Published OnlineFirst September 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3622-T Cancer Therapeutics, Targets, and Chemical Biology Research Antitumor Efficacy of a Bispecific Antibody That Targets HER2 and Activates T Cells Teemu T. Junttila, Ji Li, Jennifer Johnston, Maria Hristopoulos, Robyn Clark, Diego Ellerman, Bu-Er Wang, Yijin Li, Mary Mathieu, Guangmin Li, Judy Young, Elizabeth Luis, Gail Lewis Phillips, Eric Stefanich, Christoph Spiess, Andrew Polson, Bryan Irving, Justin M. Scheer, Melissa R. Junttila, Mark S. Dennis, Robert Kelley, Klara Totpal, and Allen Ebens Abstract Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2- expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti–PD-L1 treatment. Thus, combining HER2-TDB with anti– PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response. Cancer Res; 74(19); 1–11. Ó2014 AACR. Introduction implemented in clinical use. In 2009, the European Medicines  The recent approval of ipilimumab (1) and exciting Agency (EMA) approved the use of a trifunctional EpCam fi responses observed during clinical trials of PD-1 and PD-L1 CD3 bispeci c antibody, catumaxomab (Removab), for the antibodies (2) clearly illustrate the potential of T cell–targeting intraperitoneal treatment of malignant ascites (5). Promising  cancer immunotherapies. The success of strategies that rein- clinical activity has also been demonstrated with a CD19 – fi vigorate T-cell activity depends on modulation of multiple CD3 targeting bispeci c scFv antibody fragment, blinatumo- stimulatory and inhibitory events that enable an antitumor mab (3). Both catumaxomab and blinatumomab illustrate how fi immune response (2). An attractive alternative of leveraging T- technological challenges affect clinical use of bispeci c anti- cell activity to eliminate cancer is to not rely on existing tumor bodies. Catumaxomab is a mouse IgG2a/rat IgG2b hybrid, and immune response, but rather to induce T cells to kill tumor thus it is highly immunogenic in human. Dosing of catumax- cells directly by generating new tumor specificities. Bispecific omab is limited to maximum of four doses and 20 days. antibodies can be used to broadly harness the antitumor Because of the extremely short serum half-life (1.25 hours) of capacity of T-cell immunity (3). However, successful clinical blinatumomab (3), it is continuously infused via pump use of modified and reengineered antibodies and antibody throughout the treatment cycle. – fragments is far from trivial (4). To progress a drug candidate Here, we report the properties of a T cell dependent bis- fi from laboratory to clinic, a vast array of requirements needs to peci c antibody targeting HER2 (HER2-TDB), which can be met in regard to "drug-like" properties. Immunogenicity and induce a polyclonal T-cell response to tumors. The response short serum half-life are additional problems for bispecific does not require a preexisting tumor immune response and it molecules with modified antibody sequences and antibody has reduced potential for immune escape (e.g., loss of MHC fragment–based platforms. expression). The modality combines extreme potency of the T- Although several tumor targets and bispecific antibody cell activity with favorable drug-like properties of IgG1 mol- ecule, including long half-life. platforms have demonstrated general flexibility and preclinical þ feasibility for this approach, very few molecules have been Despite recent advances in treatment of HER2 breast cancer (6–9), several resistance mechanisms have been iden- tified that engage redundant survival signaling pathways (10– Genentech, Inc., South San Francisco, California. 12). De novo or acquired resistance is an expected outcome Note: Supplementary data for this article are available at Cancer Research also for a subset of T-DM1 (ado-trastuzumab emtansine, Online (http://cancerres.aacrjournals.org/). Kadcyla)–treated patients. Therefore, a significant unmet þ medical need remains for HER2 breast cancer. HER2-TDB Corresponding Author: Teemu T. Junttila, Genentech, Inc., 1 DNA Way, þ Mailstop 231a, South San Francisco, CA 94080. Phone: 650-225-1000; kills all tested HER2 tumor cells with low picomolar EC50, Fax: 650-225-5214; E-mail: [email protected] and due to its different mechanism of action, effectively kills doi: 10.1158/0008-5472.CAN-13-3622-T cells that are refractory to trastuzumab, lapatinib, and T- Ó2014 American Association for Cancer Research. DM1. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst September 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3622-T Junttila et al. An important outstanding question with T cell–engaging In vitro cytotoxicity assays (in vitro ADCC, T-cell killing) bispecific antibodies is whether, they too may be susceptible to In vitro cytotoxicity assays [Cytotoxicity Detection Kit; lac- T cell–suppressive resistance mechanisms following the initial tate dehydrogenase (LDH); Roche] were performed as previ- T-cell response? Our results demonstrate that PD-1/PD-L1 can ously described (21). Alternatively, in vitro cytotoxicity was inhibit T-cell killing activity induced by bispecific antibodies. monitored by flow cytometry. Target cells were labeled with þ Our results using immunocompetent huCD3-transgenic ani- CFSE (Invitrogen; #C34554). The labeled target cells and CD8 mal model further suggest that combining T cell–recruiting cells were mixed with or without TDB for 4 to 26 hours. At the antibodies with anti–PD-L1 antibodies improves outcome of end of the incubation, the cells were lifted by trypsin and the treatment. collected from the plate. The cells were resuspended in equal volume of PBS þ 2% FBS þ 1 mmol/L EDTA þ propidium Materials and Methods iodine (PI). Flow cytometry analysis was done on a FACSCa- fi libur in automation format. The number of live target cells was Antibody expression and puri cation þ À The "knob" arm of HER2 huIgG1 TDB is humanized anti- counted by gating on CFSE /PI cells. The percentage of HER2 4D5 (trastuzumab; ref. 13) and "hole" arm is humanized cytotoxicity was calculated as follows: %cytotoxicity (live À anti-CD3 UCHT1.v9 (14, 15). The huIgG1 bispecific antibodies target cell number without TDB live target cell number  were produced by two different approaches as described with TDB)/(live target cell number without TDB) 100. iCell earlier (16): coculture of bacteria expressing each of the two cardiomyocytes (Cellular Dynamics International) were antibody arms, or by expressing each arm separately and then revived from liquid nitrogen and plated at 27,000 cells per annealing them in vitro. 96-well 7 days before the assay and treated as per the man- To avoid immune response toward the TDB, a murine IgG2a ufacturer's instructions. CD8 cells were added in 3:1 ratio on (knob-hole, D265A, and N297G) isotype HER2-TDB expressed iCells for 24 hours together with the treatment. After 24 hours, in Chinese hamster ovary (CHO) cells was used in experiments cells were gently washed twice with PBS to remove T cells and with immunocompetent mice. In muIgG2a HER2-TDBs, the viability was measured using the CellTiter-Glo Assay. "knob" arm is murine anti-HER2 4D5 and the "hole" is either chimeric anti-murine CD3 2C11 (4D5/2C11-TDB; ref. 17) or Analysis of T-cell activation mouse anti-hu CD3 SP34 (4D5/SP34-TDB; ref. 18). Bispecific Cells were stained with CD8-FITC (BD Biosciences; 555634), antibody purification is described elsewhere (16). CD69-PE (BD Biosciences; 555531), and CD107a-Alexa Fluor 647 (eBioscience; 51-1079). Alternatively, cells were fixed Antibody characterization and permeabilized with Cytofix/CytoPerm solution (BD Bio- The molecular weight of the bispecific antibody was ana- sciences; 554722) and stained with anti-granzyme B-Alexa lyzed by mass spectrometry [liquid chromatography-electro- Fluor 647 (BD Biosciences; 560212). spray ionization/time of flight (LC-ESI/TOF)] as described before (19). The antibodies were also analyzed by analytic size Detection of soluble granzymes and perforin exclusion chromatography in a Zenix SEC-300 column (Sepax Soluble perforin (Cell Sciences), granzyme A, and granzyme Technologies) using an Agilent 1100 HPLC system (Agilent B (eBioscience) were detected from growth media by ELISA Technologies). The presence of residual antibody fragments according to the manufacturer's protocols. was quantified by electrophoresis using a 2100 Bioanalyzer and a Protein 230 Chip (Agilent Technologies). PD-1 induction and effect of PD-L1 expression on TDB activity þ HER2-TDB affinity Purified CD8 T cells from human peripheral blood were The competitive Scatchard assay is described in detail primed with 100 mg/mL of HER2-TDB and SKBR3 cell at 3:1 elsewhere (20). ratio for 24 hours. After 24 hours of incubation, the cell pellet was digested with Non-Enzyme Cell Dissociation Solution þ Breast cancer cell proliferation (Sigma; #C5789) at 37C for 10 minutes and CD8 T cells were þ Breast cancer cell proliferation/viability was detected recovered using Human CD8 MicroBeads (Miltenyi Biotec; þ using the CellTiter-Glo Luminescent Cell Viability Assay #130-045-201). The primed-CD8 T cells were used for in vitro (Promega). For the assay, 5  103 cells per well were plated cytotoxicity assay. In flat-bottomed 96-well plate, CFSE-labeled in 96-well plates and incubated overnight for cell attachment 293 cells or 293-PD-L1 cells were mixed with primed effector before treatments.