MGD011, A CD19 x CD3 Dual-Afﬁnity Retargeting Bi-speciﬁc ... · ination by CD3-expressing T lymphocytes as effector cells. MGD011 was engineered with a human immunoglobulin G1

Cancer Therapy: Preclinical

MGD011, A CD19 x CD3 Dual-Affinity RetargetingBi-specific Molecule Incorporating ExtendedCirculating Half-life for the Treatment ofB-Cell MalignanciesLiqin Liu, Chia-Ying K. Lam, Vatana Long, Lusiana Widjaja, Yinhua Yang, Hua Li,Linda Jin, Steve Burke, Sergey Gorlatov, Jennifer Brown, Ralph Alderson,Margaret D. Lewis, Jeffrey L. Nordstrom, Scott Koenig, Paul A. Moore,Syd Johnson, and Ezio Bonvini

Abstract

Purpose: CD19, a B-cell lineage-specific marker, is highlyrepresented in B-cell malignancies and an attractive target fortherapeutic interventions. MGD011 is a CD19 x CD3 DARTbispecific protein designed to redirect T lymphocytes to eliminateCD19-expressing cells. MGD011 has been engineered with amodified human Fc domain for improved pharmacokinetic (PK)properties and designed to cross-react with the correspondingantigens in cynomolgus monkeys. Here, we report on the preclin-ical activity, safety and PK properties of MGD011.

Experimental Design: The activity of MGD011 was evaluatedin several in vitro and in vivo models. PK, safety and pharmaco-dynamic activity was also assessed in dose-escalation and repeat-dose studies of MGD011 administered once weekly in cynomol-gus monkeys.

Results:MGD011 mediated killing of human B-cell lympho-ma lines by human or cynomolgus monkey PBMCs as well as

autologous B-cell depletion in PBMCs from both species.MGD011-mediated killing was accompanied by target-depen-dent T-cell activation and expansion, cytokine release andupregulation of perforin and granzyme B. MGD011 demon-strated antitumor activity against localized and disseminatedlymphoma xenografts reconstituted with human PBMCs. Incynomolgus monkeys, MGD011 displayed a terminal half-lifeof 6.7 days; once weekly intravenous infusion of MGD011 atdoses up to 100 mg/kg, the highest dose tested, was welltolerated and resulted in dose-dependent, durable decreasesin circulating B cells accompanied by profound reductions of Blymphocytes in lymphoid organs.

Conclusions: The preclinical activity, safety and PK pro-file support clinical investigation of MGD011 as a thera-peutic candidate for the treatment of B-cell malignancies.Clin Cancer Res; 23(6); 1506–18. �2016 AACR.

IntroductionB-cell malignancies represent a heterogeneous group of dis-

orders with varying characteristics and clinical behaviors (1).Although systemic chemotherapy is still the mainstay of treat-ment for B-cell malignancies, kinase inhibitors that selectivelytarget molecules at the core of the transformation process andantibody therapy are now well established tools (2). Among thelatter category, rituximab (Rituxan), a monoclonal antibody(mAb) that targets the B-cell antigen CD20, induces directtumor cell apoptosis as well as complement- and antibody-dependent cytotoxicity (2, 3). These orthogonal mechanisms ofaction form the basis for therapeutic combinations that have

improved outcome in advanced B-cell malignancies. Yet,approximately 20,000 patients die of lymphoma every year inthe United States alone.

Providing T lymphocytes (CTL) with the ability to recognizeand destroy tumor cells has shown promise in advanced formsof leukemia and lymphoma. In the form of chimeric antigenreceptor (CAR) T-cell therapy, such an approach requires ex vivoisolation, transduction, and reinfusion of the patient's T cells.This complexity can be overcome with bispecific antibodiesthat bind simultaneously to an antigen expressed by malignantB cells and an activation molecule on T lymphocytes (2). Thepan B-cell marker, CD19, has emerged as a promising antigenfor targeting B-cell malignancies because of its broader expres-sion profile and lower rate of downregulation compared withother B-cell antigens (3). Its expression is highly conserved inthe majority of B-cell tumors (4), with normal to high levels ofexpression in 80% of acute lymphoblastic leukemia (ALL), 88%of B-cell lymphomas, and all chronic lymphocytic leukemias(CLL; refs. 5, 6).

Redirection of CTL to CD19þ leukemia cells via the bispecificT-cell engager (BiTE) blinatumomab (Blincyto; ref. 7) is effec-tive in patients with B-cell malignancies whose disease did notrespond to standard chemo-immunotherapies and has been

Research, MacroGenics, Inc., Rockville, Maryland.

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

Corresponding Author: Ezio Bonvini, MacroGenics Inc., 9704 Medical CenterDrive, Rockville, MD 20850. Phone: 301-354-2638; Fax: 301-251-5321; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-16-0666

�2016 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 23(6) March 15, 20171506

on October 12, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 23, 2016; DOI: 10.1158/1078-0432.CCR-16-0666

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approved by the FDA for the treatment of patients with Phi-ladelphia chromosome-negative relapsed or refractory B-cellprecursor ALL. DART proteins are bispecific, antibody-basedmolecules with favorable stability, manufacturability, andpotency; furthermore, a CD19 x CD3 DART protein comparedfavorably with a BiTE of the same pair of VH and VL sequencesin redirected cytolysis assays (8, 9). MGD011 (also known asJNJ-64052781) is another CD19 x CD3 DART protein designedto simultaneously target CD19þ cells for recognition and elim-ination by CD3-expressing T lymphocytes as effector cells.MGD011 was engineered with a human immunoglobulinG1 (IgG1) Fc domain to bind the neonatal Fc receptor (FcRn)and engage the IgG salvage pathway, thus conferring prolongedcirculating half-life and the resultant dosing convenience. Toavoid unintended (target independent) CD3-mediated T-cellactivation via interaction with Fc gamma receptors (FcgR), theFc domain was mutated to greatly reduce or eliminate bindingto these receptors as well as complement. Unlike blinatumo-mab, which reacts only with human or chimpanzee's antigens(10), MGD011 cross-reacts with both CD19 and CD3 mole-cules in macaques, enabling preclinical evaluation in a relevantmodel. Here, we report on MGD011 engineering and charac-terization as well as on its preclinical safety assessment, phar-macokinetics (PK), and dose optimization in cynomolgusmonkeys. The results show robust activity in multiple modelsand predict effective dosing in humans at once weekly or longerintervals.

Materials and MethodsDART protein engineering, production, and purification

MGD011, an Fc-bearing CD19 x CD3 DART protein, wasconstructed as described (11) using VL and VH sequences fromhumanized anti-CD19mAbBU12 (12) and humanized anti-CD3mAb XR32 (13). The IgG1-derived Fc segment was modified toencode the L234A/L235Amutation to greatly reduce or eliminate

FcgR and C1q binding (14). Control molecules in which thevariable domain sequences of an anti-fluorescein mAb 4-4-20(15) replaced either of the DART protein arms (Fluo x CD3 orCD19 x Fluo) were engineered in a similar manner. The DARTproteins were expressed transiently in CHO-S cells (8) and puri-fied to greater than 99% purity using protein A and either size-exclusion chromatography (SEC) or other polishing steps. Thepurified DARTs have very low levels (less than 1%) of highmolecular weight (HMW) protein present and have an apparentmolecular weight of approximately 110 kDa (Supplementary Fig.S1A and B).

Other reagents, cell lines, and tissue samplesRecombinant soluble human and cynomolgus CD3e/d chi-

meric proteins, as well as human and cynomolgus CD19 andCD19-His proteins, were expressed in CHO-S cells. MOLM-13and JIMT-1 were obtained from DSMZ (Braunschweig, Ger-many); Jeko-1 cells were from the ATCC and HBL-2 from theNational Institutes of Health (Bethesda, MD; ref. 16). Raji/GF, aCD19þ Burkitt's B-cell lymphoma expressing luciferase andgreen fluorescent protein by stable transfection, was establishedat MacroGenics. All cell lines were passaged for less than 3months after thawing; all lines were confirmed to be free ofmycoplasma by PCR (Taconic, 2013) and were authenticatedon the basis of morphology, growth characteristics and CD19expression. Heparinized human whole blood was from Bio-logical Specialty Corporation. Cryopreserved purified primaryCLL patient samples were from AllCells, LLC. Heparinizedwhole blood from cynomolgus monkeys was from WorldwidePrimates, Inc.

Binding studiesMGD011 binding to human or cynomolgus monkey CD3 or

CD19 proteins was analyzed by ELISA or surface plasmon reso-nance (SPR) as previously described (13); binding to primaryhuman or cynomolgus monkey CD20þ, CD4þ or CD8þ cells wasanalyzed by flow cytometry.

Cell killing assayFor CTL assays, DART protein mediated killing of target cell

lines in the presence of human or cynomolgus PBMCs or purifiedT cells was determined by lactate dehydrogenase (LDH) release orluminescence assays as previously described (17).

B-cell depletion assayFor autologous B-cell depletion assays, PBMCs from normal

human donors, cryopreserved primary CLL specimens, orcynomolgus monkey were incubated with DART proteinsovernight and analyzed by flow cytometry using FSC/SSCgated lymphoid populations to determine B-cell depletion viaCD20 staining.

Cell activation studiesT-cell activation was determined by flow cytometry after stain-

ing with CD8-FITC, CD4-APC, CD25-PE, and CD69-PE-Cy5antibodies (BD Biosciences). For intracellular granzyme B andperforin determinations, T cells were stained with anti-CD4-PerCP.Cy5.5 and anti–CD8-APC antibodies (BD Biosciences),fixed, permeabilized (Cytofix/Cytoperm solution; BD Bio-sciences), and incubated with anti-granzyme B-FITC or anti-per-forin-PE antibody (BD BioSciences). T-cell proliferation was

Translational Relevance

Progress has beenmade in the clinicalmanagement of B-cellneoplasms, although most remain ultimately incurable withcurrentmodalities. Redirecting T lymphocytes to lyse lympho-ma/leukemia cells via bispecific molecules that simultaneous-ly engage CD3 on T cells with a B-cell antigen, such as CD19,has emerged as a powerful novel concept, highlighted by theclinical success of blinatumomab (Blincyto). Blinatumomab,however, requires continuous infusion, owing to its shortcirculating half-life. We report here on the preclinical devel-opment of MGD011, a bispecific DART molecule withincreased in vitro cytolytic activity compared to blinatumomaband engineered for improved circulating half-life. MGD011showed potent antitumor activity in mouse leukemia/lym-phoma models and displayed prolonged pharmacokineticproperties in cynomolgus monkeys, a cross-reacting species.MGD011 was well tolerated in monkeys, with durable andprofound B-cell depletion following weekly administrations.MGD011's potent activity and pharmacokinetic propertiesmay offer therapeutic convenience and applicability in thetreatment of B-cell malignancies.

A CD19 x CD3 Bi-specific Molecule with Extended Half-life

www.aacrjournals.org Clin Cancer Res; 23(6) March 15, 2017 1507




A

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sTime

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face

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kd (±SD)(S–1)

KD (±SD)(nmol/L)

Human CD3ε/δ 2.2 (±0.0) × 105 4.5 (±0.1) x 10–3 21.2 (±1.7)

Cynomolgus CD3ε/δ 2.0 (±0.1) × 105 4.3 (±0.2) x 10–3 21.9 (±1.2)

Human CD19 2.8 (±0.3) × 105 0.56 (±0.6) x 10–3 2.0 (±0.2)

Cynomolgus CD19 2.4 (±0.1) × 105 4.7 (±1.1) x 10–3 20.3 (±1.3)

D

H

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H2 HOOCN Fc KnobSSCD19VL CD3VH ES

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Clin Cancer Res; 23(6) March 15, 2017 Clinical Cancer Research1508




determined by flow cytometry as dilution of carboxyfluoresceindiacetate, succinimidyl ester (CFSE; Invitrogen) in labeled cells.IL-2, IFN-g , and TNF-a levels in culture supernatants were quan-titated by ELISA (R&D Systems).

In vivo studies in murine modelsAll studies were reviewed and approved by MacroGenics'

Institutional Animal Care and Use Committee (IACUC).MGD011 in vivo activity was evaluated in an established HBL-2lymphoma model and an established, disseminated Raji/GFleukemia/lymphoma model in human PBMC-reconstituted NSGbeta 2 microglobulin (B2m)-deficient (NSG B2m�/�) femalemice aged 7 to 8 weeks (Jackson Laboratory). Tumor volume inthe HBL-2 lymphoma model was calculated as follows: (length xwidth2)/2. Tumor burden in the Raji/GF leukemia/lymphomamodel was evaluated by whole-body imaging on an IVIS Spec-trum imaging system (PerkinElmer).

Cynomolgus monkey studiesThe studies were performed at Charles River Laboratories

(Reno,NV)under IACUCguidelines.Naive cynomolgusmonkeys(Macaca fascicularis) of Chinese origin (2.5–6.3-years-old, 2.3–3.9kg bodyweight) were randomized byweight and sex and receivedvehicle or MGD011 via 2-hour intravenous (IV) infusion (Sup-plementary Table S1). Serum samples were collected to examinethe PK profile of MGD011 by ELISA using immobilized goat anti-hXR32 antibody (recognizes CD3 arm of MGD011) for captureand goat anti-human IgG Fc-biotin together with streptavidin-horseradish peroxidase (SA-HRP) for detection. Whole bloodsamples were collected for flow cytometry and the peripheralblood cell surface phenotype analyzed by using the followingantibodies: CD4-V450, CD8-FITC, CD20-V500-C, CD45-PerCP,CD16-PE, CD159a-APC, CD14-APC-H7, CD69-FITC, PD-1-PE,TIM3-APC, CD4-APC-H7, CD8-V500, CD3-Pacific Blue, andKi-67 Alexa647 (BD Biosciences). Absolute numbers of cells weredetermined by Trucount� (BD Biosciences). Additional serumsamples were collected to examine cytokine levels measured bythe MILLIPLEX� MAP Non-Human Primate Cytokine MagneticBead Panel 7-Plex (EMD Millipore). Formalin-fixed paraffin-embedded sections of bone marrow, lymph node and spleenwere evaluated by immunohistochemistry (IHC) for CD20. Inbrief, tissues were deparaffinized, rehydrated, antigen retrievedand incubated with a mouse anti-CD20 antibody (Dako), fol-lowed by the Envisionþ System-HRP Labeled Polymer Anti-Mouse antibody (Dako) and developed with Diaminobenzadine(DAB).

Flow cytometryFlow cytometric analyses were carried out on a FACS Calibur

flow cytometer (4-color analyses) or BD LSRFortessa cell analyzer(8-color analyses) equippedwithCellQuest ProVersion5.2.1 (BDBiosciences) and FlowJo v9.3.3 (Treestar).

Statistical analysisIn vitro assays were repeated at least three times. Nonlinear

regression analyses were used to fit curves using GraphPad Prism.For in vivo studies, intergroup differences were assessed by two-way ANOVA with a Bonferroni correction and survival curvecompared by the log-rank test. All analyses were performed usingGraphPad Prism software (version 5.02). P values of �0.05 wereconsidered statistically significant.

ResultsEngineering, physicochemical characterization, and bindingproperties of MGD011

MGD011 is an Fc-bearing DART protein composed of threepolypeptide chains covalently linked by disulfide bonds(Fig. 1A). The Fc domain was engineered to greatly reduce oreliminate FcgR and complement C1q binding (14), whileretaining binding to FcRn to prolong MGD011 circulatinghalf-life. MGD011 binds human and cynomolgus monkey CD3with nearly identical affinity (KD ¼ 21.2 and 21.9 nmol/L,respectively), while binding to CD19 is 10-fold lower in mon-keys compared with humans (KD ¼ 20.3 and 2.0 nmol/L,respectively) due to a faster dissociation rate (Fig. 1B; Supple-mentary Fig. S1C). Importantly, MGD011 binds to both anti-gens simultaneously, as shown by bispecific ELISA and SPRanalyses that employ human CD3 protein for capture andhuman CD19 protein for detection (Fig. 1C and D); moreover,it recognizes the native antigens on both human and cyno-molgus monkey B and T lymphocytes (Supplementary Fig. S2).

MGD011 redirects T-lymphocytes to kill CD19þ B lymphocytesMGD011 activity was first evaluated by assessing its ability

to mediate redirected cytolysis by using purified human primaryT lymphocytes as effector cells and several CD19þ humanB-lymphoma lines as target cells. Although MGD011 mediatedno killing activity against the CD19� cell line, MOLM-13(Fig. 1E), potent killing was observed against all CD19þ linestested, including Raji/GF (Fig. 1F and G) as well as HBL-2 andJeko-1 cells (Supplementary Fig. S3A–S3B). A control DARTprotein, in which the CD19 arm was replaced by one with anirrelevant specificity, did not mediate killing. Furthermore,

Figure 1.MGD011 structure, binding, and redirected T-cell killing activity against CD19þ target cells. A, Schematic of the disulfide-linked polypeptide chain structureof MGD011. VL and VH are variable light and heavy chain sequences, respectively; E and K are E-coil and K-coil sequences, respectively; anddotted lines represent disulfide bonds. B, Equilibrium dissociation constants (KD) for MGD011 binding to human or cynomolgus monkey CD3 or CD19proteins. C, Bispecific ELISA using human CD3 protein for MGD011 capture and human CD19 protein for detection. Control DART proteins in whichthe variable domain sequences of an anti-fluorescein mAb 4-4-20 replaced either one of the MGD011 arms (Fluo x CD3 or CD19 x Fluo) were used.D, Bispecific SPR analysis of the binding of human CD19 to MGD011 captured by immobilized human CD3. E–G, Concentration dependence ofMGD011-mediated cytotoxicity against CD19� (MOLM-13) or CD19þ (Raji/GF) target cell lines using human T lymphocytes as effectors at effector:target (E:T) cell ratio of 10:1 and LDH release (E,F) or luminescence (G) assays to measure target cell killing following 24-hour incubation, comparedto that mediated by a control DART protein (Fluo x CD3) or a replica of blinatumomab (CD19 x CD3 BiTE molecule). H, Concentration dependenceof MGD011-mediated autologous B-cell depletion in human PBMCs; the percentage of CD20þ cells was determined by flow cytometry after incubationwith MGD011, a replica of blinatumomab or a control DART protein (Fluo x CD3).






compared to blinatumomab, MGD011 demonstrated enhancedpotency, with EC50 values approximately 10-fold lower forkilling of Raji/GF cells (0.02–0.03 ng/mL for MGD011 vs.0.38–0.41 ng/mL for blinatumomab). MGD011 also inducedB-cell depletion in human PBMCs from normal donors, withendogenous T cells serving as effector cells, demonstrating anapproximately 10-fold greater potency than blinatumomab inthis assay as well (Fig. 1H).

Incubation of MGD011 with CD19þ target cells (Raji/GFcells) was associated with concomitant CD4þ and CD8þ T-cellactivation as evidenced by upregulation of CD69, but noactivation occurred with CD19-negative cells or the controlDART protein (Fig. 2A). CFSE dilution showed induction of T-cell proliferation by MGD011, but not with the control DARTprotein (Fig. 2B). A dose-dependent upregulation of granzymeB and perforin levels in both CD8þ and CD4þ T cells wasobserved following treatment with MGD011 (Fig. 2C). Theupregulation of granzyme B and perforin was higher in CD8þ

T cells compared with CD4þ T cells, consistent with theexpected higher CTL potency of CD8þ T cells. Incubation withMGD011 in the presence of CD19þ target cells also resultedin the release of cytokines, exemplified in Fig. 2D by the releaseof IL-2 and IFN-g . No T-cell activation, proliferation, or cyto-kine release was observed in the absence of co-engagement ofCD19þ target cells, attesting to the strict dependence on CD19expression for MGD011-mediated T-lymphocyte activation.

To further evaluate the cytolytic activity of MGD011 againstprimary malignant B cells, MGD011 was incubated with PBMCsfrom patients with CLL, thus relying on the endogenous residualT lymphocytes as effectors. Malignant B cells (CD20þ/CD5þ) weredepleted in the presence of MGD011, but not control DARTprotein, in a time-dependent manner (Fig. 3A; SupplementaryFig. S4A), accompanied by concomitant expansion (Fig. 3B; Sup-plementary Fig. S4B) and activation (CD25 induction) of bothCD4þ and CD8þ T cells (Fig. 3C andD; Supplementary Fig. S4C—S4D). Thus, MGD011 mediates potent tumor cell killing not onlyagainst model cell lines but also primary leukemia samples.

Antitumor activity of MGD011 in human PBMC-reconstituted,xenograft tumor-bearing mice

Redirected T-cell killing by DART molecules can be recapitu-lated inmousemodels bearing human tumor xenografts as targetsand human PBMCs as effector cells (9). Tominimize graft-versus-host disease (GVHD) associated with human T-cell engraftment,mice deficient in the expression ofMHC class I via knock-out of itsB2m component were used. Lack of B2m expression, however, isassociated with impaired FcRn expression; hence, serum half-lifeofMGD011 is short (�4 hours) in thesemice compared to that inwild-type mice (�135 hours; data not shown). Frequent dosing(every 2–4 days) was therefore employed to achieve adequateexposure in this model.

Antitumor activity of MGD011 was first evaluated in humanPBMC-reconstituted mice bearing established HBL-2 lymphomacell tumors. MGD011 treatment (500 mg/kg IV every 3–4 days for8 doses) resulted in regression of HBL-2 tumors, with no evidenceof relapse up toDay42, the last studyday (>25days after initiationof treatment; Fig. 4A). MGD011 activity was also evaluated in adisseminated Raji/GF leukemia/lymphoma model in humanPBMC-reconstituted mice. As a prophylactic treatment (Supple-mentary Fig. S5), MGD011, administered at the same time of Rajicell intravenously inoculation, inhibited tumor dissemination

and improved survival at all doses tested (0.16–500 mg/kg), withmaximal activity at �20 mg/kg. A modest, albeit statisticallysignificant effect of the CD3-targeted control DART molecule(500 mg/kg) was also observed, possibly due to micro-clusteringof CD3 in vivo at such a high dose level. In the therapeuticparadigm, treatment was delayed 2 weeks after inoculation ofRaji/GF cells to allow for establishment of disseminated lesions.Image data through day 46 and survival curves through studycompletion (day 48) are shown in Fig. 4B and C. Most vehicle-treatedmicemet the euthanasia endpoint by the endof the secondweek after Raji/GF cell inoculation and all were dead by the end ofweek 4. A slightly longer survival was observed with 100 mg/kg ofcontrol DART protein, albeit not significantly different from thevehicle-treated group in this experiment. In contrast, the tumorburden in the animals treatedwithMGD011 showeda statisticallysignificant survival at doses �4 mg/kg, with near complete tumorregression at 100 mg/kg. In all experiments, treatment withMGD011 at any dose level was not associated with body weightloss (data not shown).

MGD011 demonstrates prolonged circulating half-life incynomolgus monkeys

Two cynomolgus monkey studies were performed (Supple-mentary Table S1). Analysis of MGD011 serum concentration-time profiles across these studies (Fig. 5A and B) showed dose-proportional increases in maximum serum concentration (Cmax)across the entire dose range evaluated, indicating linear PK.Clearance was lower than the glomerular filtration rate for cyno-molgus monkeys (�125 mL/h/kg, Supplementary Table S2), asexpected for a protein of this molecular size (�110 kDa), indi-cating that virtually no elimination occurs by renal filtration.Mean initial volumes of distribution (V1) are similar to or slightlyhigher than the plasma volume in cynomolgus monkeys (�45mL/kg), suggesting little or no depletion of MGD011 by bindingto target cells. MGD011 demonstrated a prolonged beta-phasehalf-life (t1/2,b) of 161.4� 61.3 hours (6.7� 2.6 days) and meanresidence time (MRT) of 190.6 �74.0 hours (7.9 � 3.1 days),consistent with that of an IgG Fc-bearing molecule in this species.Notwithstanding the foreign nature of the molecule in thisspecies, only 3/40 MGD011-treated animals in the GLP study(one in the 2mg/kg dose group and two in the 5mg/kg dose group)showed aberrant PK profiles after the first or subsequent infusionsand were confirmed positive for anti-drug antibodies (ADA, datanot shown).

SustainedB-cell depletion in cynomolgusmonkeys treatedwithweekly doses of MGD011

MGD011was confirmed to be active with cynomolgusmonkeycells as it mediated effective autologous B-cell depletion in mon-key PBMCs in vitro (Supplementary Fig. S6A); it was, however,less potent than with human PBMCs (EC50 values of 0.016-0.69ng/mL, n ¼ 6 for human PBMCs vs. 2.44-93.38 ng/mL, n ¼ 5 formonkey PBMCs). The CD3 arm of the DART is unlikely tocontribute to this disparity, since its affinity for CD3 of bothspecies is nearly identical (Fig. 1B); furthermore, MGD011 medi-ated comparable cytotoxicity against human CD19þ target cellswith human or monkey PBMCs as effectors (Supplementary Fig.S6B and S6C). The differential potency in autologous B-celldepletion between species is likely contributed by a combinationof factors, including an approximately 10-fold lower affinity ofMGD011 for cynomolgus monkey CD19 compared with human

Liu et al.





CD19 (Fig. 1B), a lower density of CD19 on cynomolgus monkeyB cells (Supplementary Fig. S2A and S2B), and a lower average T-cell-to-B-cell (T:B) ratio in PBMCs of cynomolgus monkeys (T:B

ratio¼ 4:1, n¼ 5) comparedwith humans (T:B ratio¼ 7:1, n¼ 6).These differencesmake pharmacodynamicmodeling in cynomol-gus monkeys a conservative estimate of MGD011 potency and

Figure 2.

MGD011-mediated T-cellactivation, proliferation, andcytokine release is dependent onco-engagement of CD19þ targetcells. A, MGD011 concentration-dependent induction of T-cellactivation marker CD69 on CD4þ

and CD8þ human T cells following24-hour incubation with CD19�

JIMT-1 cells or CD19þ Raji/GFtarget cells at E:T cell ratio of 10:1.B,MGD011-dependent induction ofproliferation of CFSE-labeledhuman T cells co-cultured withCD19þ HBL-2 target cells for 72hours at E:T cell ratio of 10:1. C,MGD011 concentration-dependentupregulation of granzyme B andperforin in CD4þ and CD8þ humanT cells following 24-hourincubation with CD19þ Raji/GFtarget cells at E:T cell ratio of 10:1.D, MGD011 concentration-dependent induction of IFN-g andIL-2 following 24-hour incubationof human T cells in the absence(left) or presence (right) of CD19þ

Raji/GF target cells at E:T cell ratioof 10:1.






should be taken into consideration when projecting humandoses.

Administration of MGD011 resulted in a decrease in circu-lating CD20þ B cells by 24 hours following the start of the firstinfusion (Fig. 5C and D). Although a partial reduction incirculating B cells was noted at the 0.2 mg/kg dose, nearlycomplete depletion was observed at dose levels �0.5 mg/kg andwere maintained throughout the 4 weekly treatments. Uponcompletion of dosing, circulating B cells returned to predoselevels, albeit with a dose-proportional lag (Fig. 5C). Histopa-thology examination at terminal necropsy showed a dose-dependent decrease in follicles and germinal centers within thespleen, lymph nodes (inguinal, mandibular, and mesenteric),and gut-associated lymphoid tissue (GALT; data not shown).CD20 immunohistochemistry (IHC) showed a reduction inCD20þ B cells to nearly undetectable levels in the lymph nodes,spleen, and bone marrow at doses �10 mg/kg (Fig. 5E and F).The pharmacologic effect of MGD011 on B lymphocytes wasprolonged, but ultimately reversible, as indicated by the returnof circulating B cells to pretreatment levels and the absence of

any abnormal histological and IHC findings at the end of a 12-week recovery period (Fig. 5E). In summary, once weeklyadministration of MGD011 was sufficient to induce completeand durable B-cell depletion in both the circulation and lym-phoid organs.

Treatment of MGD011 was associated with transient, dose-dependent fluctuations of both circulating CD4þ and CD8þ Tcells, with the lowest nadir following the first infusion and with adecreasing magnitude following subsequent infusions (Fig. 6AandB).Contrary to the durable reduction inB cells, T lymphocytesrecovered quickly after each infusion and reached or exceededbaseline levels before the next dose. Similar T-cell kinetics wereobserved in cynomolgus monkeys after the administration ofMGD006, a CD123 x CD3 DART protein (13), and in humansduring blinatumomab infusion (18). Fluctuations were alsoobserved with natural killer cells and monocytes in MGD011-treated monkeys (data not shown), likely representing transientcell margination.

MGD011 treatment was also associated with upregulation ofthe T-cell activation markers, CD69 and PD-1 (Fig. 6C–F),

CD4

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Figure 3.

MGD011-dependent depletion of malignant B cells and activation of T cells with PBMCs from a CLL patient. MGD011 or a control DART protein (Fluo x CD3) at0.05 mg/mL were incubated with PBMCs from a CLL patient (E:T ¼ 1:23) and analyzed by flow cytometry at Day 0 (before DART protein addition)and days 3 and 6. A, The percentage of CD20þ/CD5þ malignant B cells in the gated lymphoid population. B, The percentage of CD4þ and CD8þ T cells.C and D, The percentage of CD25þ T-cell activation in CD4þ and CD8þ T cells, respectively.

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together with an increase of the Ki67þ proliferating fraction ofboth CD4þ and CD8þ T-cell subsets during the treatment phase(Fig. 6G and H). Therefore, MGD011 is capable of mediatingT-cell activation and expansion in vivo. The activation markerstrended toward or returned to baseline by the end of the exper-iment; however, unexplained isolated increases in one group 4animal (showing up to 10-fold higher frequency of CD4þCD69þ

cells than the remaining 3 animals) and in two vehicle controlanimals (up to 10-fold higher frequency of CD8þPD1þ cells thanthe other 2 animals) during the recovery phase contributed toswelling the average for these groups (Fig. 6C and F). An asymp-tomatic, transient, dose-dependent increase in circulating cyto-kine levels, a first-dose effect, was observed for IFN-g (Fig. 6I), IL-6(Fig. 6J), and IL-10 (Fig. 6K) and, to a lesser extent, TNF-a levels(data not shown), all occurring 2 hours following the end of thefirst infusion and returning to at or near baseline within 24 hoursof the start of the infusion. Smaller levels of cytokine increasewerenoted with subsequent infusions, which were of a magnitudecomparable with those observed following vehicle infusions,

except IL-10, which exceeded levels of the vehicle control groupin some instances. No MGD011-related changes in circulatingIL-2, IL-4, or IL-5 levels were noted.

MGD011waswell tolerated in cynomolgusmonkeys at all doselevels tested, with no compound-related toxicological effects,including cage-side observations, changes in body weight or foodconsumption, vital parameters, ophthalmology, electrocardio-grams, body temperature, blood pressure, heart rate, respirationrate, neurological examinations, coagulation, or clinical chemis-try (data not shown); furthermore, with the exception of theaforementioned hematological, bone marrow, and lymphoidorgan changes, no other gross or microscopic pathological find-ings were noted (data not shown).

DiscussionMGD011 is a bispecific DART protein developed for the treat-

ment of B-cell malignancies and designed to redirect the Tlymphocytes via their CD3 molecule to kill target cells expressing

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Figure 4.

MGD011-mediated antitumor activity in human PBMC-reconstituted xenograft tumor-bearing mice. A, Treatment of human PBMC-reconstituted micewith established intradermal HBL-2 lymphoma cell tumors. NSG B2m�/�mice (n¼ 8/group) were implanted intradermally with HBL-2 tumor cells (5 � 106) onday 0 followed by intraperitoneal injection of human PBMCs (1 � 107) on day 4. Tumor-bearing mice, randomized at day 14, were treated with MGD011or a control DART protein (Fluo x CD3) at 500 mg/kg IV every 3–4 days for a total of 8 doses beginning at Day 17. Tumor volume was measured at indicatedtimes through Day 42 and plotted as group mean � SEM. No changes in body weight were observed (data not shown). B, Treatment of human PMBC-reconstituted mice with established disseminated CD19þRaji/GF leukemia/lymphoma tumors. NSG B2m�/� mice (n ¼ 8 per group) were implantedintraperitoneally with human PBMCs (1�107) on Day -5 followed by Raji/GF tumor cells (1 � 106) injected intravenously on day 0. Tumor-bearing mice,randomized at day 14, were treated with MGD011, a control DART protein (Fluo x CD3) or vehicle, intravenously every 2-4 days for a total of 9 dosesbeginning at Day 15 and tumor burden was monitored by whole-body imaging. Each row under each treatment group represents an individual animal; "X"indicates the animal was found dead or sacrificed. No changes in body weight were observed (data not shown). C, Survival curves from the treatment groupsin B. � , P < 0.001 and �� , P ¼ 0.03 by log-rank test comparison to the vehicle-treated group.






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CD19. MGD011 was shown to eliminate normal or pathogenicCD19þ cells in vitro and in vivo. Furthermore, weekly administra-tion of MGD011 to cynomolgus monkeys resulted in completeand durable depletion of B cells in the circulation and in lym-phoid tissues. MGD011 was well tolerated in toxicology studiesunder treatment regimens that exaggerated potential clinicalsetting scenarios in patients. Thanks to its favorable PK properties,convenient once-a-week dosing or longer interval is predicted tobe clinically feasible in humans.

The structure of MGD011 represents a refinement of a previ-ously reported format (8), where the DART chains (Chain 1 andChain 2) preserve the original C-terminal stabilizing disulfidelinkage, while opposite E/K-coil sequences were added that fur-ther improve heterodimer formation. To promote the desiredheterodimerization of Chain 1 and the Fc-domain Chain 3, knob-into-hole mutations were incorporated in the CH3 region. Tofacilitate the removal of residual Chain 3 dimers during purifi-cation, a H435R mutation was included in this chain to abolishprotein A binding. MGD011 was engineered with humanizedantibody arms displaying 10-fold greater affinity for humanCD19 than for human CD3, thus providing for preferential initialbinding to target cells, while minimizing CD3 engagement in theabsence of target cells. MGD011 exhibits binding tomonkey CD3and CD19, allowing for preclinical modeling in this species.Specifically,MGD011 cross-reacts with cynomolgusmonkeyCD3with nearly identical affinity as for humanCD3 andwith a 10-foldlower affinity for monkey CD19. Consistent with its bindingproperties, MGD011 mediated similar levels of cytotoxicityagainst human CD19þ Raji/GF target cells by either human orcynomolgus monkey PBMCs. MGD011 mediated also a dose-dependent, autologous ex vivoB-cell depletion inbothhumanandcynomolgus monkey PBMCs, albeit with a decreased potency inthe latter, due, at least in part, to its lower affinity for monkeyCD19. Because of the lower CD19 affinity and reduced potency incynomolgus monkeys, this species may offer a conservative esti-mate of potency that needs to be taken into consideration inextrapolating non-human primate data to potential clinicalsettings.

To confer prolonged circulating half-life, MGD011 was alsoengineered with an Fc-domain; with an estimated terminal half-life of approximately 161 hours (6.7 days) in monkeys and apredicted half-life of 343-495 hours (14.3–20.6 days) in humans,which is similar to that of conventional mAbs, deliveringMGD011 as an intermittent dosing regimen appears feasible. Incomparison, blinatumomab, which is engineered as a single-chain Fv pair, is administered by continuous intravenous infusionfor repeated 4-week courses, owing to its short approximately 2-hour half-life.

MGD011 administered to cynomolgus monkeys by a 2-hourintravenous infusion on a weekly schedule at doses rangingfrom 0.2 to 100 mg/kg was safe and well tolerated. Dose-dependent, on-target activity with nearly complete depletionof circulating CD20þ B cells was observed at doses �0.5 mg/kg.Histologic evaluation confirmed a dose-dependent decreasein B-cell zones in lymphoid tissues at the terminal necropsy.Although bone marrow showed no obvious histologicalchanges, B-cell depletion was observed by IHC, further attestingto the specificity of the on-target effects of MGD011. Thepharmacologic effect of MGD011 on B lymphocytes was revers-ible, as indicated by flow cytometry and IHC analysis in addi-tion to hematology and histopathology (data not shown). Eachof these parameters returned to baseline, with no differentia-tion between the animals receiving MGD011 or control articleat the end of the 12-week recovery period.

Maximal cytolytic activity against B-cell lines and in patientsamples ex vivowere observed atMGD011 concentrations of 50 to100 ng/mL. These levels are at or below the Cmax observed inmonkeys at�5 mg/kg or below trough levels at�50 mg/kg. Eitherdose level resulted innear complete depletionof B lymphocytes inthe circulation and lymphoid tissues in monkeys, attesting to thepotency of the DART molecule, particularly in account itdecreased potency in this species compared with humans. InB-cell malignancies, a low effector-to-target (E:T) cell ratio couldlimit the effectiveness of redirected cytolysis. MGD011, however,was able to eliminate leukemic cells in CLL patient samples byengaging and expanding the patient's residual T cells in vitro froma low E:T cell ratio to one exceeding the target cell population.These data also confirm that CLL T cells can be redirected tobecome cytolytic against leukemic cells in the presence ofMGD011, as previously reported for blinatumomab (19), eventhough T-cell dysfunction in CLL, such as defects in immunesynapse formation, co-stimulatory/accessory molecule expres-sion, and cytokine release have been reported (20–22).

A safety concern associated with CD3-targeting therapies iscytokine release. The monovalent nature of the binding arm ofMGD011 ensures that T-cell activation and cytokine releasedepend exclusively on target-cell engagement. Consistent withits desired design, no T-cell activation or cytokine release wereobserved in vitro in the absence of CD19þ target cells or with acontrol DART protein that includes only the CD3-targeting arm.No cytokine stormwas observed inMGD011-treated cynomolgusmonkeys. Transient dose-dependent increases in IFN-g , IL-6, andIL-10 were observed following the first dose; with subsequentdoses, they were generally no greater than those observed inanimals receiving vehicle. There were no MGD011-relatedchanges in circulating IL-2, IL-4, or IL-5 levels and only small,

Figure 5.Serum concentrations and B-cell depletion in cynomolgus monkeys following weekly doses of MGD011. A–B, MGD011 serum concentration-time profiles froma repeat-dose study (A, Study 1, Supplementary Table S1) in monkeys (5/sex/group) in which vehicle or MGD011 (dose levels ranging from 0.2 to 10 mg/kg,as indicated) were administered intravenously once weekly for 4 weeks or a repeat-dose study (B, Study 2, Supplementary Table S1) in monkeys (1–2/sex/group) in which MGD011 was administered intravenously once or twice weekly at the indicated dose levels for 3 or 4 weeks. In both studies,vehicle (V) was administered one week before the start of MGD011 dosing (D) with the exception of the fixed dose 0.5 mg/kg groups in B. The assaylower limit of quantitation (1.36 ng/mL in A, Study 1 or 2.45 ng/mL in B, Study 2) is indicated by dashed lines. Note: serum concentrations for animalstreated with 5 or 50 ng/kg MGD011 were below the lower limit of quantitation at all time points evaluated. C–D, Levels of circulating CD20þ B cells in the abovestudies. E, CD20 IHC of bone marrow, spleen, and lymph node tissues collected at terminal necropsy (1 week after the last dose) or recovery necropsy(12 weeks after the last dose). F, CD20 IHC of bone marrow, spleen, and lymph node tissues collected 3 days after the last dose for the fixed (5 ng/kg� 3) dosegroup or 7 days after the last dose for the escalating (0.5 to 50 mg/kg and 2 to 100 mg/kg) dose groups.






transient increases in TNF-a, exceeding 100 pg/mL in only a fewinstances, were observed in animals that received 10 mg/kgMGD011. First-dose cytokine release events have been previously

observed in patients treated with blinatumomab (18) or with ourCD123 x CD3 DART molecule in monkeys (13); the rapid target-cell depletion following the first administration of the bispecific

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Changes in peripheral T-lymphocytesubpopulations and cytokines incynomolgus monkeys administeredweekly doses ofMGD011. Data are from therepeat-dose study 1 in monkeys (5/sex/group) in which MGD011 (dose levelsranging from 0.2 to 10 mg/kg) or vehiclewere administered intravenously onceweekly for 4 weeks (Supplementary TableS1). A–B, CD4þ and CD8þ T cells. C–D,CD69-activated CD4þ and CD8þ T cells.E–F, PD-1–activated CD4þ and CD8þ Tcells.G–H, Proliferating (Ki67þ) CD4þ andCD8þ T cells. I–K, Serum levels ofcytokines: IFN-g , IL-6, and IL-10; V, vehicleinfusion; D, MGD011 infusion.

Liu et al.





molecule may limit further T-cell engagement and explain thetransient nature of cytokine release observed.

Tumor lysis syndrome, which has been observed with blina-tumomab (7), should be anticipated as another potential safetyconcern for MGD011 and proper preventive measures be imple-ment to limit its consequences. Blinatumomab treatmentwas alsoassociated with an increased risk of serious and/or severe neuro-logical toxicities (7); a similar safety risk of neurological toxicitiesforMGD011 is therefore recognized. It should be noted, however,that no signs of neurological toxicity were observed in theMGD011 toxicology studies, although the predictive value of thecynomolgusmonkeywith respect to neurotoxicity of CD19 xCD3bispecific interventions is unknown. Similarly, no infection-relat-ed adverse events were observed in monkeys treated withMGD011, although 25% of patients receiving blinatumomabshowed signs of infection (7). Hence, patients should be mon-itored for infections and treated appropriately.

Immunogenicty remains a potential concern associated withantibody therapy. MGD011 limits this through the incorporationof humanized Fv regions and theminimal useof linkers. Althoughimmunogenicity inmonkeys does not predict immunogenicity inhumans, notably, only 3 of 40 monkeys treated with MGD011showed an ADA response; given that MGD011 targets B lympho-cytes, ADA development is expected to be low in all species.

Upregulation of activation markers, including PD-1, wasobserved amongst circulating T lymphocytes in monkeys treatedwithMGD011. Importantly, the frequency of regulatory T cells orTIM-3þ cells in both the CD4þ and CD8þ T-lymphocyte popula-tions remained low (<1%) throughout the entire dosing period(data not shown), suggesting that T-cell activation induced byMGD011was not accompanied by expansion of regulatory T cellsor T-cell exhaustion.

The off-the-shelf convenience of bispecific antibodies offerssubstantial advantages comparedwith personalizedCART cells. Apoint of contention, however, is whether a trade-off of bispecificmolecules is a decrease in potency. The impressive response toCAR T-cell therapy is associated with a significant rate of severeadverse events, resulting in a narrow therapeutic window, withlittle, if any, opportunity for response fine tuning. We shouldfurther note that MGD011, on a molar basis, is more potent thanblinatumomab and that complete depletion of B lymphocytes inboth marrow and lymphoid tissues can be achieved in cynomol-gus monkeys in the absence of adverse effects. Ultimately, onlyclinical data will be able to address this issue.

In summary, MGD011 demonstrated potent activity in redir-ecting T lymphocytes to eliminate CD19-expressing cells in vitro,induced growth inhibition and tumor regression of B-cell lym-phoma/leukemia models in mice, showed prolonged circulating

half-life and was safe and well tolerated in cynomolgus monkeysat doses that resulted in complete and durable B-cell depletion inthe circulation, bone marrow, and lymphoid organs. The datafrom these nonclinical studies provided rationale for the clinicaldevelopment of MGD011 as a treatment for patients with B-cellmalignancies; a phase I dose-escalation study of the safety, tol-erability, dose-limiting toxicity, maximal tolerated does, andrecommended phase II dose of MGD011 when administratedintravenously over a 2-hour period once every 2 weeks to subjectswith B-cell malignancies is currently recruiting patients(NCT02454270).

Disclosure of Potential Conflicts of InterestAll authors are or were employees of MacroGenics, Inc. and received

compensation from and hold ownership interest (including patents) in Macro-Genics, Inc.

Authors' ContributionsConception and design: L. Liu, C.-Y.K. Lam, R. Alderson, J.L. Nordstrom,S. Koenig, P.A. Moore, S. Johnson, E. BonviniDevelopment of methodology: L. Liu, C.-Y.K. Lam, L. Widjaja, H. Li,R. AldersonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Liu, C.-Y.K. Lam, V. Long, L. Widjaja, Y. Yang,H. Li, L. Jin, J. Brown, R. AldersonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Liu, C.-Y.K. Lam, L. Widjaja, H. Li, R. Alderson,J.L. Nordstrom, P.A. Moore, S. Johnson, E. BonviniWriting, review, and/or revision of the manuscript: L. Liu, C.-Y.K. Lam,M.D. Lewis, J.L. Nordstrom, S. Koenig, P.A. Moore, S. Johnson, E. BonviniAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Widjaja, S. Burke, S. GorlatovStudy supervision: L. Liu, P.A. Moore, E. Bonvini

AcknowledgmentsWewould like to thankCynthia Sung (RainbowPharmaConsulting) for data

analysis, Robert Burns, Qin Tang, Nancy O'Gwin, and Xioqi Gong for technicalassistance, Melinda Hanson for administrative and editorial support, TimothyMayer and James Karrels for critical discussions and review of the manuscript.We thank the teams at the Charles River Laboratories, Inc. for the diligentconduct of the cynomolgus monkey studies.

Grant SupportThe research funding was provided by MacroGenics.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 11, 2016; revised August 8, 2016; accepted September 1,2016; published OnlineFirst September 23, 2016.

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Liu et al.




2017;23:1506-1518. Published OnlineFirst September 23, 2016.Clin Cancer Res Liqin Liu, Chia-Ying K. Lam, Vatana Long, et al. Treatment of B-Cell MalignanciesMolecule Incorporating Extended Circulating Half-life for the MGD011, A CD19 x CD3 Dual-Affinity Retargeting Bi-specific

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MGD011, A CD19 x CD3 Dual-Afﬁnity Retargeting Bi-speciﬁc ... · ination by CD3-expressing T lymphocytes as effector cells. MGD011 was engineered with a human immunoglobulin G1