MGD011, a CD19 x CD3 Dual Affinity Re-Targeting Bi ... · MGD011, a CD19 x CD3 Dual Affinity Re-Targeting Bi-specific Molecule Incorporating Extended Circulating Half-life for the

Clinical Cancer Research – Revised Version: Draft for Review 20160712

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MGD011, a CD19 x CD3 Dual Affinity Re-Targeting 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

Research, MacroGenics, Inc., Rockville, MD 20850

RUNNING TITLE: A CD19 x CD3 bi-specific molecule with extended half-life

KEY WORDS: Leukemia/lymphoma, surface molecules as targets for therapy, antibodies

FINANCIAL SUPPORT: The research funding provided by MacroGenics

CORRESPONDING AUTHOR: Ezio Bonvini, MacroGenics, 9704 Medical Center

Drive, Rockville, MD20850. Phone: 301-354-2638; Fax: 301-251-5321; Email:

[email protected]

DISCLOSURE OF CONFLICITS OF INTEREST: All authors are employed by and own

stock and/or shares in MacroGenics, Inc. SK is a member of the Board of Directors of

MacroGenics, Inc.

WORD COUNTS: Translational relevance - 149; Abstract - 246; Body - 4876

TOTAL NUMBERS OF FIGURES: 6

Research. on September 13, 2020. © 2016 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 23, 2016; DOI: 10.1158/1078-0432.CCR-16-0666

http://clincancerres.aacrjournals.org/

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Statement of Translational Relevance Progress has been made in the clinical management of B-cell neoplasms, although most

remain ultimately incurable with current modalities. Redirecting T lymphocytes to lyse

lymphoma/leukemia cells via bispecific molecules that simultaneously engage CD3 on T

cells with a B-cell antigen, such as CD19, has emerged as a powerful novel concept,

highlighted by the clinical success of blinatumomab (Blincyto™). Blinatumomab,

however, requires continuous infusion, owing to its short circulating half-life. We report

here on the preclinical development of MGD011, a bispecific DART® molecule with

increased in vitro cytolytic activity compared to blinatumomab and engineered for

improved circulating half-life. MGD011 showed potent anti-tumor activity in mouse

leukemia/lymphoma models and displayed prolonged pharmacokinetic in cynomolgus

monkeys, a cross-reacting species. MGD011 was well tolerated in monkeys, with durable

and profound B-cell depletion following weekly administrations. MGD011’s potent

activity and pharmacokinetic properties may offer therapeutic convenience and

applicability in the treatment of B-cell malignancies.




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Abstract

Purpose: CD19, a B-cell lineage-specific marker, is highly represented in B-cell

malignancies and an attractive target for therapeutic interventions. MGD011 is a CD19 x

CD3 Dual-Affinity Re-Targeting (DART®) protein designed to redirect T lymphocytes to

eliminate CD19-expressing cells. MGD011 has been engineered with a modified human

Fc domain for improved pharmacokinetic (PK) properties and designed to cross-react

with the corresponding antigens in cynomolgus monkeys. Here we report on the

preclinical activity, safety and PK properties of MGD011.

Experimental Design: The activity of MGD011 was evaluated in several in vitro and in

vivo models. PK, safety and pharmacodynamic activity was also assessed in dose-

escalation and repeat-dose studies of MGD011 administered once weekly in cynomolgus

monkeys.

Results: MGD011 mediated killing of human B-cell lymphoma 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-dependent T-cell

activation and expansion, cytokine release and upregulation of perforin and granzyme B.

MGD011 demonstrated antitumor activity against localized and disseminated lymphoma

xenografts reconstituted with human PBMCs. In cynomolgus monkeys, MGD011

displayed a terminal half-life of 6.7 days; once weekly intravenous infusion of MGD011

at doses up to 100 µg/kg, the highest dose tested, was well tolerated and resulted in dose-

dependent, durable decreases in circulating B cells accompanied by profound reductions

of B lymphocytes in lymphoid organs.




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Conclusions: The preclinical activity, safety and PK profile support clinical investigation

of MGD011 as a therapeutic candidate for the treatment of B-cell malignancies.




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Introduction

B-cell malignancies represent a heterogeneous group of disorders with varying

characteristics and clinical behaviors (1). While systemic chemotherapy is still the

mainstay of treatment for B-cell malignancies, kinase inhibitors that selectively target

molecules at the core of the transformation process and antibody therapy are now well

established tools (2). Among the latter category, rituximab (Rituxan®), a monoclonal

antibody (mAb) that targets the B-cell antigen CD20, induces direct tumor cell apoptosis

as well as complement- and antibody-dependent cytotoxicity (2)(3). These orthogonal

mechanisms of action 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 in the US alone.

Providing T lymphocytes (CTL) with the ability to recognize and destroy tumor cells has

shown promise in advanced forms of leukemia and lymphoma. In the form of chimeric

antigen receptor (CAR) T-cell therapy, such an approach requires ex vivo isolation,

transduction, and reinfusion of the patient’s T cells. This complexity can be overcome

with bispecific antibodies that bind simultaneously to an antigen expressed by malignant

B cells and an activation molecule on T lymphocytes (2). The pan B-cell marker, CD19,

has emerged as a promising antigen for targeting B-cell malignancies because of its

broader expression profile and lower rate of down-regulation compared to other B-cell

antigens (3). Its expression is highly conserved in the majority of B-cell tumors (4), with

normal to high levels of expression in 80% of acute lymphoblastic leukemia (ALL), 88%

of B-cell lymphomas, and all chronic lymphocytic leukemias (CLL) (5, 6).




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Redirection of CTL to CD19+ leukemia cells via the bispecific T-cell engager (BiTE)

blinatumomab (Blincyto™)(7) is effective in patients with B-cell malignancies whose

disease did not respond to standard chemo-immunotherapies and has been approved by

the US Food and Drug Administration for the treatment of patients with Philadelphia

chromosome-negative relapsed or refractory B-cell precursor ALL. Dual-Affinity Re-

Targeting (DART®) proteins are bispecific, antibody-based molecules with favorable

stability, manufacturability, and potency; furthermore, a CD19 x CD3 DART protein

compared favorably to a BiTE of the same pair of VH and VL sequences in redirected

cytolysis assays (8, 9). MGD011 (also known as JNJ-64052781) is another CD19 x CD3

DART protein designed to simultaneously target CD19+ cells for recognition and

elimination by CD3-expressing T lymphocytes as effector cells. MGD011 was

engineered with a human immunoglobulin G1 (IgG1) Fc domain in order to bind the

neonatal Fc receptor (FcRn) and engage the IgG salvage pathway, thus conferring

prolonged circulating half-life and the resultant dosing convenience. To avoid unintended

(target independent) CD3-mediated T-cell activation via interaction with Fc gamma

receptors (FcγRs), the Fc domain was mutated to greatly reduce or eliminate binding to

these receptors as well as complement. Unlike blinatumomab, which reacts only with

human or chimpanzee’s antigens (10), MGD011 cross-reacts with both CD19 and CD3

molecules in macaques, enabling preclinical evaluation in a relevant model. Here we

report on MGD011 engineering and characterization as well as on its preclinical safety

assessment, pharmacokinetics (PK), and dose optimization in cynomolgus monkeys. The

results show robust activity in multiple models and predict effective dosing in humans at

once weekly or longer intervals.




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Materials and Methods

DART Protein Engineering, Production, and Purification. MGD011, an Fc-bearing

CD19 x CD3 DART protein, was constructed as described (11) using VL and VH

sequences from humanized anti-CD19 mAb BU12 (12) and humanized anti-CD3 mAb

XR32 (13). The IgG1-derived Fc segment was modified to encode the L234A/L235A

mutation to greatly reduce or eliminate FcγR and C1q binding (14). Control molecules in

which the variable domain sequences of an anti-fluorescein mAb 4-4-20 (15) replaced

either of the DART protein arms (Fluo x CD3 or CD19 x Fluo) were engineered in a

similar manner. The DART proteins were expressed transiently in CHO-S cells (8) and

purified to greater than 99% purity using protein A and either size-exclusion

chromatography (SEC) or other polishing steps. The purified DARTs have very low

levels (less than 1%) of high molecular weight (HMW) protein present and have an

apparent molecular weight of ~110 kDa (Figure S1A-B).

Other Reagents, Cell Lines, and Tissue Samples. Recombinant soluble human and

cynomolgus CD3ε/δ chimeric proteins, as well as human and cynomolgus CD19 and

CD19-His proteins, were expressed in CHO-S cells. MOLM-13 and JIMT-1 were

obtained from DSMZ (Braunschweig, Germany); Jeko-1 cells were from the American

Type Culture Collection (ATCC, Manassas, VA) and HBL-2 from the National Institutes

of Health (Bethesda, MD) (16). Raji/GF, a CD19+ Burkitt’s B-cell lymphoma expressing

luciferase and green fluorescent protein by stable transfection, was established 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




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authenticated based on morphology, growth characteristics and CD19 expression.

Heparinized human whole blood was from Biological Specialty Corporation (Colmar,

PA). Cryopreserved purified primary CLL patient samples were from AllCells, LLC

(Alameda, CA). Heparinized whole blood from cynomolgus monkeys was from

Worldwide Primates, Inc. (Miami, FL).

Binding Studies. MGD011 binding to human or cynomolgus monkey CD3 or CD19

proteins was analyzed by ELISA or surface plasmon resonance (SPR) as previously

described (13); binding to primary human or cynomolgus monkey CD20+, CD4+ or CD8+

cells was analyzed by flow cytometry.

Cell Killing Assay. For CTL assays, DART protein mediated killing of target cell lines

in the presence of human or cynomolgus PBMCs or purified T cells was determined by

lactate dehydrogenase (LDH) release or luminescence assays as previously described

(17).

B-cell Depletion Assay. For autologous B-cell depletion assays, PBMCs from normal

human donors, cryopreserved primary CLL specimens, or cynomolgus monkey were

incubated with DART proteins overnight and analyzed by flow cytometry using

FSC/SSC gated lymphoid populations to determine B-cell depletion via CD20 staining.

Cell Activation Studies. T-cell activation was determined by flow cytometry after

staining with CD8-FITC, CD4-APC, CD25-PE, and CD69-PE-Cy5 antibodies (BD

Biosciences). For intracellular granzyme B and perforin determinations, T cells were

stained with anti-CD4-PerCP.Cy5.5 and anti-CD8-APC antibodies (BD BioSciences),




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fixed, permeabilized (Cytofix/Cytoperm solution; BD Biosciences), and incubated with

anti-granzyme B-FITC or anti-perforin-PE antibody (BD BioSciences). T-cell

proliferation was determined by flow cytometry as dilution of carboxyfluorescein

diacetate, succinimidyl ester (CFSE; Invitrogen) in labeled cells. IL-2, IFN-γ, and TNF-α

levels in culture supernatants were quantitated by ELISA (R&D Systems).

In Vivo Studies in Murine Models. All studies were reviewed and approved by

MacroGenics’ Institutional Animal Care and Use Committee (IACUC). MGD011 in vivo

activity was evaluated in an established HBL-2 lymphoma model and an established,

disseminated Raji/GF leukemia/lymphoma model in human PBMC-reconstituted NSG

beta 2 microglobulin (B2m)-deficient (NSG B2m-/-) female mice aged 7-8 weeks

(Jackson Laboratory). Tumor volume in the HBL-2 lymphoma model was calculated as

follows: (length x width2)/2. Tumor burden in the Raji/GF leukemia/lymphoma model

was evaluated by whole-body imaging on an IVIS Spectrum imaging system

(PerkinElmer).

Cynomolgus Monkey Studies. The studies were performed at Charles River

Laboratories (Reno, NV) under IACUC guidelines. Naive cynomolgus monkeys (Macaca

fascicularis) of Chinese origin (2.5-6.3 years old, 2.3-3.9 kg body weight) were

randomized by weight and sex and received vehicle or MGD011 via 2-hour intravenous

(IV) infusion (Table S1). Serum samples were collected to examine the PK profile of

MGD011 by ELISA using immobilized goat anti-hXR32 antibody (recognizes CD3 arm

of MGD011) for capture and goat anti-human IgG Fc-biotin together with streptavidin-

horseradish peroxidase (SA-HRP) for detection. Whole blood samples were collected for

flow cytometry and the peripheral blood cell surface phenotype analyzed by using the




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following antibodies: 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, and Ki-67 Alexa647 (BD Biosciences). Absolute numbers of

cells were determined by Trucount™ (BD Biosciences). Additional serum samples were

collected to examine cytokine levels measured by the MILLIPLEX® MAP Non-Human

Primate Cytokine Magnetic Bead Panel 7-Plex (EMD Millipore). Formalin-fixed

paraffin-embedded sections of bone marrow, lymph node and spleen were evaluated by

immunohistochemistry (IHC) for CD20. In brief, tissues were deparaffinized, rehydrated,

antigen retrieved and incubated with a mouse anti-CD20 antibody (Dako), followed by

the Envision+ System-HRP Labeled Polymer Anti-Mouse antibody (Dako) and

developed with Diaminobenzadine (DAB).

Flow Cytometry. Flow cytometric analyses were carried out on a FACS Calibur flow

cytometer (4-color analyses) or BD LSRFortessa cell analyzer (8-color analyses)

equipped with CellQuest Pro Version 5.2.1 (BD Biosciences) and FlowJo v9.3.3

(Treestar).

Statistical Analysis. In 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 analysis of variance (ANOVA) with a

Bonferroni correction and survival curve compared by logrank test. All analyses were

performed using GraphPad Prism software (version 5.02). P values of ≤ 0.05 were

considered statistically significant.




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Results

Engineering, Physicochemical Characterization, and Binding Properties of

MGD011

MGD011 is an Fc-bearing DART protein composed of three polypeptide chains

covalently linked by disulfide bonds (Figure 1A). The Fc domain was engineered to

greatly reduce or eliminate FcγR and complement C1q binding (14), while retaining

binding to FcRn to prolong MGD011 circulating half-life. MGD011 binds human and

cynomolgus monkey CD3 with nearly identical affinity (KD = 21.2 and 21.9 nM,

respectively), while binding to CD19 is 10-fold lower in monkeys compared to humans

(KD = 20.3 and 2.0 nM, respectively) due to a faster dissociation rate (Figures 1B and

S1C). Importantly, MGD011 binds to both antigens simultaneously, as shown by

bispecific ELISA and SPR analyses that employ human CD3 protein for capture and

human CD19 protein for detection (Figure 1C-D); moreover, it recognizes the native

antigens on both human and cynomolgus monkey B and T lymphocytes (Figure S2).

MGD011 Redirects T-lymphocytes to Kill CD19+ B Lymphocytes

MGD011 activity was first evaluated by assessing its ability to mediate redirected

cytolysis by using purified human primary T lymphocytes as effector cells and several

CD19+ human B-lymphoma lines as target cells. While MGD011 mediated no killing

activity against the CD19- cell line, MOLM-13 (Figure 1E), potent killing was observed

against all CD19+ lines tested, including Raji/GF (Figure 1F-G) as well as HBL-2 and

Jeko-1 cells (Figure S3A-B). A control DART protein, in which the CD19 arm was




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replaced by one with an irrelevant specificity, did not mediate killing. Furthermore,

compared to blinatumomab, MGD011 demonstrated enhanced potency, with EC50 values

~10-fold lower for killing of Raji/GF cells (0.02-0.03 ng/mL for MGD011 vs. 0.38-0.41

ng/mL for blinatumomab). MGD011 also induced B-cell depletion in human PBMCs

from normal donors, with endogenous T cells serving as effector cells, demonstrating a

~10-fold greater potency than blinatumomab in this assay as well (Figure 1H).

Incubation of MGD011 with CD19+ target cells (Raji/GF cells) was associated with

concomitant CD4+ and CD8+ T-cell activation as evidenced by upregulation of CD69, but

no activation occurred with CD19-negative cells or the control DART protein

(Figure 2A). CFSE dilution showed induction of T-cell proliferation by MGD011, but

not with the control DART protein (Figure 2B). A dose-dependent upregulation of

granzyme B and perforin levels in both CD8+ and CD4+ T cells was observed following

treatment with MGD011 (Figure 2C). The upregulation of granzyme B and perforin was

higher in CD8+ T cells compared with CD4+ T cells, consistent with the expected higher

CTL potency of CD8+ T cells. Incubation with MGD011 in the presence of CD19+ target

cells also resulted in the release of cytokines, exemplified in Figure 2D by the release of

IL-2 and IFN-γ. No T-cell activation, proliferation, or cytokine release was observed in

the absence of co-engagement of CD19+ target cells, attesting to the strict dependence on

CD19 expression for MGD011-mediated T-lymphocyte activation.

To further evaluate the cytolytic activity of MGD011 against primary malignant B cells,

MGD011 was incubated with PBMCs from patients with CLL, thus relying on the

endogenous residual T lymphocytes as effectors. Malignant B cells (CD20+/CD5+) were




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depleted in the presence of MGD011, but not control DART protein, in a time-dependent

manner (Figure 3A and S4A), accompanied by concomitant expansion (Figure 3B and

S4B) and activation (CD25 induction) of both CD4+ and CD8+ T cells (Figure 3C-D and

S4C-D). Thus, MGD011 mediates potent tumor cell killing not only against 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 recapitulated in mouse models

bearing human tumor xenografts as targets and human PBMCs as effector cells (9). To

minimize graft-versus-host disease (GVHD) associated with human T-cell engraftment,

mice deficient in the expression of MHC class I via knock-out of its B2m component

were used. Lack of B2m expression, however, is associated with impaired FcRn

expression; hence, serum half-life of MGD011 is short (~4 hours) in these mice

compared to that in wild-type mice (~135 hours) (data not shown). Frequent dosing

(every 2-4 days) was therefore employed to achieve adequate exposure in this model.

Antitumor activity of MGD011 was first evaluated in human PBMC-reconstituted mice

bearing established HBL-2 lymphoma cell tumors. MGD011 treatment (500 µg/kg IV

every 3-4 days for 8 doses) resulted in regression of HBL-2 tumors, with no evidence of

relapse up to Day 42, the last study day (>25 days after initiation of treatment) (Figure

4A). MGD011 activity was also evaluated in a disseminated Raji/GF

leukemia/lymphoma model in human PBMC-reconstituted mice. As a prophylactic

treatment (Figure S5), MGD011, administered at the same time of Raji cell IV




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inoculation, inhibited tumor dissemination and improved survival at all doses tested

(0.16-500 µg/kg), with maximal activity at ≥20 µg/kg. A modest, albeit statistically

significant effect of the CD3-targeted control DART molecule (500 µg/kg) was also

observed, possibly due to micro-clustering of CD3 in vivo at such a high dose level. In

the therapeutic paradigm, treatment was delayed 2 weeks after inoculation of Raji/GF

cells to allow for establishment of disseminated lesions. Image data through day 46 and

survival curves through study completion (day 48) are shown in Figure 4B and C. Most

vehicle-treated mice met the euthanasia endpoint by the end of the second week after

Raji/GF cell inoculation and all were dead by the end of week 4. A slightly longer

survival was observed with 100 μg/kg of control DART protein, albeit not significantly

different from the vehicle-treated group in this experiment. In contrast, the tumor burden

in the animals treated with MGD011 showed a statistically significant survival at doses

≥4 μg/kg, with near complete tumor regression at 100 μg/kg. In all experiments,

treatment with MGD011 at any dose level was not associated with body weight loss (data

not shown).

MGD011 Demonstrates Prolonged Circulating Half-life in Cynomolgus Monkeys

Two cynomolgus monkey studies were performed (Table S1). Analysis of MGD011

serum concentration-time profiles across these studies (Figure 5A-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 cynomolgus monkeys (~125 mL/h/kg, Table S2), as expected for a protein of this

molecular size (~110 kDa), indicating that virtually no elimination occurs by renal




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filtration. Mean initial volumes of distribution (V1) are similar to or slightly higher than

the plasma volume in cynomolgus monkeys (~45 mL/kg), suggesting little or no

depletion of MGD011 by binding to target cells. MGD011 demonstrated a prolonged

beta-phase half-life (t1/2,β) of 161.4 ±61.3 hours (6.7 ±2.6 days) and mean residence 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 this

species, only 3/40 MGD011-treated animals in the GLP study (one in the 2 μg/kg dose

group and two in the 5 μg/kg dose group) showed aberrant PK profiles after the first or

subsequent infusions and were confirmed positive for anti-drug antibodies (ADA, data

not shown).

Sustained B-cell Depletion in Cynomolgus Monkeys Treated with Weekly Doses of

MGD011

MGD011 was confirmed to be active with cynomolgus monkey cells as it mediated

effective autologous B-cell depletion in monkey PBMCs in vitro (Figure S6A); it was,

however, less potent than with human PBMCs (EC50 values of 0.016-0.69 ng/mL, n = 6

for human PBMCs vs. 2.44-93.38 ng/mL, n = 5 for monkey PBMCs). The CD3 arm of

the DART is unlikely to contribute to this disparity, since its affinity for CD3 of both

species is nearly identical (Figure 1B); furthermore, MGD011 mediated comparable

cytotoxicity against human CD19+ target cells with human or monkey PBMCs as

effectors (Figure S6B-C). The differential potency in autologous B-cell depletion

between species is likely contributed by a combination of factors, including a ~10-fold

lower affinity of MGD011 for cynomolgus monkey CD19 compared to human CD19




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(Figure 1B), a lower density of CD19 on cynomolgus monkey B cells (Figure S2A-B),

and a lower average T cell-to-B cell (T:B) ratio in PBMCs of cynomolgus monkeys (T:B

ratio = 4:1, n=5) compared to humans (T:B ratio = 7:1, n=6). These differences make

pharmacodynamic modeling in cynomolgus monkeys a conservative estimate of

MGD011 potency and should be taken into consideration when projecting human doses.

Administration of MGD011 resulted in a decrease in circulating CD20+ B cells by

24 hours following the start of the first infusion (Figure 5C-D). While a partial reduction

in circulating B cells was noted at the 0.2 μg/kg dose, nearly complete depletion was

observed at dose levels ≥0.5 μg/kg and were maintained throughout the 4 weekly

treatments. Upon completion of dosing, circulating B cells returned to predose levels,

albeit with a dose-proportional lag (Figure 5C). Histopathology examination at terminal

necropsy showed a dose-dependent decrease in follicles and germinal centers within the

spleen, lymph nodes (inguinal, mandibular, and mesenteric), and gut-associated lymphoid

tissue (GALT) (data not shown). CD20 immunohistochemistry (IHC) showed a reduction

in CD20+ B cells to nearly undetectable levels in the lymph nodes, spleen, and bone

marrow at doses ≥10 μg/kg (Figure 5E-F). The pharmacological effect of MGD011 on B

lymphocytes was prolonged, but ultimately reversible, as indicated by the return of

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 (Figure 5E). In summary, once

weekly administration of MGD011 was sufficient to induce complete and durable B-cell

depletion in both the circulation and lymphoid organs.




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Treatment of MGD011 was associated with transient, dose-dependent fluctuations of

both circulating CD4+ and CD8+ T cells, with the lowest nadir following the first infusion

and with a decreasing magnitude following subsequent infusions (Figure 6A-B).

Contrary to the durable reduction in B cells, T lymphocytes recovered quickly after each

infusion and reached or exceeded baseline levels prior to the next dose. Similar T-cell

kinetics were observed in cynomolgus monkeys after the administration of MGD006, a

CD123 x CD3 DART protein (13), and in humans during blinatumomab infusion (18).

Fluctuations were also observed with natural killer cells and monocytes in MGD011-

treated monkeys (data not shown), likely representing transient cell margination.

MGD011 treatment was also associated with upregulation of the T-cell activation

markers, CD69 and PD-1 (Figure 6C-F), together with an increase of the Ki67+

proliferating fraction of both CD4+ and CD8+ T-cell subsets during the treatment phase

(Figure 6G-H). Therefore, MGD011 is capable of mediating T-cell activation and

expansion in vivo. The activation markers trended toward or returned to baseline by the

end of the experiment; however, unexplained isolated increases in one group 4 animal

(showing up to 10-fold higher frequency of CD4+CD69+ cells than the remaining 3

animals) and in two vehicle control animals (up to 10-fold higher frequency of

CD8+PD1+ cells than the other 2 animals) during the recovery phase contributed to

swelling the average for these groups (Figure 6C and 6F). An asymptomatic, transient,

dose-dependent increase in circulating cytokine levels, a first-dose effect, was observed

for IFN-γ (Figure 6I), IL-6 (Figure 6J), and IL-10 (Figure 6K) and, to a lesser extent,

TNF-α 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 of the start of the infusion.




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Smaller levels of cytokine increase were noted with subsequent infusions, which were of

a magnitude comparable to those observed following vehicle infusions, except IL-10,

which exceeded levels of the vehicle control group in some instances. No MGD011-

related changes in circulating IL-2, IL-4, or IL-5 levels were noted.

MGD011 was well tolerated in cynomolgus monkeys at all dose levels tested, with no

compound-related toxicological effects, including cage-side observations, changes in

body weight or food consumption, vital parameters, ophthalmology, electrocardiograms,

body temperature, blood pressure, heart rate, respiration rate, neurological examinations,

coagulation, or clinical chemistry (data not shown); furthermore, with the exception of

the aforementioned hematological, bone marrow, and lymphoid organ changes, no other

gross or microscopic pathological findings were noted (data not shown).




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Discussion

MGD011 is a bispecific DART protein developed for the treatment of B-cell

malignancies and designed to redirect the T lymphocytes via their CD3 molecule to kill

target cells expressing CD19. MGD011 was shown to eliminate normal or pathogenic

CD19+ cells in vitro and in vivo. Furthermore, weekly administration of MGD011 to

cynomolgus monkeys resulted in complete and durable depletion of B cells in the

circulation and in lymphoid tissues. MGD011 was well tolerated in toxicology studies

under treatment regimens that exaggerated potential clinical setting scenarios in patients.

Thanks to its favorable PK properties, convenient once-a-week dosing or longer interval

is predicted to be clinically feasible in humans.

The structure of MGD011 represents a refinement of a previously reported format (8),

where the DART chains (Chain 1 and Chain 2) preserve the original C-terminal

stabilizing disulfide linkage, while opposite E/K-coil sequences were added that further

improve heterodimer formation. To promote the desired heterodimerization of Chain 1

and the Fc-domain Chain 3, knob-into-hole mutations were incorporated in the CH3

region. To facilitate the removal of residual Chain 3 dimers during purification, a H435R

mutation was included in this chain to abolish protein A binding. MGD011 was

engineered with humanized antibody arms displaying 10-fold greater affinity for human

CD19 than for human CD3, thus providing for preferential initial binding to target cells,

while minimizing CD3 engagement in the absence of target cells. MGD011 exhibits

binding to monkey CD3 and CD19, allowing for preclinical modeling in this species.

Specifically, MGD011 cross-reacts with cynomolgus monkey CD3 with nearly identical




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affinity as for human CD3 and with a 10-fold lower affinity for monkey CD19.

Consistent with its binding properties, MGD011 mediated similar levels of cytotoxicity

against human CD19+ Raji/GF target cells by either human or cynomolgus monkey

PBMCs. MGD011 mediated also a dose-dependent, autologous ex vivo B-cell depletion

in both human and cynomolgus monkey PBMCs, albeit with a decreased potency in the

latter, due, at least in part, to its lower affinity for monkey CD19. Because of the lower

CD19 affinity and reduced potency in cynomolgus monkeys, this species may offer a

conservative estimate of potency that needs to be taken into consideration in

extrapolating non-human primate data to potential clinical settings.

To confer prolonged circulating half-life, MGD011 was also engineered with an Fc-

domain; with an estimated terminal half-life of ~161 hours (6.7 days) in monkeys and a

predicted half-life of 343-495 hours (14.3-20.6 days) in humans, which is similar to that

of conventional mAbs, delivering MGD011 as an intermittent dosing regimen appears

feasible. In comparison, blinatumomab, which is engineered as a single-chain Fv pair, is

administered by continuous IV infusion for repeated 4-week courses, owing to its short

~2-hour half-life.

MGD011 administered to cynomolgus monkeys by a 2-hour IV infusion on a weekly

schedule at doses ranging from 0.2 to 100 μg/kg was safe and well tolerated. Dose-

dependent, on-target activity with nearly complete depletion of circulating CD20+ B cells

was observed at doses ≥0.5 μg/kg. Histological evaluation confirmed a dose-dependent

decrease in B-cell zones in lymphoid tissues at the terminal necropsy. While bone

marrow showed no obvious histological changes, B-cell depletion was observed by IHC,




Liu et al. - CCR

21

further attesting to the specificity of the on-target effects of MGD011. The

pharmacological effect of MGD011 on B lymphocytes was reversible, as indicated by

flow cytometry and IHC analysis in addition to hematology and histopathology (data not

shown). Each of these parameters returned to baseline, with no differentiation between

the animals receiving MGD011 or control article at the end of the 12-week recovery

period.

Maximal cytolytic activity against B-cell lines and in patient samples ex vivo were

observed at MGD011 concentrations of 50-100 ng/mL. These levels are at or below the

Cmax observed in monkeys at ≥5 μg/kg or below trough levels at ≥50 μg/kg. Either dose

level resulted in near complete depletion of B lymphocytes in the circulation and

lymphoid tissues in monkeys, attesting to the potency of the DART molecule, particularly

in account it decreased potency in this species compared to humans. In B-cell

malignancies, a low effector-to-target (E:T) cell ratio could limit the effectiveness of

redirected cytolysis. MGD011, however, was able to eliminate leukemic cells in CLL

patient samples by engaging and expanding the patient’s residual T cells in vitro from a

low E:T cell ratio to one exceeding the target cell population. These data also confirm

that CLL T cells can be redirected to become cytolytic against leukemic cells in the

presence of MGD011, as previously reported for blinatumomab (19), even though T-cell

dysfunction in CLL, such as defects in immune synapse formation, co-

stimulatory/accessory molecule expression, and cytokine release have been reported (20-

22).




Liu et al. - CCR

22

A safety concern associated with CD3-targeting therapies is cytokine release. The

monovalent nature of the binding arm of MGD011 ensures that T-cell activation and

cytokine release depend exclusively on target-cell engagement. Consistent with its

desired design, no T-cell activation or cytokine release were observed in vitro in the

absence of CD19+ target cells or with a control DART protein that includes only the

CD3-targeting arm. No cytokine storm was observed in MGD011-treated cynomolgus

monkeys. Transient dose-dependent increases in IFN-γ, IL-6 and IL-10 were observed

following the first dose; with subsequent doses, they were generally no greater than those

observed in animals receiving vehicle. There were no MGD011-related changes in

circulating IL-2, IL-4, or IL-5 levels and only small, transient increases in TNF-α,

exceeding 100 pg/mL in only a few instances, were observed in animals that received

10 μg/kg MGD011. First-dose cytokine release events have been previously observed in

patients treated with blinatumomab (18) or with our CD123 x CD3 DART molecule in

monkeys (13); the rapid target-cell depletion following the first administration of the

bispecific molecule may limit further T-cell engagement and explain the transient nature

of cytokine release observed.

Tumor lysis syndrome, which has been observed with blinatumomab (7), should be

anticipated as another potential safety concern for MGD011 and proper preventive

measures be implement to limit its consequences. Blinatumomab treatment was also

associated with an increased risk of serious and/or severe neurological toxicities (7); a

similar safety risk of neurological toxicities for MGD011 is therefore recognized. It

should be noted, however, that no signs of neurological toxicity were observed in the

MGD011 toxicology studies, although the predictive value of the cynomolgus monkey




Liu et al. - CCR

23

with respect to neurotoxicity of CD19 x CD3 bispecific interventions is unknown.

Similarly, no infection-related adverse events were observed in monkeys treated with

MGD011, although 25% of patients receiving blinatumomab showed signs of infection

(7). Hence, patients should be monitored for infections and treated appropriately.

Immunogenicty remains a potential concern associated with antibody therapy. MGD011

limits this through the incorporation of humanized Fv regions and the minimal use of

linkers. While immunogenicity in monkeys does not predict immunogenicity in humans,

notably, only 3/40 monkeys treated with MGD011 showed an ADA response; given that

MGD011 targets B lymphocytes, ADA development is expected to be low in all species.

Upregulation of activation markers, including PD-1, was observed amongst circulating

T lymphocytes in monkeys treated with MGD011. Importantly, the frequency of

regulatory T cells or TIM-3+ cells in both the CD4+ and CD8+ T-lymphocyte populations

remained low (< 1%) throughout the entire dosing period (data not shown), suggesting

that T-cell activation induced by MGD011 was not accompanied by expansion of

regulatory T cells or T-cell exhaustion.

The off-the-shelf convenience of bispecific antibodies offers substantial advantages

compared with personalized CAR T cells. A point of contention, however, is whether a

trade-off of bispecific molecules is a decrease in potency. The impressive response to

CAR T-cell therapy is associated with a significant rate of severe adverse events,

resulting in a narrow therapeutic window, with little, if any, opportunity for response fine

tuning. We should further note that MGD011, on a molar basis, is more potent than

blinatumomab and that complete depletion of B lymphocytes




Liu et al. - CCR

24

in both marrow and lymphoid tissues can be achieved in cynomolgus monkeys in the

absence of adverse effects. Ultimately, only clinical data will be able to address this

issue.

In summary, MGD011 demonstrated potent activity in redirecting T lymphocytes to

eliminate CD19-expressing cells in vitro, induced growth inhibition and tumor regression

of B-cell lymphoma/leukemia models in mice, showed prolonged circulating half-life and

was safe and well tolerated in cynomolgus monkeys at doses that resulted in complete

and durable B-cell depletion in the circulation, bone marrow, and lymphoid organs. The

data from these nonclinical studies provided rationale for the clinical development of

MGD011 as a treatment for patients with B-cell malignancies; a Phase 1 dose escalation

study of the safety, tolerability, dose-limiting toxicity, maximal tolerated does and

recommended Phase 2 dose of MGD011 when administrated intravenously over a 2-hour

period once every 2 weeks to subjects with B-cell malignancies is currently recruiting

patients (NCT02454270).




Liu et al. - CCR

25

Acknowledgements

We would like to thank Cynthia Sung (Rainbow Pharma Consulting) for data analysis,

Robert Burns, Qin Tang, Nancy O’Gwin, and Xioqi Gong for technical assistance,

Melinda Hanson for administrative and editorial support, Timothy Mayer and James

Karrels for critical discussions and review of the manuscript. We thank the teams at the

Charles River Laboratories, Inc. (Reno, NV) for the diligent conduct of the cynomolgus

monkey studies.




Liu et al. - CCR

26

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Figure Legends

Figure 1. MGD011 structure, binding, and redirected T-cell killing activity against

CD19+ 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 CD19+ (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, 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; percentage of CD20+

cells was determined by flow cytometry after incubation with MGD011, a replica of

blinatumomab or a control DART protein (Fluo x CD3).

Figure 2. MGD011-mediated T-cell activation, proliferation, and cytokine release is

dependent on co-engagement of CD19+ target cells. A, MGD011 concentration-




Liu et al. - CCR

31

dependent induction of T-cell activation marker CD69 on CD4+ and CD8+ human T cells

following 24-hour incubation with CD19- JIMT-1 cells or CD19+ 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 CD19+ 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

CD4+ and CD8+ human T cells following 24-hour incubation with CD19+ Raji/GF target

cells at E:T cell ratio of 10:1. D, MGD011 concentration-dependent induction of IFN-γ

and IL-2 following 24-hour incubation of human T cells in the absence (left) or presence

(right) of CD19+ Raji/GF target cells at E:T cell ratio of 10:1.

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 µg/mL were incubated with PBMCs from a CLL patient (E:T = 1:23) and

analyzed by flow cytometry at Day 0 (prior to DART protein addition) and Days 3 and 6.

A, percentage of CD20+/CD5+ malignant B cells in the gated lymphoid population. B,

percentage of CD4+ and CD8+ T cells. C and D, percentage of CD25+ T-cell activation in

CD4+ and CD8+ T cells, respectively.

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 = 8 per

group) were implanted intradermally with HBL-2 tumor cells (5 x 106) on Day 0

followed by intraperitoneal (IP) injection of human PBMCs (1 x 107) on Day 4. Tumor-

bearing mice, randomized at Day 14, were treated with MGD011 or a control DART




Liu et al. - CCR

32

protein (Fluo x CD3) at 500 µg/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 CD19+

Raji/GF leukemia/lymphoma tumors. NSG B2m-/- mice (n = 8 per group) were implanted

IP with human PBMCs (1 x 107) on Day -5 followed by Raji/GF tumor cells (1 x 106)

injected IV on Day 0. Tumor-bearing mice, randomized at Day 14, were treated with

MGD011, a control DART protein (Fluo x CD3) or vehicle, IV 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 panel B. * p

<0.001 and ** p = 0.03 by logrank test comparison to the vehicle-treated group.

Figure 5. Serum concentrations and B-cell depletion in cynomolgus monkeys

following weekly doses of MGD011. A-B, MGD011 serum concentration-time profiles

from a repeat-dose study (Panel A, Study 1, Table S1) in monkeys (5/sex/group) in

which vehicle or MGD011 (dose levels ranging from 0.2 to 10 µg/kg, as indicated) were

administered IV once weekly for 4 weeks or a repeat-dose study (Panel B, Study 2, Table

S1) in monkeys (1-2/sex/group) in which MGD011 was administered IV once or twice

weekly at the indicated dose levels for 3 or 4 weeks. In both studies, vehicle (V) was

administered one week prior to the start of MGD011 dosing (D) with the exception of the

fixed dose 0.5 µg/kg groups in panel B. The assay lower limit of quantitation

(1.36 ng/mL Panel A, Study 1 or 2.45 ng/mL Panel B, Study 2) is indicated by dashed




Liu et al. - CCR

33

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 CD20+ 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 x 3)

dose group or 7 days after the last dose for the escalating (0.5 to 50 µg/kg and 2 to

100 µg/kg) dose groups.

Figure 6. Changes in peripheral T-lymphocyte subpopulations and cytokines in

cynomolgus monkeys administered weekly doses of MGD011. Data are from the

repeat-dose study 1 in monkeys (5/sex/group) in which MGD011 (dose levels ranging

from 0.2 to 10 µg/kg) or vehicle were administered IV once weekly for 4 weeks

(Table S1). A-B, CD4+ and CD8+ T cells. C-D, CD69-activated CD4+ and CD8+ T cells.

E-F, PD-1-activated CD4+ and CD8+ T cells. G-H, Proliferating (Ki67+) CD4+ and CD8+

T cells. I-K, Serum levels of cytokines: IFN-γ, IL-6, and IL-10. V = vehicle infusion; D =

MGD011 infusion.




Liu et al - Figure 1

A

B

-100

0

100

200

300

400

500

600

-50 50 150 250 350

RU

s Time

MG

D0

11

hCD19

hC

D3

su

rfac

e

Buffer Buffer

Antigens ka (± SD) (M-1S-1)

kd (± SD) (S-1)

KD (± SD) (nM)

Human CD3ε/δ 2.2 (±0.0) x105 4.5 (±0.1) x10-3 21.2 (±1.7)

Cynomolgus CD3ε/δ 2.0 (±0.1) x105 4.3 (±0.2) x10-3 21.9 (±1.2)

Human CD19 2.8 (±0.3) x105 0.56 (±0.6) x10-3 2.0 (±0.2)

Cynomolgus CD19 2.4 (±0.1) x105 4.7 (±1.1) x10-3 20.3 (±1.3)

D

H

Chain 1

Chain 2

Chain 3 H2N COOH Fc Hole S S

H2N COOH Fc Knob S S CD19VL CD3VH E S

H2N COOH K CD3VL CD19VH S

F E

G

C




Co

un

ts

T=0 hr

Control DART

MGD011

T = 72 hr

Co

un

ts


A

B

C

D





CD4

CD

8

CD25

CD

8

CD25

CD

4

CD5

CD

20

Control DART MGD011

Day 3

Control DART MGD011

Day 6

Untreated

Day 0

0

1.2

73

25

0

1.6

71

28

0

0.2

75

25 1.4 58

0 41 0.1

2.4

11

86

2

95

0

3

2

97

0

1

2

94

0

4

22

6

14

57

83

16

1

0

12

0

88

0

79

0

21

0

17

0

83

0

A

B

C

D




A B


X

X

X

X

X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Control DART (100 µg/kg)

Study Day

7 14 21 28 46

Vehicle

Study Day

7 14 21 28

MGD011 (0.16 µg/kg)

Study Day

7 14 21 28 46

MGD011 (0.8 µg/kg)

Study Day

7 14 21 28 46

MGD011 (4 µg/kg)

Study Day

7 14 21 28 46

MGD011 (20 µg/kg)

Study Day

7 14 21 28 46

MGD011 (100 µg/kg)

Study Day

7 14 21 28 46

C





A B

C D

E F 2→10→100→100

μg/kg 5→5→5

ng/kg

MGD011

0.5→5→50→50 μg/kg

Lym

ph

No

de

B

on

e M

arro

w

Term

inal N

ecro

psy

Sple

en

100 µm

6 mm

600 µm

Sple

en

Ly

mp

h n

od

e

Vehicle 4 x 0.2 μg/kg 4 x 5 μg/kg 4 x 10 μg/kg

MGD011

Sple

en

Term

inal N

ecro

psy

Re

cove

ry Ne

crop

sy

Lym

ph

no

de

B

on

e M

arro

w

Bo

ne

Mar

row

2 mm

1 mm

200 µm

2 mm

1 mm

200 µm





A B

C D

E F

G H

I J K




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 Re-Targeting Bi-specific

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