CD20-TCB with Obinutuzumab Pretreatment as Next-Generation ... · CD20-TCB was ﬂuorescently labeled using Alexa Fluor 647 (white signal). B, Quantiﬁcation of T-cell speed, synapse

Translational Cancer Mechanisms and Therapy

CD20-TCB with Obinutuzumab Pretreatment asNext-Generation Treatment of HematologicMalignanciesMarina Bacac1, Sara Colombetti1, Sylvia Herter1, Johannes Sam1, Mario Perro1,Stanford Chen1, Roberta Bianchi1, Marine Richard1, Anne Schoenle1, Valeria Nicolini1,Sarah Diggelmann1, Florian Limani1, Ramona Schlenker1, Tamara H€usser1,Wolfgang Richter2, Katharine Bray-French2, Heather Hinton1, Anna Maria Giusti1,Anne Freimoser-Grundschober1, Laurent Lariviere3, Christiane Neumann1,Christian Klein1, and Pablo Uma~na1

Abstract

Purpose: Despite promising clinical activity, T-cell–engag-ing therapies including T-cell bispecific antibodies (TCB) areassociated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present anext-generation CD20-targeting TCB (CD20-TCB) with signif-icantly higher potency and a novel approach enabling saferadministration of such potent drug.

Experimental Design: We developed CD20-TCB based onthe 2:1 TCB molecular format and characterized its activitypreclinically. We also applied a single administration of obi-nutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche)prior to the first infusion of CD20-TCB as a way to safelyadminister such a potent drug.

Results: CD20-TCB is associated with a long half-life andhigh potency enabled by high-avidity bivalent binding toCD20 and head-to-tail orientation of B- and T-cell–bindingdomains in a 2:1 molecular format. CD20-TCB displays

considerably higher potency than other CD20-TCB antibo-dies in clinical development and is efficacious on tumorcells expressing low levels of CD20. CD20-TCB also displayspotent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumorsof aggressive lymphoma models. Gpt enables profoundB-cell depletion in peripheral blood and secondary lym-phoid organs and reduces T-cell activation and cytokinerelease in the peripheral blood, thus increasing the safety ofCD20-TCB administration. Gpt is more efficacious and saferthan SUD.

Conclusions: CD20-TCB and Gpt represent a potent andsafer approach for treatment of lymphoma patients and arecurrently being evaluated in phase I, multicenter study inpatients with relapsed/refractory non-Hodgkin lymphoma(NCT03075696). Clin Cancer Res; 24(19); 4785–97. �2018 AACR.

See related commentary by Prakash and Diefenbach, p. 4631

IntroductionThe introduction of rituximab over 20 years ago marked the

beginning of a new era in the targeted treatment of B-cell non-Hodgkin lymphomas (NHL). Its combination with chemother-apy (R-CHOP) remains a standard of care for follicular NHL(fNHL), diffuse large B-cell lymphoma (DLBCL), and chroniclymphocytic leukemia (CLL; ref. 1), but prognosis is poor in

patients who fail to respond to first-line treatment or developresistance.Multiple attempts have beenmade to improve on anti–B-cell therapeutic antibody therapy, including targeting alterna-tive CD20 epitopes and alternative B-cell surface targets (e.g.,CD19 and CD22), modification of the Fc region, and develop-ment of antibody–drug conjugates. To date, only obinutuzumab(Gazyva; Genentech/Roche), a glycoengineered Type II CD20humanized antibody (2), has shown an advantage over rituximabin phase III chemo-immunotherapy trials, demonstrating super-ior efficacy infirst-lineCLL and fNHL (3, 4), but failing to improveprogression-free survival versus rituximab in first-line DLBCL (5).Despite these efforts, and parallel development of small-mole-cule–based targeted therapies (6–9), a significant unmet needremains for improved therapeutic options in NHL (10).

A more recent therapeutic approach involves redirecting T cellsto attack B cells, using either bispecific antibodies that bind to a B-cell surface target and common surface component of the T-cellreceptor (TCR; e.g., CD3e; refs. 11, 12), or chimeric antigenreceptor-modified T cells (CAR-T cells) that are genetically mod-ified to express the target-binding domain of an anti–B-cellantibody fused to a TCR intracellular signaling domain (13,14). Blinatumomab, a T-cell bispecific (TCB) antibody targetingCD19 and CD3e, is approved in relapsed/refractory B-cell acutelymphoblastic leukemia (B-ALL; ref. 15) and in clinical trials for

1Roche Innovation Center Zurich, Roche Pharmaceutical Research and EarlyDevelopment, pRED, Zurich, Switzerland. 2Roche Innovation Center Basel,Roche Pharmaceutical Research and Early Development, pRED, Basel, Switzer-land. 3Roche Innovation Center Munich, Roche Pharmaceutical Research andEarly Development, pRED, Munich, Germany.

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

S. Colombetti and S. Herter contributed equally to this article.

Corresponding Authors:Marina Bacac, Roche Innovation Center Zurich, Wagis-trasse 10, Schlieren, 8952 Zurich, Switzerland. Phone: 41 43 215 16 24; Fax 41 43215 10 01; E-mail: [email protected]; and Pablo Uma~na, Phone: 41 43 21510 00; [email protected]

doi: 10.1158/1078-0432.CCR-18-0455

�2018 American Association for Cancer Research.

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NHL. TwoCAR-T cell therapies targeting CD19 are also approved:tisagenlecleucel-T for relapsed/refractory B-ALL (16), and axicab-tagene ciloleucel for relapsed/refractory large B-cell lymphomaafter �2 lines of systemic therapy (17). Although T-cell–redir-ected approaches are promising as their mode of action ispotentially complementary to classical IgG1 antibodies, theiruse in the broader NHL population may be limited. Blinatumo-mab, for example, must be administered by continuous infusiondue to its short half-life. Further, step-up-dosing (SUD) isrequired to mitigate side effects associated with initial TCBantibody infusions. Even with SUD, infusion-related reactionsand CNS toxicity are still issues for blinatumomab in DLBCL(12). CAR-T cell administration involves a 3-week turnaroundtimebefore administration—challenging in an aggressive diseasesetting—and specialized centers are required for its administra-tion and management of associated neurological side effects/severe infusion reactions. Several IgG-based anti–CD20-directedTCB antibodies, all of which are monovalent for CD20, are inearly clinical development (18–20). Although these agents havethe potential to overcome the frequent dosing limitations seenwith blinatumomab, the need for SUD remains and their com-parative efficacy with blinatumomab and CAR-T cells has yet tobe determined.

Translational Relevance

Redirecting T cells to hematologic malignancies with bis-pecific antibodies (TCB) is an attractive strategy to improveoutcome of patients with recurrent and/or refractory disease.However, such approaches frequently lead to severe sideeffects upon infusion requiring the introduction of step-up-dosing (SUD) regimens and steroid pretreatment to enhancesafety. We developed a potent, next-generation, half-lifeextended CD20-targeting TCB antibody (CD20-TCB), basedon the 2:1 molecular format, which has significantly superiorpotency compared with other CD20-targeting TCBs in clinicaldevelopment. In addition, we present a novel approachenabling safer administration of such a potent drug consistingof a single administration of obinutuzumab (Gazyva pretreat-ment, Gpt) prior to the first infusion of CD20-TCB. Our dataprovide evidence that Gpt abrogated cytokine release of thefirst CD20-TCB administration more efficiently than SUDregimen, thus representing an attractive, safer, and clinicallyrelevant approach for treatment of patients with hematologicmalignancies.

Figure 1.

In vitro and ex vivo tumor cell lysis mediated by different CD20-TCB variants. A, Tumor cell lysis mediated by 2:1 CD20-TCB, 1:1 IgG CD20-TCB, and1:1 OA CD20-TCB, determined using LDH release after 20- to 24-hour incubation of human PBMCs with tumor targets and indicated TCB concentrations (E:T5:1). One representative donor of 3 total donors is shown (other donors are presented in Supplementary Fig. S2A). B, Tumor cell lysis mediated byobinutuzumab-based 2:1 CD20-TCB as compared with 1:1 CD20-TCB having alternative anti-CD20, anti-CD3, and Fc sequences (VH-A-1242-1250;WO2014/047231 A1). One representative donor of 3 total donors is shown (other donors are presented in Supplementary Fig. S2B). C, EC50 values of2:1 CD20-TCB–mediated tumor cell lysis in bone marrow aspirates derived from patients with aggressive lymphoma and leukemia (n ¼ 17 patients).Each dot represents the average EC50 per patient derived from different time points (24–120 hours). D, Examples of concentration-dependent B-celldepletion curves mediated by 2:1 CD20-TCB in 3 different patients (P4, P8, and P17).

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Clin Cancer Res; 24(19) October 1, 2018 Clinical Cancer Research4786




Figure 2.

Mode of action of CD20-TCB. A, In vitro visualization of the dynamics of CD20-TCB (white) during interactions between CD8þ T cells (green) and WSU DLBCLtumor cells (blue). Image sequence highlights CD20-TCB localization in immunologic synapse areas along with tumor cell killing evidenced by the formationof tumor cell blebs. CD20-TCB was fluorescently labeled using Alexa Fluor 647 (white signal). B, Quantification of T-cell speed, synapse area, and CD20-TCBintensity at synapse. C, Flow cytometry analysis of samples collected within 1 to 3 days of tumor cell lysis shows the kinetics of expression of T-cell activation(CD25, 4-1BB, Ox40, ICOS) and exhaustion markers (PD-1, Lag3).

CD20-TCB with Gpt in Hematologic Malignancies

www.aacrjournals.org Clin Cancer Res; 24(19) October 1, 2018 4787




Here, we present a novel CD20-targeting TCB (CD20-TCB;RG6026), which has a long half-life and high potency enabledby high-avidity bivalent binding to CD20 and head-to-tail ori-entation of B- and T-cell–binding domains in a previously char-acterized 2:1 TCB molecular format (21, 22). Our preclinicalinvestigations indicate that a single administration of obinutu-zumab prior to the first infusion of CD20-TCB (Gazyva pretreat-ment, Gpt) may represent a more robust approach to safelyadminister such potent drugs.

Materials and MethodsCell culture for in vitro assays

CCRF SB, Pfeiffer, SU-DHL-2, SU-DHL-5, Toledo cells [allAmerican Type Culture Collection (ATCC)], as well as Jurkat,DoHH-2, OCI-Ly18, U2932, and WSUDLCL2 cells [all DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ)] and Z138 cells (gifted from the University of Leicester,Leicester, United Kingdom) were cultivated in RPMI 1640

medium(Gibco/Lubioscience; #42401-042) containing 10%FCS(Gibco) and 1% Glutamax (Invitrogen/Gibco; #35050-038).RC-K8 and SU-DHL-6 cells (both DSMZ) were cultivated in RPMI1640medium containing 15%FCS and1%Glutamax. SU-DHL-8and ULA cells (both DSMZ) were cultivated in RPMI 1640medium containing 20% FCS and 1%Glutamax. SU-DHL-4 cells(DSMZ) were cultivated in DMEM (Invitrogen/Gibco; #42430-082) containing 10% FCS and 1% Glutamax.

Cell lines provided by the ATCC and DSMZ are routinelyauthenticated by short tandem repeat profiling prior to delivery.Upon receipt, cells were expanded and frozen; cells were notpassaged for more than 6 months after resuscitation. No furtherauthentication of these cell lines was conducted. The Z138 cellswere not authenticated.

Binding to CD20- and CD3-expressing cellsMedian fluorescence intensities were calculated for binding of

CD20-TCB to B cells and primary CD4þ and CD8þ T cells freshlyisolated from human and cynomolgus monkey peripheral blood

Figure 3.

In vivo activity of CD20-TCB. A, Nontumor bearing HSC-NSG mice injected with CD20-TCB (0.5 mg/kg) or with PBS (vehicle) at study days 0and 7. Mice were bled at baseline (i.e., prior to the first CD20-TCB injection) and at indicated time points for PK and pharmacodynamics (PD) analysis. B, Kinetics ofperipheral blood B-cell and T-cell counts. Flow cytometry analysis of CD19þ B cells (left) and CD3þ T cells (right) in blood of vehicle- and CD20-TCB–treated mice(0.5 mg/kg) over time. C, PK results of CD20-TCB in HSC-NSG mice and NSG mice after CD20-TCB single-dose injection (0.5 mg/kg). Singlemice are shown for each of the two mouse models (n ¼ 3). D, T-cell phenotype analysis in the peripheral blood at 72 hours after first CD20-TCB injection.Flow cytometry analysis of CD25, 4-1BB, PD-1, Ki-67, and GZB on CD4þ and CD8þ T cells. Frequency of marker-positive cells is reported as % for vehicle- (gray) andCD20-TCB–treated mice (red). E, Multiplex analysis of cytokines in blood of vehicle- and CD20-TCB–treated mice over time displaying the intensity ofcytokine release at 24 and72 hours after first and 72 hours after secondCD20-TCB injection (labeled as 1d, 3d, and 10d, respectively). The absolute values of cytokinesare reported in Supplementary Table S4. F, Immunohistochemical analysis (CD19 staining) of human B cells in spleen and lymph nodes (LN) of vehicle- andCD20-TCB–treated mice at study termination (d10 corresponding to 3 days after the second CD20-TCB injection).

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mononuclear cells (PBMC). Binding of CD20-TCB to humanJurkat T cells, cynomolgus monkey HSC-F T cells, and humanmantle cell lymphoma tumor line Z-138 was measured by flowcytometry as described in the Supplementary Methods.

Tumor cell lysis in vitroB-cell–depleted PBMCs derived from healthy donors were

prepared using standard density-gradient isolation followed byB-cell depletion with CD20 Microbeads (Miltenyi Biotec) andincubated with CD20-TCB or untargeted-TCB, tumor targets(Toledo, U2932 or Z-138) at ratio of 5:1 for 20 to 24 hours. Thereason of using B-cell–depleted PBMCs as effector cells was toenable an exclusive assessment and quantification of tumor targetcell lysis, without the additional signal coming from the lysis ofnormal B cells (present within PBMCs), which are expressingCD20 and thus represent an additional target for CD20-TCB.Samples were incubated at 37�C and 5% CO2 in a humidifiedincubator for indicated time points (all in triplicate). Lactatedehydrogenase (LDH) release was measured using the LDHCytotoxicity Detection Kit (Roche Applied Science). Antibody-dependent cellular cytotoxicity (ADCC) was calculated asfollows:

Percentage ADCC ¼ sample release� spontaneous releasemaximal release� spontaneous release

� �� 100:

Spontaneous release, corresponding to target cells incubatedwith effector cells without TCB, was defined as 0% cytotoxicityand maximal release (target cells lysed with 2% Triton X-100)defined as 100% cytotoxicity. The average percentage of ADCCand standard deviations of the triplicates of each experiment werecalculated.

Flow cytometry was performed on PBMCs collected within 1 to3days of tumor cell lysis stainedwith FACSantibodies to show thein vitro kinetics of expression of T-cell activation (CD25, 4-1BB,Ox40, ICOS) and exhaustionmarkers (PD-1, Lag3). Supernatantsderived from20- to 24-hour incubated tumor cell lysis assayswithZ-138 and isolated pan T cells (Miltenyi Biotec; effector:target 3:1)were analyzed by Cytometric Bead Array (BD Biosciences) toquantify cytokine release. T-cell proliferation and endothelialadhesion/activation in vitro were also assessed (see the Supple-mentary Methods for further details).

Live in vitro imagingWSU-DLCL2 cells were plated overnight onRetronectin-treated

(2 hours at room temperature using 5 mg/cm2) 8-well GlassBottom slides (Ibidi #07/2019) at 37�C to adhere and stainedwith CMAC dye (Molecular Probes, Life Technologies). Beforeacquisition, CD3/CD28-activated CD8-positive T cells werestained with CMTMR (Molecular Probes, Life Technologies) andplated together with tumor cells. Alexa647-labeled CD20-TCB(500 ng/mL) was added directly into the growth media. Slides

Figure 4.

In vivo antitumor activity in a DLBCL xenograft model. A, Flow cytometry analysis of CD19þ B cells and CD3þ T cells in peripheral blood of vehicle- andCD20-TCB–treated mice (0.5 mg/kg, 0.15 mg/kg, and 0.05 mg/kg i.v. once weekly) measured 24 hours after first administration. Baseline indicates the level ofB-cell and T-cell counts in mice prior to treatment. B, Multiplex analysis of cytokines released in the peripheral blood of vehicle- and CD20-TCB–treated micedetected 24 hours after the first administration. The absolute values of cytokines detected are reported in Supplementary Table S4. C, Antitumor activityof CD20-TCB at different doses (0.5 mg/kg, 0.15 mg/kg, and 0.05 mg/kg i.v. once weekly) in HSC-NSG mice bearing WSU-DLCL2 (DLBCL) xenografts.Red arrows indicate therapy injection once per week (n ¼ 10 animals/group).






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were imaged with a confocal microscope (inverted LSM 700,Zeiss) with a temperature and CO2-controlled stage. Live acqui-sition was performed with a 20x objective. Movies were collectedusing Zen software (Zeiss) coupled to the microscope, analyzedwith Imaris (Bitplane; Oxford Instruments), and plotted usingGraphPad Prism.

Ex vivo assays using primary tumor samplesEx vivo assays were performed by Vivia Biotech incubating fresh

and frozen bone marrow samples from adult leukemia andlymphoma patients with selected TCBs. Studies were conductedby Vivia Biotech in accordance with recognized ethical guidelines.T cells and target cells were analyzed by flow cytometry and targetcell death determined by Annexin V.

In vivo studies in mice and cynomolgus monkeysThe routine care of all animals used in this study is described

in detail in the Supplementary Methods. Mice were maintainedunder specific pathogen-free condition with daily cycles of 12-hour light/12-hour darkness according to international (Federa-tion of European Laboratory Animal Science Associations) andnational [Gesellschaft f€ur Versuchstierkunde/Society of Labora-tory Animal Science (GV-Solas) and Tierschutzgesetz (TierSchG]guidelines. The study protocol was reviewed and approved bylocal government (license ZH193/2014).

CD20-expressing WSU-DLCL2 or OCI-LY18 cells were culturedat 37�C in a water-saturated atmosphere at 5% CO2. Cells wereharvested, washed once with RPMI, resuspended in RPMI, andinjected into female stem cell humanized NSG mice (HSC-NSG,also called fully humanized mice) SC at 0.1 mL/mouse (1.5� 106

WSU-DLCL2 or 10 � 106 OCI-LY18 cells in 50% growth factor–reduced Matrigel admixed with 50% RPMI). CD20-TCB treatmentstarted when tumor volume reached 100 to 150 mm3 and wasadministered i.v. onceweekly at thedoses described later in the text.In some studies, mice received obinutuzumab pretreatment (Gpt)at the dose of 10 mg/kg i.v., 1 week prior to CD20-TCB adminis-tration. Antibody dilutions were freshly prepared prior to use.

Purpose-bred, na€�ve, Cynomolgus monkey (Macaca fascicu-laris)were obtained fromCharles River Laboratories. Animalwere3.5 to 4.7 years old andweighed3.5 to 6.1 kg at the timeof dosing.Animals were housed in stainless-steel cages. Primary enclosureswere as specified in the USDA AnimalWelfare Act (9 CFR, Parts 1,2, and 3) and as described in the most current Guide for the Careand Use of Laboratory Animals (National Research Council.

Guide for the Care and Use of Laboratory Animals. Most currentedition. Washington, DC: National Academies Press. 2011).

Cynomolgus monkeys were treated with 50 mg/kg i.v. obinu-tuzumab to deplete B cells, 0.1 mg/kg CD20-TCB i.v., or vehiclecontrol. IFNg in plasma samples was measured 4 hours aftertreatment by bead-based sandwich immunoassay measured ona Luminex platform. Low limit of quantification (LLOQ) was75 pg/mL.

ResultsGeneration and characterization of CD20-TCB biological activity

Three different TCB structural variants were generated, andtheir biological activity was compared (Fig. 1A; SupplementaryTable S1). The first, 2:1 CD20-TCB, based on the 2:1 TCB format(21, 22), had two anti-CD20 Fabs and one anti-CD3 epsilonsubunit (CD3e) Fab with one of the CD20 Fabs fused directly in a"head-to-tail" fashion to the anti-CD3e Fab via a flexible linker. Aheterodimeric human IgG1 Fc region carrying the "PG LALA"mutations (23) was incorporated to abolish binding to Fcgreceptors and to complement component C1qwhilemaintainingneonatal Fc receptor (FcRn) binding, enabling a long circulatoryhalf-life. Two additional TCBs were based on 1:1 molecularformats (i.e., having one anti-CD20 Fab and one anti-CD3e Fab)named the 1:1 IgGCD20-TCB, an IgG-likemoleculewith each Fabfused to either N-terminus of the IgG1 hinge region, and 1:1 OACD20-TCB, a one-armed OA molecule with both Fabs fused toone terminus of the hinge region in a head-to-tail fashion. Thetype II anti-CD20, humanized Fab region was from obinutuzu-mab. Both anti-CD20 and anti-CD3e Fabs recognized theirrespective targets with similar affinity between human and cyno-molgusmonkey cells, e.g., binding EC50 of 4.8 versus 3.3 nmol/L,for primary human versus cynomolgus monkey B cells, respec-tively (Supplementary Fig. S1).

The superior biological activity of the 2:1 molecular formatover both 1:1 TCB formats was demonstrated in tumorlysis experiments using target cell lines expressing variouslevels of CD20 antigen (Supplementary Table S2) andhealthy donor PBMCs derived from several individuals. Notethat 2:1 CD20-TCB displayed on average a 40-fold strongerpotency (range between 12- and 95-fold) than the 1:1CD20-TCBs (Fig. 1A; Supplementary Fig. S2A; SupplementaryTable S2), supporting the relevance of CD20 avidity/bivalency.Notably, the 1:1 head-to-tail format was more potent than the

Figure 5.Effect of obinutuzumab (Gazyva) pretreatment (Gpt) on T-cell activation by CD20-TCB and cytokine release. A, Antitumor activity of vehicle; CD20-TCBmonotherapy (0.5 mg/kg i.v. once weekly); obinutuzumab (Gazyva) monotherapy (10 mg/kg i.v. once weekly); and obinutuzumab (Gazyva) pretreatment (Gpt,10 mg/kg single injection) followed by CD20-TCB (0.5 mg/kg i.v. once weekly) in WSU-DLCL2 tumor–bearing HSC-NSG mice. Arrows indicatetherapy injections (red arrow: CD20-TCB; blue arrow: obinutuzumab; number of animals/group 8 < n < 10). B, Immunohistochemical staining showing intratumorT-cell infiltration (CD3 staining, brown) at indicated time points and for indicated groups. Histograms denote the quantification of the intratumor CD3 T-cellinfiltrate among the various groups. C, Flow cytometry analysis of CD19þ B cells (left) and CD3þ T cells (right) in the peripheral blood of vehicle- andCD20-TCB–treated WSU-DLCL2 tumor–bearing HSC-NSG mice, 24 and 72 hours after first- and second-therapy injections as indicated by the legend.D, Cytokines released in peripheral blood among the different treatment groups 24 hours after the first and second treatments. The absolute values ofcytokines detected are reported in Supplementary Table S4. E, Toxicology study in cynomolgus monkey with administration of vehicle or obinutuzumab(50 mg/kg i.v.) at day 1 to reduce B-cell load. Five days later, animals were treated with either vehicle or CD20-TCB (0.1 mg/kg i.v.), and IFNg in plasma wasmeasured after 4 hours. F, Antitumor activity obtained upon CD20-TCB, obinutuzumab (Gazyva), or CD20-TCB þ obinutuzumab (Gazyva) treatment inWSU-DLCL2 and OCI-Ly18 DLBCL tumor models in HSC-NSG mice. Doses are indicated on figure plots; the number of injections was n ¼ 7 for WSU-DLCL2 andn ¼ 2 for OCI-Ly18. All therapies were administered once weekly; the combination of CD20-TCB and obinutuzumab was performed by concomitantadministrationof both antibodies at each treatment cycle. Redarrow indicates the start of therapy injection; treatments started at tumor size of 520 to 550mm3 in theWSU-DLCL2 tumor model (study day 15 upon tumor-cell inoculation), and 360 to 380 mm3 in the OCI-Ly18 tumor model (study day 15 upon tumor-cellinoculation). Number of animals/group, 8 < n < 10.






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classical 1:1 IgG format, supporting the benefit of bringingboth Fabs in close spatial proximity via a flexible linkercompared with separating them on either side of an IgG1hinge region.

The activity of 2:1 CD20-TCB was also compared with a CD20-targeting 1:1 TCB antibody with alternative anti-CD20, anti-CD3,and Fc sequences (derived from a TCB currently in phase I trials)(19) and showed on average a 40-fold superior potency (rangebetween 4- and 153-fold; Fig. 1B; Supplementary Fig. S2B; Sup-plementary Table S2).

Note that 2:1CD20-TCB alsodisplayed strong activity ex vivoonprimary tumor samples derived from aggressive lymphoma andleukemia patients (Fig. 1C and D; Supplementary Table S3). Evenunder low effector-to-target (E:T) cell ratios (0.02–0.8), andusing only the existing T cells present in the primary samples,2:1 CD20-TCB displayed high potency as illustrated by the lowEC50 values of target cell killing (Fig. 1C) and profound targetcell depletion (Fig. 1D). Flow cytometric analyses of patientsamples revealed efficient target cell depletion occurring as earlyas 24 hours of incubation, which continued at subsequent timepoints (Supplementary Fig. S3).

Together, the 2:1 CD20-TCB displayed superior potency com-pared with CD20-TCB antibodies based on the classical 1:1 IgGformat and was therefore selected for further development. Thedevelopment molecule is referred as CD20-TCB in the remainderof the article.

Insights into the mode of action of CD20-TCBLive in vitro imaging of cocultures of CD20-expressing lym-

phoma cells (WSU-DLCL2), preactivated CD8 cytotoxic T cells,and fluorescently labeled CD20-TCB enabled visualizationof antibody binding and tumor cell lysis. Incubation withCD20-TCB notably reduced the speed of T-cell movement andincreased tumor/T cell synapse area, reflecting prolonged andlarger interactions between tumor cells and T cells that corre-lated with CD20-TCB localization at synaptic areas (Fig. 2A andB; Supplementary Fig. S4A; Supplementary Video S1 and S2).Tumor lysis was observed already at 4 hours with multiple Tcells contributing to killing of a single target cell (Supplemen-tary Video S3). Flow cytometric analysis of samples collected atdifferent time points confirmed T-cell activation and expansionassociated with tumor killing as evidenced by the expression of

a broad panel of T-cell activation and exhaustion markers (Fig.2C; Supplementary Fig. S4B), cytotoxic granule and cytokinesecretion, and proliferation (Supplementary Fig. S5A and S5B).Both CD4 and CD8 T-cell subsets expanded following tumorkilling, but CD8 T cells underwent stronger proliferation asshown by higher number of proliferation cycles compared withCD4 T cells (Supplementary Fig. S5B).

In vivo activity of CD20-TCBIn vivo pharmacology studies performed in stem cell human-

ized NSG mice (HSC-NSG) (Fig. 3A) showed that peripheralblood B cells were largely depleted within 24 hours of the firstCD20-TCB administration (0.5 mg/kg, once weekly i.v.) andremained undetectable for the rest of the study (Fig. 3B). Phar-macokinetic (PK)data showed target-mediated clearance byB-cellbinding during the first TCB administration and IgG-like TCB PKfor a subsequent administration once peripheral B cells weredepleted (Fig. 3C). Peripheral blood T cells decreased transientlywithin 24 hours of the first CD20-TCB administration butreturned to baseline levels within 72 hours and remained stablefor the rest of the study (Fig. 3B). The second and subsequentadministrations of CD20-TCB did not induce such T-cell decreasein the peripheral blood.

Flow cytometry showed that, 72 hours after the first CD20-TCBadministration, peripheral blood T cells were activated and bothCD4þ andCD8þ T cell subsets were proliferating as demonstratedby the upregulation of CD25, 4-1BB, PD-1, Granzyme B, andKi67(all hallmarks of T-cell activation through TCR complex cross-linking; Fig. 3D). Initial T-cell activation in peripheral blood,during CD20-TCB–mediated B-cell depletion, was further exem-plified by transient cytokine release (IFNg , TNFa,MCP-1,MIP-1b,IL2, IL5, IL6, IL8, IL10) in blood of treated mice (measured at 24hours after the first CD20-TCB injection and normalizing tonearly baseline levels by 72 hours; Fig. 3E; Supplementary TableS4). Importantly, cytokine levels did not increase after a secondadministration of CD20-TCB (day 10), consistent with the almostcomplete B-cell depletion in the peripheral blood upon the firstCD20-TCB administration (Fig. 3B and E; SupplementaryTable S4), as CD20 binding is a prerequisite for CD3 cross-linkingon T cells by CD20-TCB. In addition to peripheral blood, CD20-TCB also efficiently eliminated B cells in the spleen and lymphnodes (Fig. 3F).

Figure 6.Safety and efficacy of CD20-TCB with obinutuzumab pretreatment (Gpt) or as SUD in tumor-bearing WSU-DLCL2 HSC-NSG mice. Comparison ofobinutuzumab pretreatment (Gpt) to SUD approach in tumor-bearing WSU-DLCL2 (SC) in HSC-NSG mice. Mice received the first therapy either as SUD ofCD20-TCB (0.15, 0.05, and 0.015 mg/kg i.v.) or obinutuzumab pretreatment (Gpt, 10 mg/kg), followed by a full dose of CD20-TCB (0.5 mg/kg) 7 dayslater for 5 treatment cycles (i.e., 5 weeks). A, Flow cytometry analysis of CD19þ B cells and CD3þ T cells in the peripheral blood and spleen of vehicle, SUD, andGpt-treated WSU-DLCL2 tumor–bearing HSC-NSG mice 7 days after first therapy injection. B, Cytokines released in peripheral blood among the differenttreatment groups 24 hours after the first and second treatments. The absolute values of cytokines detected are reported in Supplementary Table S4.C, Antitumor activity of SUD versus Gpt þ CD20-TCB approach. Arrows indicate therapy injections (red arrow: CD20-TCB; blue arrow: obinutuzumab). Number ofanimals/group (n ¼ 9 or 10). D, Perivascular T-cell localization in lungs of WSU-DLCL2 tumor–bearing HSC-NSG mice 7 days after the first treatment and24 hours after the second treatment. Lung sections were immunohistochemically stained with anti-CD3 antibody (brown); nuclei were counterstained withhematoxylin. Magnification, �20. Supplementary Table S5 shows the average score of perivascular CD3þ T cells in the lung (performed by blinded pathologistanalysis). E, Schematic representation of the assay used to test the effect of CD20-TCB conditioned media from tumor lysis experiments on endothelial cellactivation and T-cell adhesion. F, Human endothelial cells (HUVEC) were stimulated for 6 hours with conditioned media from tumor lysis experiments (PBMCsplus Z-138 tumor cells incubatedwith CD20-TCBor untargeted TCB for 24 hours), and adhesionmoleculesweremeasured byflowcytometry. CD20-TCB conditionedmedia potently upregulated ICAM, VCAM, and E-selectin. TNFa (10 ng/mL) was used as positive control. Data are representative of >3 experiments. G, Activated Tcells upregulate LFA-1 expression upon CD20-TCB–mediated tumor lysis. H, T cells isolated from tumor lysis experiments in the presence of CD20-TCB werefluorescently labeled and left to adhere for 1 hour on HUVEC monolayers stimulated with conditioned media collected from the same tumor lysis experiment asdepicted in E. T-cell adhesion (measured as fluorescence units, FU) was increased on endothelium stimulated with CD20-TCB conditioned media as compared withuntargeted TCB conditioned media. Data are representative of two independent experiments. Statistical analysis, one-way ANOVA.






The in vivo antitumor activity of CD20-TCB was assessed inHSC-NSG mice bearing an aggressive DLBCL xenograft model(WSU-DLCL2), poorly responsive to anti-CD20 IgG1 antibodiesandother therapies (Fig. 4; ref. 24). Threedoses ofCD20-TCBwerecompared (0.5, 0.15, 0.05 mg/kg, i.v. all given once weekly).Whereas all dosesmediated efficient B-cell depletionand transientT-cell decrease in the peripheral blood (Fig. 4A), along withcytokine increase measured 24 hours after the first TCB admin-istration (Fig. 4B; Supplementary Table S4), only the intermediate(0.15 mg/kg) and the highest (0.5 mg/kg) doses induced dose-dependent regression of established tumors (Fig. 4C). Together,CD20-TCB treatment led to efficient B-cell depletion in theperipheral blood, spleen, and lymph nodes along with tumorregression. At least 5- to 10-fold higher TCB doses were requiredfor the regression of established tumor lesions compared withthose required for normalB-cell depletion in theperipheral blood.

Obinutuzumab (Gazyva) pretreatment mitigates cytokine-release syndrome associated with CD20-TCB administration

Wehypothesized that initial B-cell depletion by obinutuzumab(Gazyva) pretreatment (Gpt) prior to CD20-TCB administrationcould safely reduce T-cell activation by TCB in peripheralblood or other normal tissues, thus reducing the risk of cyto-kine-release syndrome (CRS) and other potential side effects.This was evaluated by administering CD20-TCB (0.5mg/kg, onceweekly) after a single injection of obinutuzumab (10 mg/kg i.v. 7days before CD20-TCB) in WSU-DLCL2–bearing HSC-NSGmice(Fig. 5). The antitumor activity of CD20-TCB administered afterGpt was comparable with that of CD20-TCB alone, confirmingthe combinability of the two agents at these doses despite bindingto the sameCD20 epitope (Fig. 5A). Histologic analysis of tumorscollected 7 days after CD20-TCB or Gpt administration showedthat both treatments led to comparable increase of intratumor Tcells (Fig. 5B, left). In addition, CD20-TCB monotherapy andGptþCD20-TCB achieved the same intratumor T-cell infiltrationas demonstrated at study termination (Fig. 5B, right).

Interestingly, whereas all treatments led to comparable B-celldepletion in peripheral blood (Fig. 5C and B cell panel), there wasa clear difference in the transient decline of T-cell counts betweenthe groups (Fig. 5C, T-cell panel). As reported earlier, T cellsexperienced a strong transient decline in peripheral blood fol-lowing the first CD20-TCB administration, but their numbers didnot change in obinutuzumab and GptþCD20-TCB treatmentgroups, suggesting that T cells are not affected by obinutuzumabtreatment in mice. Most importantly, Gpt significantly reducedthe cytokine peak associated with the first CD20-TCB adminis-tration (Fig. 5D; Supplementary Table S4). This finding wasreproduced in toxicology studies in cynomolgus monkeys (Fig.5E), further confirming that Gpt enables a safe depletion ofperipheral blood and secondary lymphoid organ B cells andprevents the strong T-cell–mediated cytokine release associatedwith the first CD20-TCB administration.

CD20-TCB combination with obinutuzumab beyond GptThe combinability of CD20-TCB and obinutuzumab was fur-

ther explored by combining the two antibodies for several admin-istration cycles in two different DLBCL models, WSU-DLCL2 andOCI-Ly18 (Fig. 5F). The synergy of this combination is evidencedby a rapid tumor regression in all animals and in both tumormodels as compared with the corresponding single agents.

Gpt versus SUD: superior safety and efficacyGpt was compared with SUD, an approach frequently

used in the clinic as a means of mitigating the CRS of thefirst TCB administration (Fig. 6). SUD corresponded to 1/30(0.015 mg/kg), 1/10 (0.05 mg/kg), and 1/3 (0.15 mg/kg) ofthe full CD20-TCB dose (0.5 mg/kg). Seven days after the SUD,all animals received the full CD20-TCB dose (0.5 mg/kg). Gptwas either superior (vs. 0.015 mg/kg) or comparable (vs. 0.05and 0.15 mg/kg) to SUD doses in terms of the efficiency ofperipheral blood and spleen B-cell depletion (Fig. 6A; Sup-plementary Table S4). Importantly, Gpt was superior to SUDin terms of reducing cytokine release upon CD20-TCB admin-istration, indicating that Gpt provides a safer B-cell depletionapproach than SUD (Fig. 6B, left; Supplementary Table S4).Moreover, administration of CD20-TCB full dose (0.5 mg/kg)7 days after 0.015 and 0.05 mg/kg SUD led to a substantialcytokine peak, which was not observed with Gpt or the highestSUD tested (0.15 mg/kg), consistent with efficacious B-celldepletion in peripheral blood during the first cycle by thesetwo approaches (Fig. 6B, right; Supplementary Table S4).However, as the 0.15 mg/kg SUD was associated with signif-icant cytokine release during the first cycle, Gpt provided anoverall safer approach for CD20-TCB administration.

Gpt was also superior to all SUD groups in terms ofantitumor activity. Whereas all treatment options led to com-parable tumor growth inhibition (Fig. 6C), there were moretumor-free animals (defined as those having tumor lesion size<10 mm3 and not having malignant tumor cells assessed byhistology) at study termination in the Gpt group (n ¼ 6) thanin the SUD groups (n ¼ 4 in 0.015 mg/kg; n ¼ 2 in 0.05 mg/kg;n ¼ 4 in 0.15 mg/kg).

Histologic analysis of lungs collected from mice 7 daysafter first treatment revealed a prominent perivascular T-celllocalization in all animals treated with SUD (0.15 and0.05 mg/kg) but not with Gpt nor with the lowest SUD(0.015 mg/kg; Fig. 6D, left; Supplementary Table S5). Perivas-cular T-cell localization persisted at later time points [24 hoursafter administration of the full CD20-TCB dose (0.5 mg/kg)] inall animals that received SUD, but not in the ones with Gpt(Fig. 6D, right; Supplementary Table S5).

Conditioned media collected from tumor lysis experiments(containing a number of cytokines secreted by CD20-TCB–acti-vated T cells; Supplementary Fig. S5A) induced the activation ofendothelial cells, as shown by strong upregulation of endothelialcell adhesionmarkers (E-selectin, VCAM, ICAM;Fig. 6E andF). Thesame was true formouse sera derived from a single treatment withCD20-TCB, but not from those receiving obinutuzumab (Supple-mentary Fig. S6A), consistent with the high levels of cytokinessecreted in the peripheral blood upon CD20-TCB but not obinu-tuzumab administration (Fig. 5D). In addition, CD20-TCB–activated T cells upregulated LFA-1 (ICAM-1 ligand; Fig. 6G) andhad a high constitutive expression of VLA-4 (VCAM ligand; Sup-plementary Fig. S6B), thus increasingT-cell adhesion toCD20-TCBconditioned media–activated endothelium (Fig. 6H). Blockingexperiments revealed that TNFa plays an important role in endo-thelial activation as TNFa-neutralizing antibodies significantlyreduced upregulation of adhesion molecules by human endothe-lial cells (Supplementary Fig. S6C). Together, cytokines released byactivated T cells during CD20-TCB–mediated B-cell killing caninduce endothelial activation and promote T-cell adhesion. TNFaappears to play a key role in orchestrating this process.

Bacac et al.





DiscussionDespite generating promising clinical activity and offering

curative solutions for a proportion of patients, T-cell–engagingapproaches, including TCB antibodies, are associated with severeside effects upon infusion requiring the introduction ofSUD regimens to mitigate safety. Even newly developed, half-life–extended TCB antibodies with IgG-like TCB 1:1 molecularformat have similar issues, in addition to having a suboptimalformat that hampers maximal potency.

Here, we present a novel approach that addresses both short-comings. We developed a potent CD20-targeting TCB antibody(CD20-TCB) with a long half-life and strong potency enabledby head-to-tail orientation and high-avidity binding to CD20 inthe 2:1 molecular format (21–23), along with a safer adminis-tration regimen for such potent drugs consisting of a singleadministration of obinutuzumab prior to the first infusion ofCD20-TCB (Gpt).

CD20-TCB displayed significant potency and antitumor activ-ity in vitro (even on cells expressing low levels of CD20), ex vivo(using tumor-infiltrated primary bonemarrow aspirates of aggres-sive lymphoma and leukemia patients), and in vivo (where itregressed established tumors of an aggressive DLBCL model).Interestingly, potent killing activity was observed even at very lowE:T cell ratios, including those in primary tumor samples, whichmay be reflective of both the serial killing mediated by TCB-engaged T cells and their rapid amplification as a result of TCB-mediated activation (25). The stronger potency of CD20-TCBrelative to 1:1CD20-TCB antibodies (someofwhich are in phase Iclinical development) highlighted the relevance of having theCD20 binder and CD3 binder in close proximity (enabled by ashort flexible linker), as opposed to having them distant as in theclassical 1:1 IgG-based TCB format. Such superior killing potencyhas been observed for TCBs against other cancer cell targets, andwe hypothesize that it may be related to a tighter andmore stableT-cell cancer cell synapse induced by the head-to-tail fusionmolecular format (26).

The increased potency of CD20-TCB over other TCBs increasesthe risk of side effects due to higher levels of cytokine releaseresulting from stronger T-cell activation and more potent B-cellkilling. If not adequatelymitigated, thismay lead to severe toxicityand limit the doses required for efficacious tumor control. Steroidpretreatment is used clinically to reduce systemic T-cell activationand cytokine release; however, it is currently unclear whetherhigh-dose and prolonged steroid treatment might affect T-cellactivation and functionality (12, 27–29). Alternative clinicalapproaches include use of the IL6 receptor blocking antibody(tocilizumab) or fractionation of the first TCB dose (so-calledSUD approach). Here, we present an alternative and novelapproach consisting of a single obinutuzumab pretreatment(Gpt) 7 days prior to the first CD20-TCB administration. Thisapproach not only was more efficacious than SUD, achievinggreater B-cell depletion (de-bulking) in both peripheral bloodand secondary lymphoid organs, but also increased safety, as itabrogated the initial strong cytokine release associated with T-cellactivation. Such T-cell activation during peripheral blood andnormal tissue B-cell depletion makes SUD inherently less robustas a pretreatment approach, as evidenced by the proportion ofpatients that still experience significant side effects after SUD (12).Because of lower cytokine release, obinutuzumab pretreatmentalso prevented the transient T-cell margination and extravasation

into peripheral tissues, possibly via reducing cytokine-mediatedendothelial cell activation.

Importantly, despite competing for the same target antigen,obinutuzumab and CD20-TCB could be combined in vitroand in vivo, yielding promising antitumor efficacy. The 2:1 TCBmolecular format of CD20-TCB provides twoCD20-binding Fabs(as in obinutuzumab), thus conferring to the two molecules thesame binding avidity to CD20 antigen, enabling CD20-TCBbinding even in the presence of saturating concentrations ofobinutuzumab. Indeed, the same avidity for CD20 confers thepossibility for CD20-TCB to outcompete prebound obinutuzu-mab, which is a significant challenge for 1:1 IgG CD20-targetingTCB antibodies that have only a single low-affinity CD20-bindingFab. This may be relevant in future front-line settings whereCD20-TCBs might be combined with standard-of-care therapieswith a rituximab or obinutuzumab backbone (e.g., R-CHOP inDLBCL; obinutuzumab in CLL; obinutuzumabþchemotherapyin FL). Combining T-cell–redirected and IgG1-antibody–mediat-ed approaches is attractive in cancer treatment due to synergy andcomplementarity of different modes of action that combine T-cell–mediated cytotoxicity (of TCB antibodies) with ADCC,phagocytosis (30, 31), complement-dependent cellular cytotox-icity, and direct cell death induction of type II anti-CD20 anti-bodies. Consistent with this, the sustained and simultaneouscombination of CD20-TCB and obinutuzumab for several treat-ment cycles did not affect CD20-TCB activity and translated into arapid and more profound antitumor efficacy with tumor regres-sion in all animals as compared with single-agent treatment arms.

In conclusion, we present a novel, more efficacious, and saferapproach for treatment ofNHL, consisting of a singlefixed dose ofobinutuzumab prior to CD20-TCB therapy. This approach iscurrently under evaluation in a phase I, multicenter, open-label,dose-escalation study in patients with relapsed/refractory NHL(NCT03075696). Clinical data will clarify whether the strongefficacy seen in preclinicalmodels translates into clinical responseand more favorable safety compared with approaches currentlyused in the clinic.

Disclosure of Potential Conflicts of InterestM. Bacac, S. Colombetti, A. Freimoser-Grundschober, H. Hinton, C. Klein, C.

Neumann, and P.Uma~na are listed as co-inventors onHoffmann-La Roche Inc’sprovisional patent application on T-cell bispecific antibodies. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Bacac, S. Colombetti, J. Sam, K. Bray-French,H. Hinton, C. Neumann, C. Klein, P. Uma~naDevelopment ofmethodology: S. Colombetti, J. Sam,M. Perro, K. Bray-French,H. Hinton, C. KleinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Colombetti, S. Herter, J. Sam, M. Perro, S. Chen,R. Bianchi, M. Richard, A. Schoenle, V. Nicolini, S. Diggelmann, F. Limani,R. Schlenker, T. H€usser, K. Bray-FrenchAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Bacac, S. Colombetti, S. Herter, J. Sam, M. Perro,S. Chen, R. Bianchi, M. Richard, A. Schoenle, V. Nicolini, R. Schlenker,W. Richter, K. Bray-French, H. Hinton, A.M. Giusti, C. NeumannWriting, review, and/or revision of the manuscript: M. Bacac, S. Colombetti,S. Herter, J. Sam, M. Perro, R. Bianchi, W. Richter, K. Bray-French, A.M. Giusti,L. Lariviere, C. Neumann, C. Klein, P. Uma~naAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Sam, A. Freimoser-Grundschober, L. Lariviere,C. NeumannStudy supervision: M. Bacac, J. Sam, M. Perro, H. Hinton, P. Uma~na






AcknowledgmentsThe authors thankBetsyQuackenbush, Cristiano Ferlini, TomMoore, and all

members of the CD20-TCB project team for critical reading of the article; allmembers from Cancer Immunotherapy, Oncology, Large Molecule Research,and Pharmacology at Roche Pharma Research and Early Development (pRED)who contributed to the development of the program; Oncology DTA and pREDleadership for support during all phases of the preclinical and clinical devel-opment of the program; and Vivia Biotec and all scientists involved in the studyusing patient tumor samples.

The study was conducted by Roche. Jamie Ashman (Prism Ideas), a medicalwriter supported by funding from Roche, provided editorial assistance to the

authors during preparation of the article. This study and editorial support for thepreparation of this article were funded by Roche.

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 February 13, 2018; revisedMarch 24, 2018; accepted April 25, 2018;published first May 1, 2018.

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2018;24:4785-4797. Published OnlineFirst May 1, 2018.Clin Cancer Res Marina Bacac, Sara Colombetti, Sylvia Herter, et al. Treatment of Hematologic MalignanciesCD20-TCB with Obinutuzumab Pretreatment as Next-Generation

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