MGD011, a CD19 X CD3 Dual-Affinity Retargeting Bi-Specific Molecule Incorporating Extended Circulating Half-Life for the Treatment of B-Cell Malignancies

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

Cancer Therapy: Preclinical Clinical Cancer Research MGD011, A CD19 x CD3 Dual-Afﬁnity Retargeting Bi-speciﬁc Molecule Incorporating Extended Circulating Half-life for the Treatment of B-Cell Malignancies Liqin 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 highly autologous B-cell depletion in PBMCs from both species. represented in B-cell malignancies and an attractive target for MGD011-mediated killing was accompanied by target-depen- therapeutic interventions. MGD011 is a CD19 x CD3 DART dent T-cell activation and expansion, cytokine release and bispecific protein designed to redirect T lymphocytes to eliminate upregulation of perforin and granzyme B. MGD011 demon- CD19-expressing cells. MGD011 has been engineered with a strated antitumor activity against localized and disseminated modified human Fc domain for improved pharmacokinetic (PK) lymphoma xenografts reconstituted with human PBMCs. In properties and designed to cross-react with the corresponding cynomolgus monkeys, MGD011 displayed a terminal half-life antigens in cynomolgus monkeys. Here, we report on the preclin- of 6.7 days; once weekly intravenous infusion of MGD011 at ical activity, safety and PK properties of MGD011. dosesupto100mg/kg, the highest dose tested, was well Experimental Design: The activity of MGD011 was evaluated tolerated and resulted in dose-dependent, durable decreases in several in vitro and in vivo models. PK, safety and pharmaco- in circulating B cells accompanied by profound reductions of B dynamic activity was also assessed in dose-escalation and repeat- lymphocytes in lymphoid organs. dose studies of MGD011 administered once weekly in cynomol- Conclusions: The preclinical activity, safety and PK pro- gus monkeys. file support clinical investigation of MGD011 as a thera- Results: MGD011 mediated killing of human B-cell lympho- peutic candidate for the treatment of B-cell malignancies. ma lines by human or cynomolgus monkey PBMCs as well as Clin Cancer Res; 1–13. 2016 AACR.

Introduction improved outcome in advanced B-cell malignancies. Yet, approximately 20,000 patients die of lymphoma every year in B-cell malignancies represent a heterogeneous group of dis- the United States alone. orders with varying characteristics and clinical behaviors (1). Providing T lymphocytes (CTL) with the ability to recognize Although systemic chemotherapy is still the mainstay of treat- and destroy tumor cells has shown promise in advanced forms ment for B-cell malignancies, kinase inhibitors that selectively of leukemia and lymphoma. In the form of chimeric antigen target molecules at the core of the transformation process and receptor (CAR) T-cell therapy, such an approach requires ex vivo antibody therapy are now well established tools (2). Among the isolation, transduction, and reinfusion of the patient's T cells. latter category, rituximab (Rituxan), a monoclonal antibody This complexity can be overcome with bispecific antibodies (mAb) that targets the B-cell antigen CD20, induces direct that bind simultaneously to an antigen expressed by malignant tumor cell apoptosis as well as complement- and antibody- BcellsandanactivationmoleculeonTlymphocytes(2).The dependent cytotoxicity (2, 3). These orthogonal mechanisms of pan B-cell marker, CD19, has emerged as a promising antigen action form the basis for therapeutic combinations that have for targeting B-cell malignancies because of its broader expression profile and lower rate of downregulation compared with other B-cell antigens (3). Its expression is highly conserved in Research, MacroGenics, Inc., Rockville, Maryland. the majority of B-cell tumors (4), with normal to high levels of Note: Supplementary data for this article are available at Clinical Cancer expression in 80% of acute lymphoblastic leukemia (ALL), 88% Research Online (http://clincancerres.aacrjournals.org/). of B-cell lymphomas, and all chronic lymphocytic leukemias Corresponding Author: Ezio Bonvini, MacroGenics Inc., 9704 Medical Center (CLL; refs. 5, 6). þ Drive, Rockville, MD 20850. Phone: 301-354-2638; Fax: 301-251-5321; E-mail: Redirection of CTL to CD19 leukemia cells via the bispecific [email protected] T-cell engager (BiTE) blinatumomab (Blincyto; ref. 7) is effec- doi: 10.1158/1078-0432.CCR-16-0666 tive in patients with B-cell malignancies whose disease did not 2016 American Association for Cancer Research. respondtostandardchemo-immunotherapies and has been

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FcgR and C1q binding (14). Control molecules in which the Translational Relevance variable domain sequences of an anti-fluorescein mAb 4-4-20 Progress has been made in the clinical management of B-cell (15) replaced either of the DART protein arms (Fluo x CD3 or neoplasms, although most remain ultimately incurable with CD19 x Fluo) were engineered in a similar manner. The DART current modalities. Redirecting T lymphocytes to lyse lympho- proteins were expressed transiently in CHO-S cells (8) and puri- ma/leukemia cells via bispecific molecules that simultaneous- fied to greater than 99% purity using protein A and either size- ly engage CD3 on T cells with a B-cell antigen, such as CD19, exclusion chromatography (SEC) or other polishing steps. The has emerged as a powerful novel concept, highlighted by the purified DARTs have very low levels (less than 1%) of high clinical success of blinatumomab (Blincyto). Blinatumomab, molecular weight (HMW) protein present and have an apparent however, requires continuous infusion, owing to its short molecular weight of approximately 110 kDa (Supplementary Fig. circulating half-life. We report here on the preclinical devel- S1A and B). opment of MGD011, a bispecific DART molecule with increased in vitro cytolytic activity compared to blinatumomab Other reagents, cell lines, and tissue samples and engineered for improved circulating half-life. MGD011 Recombinant soluble human and cynomolgus CD3e/d chi- showed potent antitumor activity in mouse leukemia/lym- meric proteins, as well as human and cynomolgus CD19 and phoma models and displayed prolonged pharmacokinetic CD19-His proteins, were expressed in CHO-S cells. MOLM-13 properties in cynomolgus monkeys, a cross-reacting species. and JIMT-1 were obtained from DSMZ (Braunschweig, Ger- MGD011 was well tolerated in monkeys, with durable and many); Jeko-1 cells were from the ATCC and HBL-2 from the profound B-cell depletion following weekly administrations. NationalInstitutesofHealth(Bethesda,MD;ref.16).Raji/GF,a þ MGD011's potent activity and pharmacokinetic properties CD19 Burkitt's B-cell lymphoma expressing luciferase and may offer therapeutic convenience and applicability in the green fluorescent protein by stable transfection, was established treatment of B-cell malignancies. at MacroGenics. All cell lines were passaged for less than 3 months after thawing; all lines were confirmed to be free of mycoplasma by PCR (Taconic, 2013) and were authenticated on the basis of morphology, growth characteristics and CD19 approved by the FDA for the treatment of patients with Phi- expression. Heparinized human whole blood was from Bio- fi ladelphia chromosome-negative relapsed or refractory B-cell logical Specialty Corporation. Cryopreserved puri ed primary precursor ALL. DART proteins are bispecific, antibody-based CLL patient samples were from AllCells, LLC. Heparinized molecules with favorable stability, manufacturability, and whole blood from cynomolgus monkeys was from Worldwide potency; furthermore, a CD19 x CD3 DART protein compared Primates, Inc. favorably with a BiTE of the same pair of VH and VL sequences in redirected cytolysis assays (8, 9). MGD011 (also known as Binding studies JNJ-64052781) is another CD19 x CD3 DART protein designed MGD011 binding to human or cynomolgus monkey CD3 or þ to simultaneously target CD19 cells for recognition and elim- CD19 proteins was analyzed by ELISA or surface plasmon reso- nance (SPR) as previously described (13); binding to primary ination by CD3-expressing T lymphocytes as effector cells. þ þ þ MGD011 was engineered with a human immunoglobulin human or cynomolgus monkey CD20 , CD4 or CD8 cells was fl G1 (IgG1) Fc domain to bind the neonatal Fc receptor (FcRn) analyzed by ow cytometry. and engage the IgG salvage pathway, thus conferring prolonged circulating half-life and the resultant dosing convenience. To Cell killing assay avoid unintended (target independent) CD3-mediated T-cell For CTL assays, DART protein mediated killing of target cell fi activation via interaction with Fc gamma receptors (FcgR), the lines in the presence of human or cynomolgus PBMCs or puri ed Fc domain was mutated to greatly reduce or eliminate binding T cells was determined by lactate dehydrogenase (LDH) release or to these receptors as well as complement. Unlike blinatumo- luminescence assays as previously described (17). mab, which reacts only with human or chimpanzee's antigens (10), MGD011 cross-reacts with both CD19 and CD3 mole- B-cell depletion assay cules in macaques, enabling preclinical evaluation in a relevant For autologous B-cell depletion assays, PBMCs from normal model. Here, we report on MGD011 engineering and charac- human donors, cryopreserved primary CLL specimens, or terization as well as on its preclinical safety assessment, phar- cynomolgus monkey were incubated with DART proteins fl macokinetics (PK), and dose optimization in cynomolgus overnight and analyzed by ow cytometry using FSC/SSC monkeys. The results show robust activity in multiple models gated lymphoid populations to determine B-cell depletion via and predict effective dosing in humans at once weekly or longer CD20 staining. intervals. Cell activation studies fl Materials and Methods T-cell activation was determined by ow cytometry after staining with CD8-FITC, CD4-APC, CD25-PE, and CD69-PE-Cy5 DART protein engineering, production, and purification antibodies (BD Biosciences). For intracellular granzyme B and MGD011, an Fc-bearing CD19 x CD3 DART protein, was perforin determinations, T cells were stained with anti-CD4- constructed as described (11) using VL and VH sequences from PerCP.Cy5.5 and anti–CD8-APC antibodies (BD Biosciences), humanized anti-CD19 mAb BU12 (12) and humanized anti-CD3 fixed, permeabilized (Cytofix/Cytoperm solution; BD Bio- mAb XR32 (13). The IgG1-derived Fc segment was modified to sciences), and incubated with anti-granzyme B-FITC or anti-per- encode the L234A/L235A mutation to greatly reduce or eliminate forin-PE antibody (BD BioSciences). T-cell proliferation was

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determined by flow cytometry as dilution of carboxyfluorescein Results diacetate, succinimidyl ester (CFSE; Invitrogen) in labeled cells. Engineering, physicochemical characterization, and binding IL-2, IFN-g, and TNF-a levels in culture supernatants were quan- properties of MGD011 titated by ELISA (R&D Systems). MGD011 is an Fc-bearing DART protein composed of three polypeptide chains covalently linked by disulfide bonds In vivo studies in murine models (Fig. 1A). The Fc domain was engineered to greatly reduce or All studies were reviewed and approved by MacroGenics' eliminate FcgR and complement C1q binding (14), while Institutional Animal Care and Use Committee (IACUC). retaining binding to FcRn to prolong MGD011 circulating MGD011 in vivo activity was evaluated in an established HBL-2 half-life. MGD011 binds human and cynomolgus monkey CD3 lymphoma model and an established, disseminated Raji/GF with nearly identical affinity (K ¼ 21.2 and 21.9 nmol/L, leukemia/lymphoma model in human PBMC-reconstituted NSG D respectively), while binding to CD19 is 10-fold lower in mon- beta 2 microglobulin (B2m)-deficient (NSG B2m / ) female keys compared with humans (K ¼ 20.3 and 2.0 nmol/L, mice aged 7 to 8 weeks (Jackson Laboratory). Tumor volume in D respectively) due to a faster dissociation rate (Fig. 1B; Supple- the HBL-2 lymphoma model was calculated as follows: (length x mentary Fig. S1C). Importantly, MGD011 binds to both anti- width2)/2. Tumor burden in the Raji/GF leukemia/lymphoma gens simultaneously, as shown by bispecificELISAandSPR model was evaluated by whole-body imaging on an IVIS Spec- analyses that employ human CD3 protein for capture and trum imaging system (PerkinElmer). human CD19 protein for detection (Fig. 1C and D); moreover, Cynomolgus monkey studies it recognizes the native antigens on both human and cyno- The studies were performed at Charles River Laboratories molgus monkey B and T lymphocytes (Supplementary Fig. S2). (Reno, NV) under IACUC guidelines. Naive cynomolgus monkeys þ (Macaca fascicularis) of Chinese origin (2.5–6.3-years-old, 2.3–3.9 MGD011 redirects T-lymphocytes to kill CD19 B lymphocytes kg body weight) were randomized by weight and sex and received MGD011 activity was first evaluated by assessing its ability vehicle or MGD011 via 2-hour intravenous (IV) infusion (Sup- to mediate redirected cytolysis by using purified human primary þ plementary Table S1). Serum samples were collected to examine T lymphocytes as effector cells and several CD19 human the PK profile of MGD011 by ELISA using immobilized goat anti- B-lymphoma lines as target cells. Although MGD011 mediated hXR32 antibody (recognizes CD3 arm of MGD011) for capture no killing activity against the CD19 cell line, MOLM-13 þ and goat anti-human IgG Fc-biotin together with streptavidin- (Fig. 1E), potent killing was observed against all CD19 lines horseradish peroxidase (SA-HRP) for detection. Whole blood tested, including Raji/GF (Fig. 1F and G) as well as HBL-2 and samples were collected for flow cytometry and the peripheral Jeko-1 cells (Supplementary Fig. S3A–S3B). A control DART blood cell surface phenotype analyzed by using the following protein, in which the CD19 arm was replaced by one with an antibodies: CD4-V450, CD8-FITC, CD20-V500-C, CD45-PerCP, irrelevant specificity, did not mediate killing. Furthermore, CD16-PE, CD159a-APC, CD14-APC-H7, CD69-FITC, PD-1-PE, compared to blinatumomab, MGD011 demonstrated enhanced fi TIM3-APC, CD4-APC-H7, CD8-V500, CD3-Paci c Blue, and potency, with EC50 values approximately 10-fold lower for Ki-67 Alexa647 (BD Biosciences). Absolute numbers of cells were killing of Raji/GF cells (0.02–0.03 ng/mL for MGD011 vs. determined by Trucount (BD Biosciences). Additional serum 0.38–0.41 ng/mL for blinatumomab). MGD011 also induced samples were collected to examine cytokine levels measured by B-cell depletion in human PBMCs from normal donors, with the MILLIPLEX MAP Non-Human Primate Cytokine Magnetic endogenous T cells serving as effector cells, demonstrating an Bead Panel 7-Plex (EMD Millipore). Formalin-fixed paraffin- approximately 10-fold greater potency than blinatumomab in embedded sections of bone marrow, lymph node and spleen this assay as well (Fig. 1H). þ were evaluated by immunohistochemistry (IHC) for CD20. In Incubation of MGD011 with CD19 target cells (Raji/GF þ þ brief, tissues were deparaffinized, rehydrated, antigen retrieved cells) was associated with concomitant CD4 and CD8 T-cell and incubated with a mouse anti-CD20 antibody (Dako), fol- activation as evidenced by upregulation of CD69, but no lowed by the Envisionþ System-HRP Labeled Polymer Anti- activation occurred with CD19-negative cells or the control Mouse antibody (Dako) and developed with Diaminobenzadine DART protein (Fig. 2A). CFSE dilution showed induction of T- (DAB). cell proliferation by MGD011, but not with the control DART protein (Fig. 2B). A dose-dependent upregulation of granzyme þ þ Flow cytometry B and perforin levels in both CD8 and CD4 Tcellswas Flow cytometric analyses were carried out on a FACS Calibur observed following treatment with MGD011 (Fig. 2C). The þ flow cytometer (4-color analyses) or BD LSRFortessa cell analyzer upregulation of granzyme B and perforin was higher in CD8 þ (8-color analyses) equipped with CellQuest Pro Version 5.2.1 (BD T cells compared with CD4 T cells, consistent with the þ Biosciences) and FlowJo v9.3.3 (Treestar). expected higher CTL potency of CD8 T cells. Incubation with þ MGD011inthepresenceofCD19 target cells also resulted Statistical analysis in the release of cytokines, exemplified in Fig. 2D by the release In vitro assays were repeated at least three times. Nonlinear of IL-2 and IFN-g. No T-cell activation, proliferation, or cyto- regression analyses were used to fit curves using GraphPad Prism. kine release was observed in the absence of co-engagement of þ For in vivo studies, intergroup differences were assessed by two- CD19 target cells, attesting to the strict dependence on CD19 way ANOVA with a Bonferroni correction and survival curve expression for MGD011-mediated T-lymphocyte activation. compared by the log-rank test. All analyses were performed using To further evaluate the cytolytic activity of MGD011 against GraphPad Prism software (version 5.02). P values of 0.05 were primary malignant B cells, MGD011 was incubated with PBMCs considered statistically significant. from patients with CLL, thus relying on the endogenous residual T

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A Chain 1 H2N CD19VL CD3VHS E SS Fc Knob HOOC

Chain 2 H2N CD3VL CD19VH S K HOOC

Chain 3 H2N SS Fc Hole HOOC

B k (±SD) k (±SD) K (±SD) Angens a d D (M–1S–1) (S–1) (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)

C D RU 600 Buﬀer Buﬀer 500 400 300 200 MGD011 hCD19 100

OD 450 nmol/L (RLU) OD 450 nmol/L 0 –100

hCD3 Surface –50 50 150 250 350 s Time

E F

G H B Cells %

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þ þ lymphocytes as effectors. Malignant B cells (CD20 /CD5 ) were burden in the animals treated with MGD011 showed a statistically depleted in the presence of MGD011, but not control DART significant survival at doses 4 mg/kg, with near complete tumor protein, in a time-dependent manner (Fig. 3A; Supplementary regression at 100 mg/kg. In all experiments, treatment with Fig. S4A), accompanied by concomitant expansion (Fig. 3B; MGD011 at any dose level was not associated with body weight Supplementary Fig. S4B) and activation (CD25 induction) of loss (data not shown). þ þ both CD4 and CD8 T cells (Fig. 3C and D; Supplementary Fig. S4C—S4D). Thus, MGD011 mediates potent tumor cell MGD011 demonstrates prolonged circulating half-life in killing not only against model cell lines but also primary leukemia cynomolgus monkeys samples. Two cynomolgus monkey studies were performed (Supple- mentary Table S1). Analysis of MGD011 serum concentration- Antitumor activity of MGD011 in human PBMC-reconstituted, time profiles across these studies (Fig. 5A and B) showed dose- xenograft tumor-bearing mice proportional increases in maximum serum concentration (Cmax) Redirected T-cell killing by DART molecules can be recapitu- across the entire dose range evaluated, indicating linear PK. lated in mouse models bearing human tumor xenografts as targets Clearance was lower than the glomerular filtration rate for cyno- and human PBMCs as effector cells (9). To minimize graft-versus- molgus monkeys (125 mL/h/kg, Supplementary Table S2), as host disease (GVHD) associated with human T-cell engraftment, expected for a protein of this molecular size (110 kDa), indi- mice deficient in the expression of MHC class I via knock-out of its cating that virtually no elimination occurs by renal filtration. B2m component were used. Lack of B2m expression, however, is Mean initial volumes of distribution (V1) are similar to or slightly associated with impaired FcRn expression; hence, serum half-life higher than the plasma volume in cynomolgus monkeys (45 of MGD011 is short (4 hours) in these mice compared to that in mL/kg), suggesting little or no depletion of MGD011 by binding wild-type mice (135 hours; data not shown). Frequent dosing to target cells. MGD011 demonstrated a prolonged beta-phase (every 2–4 days) was therefore employed to achieve adequate half-life (t1/2,b) of 161.4 61.3 hours (6.7 2.6 days) and mean exposure in this model. residence time (MRT) of 190.6 74.0 hours (7.9 3.1 days), Antitumor activity of MGD011 was first evaluated in human consistent with that of an IgG Fc-bearing molecule in this species. PBMC-reconstituted mice bearing established HBL-2 lymphoma Notwithstanding the foreign nature of the molecule in this cell tumors. MGD011 treatment (500 mg/kg IV every 3–4 days for species, only 3/40 MGD011-treated animals in the GLP study 8 doses) resulted in regression of HBL-2 tumors, with no evidence (one in the 2 mg/kg dose group and two in the 5 mg/kg dose group) of relapse up to Day 42, the last study day (>25 days after initiation showed aberrant PK profiles after the first or subsequent infusions of treatment; Fig. 4A). MGD011 activity was also evaluated in a and were confirmed positive for anti-drug antibodies (ADA, data disseminated Raji/GF leukemia/lymphoma model in human not shown). PBMC-reconstituted mice. As a prophylactic treatment (Supple- mentary Fig. S5), MGD011, administered at the same time of Raji Sustained B-cell depletion in cynomolgus monkeys treated with cell intravenously inoculation, inhibited tumor dissemination weekly doses of MGD011 and improved survival at all doses tested (0.16–500 mg/kg), with MGD011 was confirmed to be active with cynomolgus monkey maximal activity at 20 mg/kg. A modest, albeit statistically cells as it mediated effective autologous B-cell depletion in mon- significant effect of the CD3-targeted control DART molecule key PBMCs in vitro (Supplementary Fig. S6A); it was, however, (500 mg/kg) was also observed, possibly due to micro-clustering less potent than with human PBMCs (EC50 values of 0.016-0.69 of CD3 in vivo at such a high dose level. In the therapeutic ng/mL, n ¼ 6 for human PBMCs vs. 2.44-93.38 ng/mL, n ¼ 5 for paradigm, treatment was delayed 2 weeks after inoculation of monkey PBMCs). The CD3 arm of the DART is unlikely to Raji/GF cells to allow for establishment of disseminated lesions. contribute to this disparity, since its affinity for CD3 of both Image data through day 46 and survival curves through study species is nearly identical (Fig. 1B); furthermore, MGD011 medi- þ completion (day 48) are shown in Fig. 4B and C. Most vehicle- ated comparable cytotoxicity against human CD19 target cells treated mice met the euthanasia endpoint by the end of the second with human or monkey PBMCs as effectors (Supplementary Fig. week after Raji/GF cell inoculation and all were dead by the end of S6B and S6C). The differential potency in autologous B-cell week 4. A slightly longer survival was observed with 100 mg/kg of depletion between species is likely contributed by a combination control DART protein, albeit not significantly different from the of factors, including an approximately 10-fold lower affinity of vehicle-treated group in this experiment. In contrast, the tumor MGD011 for cynomolgus monkey CD19 compared with human

Figure 1. MGD011 structure, binding, and redirected T-cell killing activity against CD19þ target cells. A, Schematic of the disulfide-linked polypeptide chain structure of MGD011. VL and VH are variable light and heavy chain sequences, respectively; E and K are E-coil and K-coil sequences, respectively; and dotted lines represent disulfide bonds. B, Equilibrium dissociation constants (KD) for MGD011 binding to human or cynomolgus monkey CD3 or CD19 proteins. C, Bispecific ELISA using human CD3 protein for MGD011 capture and human CD19 protein for detection. Control DART proteins in which the 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 of MGD011-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)orluminescence(G) assays to measure target cell killing following 24-hour incubation, compared to that mediated by a control DART protein (Fluo x CD3) or a replica of blinatumomab (CD19 x CD3 BiTE molecule). H, Concentration dependence of MGD011-mediated autologous B-cell depletion in human PBMCs; the percentage of CD20þ cells was determined by flow cytometry after incubation with MGD011, a replica of blinatumomab or a control DART protein (Fluo x CD3).

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Figure 2. MGD011-mediated T-cell activation, proliferation, and cytokine release is dependent on co-engagement of CD19þ target cells. A, MGD011 concentration- dependent induction of T-cell activation marker CD69 on CD4þ and CD8þ human T cells following 24-hour incubation with CD19 JIMT-1 cells or CD19þ Raji/GF target cells at E:T cell ratio of 10:1. B, MGD011-dependent induction of proliferation of CFSE-labeled human T cells co-cultured with CD19þ HBL-2 target cells for 72 hours at E:T cell ratio of 10:1. C, MGD011 concentration-dependent upregulation of granzyme B and perforin in CD4þ and CD8þ human T cells following 24-hour incubation with CD19þ Raji/GF target cells at E:T cell ratio of 10:1. D, MGD011 concentration- dependent induction of IFN-g and IL-2 following 24-hour incubation of human T cells in the absence (left) or presence (right) of CD19þ Raji/GF target cells at E:T cell ratio of 10:1.

CD19 (Fig. 1B), a lower density of CD19 on cynomolgus monkey ratio ¼ 4:1, n ¼ 5) compared with humans (T:B ratio ¼ 7:1, n ¼ 6). B cells (Supplementary Fig. S2A and S2B), and a lower average T- These differences make pharmacodynamic modeling in cynomol- cell-to-B-cell (T:B) ratio in PBMCs of cynomolgus monkeys (T:B gus monkeys a conservative estimate of MGD011 potency and

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Day 0 Day 3 Day 6

Untreated Control DART MGD011 Control DART MGD011 A 0 73 0 71 0 410 75 0.1 11

1.2 25 1.6 28 1.4 58 0.2 25 2.4 86 CD20 CD5 B 2 0 2 0 2 0 22 14

95 3 97 1 94 4 6 57 CD8

CD4 C 83 1 12 88 CD4 16 0 0 0 CD25 79 21 17 83 D CD8 0 0 0 0 CD25

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) at 0.05 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) anddays3and6.A, ThepercentageofCD20þ/CD5þ malignant B cells in the gated lymphoid population. B, ThepercentageofCD4þ and CD8þ T cells. C and D, The percentage of CD25þ T-cell activation in CD4þ and CD8þ T cells, respectively. should be taken into consideration when projecting human any abnormal histological and IHC findings at the end of a 12- doses. week recovery period (Fig. 5E). In summary, once weekly Administration of MGD011 resulted in a decrease in circu- administration of MGD011 was sufficient to induce complete þ lating CD20 B cells by 24 hours following the start of the first and durable B-cell depletion in both the circulation and lym- infusion (Fig. 5C and D). Although a partial reduction in phoid organs. circulating B cells was noted at the 0.2 mg/kg dose, nearly Treatment of MGD011 was associated with transient, dose- þ þ complete depletion was observed at dose levels 0.5 mg/kg and dependent fluctuations of both circulating CD4 and CD8 T were maintained throughout the 4 weekly treatments. Upon cells, with the lowest nadir following the first infusion and with a completion of dosing, circulating B cells returned to predose decreasing magnitude following subsequent infusions (Fig. 6A levels, albeit with a dose-proportional lag (Fig. 5C). Histopa- and B). Contrary to the durable reduction in B cells, T lymphocytes thology examination at terminal necropsy showed a dose- recovered quickly after each infusion and reached or exceeded dependent decrease in follicles and germinal centers within the baseline levels before the next dose. Similar T-cell kinetics were spleen, lymph nodes (inguinal, mandibular, and mesenteric), observed in cynomolgus monkeys after the administration of and gut-associated lymphoid tissue (GALT; data not shown). MGD006, a CD123 x CD3 DART protein (13), and in humans CD20 immunohistochemistry (IHC) showed a reduction in during blinatumomab infusion (18). Fluctuations were also þ CD20 B cells to nearly undetectable levels in the lymph nodes, observed with natural killer cells and monocytes in MGD011- spleen, and bone marrow at doses 10 mg/kg (Fig. 5E and F). treated monkeys (data not shown), likely representing transient The pharmacologic effect of MGD011 on B lymphocytes was cell margination. prolonged, but ultimately reversible, as indicated by the return MGD011 treatment was also associated with upregulation of of circulating B cells to pretreatment levels and the absence of the T-cell activation markers, CD69 and PD-1 (Fig. 6C–F),

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Control DART MGD011 MGD011 MGD011 MGD011 MGD011 A B Vehicle (100 μg/kg) (0.16 μg/kg) (0.8 μg/kg) (4 μg/kg) (20 μg/kg) (100 μg/kg) Study day Study day Study day Study day Study day Study day Study day 7142128 714212846 714212846 714212846 714212846 714212846 714212846

) X X 3

X X X

X X X X Tumor volume Tumor

(mean ± SEM mm X X X X X

X X X X X

X X X X X Randomization Tumor cells PBMC

X X X X X X X

C Survival (%)

Study days

Figure 4. MGD011-mediated antitumor activity in human PBMC-reconstituted xenograft tumor-bearing mice. A, Treatment of human PBMC-reconstituted mice with established intradermal HBL-2 lymphoma cell tumors. NSG B2m/ mice (n ¼ 8/group) were implanted intradermally with HBL-2 tumor cells (5 106)on day 0 followed by intraperitoneal injection of human PBMCs (1 107) on day 4. Tumor-bearing mice, randomized at day 14, were treated with MGD011 or 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 indicated times 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 implanted intraperitoneally with human PBMCs (1107) 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 doses beginning 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 groups in B. , P < 0.001 and , P ¼ 0.03 by log-rank test comparison to the vehicle-treated group.

þ together with an increase of the Ki67 proliferating fraction of except IL-10, which exceeded levels of the vehicle control group þ þ both CD4 and CD8 T-cell subsets during the treatment phase in some instances. No MGD011-related changes in circulating (Fig. 6G and H). Therefore, MGD011 is capable of mediating IL-2, IL-4, or IL-5 levels were noted. T-cell activation and expansion in vivo. The activation markers MGD011 was well tolerated in cynomolgus monkeys at all dose trended toward or returned to baseline by the end of the exper- levels tested, with no compound-related toxicological effects, iment; however, unexplained isolated increases in one group 4 including cage-side observations, changes in body weight or food þ þ animal (showing up to 10-fold higher frequency of CD4 CD69 consumption, vital parameters, ophthalmology, electrocardio- cells than the remaining 3 animals) and in two vehicle control grams, body temperature, blood pressure, heart rate, respiration þ þ animals (up to 10-fold higher frequency of CD8 PD1 cells than rate, neurological examinations, coagulation, or clinical chemis- the other 2 animals) during the recovery phase contributed to try (data not shown); furthermore, with the exception of the swelling the average for these groups (Fig. 6C and F). An asymp- aforementioned hematological, bone marrow, and lymphoid tomatic, transient, dose-dependent increase in circulating cyto- organ changes, no other gross or microscopic pathological find- kine levels, a first-dose effect, was observed for IFN-g (Fig. 6I), IL-6 ings were noted (data not shown). (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 the first infusion and returning to at or near baseline within 24 hours Discussion of the start of the infusion. Smaller levels of cytokine increase were MGD011 is a bispecific DART protein developed for the treat- noted with subsequent infusions, which were of a magnitude ment of B-cell malignancies and designed to redirect the T comparable with those observed following vehicle infusions, lymphocytes via their CD3 molecule to kill target cells expressing

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

A B ng/mL ng/mL (Mean ± SEM) (Mean ± SEM)

Study days Study days C D Cells/ m L Cells/ m L (mean ± SEM) (mean ± SEM)

Study days Study days E MGD011 F MGD011 Vehicle 4 × 0.2 μg/kg 4 × 5 μg/kg 4 × 10 μg/kg 5→5→5 0.5→5→50→50 2→10→100→100 ng/kg μg/kg μg/kg Terminal necropsy 200 μm Bone marrow 100 μm Bone marrow Bone Terminal necropsy

Spleen 1 mm

Spleen 600 μm

2 mm Lymph node

6 mm Lymph node 200 μm Recovery necropsy Bone marrow

Spleen 1 mm

2 mm Lymph node

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CD19. MGD011 was shown to eliminate normal or pathogenic MGD011 administered to cynomolgus monkeys by a 2-hour þ CD19 cells in vitro and in vivo. Furthermore, weekly administra- intravenous infusion on a weekly schedule at doses ranging tion of MGD011 to cynomolgus monkeys resulted in complete from 0.2 to 100 mg/kg was safe and well tolerated. Dose- and durable depletion of B cells in the circulation and in lym- dependent, on-target activity with nearly complete depletion þ phoid tissues. MGD011 was well tolerated in toxicology studies of circulating CD20 B cells was observed at doses 0.5 mg/kg. under treatment regimens that exaggerated potential clinical Histologic evaluation confirmed a dose-dependent decrease setting scenarios in patients. Thanks to its favorable PK properties, in B-cell zones in lymphoid tissues at the terminal necropsy. convenient once-a-week dosing or longer interval is predicted to Although bone marrow showed no obvious histological be clinically feasible in humans. changes, B-cell depletion was observed by IHC, further attesting The structure of MGD011 represents a refinement of a previ- to the specificity of the on-target effects of MGD011. The ously reported format (8), where the DART chains (Chain 1 and pharmacologic effect of MGD011 on B lymphocytes was revers- Chain 2) preserve the original C-terminal stabilizing disulfide ible, as indicated by flow cytometry and IHC analysis in addi- linkage, while opposite E/K-coil sequences were added that fur- tion to hematology and histopathology (data not shown). Each ther improve heterodimer formation. To promote the desired of these parameters returned to baseline, with no differentia- heterodimerization of Chain 1 and the Fc-domain Chain 3, knob- tion between the animals receiving MGD011 or control article into-hole mutations were incorporated in the CH3 region. To at the end of the 12-week recovery period. facilitate the removal of residual Chain 3 dimers during purifi- Maximal cytolytic activity against B-cell lines and in patient cation, a H435R mutation was included in this chain to abolish samples ex vivo were observed at MGD011 concentrations of 50 to protein A binding. MGD011 was engineered with humanized 100 ng/mL. These levels are at or below the Cmax observed in antibody arms displaying 10-fold greater affinity for human monkeys at 5 mg/kg or below trough levels at 50 mg/kg. Either CD19 than for human CD3, thus providing for preferential initial dose level resulted in near complete depletion of B lymphocytes in binding to target cells, while minimizing CD3 engagement in the the circulation and lymphoid tissues in monkeys, attesting to the absence of target cells. MGD011 exhibits binding to monkey CD3 potency of the DART molecule, particularly in account it and CD19, allowing for preclinical modeling in this species. decreased potency in this species compared with humans. In Specifically, MGD011 cross-reacts with cynomolgus monkey CD3 B-cell malignancies, a low effector-to-target (E:T) cell ratio could with nearly identical affinity as for human CD3 and with a 10-fold limit the effectiveness of redirected cytolysis. MGD011, however, lower affinity for monkey CD19. Consistent with its binding was able to eliminate leukemic cells in CLL patient samples by properties, MGD011 mediated similar levels of cytotoxicity engaging and expanding the patient's residual T cells in vitro from þ against human CD19 Raji/GF target cells by either human or a low E:T cell ratio to one exceeding the target cell population. cynomolgus monkey PBMCs. MGD011 mediated also a dose- These data also confirm that CLL T cells can be redirected to dependent, autologous ex vivo B-cell depletion in both human and become cytolytic against leukemic cells in the presence of cynomolgus monkey PBMCs, albeit with a decreased potency in MGD011, as previously reported for blinatumomab (19), even the latter, due, at least in part, to its lower affinity for monkey though T-cell dysfunction in CLL, such as defects in immune CD19. Because of the lower CD19 affinity and reduced potency in synapse formation, co-stimulatory/accessory molecule expres- cynomolgus monkeys, this species may offer a conservative esti- sion, and cytokine release have been reported (20–22). mate of potency that needs to be taken into consideration in A safety concern associated with CD3-targeting therapies is extrapolating non-human primate data to potential clinical cytokine release. The monovalent nature of the binding arm of settings. MGD011 ensures that T-cell activation and cytokine release To confer prolonged circulating half-life, MGD011 was also depend exclusively on target-cell engagement. Consistent with engineered with an Fc-domain; with an estimated terminal half- its desired design, no T-cell activation or cytokine release were þ life of approximately 161 hours (6.7 days) in monkeys and a observed in vitro in the absence of CD19 target cells or with a predicted half-life of 343-495 hours (14.3–20.6 days) in humans, control DART protein that includes only the CD3-targeting arm. which is similar to that of conventional mAbs, delivering No cytokine storm was observed in MGD011-treated cynomolgus MGD011 as an intermittent dosing regimen appears feasible. In monkeys. Transient dose-dependent increases in IFN-g, IL-6, and comparison, blinatumomab, which is engineered as a single- IL-10 were observed following the first dose; with subsequent chain Fv pair, is administered by continuous intravenous infusion doses, they were generally no greater than those observed in for repeated 4-week courses, owing to its short approximately 2- animals receiving vehicle. There were no MGD011-related hour half-life. changes 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 from arepeat-dosestudy(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.Theassay lower 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 animals treated 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 above studies. 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) dose group or 7 days after the last dose for the escalating (0.5 to 50 mg/kg and 2 to 100 mg/kg) dose groups.

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

+ CD4+ Cells CD8 Cells AB5,000 Vehicle 5,000 Vehicle 0.2 μg/kg 0.2 μg/kg 2 μg/kg 2 μg/kg 5 μg/kg 4,000 4,000 5 μg/kg 10 μg/kg 10 μg/kg 3,000 3,000 Cells/ μ L Cells/ μ L 2,000 2,000 (mean ± SEM) (mean ± SEM)

1,000 1,000

0 0 –7 0 7 14212835424956637077849198105126 –7 0 7 14212835424956637077849198105126

V DDDD V DDDD Study days Study days

CD4+CD69+ Cells CD8+CD69+ Cells CD50 50 Vehicle Vehicle 0.2 μg/kg 0.2 μg/kg 2 μg/kg μ 40 40 2 g/kg 5 μg/kg 5 μg/kg 10 μg/kg 10 μg/kg 30 30

20 20 (mean ± SEM) (mean ± SEM) % of Gated cells % of Gated cells 10 10

0 0 –7 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98105 126 –7 0 7 14212835424956637077849198105 126

Figure 6. V DDDD V DDDD Changes in peripheral T-lymphocyte Study days Study days + + subpopulations and cytokines in EFCD4 PD-1 Cells CD8+PD-1+ Cells 50 cynomolgus monkeys administered Vehicle 50 Vehicle 0.2 μg/kg μ μ 0.2 g/kg weekly doses of MGD011. Data are from the 40 2 g/kg 2 μg/kg 5 μg/kg 40 μ repeat-dose study 1 in monkeys (5/sex/ μ 5 g/kg 10 g/kg 10 μg/kg group) in which MGD011 (dose levels 30 30 ranging from 0.2 to 10 mg/kg) or vehicle were administered intravenously once 20 20 (mean ± SEM) (mean ± SEM) % of Gated cells weekly for 4 weeks (Supplementary Table % of Gated cells 10 10 S1). A–B, CD4þ and CD8þ T cells. C–D, þ þ CD69-activated CD4 and CD8 T cells. 0 0 E–F, PD-1–activated CD4þ and CD8þ T –7 0 7 14212835424956637077849198105126 –7 0 7 14212835424956637077849198105126 cells. G–H, Proliferating (Ki67þ) CD4þ and V DDDD V DD D D CD8þ T cells. I–K, Serum levels of Study days Study days G + + H CD8+Ki67+ Cells cytokines: IFN-g, IL-6, and IL-10; V, vehicle CD4 Ki67 Cells 100 Vehicle 100 Vehicle infusion; D, MGD011 infusion. 0.2 μg/kg 0.2 μg/kg 2 μg/kg 2 μg/kg 80 80 5 μg/kg 5 μg/kg 10 μg/kg 10 μg/kg 60 60

40 40 (mean ± SEM) (mean ± SEM) % of Gated cells % of Gated cells 20 20

0 0 –7 0 7 142128354249 56 63 70 77 84 91 98 105126 –7 0 7 14212835424956637077849198105126

V DDDD V DD D D Study days Study days IJKIFN-g IL-6 IL-10 200 800 Vehicle 600 Vehicle Vehicle 0.2 μg/kg 0.2 μg/kg 700 0.2 μg/kg μ μ μ 150 2 g/kg 500 2 g/kg 600 2 g/kg 5 μg/kg 5 μg/kg 5 μg/kg μ μ μ 10 g/kg 400 10 g/kg 500 10 g/kg 100 400 300 pg/mL pg/mL pg/mL 300

(Mean ± SEM) (Mean ± SEM) 200 (Mean ± SEM) 50 200

100 100 0 0 0 0 7 1421283542 0 7 1421283542 0 7 1421283542

VDDDD VDDDD VDDDD Study days Study days Study days transient increases in TNF-a, exceeding 100 pg/mL in only a few observed in patients treated with blinatumomab (18) or with our instances, were observed in animals that received 10 mg/kg CD123 x CD3 DART molecule in monkeys (13); the rapid target- MGD011. First-dose cytokine release events have been previously cell depletion following the ﬁrst administration of the bispeciﬁc

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Liu et al.

molecule may limit further T-cell engagement and explain the half-life and was safe and well tolerated in cynomolgus monkeys transient nature of cytokine release observed. at doses that resulted in complete and durable B-cell depletion in Tumor lysis syndrome, which has been observed with blina- the circulation, bone marrow, and lymphoid organs. The data tumomab (7), should be anticipated as another potential safety from these nonclinical studies provided rationale for the clinical concern for MGD011 and proper preventive measures be imple- development of MGD011 as a treatment for patients with B-cell ment to limit its consequences. Blinatumomab treatment was also malignancies; a phase I dose-escalation study of the safety, tol- associated with an increased risk of serious and/or severe neuro- erability, dose-limiting toxicity, maximal tolerated does, and logical toxicities (7); a similar safety risk of neurological toxicities recommended phase II dose of MGD011 when administrated for MGD011 is therefore recognized. It should be noted, however, intravenously over a 2-hour period once every 2 weeks to subjects that no signs of neurological toxicity were observed in the with B-cell malignancies is currently recruiting patients MGD011 toxicology studies, although the predictive value of the (NCT02454270). cynomolgus monkey with respect to neurotoxicity of CD19 x CD3 fi bispeci c interventions is unknown. Similarly, no infection-relat- Disclosure of Potential Conflicts of Interest ed adverse events were observed in monkeys treated with All authors are or were employees of MacroGenics, Inc. and received MGD011, although 25% of patients receiving blinatumomab compensation from and hold ownership interest (including patents) in Macro- showed signs of infection (7). Hence, patients should be mon- Genics, Inc. itored for infections and treated appropriately. Immunogenicty remains a potential concern associated with Authors' Contributions antibody therapy. MGD011 limits this through the incorporation Conception and design: L. Liu, C.-Y.K. Lam, R. Alderson, J.L. Nordstrom, of humanized Fv regions and the minimal use of linkers. Although S. Koenig, P.A. Moore, S. Johnson, E. Bonvini immunogenicity in monkeys does not predict immunogenicity in Development of methodology: L. Liu, C.-Y.K. Lam, L. Widjaja, H. Li, humans, notably, only 3 of 40 monkeys treated with MGD011 R. Alderson showed an ADA response; given that MGD011 targets B lympho- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Liu, C.-Y.K. Lam, V. Long, L. Widjaja, Y. Yang, cytes, ADA development is expected to be low in all species. H. Li, L. Jin, J. Brown, R. Alderson Upregulation of activation markers, including PD-1, was Analysis and interpretation of data (e.g., statistical analysis, biostatistics, observed amongst circulating T lymphocytes in monkeys treated computational analysis): L. Liu, C.-Y.K. Lam, L. Widjaja, H. Li, R. Alderson, with MGD011. Importantly, the frequency of regulatory T cells or J.L. Nordstrom, P.A. Moore, S. Johnson, E. Bonvini þ þ þ TIM-3 cells in both the CD4 and CD8 T-lymphocyte popula- Writing, review, and/or revision of the manuscript: L. Liu, C.-Y.K. Lam, tions remained low (<1%) throughout the entire dosing period M.D. Lewis, J.L. Nordstrom, S. Koenig, P.A. Moore, S. Johnson, E. Bonvini Administrative, technical, or material support (i.e., reporting or organizing (data not shown), suggesting that T-cell activation induced by data, constructing databases): L. Widjaja, S. Burke, S. Gorlatov MGD011 was not accompanied by expansion of regulatory T cells Study supervision: L. Liu, P.A. Moore, E. Bonvini or T-cell exhaustion. fi The off-the-shelf convenience of bispeci c antibodies offers Acknowledgments substantial advantages compared with personalized CAR T cells. A We would like to thank Cynthia Sung (Rainbow Pharma Consulting) for data point of contention, however, is whether a trade-off of bispecific analysis, Robert Burns, Qin Tang, Nancy O'Gwin, and Xioqi Gong for technical molecules is a decrease in potency. The impressive response to assistance, Melinda Hanson for administrative and editorial support, Timothy CAR T-cell therapy is associated with a significant rate of severe Mayer and James Karrels for critical discussions and review of the manuscript. adverse events, resulting in a narrow therapeutic window, with We thank the teams at the Charles River Laboratories, Inc. for the diligent little, if any, opportunity for response fine tuning. We should conduct of the cynomolgus monkey studies. further note that MGD011, on a molar basis, is more potent than blinatumomab and that complete depletion of B lymphocytes in Grant Support both marrow and lymphoid tissues can be achieved in cynomol- The research funding was provided by MacroGenics. gus monkeys in the absence of adverse effects. Ultimately, only The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked clinical data will be able to address this issue. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate In summary, MGD011 demonstrated potent activity in redir- this fact. ecting T lymphocytes to eliminate CD19-expressing cells in vitro, induced growth inhibition and tumor regression of B-cell lym- Received March 11, 2016; revised August 8, 2016; accepted September 1, phoma/leukemia models in mice, showed prolonged circulating 2016; published OnlineFirst September 23, 2016.

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www.aacrjournals.org Clin Cancer Res; 2017 OF13

MGD011, A CD19 x CD3 Dual-Affinity Retargeting Bi-specific Molecule Incorporating Extended Circulating Half-life for the Treatment of B-Cell Malignancies

Liqin Liu, Chia-Ying K. Lam, Vatana Long, et al.

Clin Cancer Res Published OnlineFirst September 23, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-0666

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