AACR Annual Meeting 16-20 April 2016 - Poster #280 Synergistic in vitro activity of MOR209/ES414 in combination with enzalutamide 1Emergent Product Development Seattle – Seattle, WA, USA Toddy Sewell1, Jan Endell2, Johannes Weirather2, Michelle Blake1, Jane Gross1, and John W. Blankenship1 and 2MorphoSys AG – Martinsried/Planegg, Germany Combination Treatment of LNCaP Cells with Introduction Enzalutamide Drug Activity on LNCaP Cells Monitored by Fluorescence MOR209/ES414 and Enzalutamide Metastatic castrate-resistant prostate cancer (mCRPC) presents a daunting therapeutic Enzalutamide (previously known as MDV3100) is a non-steroidal MOR209/ES414 is cytotoxic to CRPC cells by a different mode of action than enzalutamide, Combinations of MOR209/ES414 and enzalutamide were tested in a 96 well plate fluorolysis challenge for patients who eventually progress after androgen ablation therapy. Although newer androgen receptor (AR) inhibitor. It was approved for oral dosing of directing T cells to lyse PSMA-bearing tumor cells. Enzalutamide inhibits the growth of assays using LNCaP(GFP) cells in culture with primary T cells. Surviving LNCaP cells were approaches to antagonize androgen receptor signaling such as enzalutamide or blockading mCRPC patients in 2012. Enzalutamide impairs AR translocation to tumor cells dependent on androgen signaling. To enable measurement of the impacts of the quantitated 4-5 days later. Data shown is from one of four similar experiments, using T cells androgen biosynthesis such as abiraterone provide a survival benefit to patients, patients the nucleus and AR binding to DNA. It is an effective antagonist of two drugs simultaneously, a multi-day growth inhibition assay was needed. At least 4 days from 3 healthy donors. treated with these therapies ultimately relapse. Thus, it is critical to identify new therapies that AR receptor signaling with no agonist activity. of target cell growth were required to provide accurate data on growth inhibition by will work in combination with AR antagonists or in settings where patients have developed enzalutamide. Enzalutamide did not interfere with the ability of MOR209/ES414 to target T cells to LNCaP cells, resistance to hormonal therapies. MOR209/ES414 is a bispecific ADAPTIRTM (modular protein In phase III trials enzalutamide was found to prolong the survival of and augmented the effect of MOR209/ES414 when used at suboptimal concentrations. technology) molecule currently under investigationinaphase1clinicaltrialinmCRPC,including men with mCRPC, reduce the risk of death, significantly improve LNCaP cells provided a PSMA-positive, enzalutamide-sensitive cell line for testing patients that have progressed on enzalutamide or abiraterone. In pre-clinical studies, quality of life measures, and demonstrate consistent safety and MOR209/ES414 in combination with enzalutamide. LNCaP cells transduced to express Green 100 MOR209/ES414 has previously been shown to redirect T-cell cytotoxicity against cells tolerability across all patient subgroups.b,c Fluorescent Protein (GFP) were obtained from AntiCancer, Inc. Fluorescence from GFP 0 nM Enzalutamide - MOR209 a expressing Prostate Specific Membrane Antigen (PSMA). Here, we aimed to determine provided a reliable method to monitor target cell growth in the presence of T-cells 80 39 nM Enzalutamide - MOR209 whether enzalutamide affects PSMA expression on CRPC cells in vitro,andcouldalterthe Despite the improved survival benefitthatenzalutamideprovides, (fluorolysis, or loss of fluorescence from lysed cells). Primary T cells were used as effector patients ultimately relapse. Therapeutic agents which can work in 156 nM Enzalutamide - MOR209 effectiveness of MOR209/ES414 at inducing T-cell mediated tumor cell death. cells. 60 combination with enzalutamide, or in patients who have developed 625 nM Enzalutamide - MOR209 resistance, are critically needed. MOR209/ES414: ADAPTIR Molecule Targeting PSMA and CD3 For fluorolysis assays, LNCaP (GFP+) were cocultured with primary T cells and 40 2500 nM Enzalutamide - MOR209 MOR209/ES414 for 3-5 days in 96 well plates. LNCaP cells are colored green in images, and cells dead % T cells are visible by blue nuclear staining from Hoechst. Images show the cell loss from 20 ADAPTIR molecules are bispecific antibody-like therapeutics 22Rv1 Prostate Cancer Cell Line is Enzalutamide Resistant redirected T cell cytotoxicity when target cells are cultured with T cells and MOR209/ES414. containing two sets of binding domains linked to immunoglobulin Fc 0 domains to extend the half-life of the molecule in vivo. PSMA scFv 0.0 0.5 1.0 1.5 2.0 2.5 To confirm the sensitivity or resistance of several MOR209/ES414 is an ADAPTIR molecule that binds both PSMA and log c MOR209 [pM] prostate cancer cell lines to enzalutamided,e,cells CD3 to redirect T-cell cytotoxicity against PSMA expressing tumor 120 cells. The anti-PSMA binding domain is a humanized single chain weregrownin96wellplatesfor7daysinthe presence of the drug. Cell number was quantitated variable fragment (scFv) derived from a murine antibody that binds effector null Fc 100 Combination Index Analysis human and non-human primate (NHP) PSMA. The anti-CD3 binding by Cell Titer Glo luminescence and expressed as a 22Rv1 ratio to signal from untreated wells. 80 domain is a humanized scFv adapted from a murine antibody that MDA-PCa-2b f binds human and NHP CD3. In order to avoid interactions with other 60 Combination Index analysis following the method of Chou and Talalay was used to determine components of the immune system that could lead to CD3 22Rv1 and MDA-PCa-2b cell lines were shown to be VCaP whether or not the combination of MOR209/ES414 with enzalutamide was additive or enzalutamide-resistant. VCaPandLNCaPcellswere 40 clustering and non-specific T cell activation, the Fc region of CD3 scFv LNCaP synergistic in vitro. When plotted on Fa-CI plots (below), the activity of MOR209/ES414 and sensitive to enzalutamide, with EC50 values of 113 enzalutamide was synergistic in vitro at most concentrations of MOR209/ES414 and MOR209/ES414, has been engineered to minimize complement % of untreated control 20 15.6 pM fixation and interaction with Fc receptors. nM and 298 nM, respectively. Growth of VCaP and untreated MOR209/ES414 enzalutamide. LNCaP cells was inhibited 60-70% over a 7 day 0 treatment period. 0 0.001 0.01 0.1 1 10 M enzalutamide 156 nM enzalutamide 625 nM enzalutamide Redirected T Cell Cytotoxicity (RTCC) by MOR209/ES414 39 nM Enzalutamide Enzalutamide Increases PSMA Expression in 22Rv1 Cells 1.5 1.5 1.5 1.0 1.0 Prostate Specific 22Rv1 cells (ATCC) are androgen-independent, 90 1.0 CI Membrane Antigen AR-positive, and PSMA-positive. Resistance of 3 wk + enza 80 (PSMA) this cell line to enzalutamide provided the 0.5 0.5 0.5 2 wk + enza opportunity to explore whether treatment of this 70 model of mCRPC with enzalutamide would affect 1 wk + enza 0.0 Elimination of 60 0.0 0.0 PSMA expression. MFI no enza 62.5 pM 250 pM tumor cells by Tumor 50 MOR209/ES414 MOR209/ES414 release of Parallel cultures of 22Rv1 cells were treated with cell pM MOR209/ES414 pM MOR209/ES414 pM MOR209/ES414 cytotoxic 10 M enzalutamide for 1-3 weeks, with twice 40 granules weekly passages into fresh media, then stained 30 Single Agent Activity of MOR209/ES414 for PSMA expression using FITC-labeled anti- 0 0.1 1 10 PSMA mAb. nM mAb and Enzalutamide on LNCaP Cells MOR209/ES414: Summary and Conclusions Bispecific An increase in cell surface PSMA expression could 22rv1 22rv1+enza molecule cross- be measured by flow cytometry after 1 week of (no enza) 3 wk MOR209/ES414 and enzalutamide were titrated individually on LNCaP(GFP) cells, using . Treatment with enzalutamide increased PSMA expression on enzalutamide-resistant 22Rv1 links tumor cell enzalutamide treatment, and a further increase in fluorolysis assay conditions designed to accommodate the different mechanisms of action of cells, and also increased the sensitivity of 22Rv1 cells to T-cell mediated lysis induced by and T cell PSMA expression was seen at 2 and 3 weeks. both drugs. LNCaP cells were cultured for 5 days in the presence of a 3:1 ratio of primary T anti-PSMA mAb cells to target cells, necessary for MOR209/ES414, and with 0.2% DMSO present, a MOR209/ES414. unstained T cell necessary control for enzalutamide. Treatment of enzalutamide-sensitive LNCaP cells with suboptimal concentrations of CD3/TCR expansion MOR209/ES414 and enzalutamide showed a synergistic increase in activity as determined by complex The EC50 for MOR209/ES414 was 14.5 pM under these conditions. Enzalutamide affected cell Combination Index analysis. growth with an EC50 of 218 nM. Five days’ culture of LNCaP(GFP) cells with enzalutamide Enzalutamide Increases Susceptibility of 22Rv1 Cells to RTCC inhibited cell growth by 25% at the maximal dose of 10 M, as opposed to 100% inhibition by MOR209/ES414 at the maximal dose of 125 pM. These studies provide a rationale for further examination of combining MOR209/ES414 with enzalutamide, even in enzalutamide-resistant settings. T cell 22Rv1 cells are efficiently lysed by RTCC using Cytotoxic 30 unstimulated primary T cells and MOR209/ES414, 100 granule despite their low level of PSMA expression. A 3 wk + enza 100 25 References chromium-51 release assay was used to examine 2 wk + enza 80 whether exposure to enzalutamide would affect the 20 90 a Sewell, T et al, anti-PSMA x anti-CD3 bispecific antibody redirects T cell cytotoxicity in castrate-resistant prostate sensitivity of 22Rv1 cells to T-cell mediated lysis. 1 wk + enza 60 cancer models. 2012 EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, Dublin, Ireland. b Scher, H., Fizazi, K., Saad, F., Taplin, M., Sternberg, C., Miller, K. et al. (2012) Increased survival with enzalutamide 15 no enza 22Rv1 cells exposed to enzalutamide were more 80 in prostate cancer after chemotherapy. N Engl J Med 367: 1187–1197. 40 c Beer, T., Armstrong, A., Rathkopf, D., Loriot, Y., Sternberg, C., Higano, C.