas 299 tumor response to ADC treatment ofcAC10-vcMMAE or nonbinding control of IgG-vcMMAE (2 mg/kg, once i.p. n ¼ 8). B, intratumoral MMAE released from cAC10-vcMMAE or IgG-vcMMAE inKarpas 299 tumors at 4 hour, 1 day, 3 day, and 10 days after dose. n ¼ 3 independent tumors per time points. Data represent mean þ SEM. The complete set ofmeasurements was repeated in an independent experiment.

Li et al.





with cAC10-vcMMAE or cAC10-vcMMAF and found the ADCsreleased comparable amount of intratumoral drug (360 nmol/LMMAE vs. 200 nmol/L MMAF; Supplementary Table S1). Con-tinuous tumor growth in the cAC10-vcMMAF treatment groupwas therefore likely because releasedMMAF could not diffuse intoneighboring CD30� cells. Collectively, these results demonstrateintratumoral releasedMMAE, but not MMAF, canmediate potentbystander killing in vivo.

Conjugation of MMAE using a glucuronide linker maintainsbystander killing in vivo

Encouraged by the result that the admixed tumor model candifferentiate a payload's bystander killing property, we then evalu-ated the role of the linkers in bystander killing by comparingMMAEconjugated to cAC10 using either peptide (cAC10-vcMMAE) orb-glucuronide (cAC10-glucMMAE; Fig. 6A; ref. 17) linkers. Com-plete remission of the admixed tumors was observed after a singledose of cAC10-vcMMAE or cAC10-glucMMAE (Fig. 6B). As a con-trol, intratumoral MMAEmeasurement confirmed 320 nmol/L and490 nmol/LMMAEwas released from cAC10-vcMMAE and cAC10-glucMMAE, respectively (Supplementary Table S2). These resultssuggest that both of the cleavable linkers are releasing MMAE andcan mediate bystander killing in vivo.

Potent bystander killing activity of PBD conjugates in vivoThe DNA cross-linking PBD dimers have empowered ADCs to

overcome the multiple drug resistance phenotype observed inacute myeloid leukemia (5, 16). Based on their membrane per-

meable nature in vitro (5), we hypothesized that PBDs may alsohave bystander killing potential. We engineered the cAC10 anti-body to enable site-specific conjugation of twoPBDmolecules perantibody by replacing the heavy chain serine residue at position239 with a cysteine (cAC10ec-PBD; Fig. 6C; ref. 5). Remarkably,cAC10ec-PBD treatment (0.1 mg/kg, once i.p.) resulted in com-plete remission of the admixed tumors in 5 of 5 (100%) animals(Fig. 6D). In contrast, nonbinding IgGec-PBDdidnot delay tumorgrowth. These data demonstrate that PBD-basedADCs can initiatepotent local bystander killing in vivo.

Antigen expression for PBD and MMAE-mediated bystanderkilling

The high potency of a PBD payload suggested that it mayrequire fewer antigen-positive cells to enable bystander killingactivity than MMAE. To test this, admixed tumors were generatedwith a titration comprising Karpas 299 cells from10% to 25%and50%. CD30 IHC of the resulting admixed tumors (200 mm3)confirmed a corresponding increase in CD30þ cell percentage(Fig. 7A). Flow cytometric analysis of dissociated tumors alsoconfirmed increasing CD30þ cell percentage in the admixedtumors (34%, 61% and 69% CD30þ cells, respectively; Fig. 7Band Supplementary Table S3). Interestingly, tumors consisting of69% and 61%CD30þKarpas 299 cells underwent remission aftercAC10-vcMMAE or cAC10ec-PBD treatment. However, tumorsconsisting of 34% Karpas 299 cells regressed subsequent tocAC10ec-PBD treatment, whereas cAC10-vcMMAE only causeda growth delay (Fig. 7C–E). These results argue that PBD ADCs

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Figure 4.Development of an admixed tumormodel with heterogeneous CD30expression. A, CD30 and CD71receptor expression in Karpas 299and Karpas-35R cells, determined byquantitative flow cytometry, andin vitro IC50 of ADCs targeting CD30or CD71 in these two cell lines,determined by a 96-hour CellTiter-Gloassay. B, tumor growth of Karpas 299,Karpas-35R, or 1:1 admixed of the twotypes of tumor cells in SCID animals.n ¼ 5 per group. C, IHC staining ofCD30 (brown) and CD71 (red) inxenograft tumors from Karpas 299,Karpas-35R, or 1:1 admixed tumors.Tumors were retrieved whensacrificed. Scale bar, 50 mm.






may require fewer CD30þ cells to mediate bystander killing invivo, presumably due to its superior potency.

DiscussionWith recent approval of ADCETRIS and KADCYLA, ADCs have

become major players in oncology drug development pipelines,as a result of the advancement in areas such as antigen selection,linker chemistry, and payload optimization (25). However, thechallenge remains to select the appropriate cancer target, payload,and linker to identify lead ADC candidates, mainly due to theincomplete understanding ofwhat factors drive of ADC activity inthe tumor microenvironment.

Released drug and ADC activity correlationHere, we studied the role of released payload within the L-82

cell line by alternating target antigen and drug loading. Thisallowed us to compare ADC activity of multiple ADCs withoutother variables such as drug sensitivity and efflux status, whichmay have confounded studies comparing multiple cell lines (8,9). While our data suggest that antigen expression and DAR canaffect ADC potency, these factors are not restrictive. In contrast,intracellular released payload appears to determine ADC potencyin vitro. Importantly, the amount of released MMAE also deter-mines the antitumor activity of ADC in vivo, independent ofantigen expression, suggesting the ADC design shall aim toimprove intratumoral payload delivery. For example, a recentconjugation method allowing higher drug antibody ratio mayhelp deliver more drug to tumors (26).

It has been challenging to predict pharmacodynamicsresponses using traditional pharmacokinetic parameters such astotal antibody and small-molecule concentrations (27, 28). Ourparallel measurement of plasma ADC, plasma MMAE, and intra-tumoral released MMAE suggests that intratumoral releasedMMAE concentration correlates best with antitumor activity inxenograft models. Although this observation is encouraging,correlation between the intratumoral released payload concen-trations and the clinical response to ADCs in patients remains tobe tested.

Modeling bystander killing in vivoPart of the remarkable clinical response to cAC10-vcMMAE has

been attributed to thebystander killing effect of ADCs (2, 29). Thiscapacity may distinguish ADCs from each other in solid cancers,where target heterogeneity is common (11, 30). There is anabsolute requirement for target expression in order to obtainantitumor activity. Therefore, several studies have tried to under-standbystander killing in vitro and in vivo (11–13, 30, 31).While invitro studies are informative, they often lack the close cell-to-cellcontact, which may limit the study of the phenotype. Admixedtumor models mimicking heterogeneous expression have beenreported (11, 13). However, we have discovered that mixing twodistinct cell types, one expressing the target of interest and theother does not, often led to outgrowth of a single-cell populationor spatial segregation of antigen-positive and -negative cells asreported in other studies [data not shown (11)]. Here, we takeadvantage of the Karpas-35R cells, which are essentially syngeneicas Karpas 299 cells except for the CD30 expression. This can be

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Figure 5.In vivo bystander killing of MMAE. A, chemical structures of MMAE andMMAF. Note that MMAF is a chargedmolecule and hence has a negative log D value. IC50 valuewas determined in Karpas 299 cells using 96-hour CellTiter-Glo assay. B, response of admixed tumors to treatment of cAC10-vcMMAE, IgG-vcMMAE,cOKT9-vcMMAE, cAC10-vcMMAF, IgG-vcMMAF, or cOKT9-vcMMAF. All ADCs were dosed 3mg/kg, once i.p. (n¼ 5), when tumor volumes reached about 100 mm3.C, IHC analysis of CD30 (brown) in tumors retrieved from ADC treatment. Note the cAC10-vcMMAE–treated tumors were not available until 35 days afterimplant. Scale bar, 50 mm.

Li et al.





critical to develop admixed tumor models. Recently, a relatedapproach employing HT-26 cells engineered to overexpressmesothelin was used to study the bystander killing of BAY 94-9343, an anti-mesothelin maytansine ADC (31). Based on theseobservations, we propose these criteria for robust bystanderkilling models: (i) the targeted cell line expresses the antigen ofinterest in their cell surface; (ii) the bystander cell line lacks thetargeted antigen and is insensitive to the testing ADC; (iii) testingdoses are within the specific range where nonbinding ADCs arenot active on either cell line; and (iv) the target-positive and target-negative cells are interspersed throughout the tumor.

The role of linker, payload, and target cells in bystander killingIntratumoral MMAE released from cAC10-vcMMAE kills both

CD30þ cells and CD30� cells in vivo, whereas another auristatinpayload, MMAF, lacked such capacity. These data argue thatmembrane permeability and the ability of the released payloadto diffuse through the tumor are required for bystander killing. Inlinewith this argument, themembrane permeable PBDdimerwasalso found tomediate bystander killing. Therefore, it is importantto evaluate engineered payload for their membrane permeabilityand bystander killing.

The role of linkers in bystander killing has been unclear, partlydue to introduction of "non-cleavable" linkers. For example, it hasbeen reported that a noncleavable linkermay abolish the bystand-er killing capacity of DM1 (11). However, it is important to notethat this noncleavable linker releases a modified payload (e.g., T-

DM1 releases lysine-MCC-DM1 instead of DM1; ref. 32); themembranepermeability andpotencyof lysine-MCC-DM1maybedrastically different from that of DM1. In the present study, wefound two different linkers (b-glucuronide and vc-PAB), bothrelease MMAE and mediate bystander killing similarly in vivo.These results demonstrate that the bystander killing property ismainly determined by the biophysical properties of the releasedpayload, as opposed to the specific details of the linker and themechanism of drug release.

The role of antigen-positive cancer cells in bystander killing isalso evaluated in this study. As expected, a higher percentage oftarget-positive cells in admixed tumors leads tomore pronouncedtumor regression. Interestingly, the extent of bystander killing canbe related to payload potency. Our results suggest that PBD-basedADCs can mediate bystander killing in admixed tumors with aslittle as 34% antigen-positive cells while MMAE-based ADCsrequired higher percentages. Hence, the abundance of antigen-positive cell required for bystander killing may vary according thepayload potency.

Thesefindings collectively argue that the locally releasedpayloadfrom the ADC determines the activity of killing of tumor cells andbystander cells. This is particularly important for selecting appro-priate payload for novel tumor antigens. Heterogeneity of antigenexpression often presents obstacle for ADC development in solidtumors; therefore, a membrane permeable payload may providemaximal killing of tumor cells (11). In contrast, when antigenexpression is homogeneous on cancer cells (e.g., CD19 andCD79b

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Figure 6.Role of linker and payload in bystander killing. A, chemical structures of cAC10-vcMMAE and cAC10-gluc-MMAE, where mAb stands for cAC10 in this experiment.B, growth of admixed tumors after the treatment of cAC10-vcMMAE and cAC10-gluc-MMAE (3 mg/kg, once i.p., n ¼ 5). Tumor volumes were about100mm3when animalswere treated. C, structure of val-ala PBD conjugated to antibody. D, growthof admixed tumors after treatmentwith cAC10-vcMMAE (3mg/kgonce i.p., n ¼ 5), cAC10ec-PBD, or IgGec-PBD (0.1 mg/kg, once i.p., n ¼ 5). PBD was conjugated to antibodies with an average DAR of 2. Tumor volumeswere about 200 mm3 at the beginning of study.






expression in non-Hodgkin lymphoma; ref. 33), a potent andnonpermeable drug linker may provide a higher therapeutic index(4). All together, these criteria can guide the design and selection ofappropriate ADC candidates for clinical development.

Disclosure of Potential Conflicts of InterestN.M. Okeley is Associate Director and has ownership interest (including

patents) in Seattle Genetics. Robert P. Lyon is Sr. Director at Seattle Genetics. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: F. Li, N.M. Okeley, R.P. Lyon, C.-L. LawDevelopment of methodology: F. Li, X. Zhang, C.-L. LawAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Li, K.K. Emmerton, M. Jonas, J.B. Miyamoto,J.R. Setter, N.D. NicholasAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Li, J.B. Miyamoto, D.R. Benjamin, C.-L. LawWriting, review, and/or revision of the manuscript: F. Li, J.B. Miyamoto,N.M. Okeley, R.P. Lyon, D.R. Benjamin, C.-L. Law

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Figure 7.Role of antigen expression in bystander killing. A, CD30 IHC analysis of tumors implanted with 0, 10%, 25%, 50%, or 100% CD30þ Karpas 299 cells (left to right).Tumors were collected when they reached approximately 200 mm3, immediately prior to ADC treatment. Scale bar, 100 mm. Two tumors from eachgroupwere analyzed by CD30 IHC. B, flow cytometry analysis of CD30 and CD45 of tumor retrieved at the same time as A. C–E, tumor growth after the treatment ofcAC10-vcMMAE (3 mg/kg, once i.p.), cAC10ec-PBD, or IgGec-PBD (0.1 mg/kg, once i.p.; n ¼ 5 per group).

Li et al.





Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.B. Miyamoto, N.M. OkeleyStudy supervision: F. Li, C.-L. Law

AcknowledgmentsThe authors thank David Meyer for help with conjugation and Dana Chace

for generating SGN-35–resistant Karpas-35R cell line. They appreciate discus-sion with many colleagues at Seattle Genetics, including Drs. Svetlana Dor-onina, Peter Senter, and Jonathan Drachman.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 2, 2015; revised January 8, 2016; accepted February 13, 2016;published OnlineFirst February 26, 2016.

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2016;76:2710-2719. Published OnlineFirst February 26, 2016.Cancer Res Fu Li, Kim K. Emmerton, Mechthild Jonas, et al. Preclinical ModelsBystander-Killing Effects of Antibody-Drug Conjugates in Intracellular Released Payload Influences Potency and

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