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. Ó2016 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 expres- sion 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 Ó2016 American Association for Cancer Research. respondtostandardchemo-immunotherapies and has been www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 23, 2016; DOI: 10.1158/1078-0432.CCR-16-0666 Liu et al. 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