A Bispecific DLL3/CD3 Igg-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer

Published OnlineFirst June 18, 2020; DOI: 10.1158/1078-0432.CCR-20-0926

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

A Bispeciﬁc DLL3/CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer A C Susanne Hipp1, Vladimir Voynov2, Barbara Drobits-Handl3, Craig Giragossian2, Francesca Trapani4, Andrew E. Nixon2, Justin M. Scheer2, and Paul J. Adam5

ABSTRACT ◥ Purpose: Small cell lung cancer (SCLC) is the most lethal and Results: Selective binding of DLL3/CD3 ITE to DLL3-positive aggressive subtype of lung carcinoma characterized by highly tumor cells and T-cells induces formation of an immunological chemotherapy-resistant recurrence in the majority of patients. synapse resulting in tumor cell lysis and activation of T-cells. In To effectively treat SCLC, we have developed a unique and novel a human T-cell engrafted xenograft model, the DLL3/CD3 ITE leads IgG-like T-cell engaging bispecific antibody (ITE) that potently to an increase in infiltration of T-cells into the tumor tissue resulting redirects T-cells to specifically lyse SCLC cells expressing Delta-like in apoptosis of the tumor cells and tumor regression. Consistent ligand 3 (DLL3), an antigen that is frequently expressed on the cell with the mode of action, the DLL3/CD3 ITE treatment led to surface of SCLC cells, with no to very little detectable expression in upregulation of PD-1, PD-L1, and LAG-3. normal tissues. Conclusions: This study highlights the ability of the DLL3/CD3 ITE Experimental Design: The antitumor activity and mode of to induce strictly DLL3-dependent T-cell redirected lysis of tumor cells action of DLL3/CD3 ITE was evaluated in vitro using SCLC cell and recruitment of T-cells into noninflamed tumor tissues leading to lines and primary human effector cells and in vivo in an SCLC tumor regression in a preclinical in vivo model. These data support clinical þ xenograft model reconstituted with human CD3 T-cells. testing of the DLL3/CD3 ITE in patients with SCLC.

Introduction endocytic compartment preventing Notch1 from reaching the cell surface (4). This finding has been confirmed in in vitro studies by Lung cancer remains the number one cause of cancer-related deaths demonstrating that DLL3 does not activate Notch signaling through in both men and women in the United States with over 220,000 new binding to Notch expressed on the cell surface, but rather acts as a cases diagnosed annually (1). Out of these, small cell lung cancer Notch antagonist by suppressing Notch signaling in a cell-autonomous (SCLC), a poorly differentiated neuroendocrine tumor, accounts for manner (5). DLL3 expression has been described in SCLC tissue roughly 10% to 15% of cases (2). SCLC is the most lethal and aggressive samples (6). In normal tissues, DLL3 is expressed during embryonal subtype of lung carcinoma with 5-year survival rates below 7% development with highest expression in fetal brain, but is absent in characterized by highly chemotherapy-resistant recurrence within adult normal tissues (6). Although in developmental processes DLL3 is 12 months in the majority of patients (2). expressed intracellularly, in SCLC tumors with DLL3 overexpression, Delta-like ligand 3 (DLL3) is a member of the Notch receptor ligand DLL3 escapes to the cell surface which makes it targetable with family that plays a critical role for Notch signaling during embryonal antibody-based therapies (6). IHC studies indicate a high prevalence development and is functionally distinct from the related Notch family of DLL3 expression in SCLC tissues from Caucasian and Japanese members DLL1 and DLL4, as demonstrated in animal studies in patients, with 76% to 88% of tumors comprising at least 1% of DLL3- xenopus laevis and mus musculus. In mice, DLL3 is expressed by positive tumor cells and 32% to 67% of patients with tumors com- postmitotic cells during somitogenesis (3). However, DLL3 is not prising more than 50% positive tumor cells (7–9). presented on the surface, but instead interacts with Notch1 in the late Bispecific T-cell engagers represent a promising class of antibody- based immunotherapy. These engineered molecules are designed to induce the formation of a cytolytic synapse in an MHC-independent 1Boehringer Ingelheim Pharmaceuticals, Inc., Cancer Immunology & Immune manner by binding concomitantly to a respective antigen on the cell Modulation, Ridgefield, Connecticut. 2Boehringer Ingelheim Pharmaceuticals, surface of tumor cells and to CD3 on T-cells, and direct their cytolytic Inc., Biotherapeutics Discovery, Ridgefield, Connecticut. 3Boehringer Ingelheim activity selectively to the tumor cells. After formation of the RCV, GmbH & Co KG., Cancer Pharmacology and Disease Positioning, Vienna, cytolytic synapse, the T-cells produce perforin and granzyme B, Austria. 4Boehringer Ingelheim RCV, GmbH & Co KG., Oncology Translational leading to apoptosis of the tumor cells. Activation of T-cells leads Science, Vienna, Austria. 5Boehringer Ingelheim RCV, GmbH & Co KG., Cancer Immunology & Immune Modulation, Vienna, Austria. to transient release of cytokines, which engages other immune cells and broadens the immune response against the tumor tissue Note: Supplementary data for this article are available at Clinical Cancer fl fl Research Online (http://clincancerres.aacrjournals.org/). leading to conversion of a nonin amed (cold) to an in amed (hot) tumor environment, infiltration and proliferation of T-cells, Corresponding Author: Susanne Hipp, Boehringer Ingelheim Pharmaceuticals, and serial killing of tumor cells (10–13). After recent clinical Inc., 900 Ridgebury Rd./P.O. Box 368, Ridgefield, CT 06877-0368. Phone: 203- fi 798-4567; E-mail: [email protected] successes of bispeci c T-cell engagers with a short half-life (BiTE) for the treatment of hematologic malignancies (14, 15), the next Clin Cancer Res 2020;XX:XX–XX generation of T-cell engagers incorporating half-life extension for doi: 10.1158/1078-0432.CCR-20-0926 increased dosing convenience for patients for the treatment of solid 2020 American Association for Cancer Research. tumors is emerging (13, 16–18).

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Cell lines and cell culture Translational Relevance All cell lines were obtained from the ATCC and cultured as Small cell lung cancer (SCLC) is the most lethal and aggressive described in the supplier's description. Recombinant human DLL1, subtype of lung carcinoma characterized by a high relapse rate to DLL4, and cynomolgus monkey DLL3-expressing HEK293 cell lines chemotherapy in a majority of patients. Delta-like ligand 3 (DLL3) were generated by transfection of HEK293 cells using Lipofectamine þ represents a promising antigen for targeted therapy of SCLC. This (Thermo Fisher Scientific) with pcDNA3.1( ) (Thermo Fisher Scien- article provides mechanistic insights into the mode of action of the tific) containing the coding sequences including an N-terminal FLAG DLL3/CD3 ITE, a novel IgG-like bispecific T-cell engaging anti- tag between the signal sequence and the extracellular domain. All cell body currently in preclinical development. The DLL3/CD3 ITE lines were tested to exclude mycoplasma contamination and main- induces highly selective lysis of tumor cells and subsequent acti- tained in culture for a maximum of 20 passages. vation and proliferation of T cells. In a preclinical in vivo model, the þ þ DLL3/CD3 ITE induces infiltration of CD4 and CD8 T-cells into mRNA sequencing of human SCLC tissues and SCLC cell lines noninflamed tumors leading to a more inflamed tumor environ- RNA was isolated using the TRI Reagent (Sigma) and RNeasy Mini ment and resulting in complete tumor regression. Kit (Qiagen). Five hundred nanograms RNA was subjected to library preparation using the TruSeq RNA Library Prep Kit v2 with poly-A selection (Illumina). The Library was then multiplexed and sequenced using a HiSeq device (Illumina) using paired end sequencing. All In this article, we describe the preclinical profile of a novel half- human SCLC tissues were obtained after receiving written-informed life extended DLL3/CD3 IgG-like T-cell engager (DLL3/CD3 ITE). consent from the patients. DLL3/CD3 ITE monotherapy treatment induces potent and strictly DLL3-dependent lysis of tumor cells and T-cell infiltration into tumor Isolation of peripheral blood mononuclear cells and T-cells tissue leading to complete tumor regression and an inflamed tumor Fresh buffy coats from healthy volunteers were obtained from the environment in vivo. Austrian Red Cross after receiving written-informed consent in accordance with the Declaration of Helsinki and with approval of the federal ethical committee in Austria. Peripheral blood mononuclear cells Materials and Methods (PBMC) were purified from buffy coats by density gradient centrifu- Engineering, expression, and purification of DLL3/CD3 ITE gation. T-cell subsets were purified from PBMCs using the respective Single B-cell technology from mice immunized with the recombi- T-cell subset isolation kits (Miltenyi Biotech) according to the nant extracellular domain of human DLL3 generated the anti-DLL3 manufacturer's instructions. Catalog numbers of T-cell subset kits are antibody. The murine anti-CD3 antibody described previously was the listed in Supplementary Table S1. source of the anti-CD3 arm used in the test article (19). Humanization and further sequence optimization of both variable regions was Cell binding analysis performed using a Fab expression vector system according to methods Cells were incubated in the presence of DLL3/CD3 ITE on ice for 25 previously described (20). In brief, closely matching human germlines minutes at 4C. Subsequently, cells were washed 2 times in FACS identified in silico and libraries of Fab variants based on these buffer and stained with phycoerythrin-conjugated goat anti-human germlines were evaluated for binding to the target, and percent human IgG secondary antibody (Sigma-Aldrich) diluted 1:200 in FACS buffer and Epivax (in silico predictive tool for potential immunogenicity) for 25 minutes at 4C. Samples were measured by flow cytometry on an scores. Subsequently, the optimized variable regions with drug-like FACS Canto II instrument (Becton Dickinson) and analyzed with properties (high percentage of human sequence, minimal chemical FlowJo V10 software (Becton Dickinson). liabilities, and reduced immunogenicity potential) were formatted in an ITE-bispecific expression construct using pTT5 expression vector Quantification of DLL3 cell surface molecules on SCLC cell (National Research Council, Canada) with common molecular biology lines techniques. The ITE is a two-chain heterodimeric bispecificanti- Quantification of DLL3 cell surface molecules expressed on SCLC body with Fab domains that contain polypeptide linkers to assure cell lines was performed by flow cytometry using the QIFIKIT correct light-heavy chain pairing and an Fc domain engineered for (Agilent) according to the manufacturer's instructions. heterodimerization and efficient purification (21). The DLL3/CD3 ITE produced in CHO-E (National Research Council, Canada) Cytotoxicity, T-cell activation, T-cell proliferation, and cytokine cells by transient transfection with DLL3:CD3 DNA plasmid mass secretion assays ratio of 1:3 enabled efficient purification of heterodimeric species Cytotoxicity, activation, degranulation, and proliferation of as follows. After 10-day culturing, the supernatant was harvested T-cells, and cytokine secretion were analyzed in multiparametric and purified by Protein A affinity chromatography using MabSelect cytotoxicity assays. column (GE Healthcare). The bispecificantibodywasfurther Redirected T-cell cytotoxicity was assessed by quantification of purifiedtohomogeneitybycationexchangechromatographyusing lactate dehydrogenase (LDH) concentrations in the cell culture super- a Poros 50 HS column (Applied Biosystems). The purified protein natant using the Cytotoxicity Detection KitPLUS (Sigma-Aldrich) was concentrated and stored in 50 mmol/L sodium acetate and according to the manufacturer's instructions. Monitoring of specificity 100 mmol/L NaCl, pH 5.0 buffer. Analytical characterization of lysis in the presence of DLL3-positive and DLL3-negative cells was by intact mass spectrometry, SDS-PAGE, and size-exclusion chroma- assessed by monitoring of depletion of cells labeled with carboxy- tography showed that the purified antibody was the expected fluorescein diacetate succinimidyl ester (CFDA-SE) or CellTrace heterodimeric mass and contained minimal high-molecular-weight Violet (CTV; both Thermo Fisher Scientific). aggregate (less than 1%) and no contaminating homodimeric For analysis of T-cell proliferation, the PBMCs and T-cells were species. labeled with CFDA-SE (Becton Dickinson), and T-cells were

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additionally stained with anti-CD3 (BioLegend). Cytokine con- HEK293 cell lines expressing human DLL1 or DLL4 (Supplementary centrations were analyzed from supernatants of cytotoxicity assays Fig. S2). DLL3/CD3 ITE–induced T-cell redirected lysis is strictly using the V-PLEX Assays (Mesoscale Discovery) following the dependent on DLL3 expression and presence of T-cells (Fig. 1B). manufacturer's instructions. Donor dependency was observed in a cytotoxicity assay with PBMCs from 23 different human donors and the SCLC cell line SHP-77 with a Mouse xenograft studies geometric mean EC50 of 5.5 ng/mL with a range from 0.7 to 20.8 ng/mL All animal studies were approved by the internal ethics committee (Fig. 1C). and the responsible local governmental committee. Antitumor activity The potency of the DLL3/CD3 ITE–induced lysis of SHP-77 cells was evaluated in 8-week-old female NOD.CgPrkdcscidIl2rgtm1Sug/ depends on the E:T ratio as indicated by the range of EC50 values JicTac mice (Taconic). Note that 2.5 106 SHP-77 cells were ranging from 1 ng/mL at an E:T of 30:1 to 129 ng/mL at an E:T of 2:1 suspended in 1 PBS/5% FCS and injected subcutaneously into the with maximal lysis activity observed at an E:T ratio of ≥ 10:1 (Fig. 1D). right dorsal flank of animals that had been sublethally irradiated with 2 The selectivity of the DLL3/CD3 ITE–induced lysis of DLL3-positive Gy. Thirteen days later, when tumors reached a size between 71 and cells was further evaluated by coculture of PBMCs, CTV-labeled 118 mm3, mice were randomized in groups, and 2 107 in vitro– DLL3-positive SHP-77 cells, and CFDA-SE–labeled DLL3-negative expanded human T-cells (T-cell isolation, activation/expansion kits HL-60 cells at a ratio of 20:1:1, and increasing concentrations of from Miltenyi Biotec) were injected into the peritoneal cavity. Treat- DLL3/CD3 ITE for 72 hours. The number of viable SHP-77 and ment with DLL3/CD3 ITE was initiated 3 days after T-cell injection HL-60 cells was determined by flow cytometry. The DLL3/CD3 ITE (Supplementary Fig. S1). Tumor size was measured by an electronic did not induce lysis of DLL3-negative HL-60 cells, when cocultured caliper and calculated according to the formula “tumor volume ¼ with DLL3-positive SHP-77 cells. The slight reduction of viable target length diameter2 p/6.” Regressions were defined as a relative cell numbers at high concentrations of DLL3/CD3 ITE is considered tumor volume < 1 when normalized to the tumor volume at the start of negligible compared with the strong reduction of SHP-77 cells treatment. One-sided nonparametric Mann–Whitney–Wilcoxon (Fig. 1E). The DLL3/CD3 ITE lyses cells through induction of U tests were applied to compare the treatment group with the vehicle apoptosis of SHP-77 cells, as demonstrated by the increase of Annexin group. The level of significance was fixed at a ¼ 5%. V binding and PI incorporation in CTV-labeled SHP-77 cells after cocultivation with PBMCs and increasing concentration of DLL3/CD3 IHC analysis ITE for 24 hours (Fig. 1F). Further analysis of the mode of action of the Mouse tumor tissues were harvested when animals were termi- DLL3/CD3 ITE confirmed DLL3-dependent secretion of MCP-1 nated, fixed overnight in 4% neutral-buffered formaldehyde, and (Supplementary Fig. S3A) and IFNg (Supplementary Fig. S3B), and embedded in paraffin. Note that 2-mm-thick serial sections were proliferation of T-cells (Fig. 1G). þ prepared on a microtome, put on glass slides, and subsequently In further cytotoxicity experiments, we confirmed that CD4 as well þ dewaxed. SHP-77 tumor tissues were stained with anti-human as CD8 T-cell subsets contributes to the DLL3/CD3 ITE–induced CD3 (Roche Diagnostics), anti-human CD4 (Roche Diagnostics), T-cell redirected lysis of SHP-77 cells in a time-dependent manner. anti-human CD8 (Roche Diagnostics), anti-human PD-1 (Cell T-cell redirected lysis by PBMCs (Fig. 2A), pan T-cells (Fig. 2B), and þ Signaling Technology), anti-human LAG-3 (Novus Biologicals), CD8 T-cells (Fig. 2D) became already apparent after 48 hours when þ anti-human PD-L1 (Abcam), anti-human cleaved Caspase 3 (CST), CD4 T-cells (Fig. 2C) still showed only minor lysis. After 72 hours, þ þ or anti-human DLL3 (Ventana SP347) antibodies. Two-sided non- both CD4 and CD8 T-cells contributed comparably with the lysis of parametric Mann–Whitney–Wilcoxon U tests were applied to SHP-77 cells. In coculture experiments with purified subsets of T-cells, þ þ compare the treatment group with the vehicle group. we demonstrated that CD4 central memory (CD4 /CD45RA / þ þ þ CD197 ), CD4 effector memory (CD4 /CD45RA /CD197 ), þ þ þ þ þ Pharmacokinetic studies CD8 memory (CD8 /CD45RA ), CD8 CD45RA (CD8 / þ þ þ þ Serum concentrations of DLL3/CD3 ITE were determined in an CD45RA /CD197 ), as well as naïve T-cells (CD4 /CD45RA and þ þ ELISA-based assay using anti-human IgG capture (Novus) and detec- CD8 CD45RA ) are involved in the DLL3/CD3 ITE–induced T-cell tion (MyBiosource) reagents. Data were treated as a naïve pool and redirected lysis of SHP-77 cells (Fig. 2E–I). fitted to a linear two-compartment model using a log-additive error model in Phoenix 64 WinNonlin 7.0. Correlation of DLL3 expression with DLL3/CD3 ITE–induced T-cell redirected lysis DLL3 mRNA expression was analyzed by RNA sequencing in Results primary tumor tissues from 20 patients with SCLC and eight SCLC Molecule discovery and mode of action of the DLL3/CD3 ITE cell lines. The mRNA expression ranged from 30 to 370 TPM The DLL3/CD3 ITE is based on a heterodimeric IgG scaffold that (transcripts per million) in tumor tissues and 20 to 230 TPM in the incorporates flexible peptide linkers between light and heavy chains cell lines (Fig. 3A). Cell pellets of three representative SCLC cell lines and is designed to bind concurrently to DLL3 on tumor cells and CD3 with low (NCI-H2286), medium (NCI-H82), and high (SHP-77) DLL3 on T-cells (Fig. 1A). expression levels were generated and analyzed by an IHC assay Binding specificity of DLL3/CD3 ITE for human DLL3 and CD3 (SP347) qualified for assessing DLL3 protein expression in early was verified by using DLL3-positive SCLC cell lines with endogenous clinical trials with Rova-T (9). DLL3 protein expression was detectable DLL3 expression, DLL3-negative cell lines, purified T-cells, and for the SHP-77 and NCI-H82 cell pellets, but not the NCI-H2286 cell recombinant cell lines expressing human DLL1 or DLL4, the closest pellet (Fig. 3B). To analyze DLL3/CD3 ITE–induced T-cell redirected homologs of DLL3. Cell-bound DLL3/CD3 ITE was detected by flow lysis of the eight SCLC cell lines, each cell line was cocultured with cytometry. DLL3/CD3 ITE binds to DLL3-positive SCLC cell lines PBMCs and increasing concentrations of DLL3/CD3 ITE. The DLL3/ SHP-77 and NCI-H82, and CD3-positive purified human T-cells, but CD3 ITE–induced T-cell redirected lysis of all tested SCLC cell lines in not to DLL3-negative cell lines RKO-E6, HL-60, and recombinant a dose-dependent manner, but the level of DLL3/CD3 ITE–induced

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A B DLL3 CD3

100 PBMCs + SHP-77 80 PBMCs + NCI-H82

60 PBMCs + RKO-E6 PBMCs 40 SHP-77 Linkers Enable 20 NCI-H82 RKO-E6

manufacturing 0 Maintain native IgG architecture

Engineered Fc relative to control % Lysis 1 0 0.1 10 0.01 100 0.001 1,000 10,000 DLL3/CD3 ITE (ng/mL) 100,000 C D 100 100 E:T 30:1 80 80 E:T 20:1 60 60 E:T 10:1 40 E:T 5:1 40 E:T 2:1 20 20 E:T 1:2 n = 23 0

% Lysis relative to control % Lysis 0

1 relative to control % Lysis 0 0.1 10 0.01 100 1 0.001 1,000 0 0.1 10 10,000 0.01 100 100,000 0.001 1,000 DLL3/CD3 ITE (ng/mL) 10,000 DLL3/CD3 ITE (ng/mL) 100,000

E F w/o DLL3/CD3 ITE 100 DLL3/CD3 ITE 300 ng/mL

105 5 80 10

PBMCs + SHP-77 + HL-60: SHP-77 4 60 10 104 PBMCs + SHP-77 + HL-60: HL-60 3 40 Pos. ctrl. (PBMCs + SHP-77) 10 103 Comp-PE-A :: PI Comp-PE-A 20 Neg. ctrl. (PBMCs + HL-60) :: PI Comp-PE-A 0 0 % Living target cells 0 0 103 104 105 0 103 104 105 1 0 0.1 10 Comp-APC-A :: Annexin V Comp-APC-A :: Annexin V 0.01 100 0.001 1,000 10,000 DLL3/CD3 ITE (ng/mL) 100,000

G PBMCs + SHP-77 PBMCs + NCI-H82 PBMCs + RKO-E6 PBMCs

74.9% DLL3/CD3 ITE 48.9% 0.19% 1.29% 30,000 ng/mL

28.4% DLL3/CD3 ITE 6.78% 0.13% 1.11% 3 ng/mL

DLL3/CD3 ITE 1.35% 0.41% 0.08% 0.63% 0 ng/mL

-103 01103 04 105 -103 01103 04 105 -103 01103 04 105 -103 01103 04 105 Comp-FITC-A Comp-FITC-A Comp-FITC-A Comp-FITC-A

Figure 1. Selectivity of DLL3/CD3 ITE. A, Schematic graphic of DLL3/CD3 ITE. B–E, Cytotoxicity was determined by LDH release relative to a control containing 3% Triton X-100 after coculture of PBMCs and SCLC cell lines at the indicated E:T ratios for 72 hours. Each datapoint represents the mean of duplicate measurements, and error bars represent the SD. B, DLL3 dependency of DLL3/CD3 ITE–induced cell lysis (E:T 10:1). C, Donor dependency of DLL3/CD3 ITE–induced lysis of SHP-77 cells (E:T 10:1). D, E:T ratio dependency of DLL3/CD3 ITE–induced lysis of SCLC cell lines after 72 hours. E, Selectivity of DLL3/CD3 ITE–induced lysis of DLL3-positive SHP-77 cells in coculture with DLL3-negative HL-60 cells. Human PBMCs were cocultivated with SHP-77 and/or HL-60 cells, and increasing concentrations of DLL3/CD3 ITE at an E:T ratio of 10:1 for 72 hours and DLL3/ITE-dependent reduction of viable target cells relative to the respective untreated coculture was monitored by ﬂow cytometry. Each datapoint represents a single measurement. F, DLL3/CD3 ITE–induced apoptosis of SCLC cells. Human PBMCs were cocultivated with DLL3-positive SHP-77 cells and increasing concentrations of DLL3/CD3 ITE at an E:T of 10:1 for 24 hours. Cells were stained with anti-CD3, Annexin V, and PI and analyzed by ﬂow cytometry. Each datapoint represents a single measurement. G, Proliferation of T-cells. Representative example.

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A B C

100 100 100 80 80 24 h 80 24 h 24 h 60 60 60 48 h 48 h 48 h 40 40 40 72 h 72 h 72 h 20 20 20

0 0 0 % Lysis relative to control % Lysis % Lysis relative to control % Lysis relative to control % Lysis 0 1 0 1 1 10 0.1 10 0 0.1 10 0.1 100 0.01 100 0.01 100 0.01 0.001 1,000 0.001 1,000 0.001 1,000 10,000 10,000 10,000 100,000 100,000 100,000 DLL3/CD3 ITE (ng/mL) DLL3/CD3 ITE (ng/mL) DLL3/CD3 ITE (ng/mL)

D E F

100 100 100

80 80 80 24 h 60 60 60 48 h 40 40 40 72 h 20 20 20

0 0 0 % Lysis relative to control % Lysis relative to control % Lysis relative to control % Lysis 0 1 0 1 1 0.1 10 0.1 10 0 0.1 10 0.01 100 0.01 100 0.01 100 0.001 1,000 0.001 1,000 0.001 1,000 10,000 10,000 10,000 100,000 100,000 100,000 DLL3/CD3 ITE (ng/mL) DLL3/CD3 ITE (ng/mL) DLL3/CD3 ITE (ng/mL)

CD4+ T-cells CD4+ T-cells CD4+ Central memory T-cells CD4+ Effector memory T-cells G H I

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0 % Lysis relative to control % Lysis relative to control % Lysis relative to control % Lysis 0 1 0 1 0 1 0.1 10 0.1 10 0.1 10 0.01 100 0.01 100 0.01 100 0.001 1,000 0.001 1,000 0.001 1,000 10,000 10,000 10,000 100,000 100,000 100,000 DLL3/CD3 ITE (ng/mL) DLL3/CD3 ITE (ng/mL) DLL3/CD3 ITE (ng/mL)

CD8+ T-cells CD8+ T-cells T-cells CD8+ Memory T-cells CD8+ CD45RA+ T-cells Naive T-cells

Figure 2. CD4þ and CD8þ T-cells contribute to DLL3/CD3 ITE–mediated T-cell redirected lysis of SHP-77 cells. Time dependency of DLL3/CD3 ITE–induced T-cell redirected lysis of SHP-77 cells by (A) PBMCs, (B) pan T-cells, (C) CD4þ,or(D) CD8þ T-cells. DLL3/CD3 ITE–induced T-cell redirected lysis of SHP-77 cells by puriﬁed human (E and F) CD4þ,(E) CD4þ central memory, (F) CD4þ effector memory, (G and H) CD8þ,(G) CD8þ memory, (H) CD8þCD45þ effector, or (I) naïve T-cells. Each datapoint represents the mean of duplicate measurements, and error bars represent the SD.

T-cell redirected lysis depended on the tumor cell line and the DLL3 In a first efficacy study (Study I), the DLL3/CD3 ITE was admin- expression levels, indicated by the range of EC50 concentrations from istered at doses of 0.025 and 0.25 mg/kg i.v., and tumor growth was 5.9 to 24 ng/mL (Fig. 3C; Supplementary Table S3). Immunohisto- monitored until day 21. Although treatment with 0.025 mg/kg induced chemically detectable DLL3 levels correlated with higher DLL3/CD3 tumor regression in 3 out of 5 animals at day 21 (P < 0.05), a stronger ITE potency in the SHP-77 and NCI-H82 cell lines. However, lysis antitumor response was observed with a dose of 0.25 mg/kg and tumor activity could also be detected using the NCI-H2286 cell line for which regression in all animals (5/5) already at day 9. Notably, four out of five no DLL3 protein could be detected immunohistochemically, indicat- tumors were still in regression at the end of the study (P < 0.05; ing the potential of DLL3/CD3 ITE to induce lysis of cells with very low Fig. 4A). In a second efficacy study (Study II) with a different human DLL3 expression levels. T-cell donor, DLL3/CD3 ITE monotherapy induced a statistically significant tumor growth inhibition (P < 0.05 at day 15) compared with DLL3/CD3 ITE–induced antitumor activity and modulation of the vehicle control group. Sustained tumor regressions could be T-cell infiltration in vivo observed for most of the tumors from day 15 (7/9 animals) until day In vivo efficacy studies were conducted in a subcutaneous human 36 (8/9 animals). Although treatment was stopped after 4 cycles (day þ SHP-77 xenograft model in CD3 T-cell humanized mice (Fig. 4A 22), tumor regression was maintained. Two out of the eight animals and B) where the DLL3/CD3 ITE was administered in a weekly (q7d) with tumor regression still showed complete tumor clearance (tumor regimen supported by its long half-life of 20 days in C57/BL6 mice not palpable anymore) at the end of the study (day 36), 14 days after the (Fig. 4C). last dose (Fig. 4B).

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Figure 3. Correlation of DLL3 expression and DLL3/ CD3 ITE–induced T-cell redirected lysis. A, DLL3 mRNA expression in 20 SCLC tissues and eight SCLC cell lines. B, IHC detection of DLL3 using cell pellets of three representative SCLC cell lines. C, Representative example of dose-dependent DLL3/CD3 ITE–induced lysis of eight SCLC cell lines (E:T 10:1). Cytotoxicity was determined by LDH release relative to a control containing 3% Triton X-100 after coculture of PBMCs and eight SCLC cell lines at an E:T ratio of 10:1 for 72 hours. Each datapoint represents the mean of duplicate measurements, and error bars represent the SD of maximal lysis versus DLL3 molecules on the cell surface.

Tumor tissues from mice treated with vehicle or 0.25 mg/kg increased PD-L1 (Fig. 6E) expression. In the tumor cells, a statistically DLL3/CD3 ITE in Study II were collected at the day of termination, significant increase of cleaved caspase 3 was detected (Fig. 4F). The and tissue sections were stained with anti-CD3, PD-1, and PD-L1 proximity of the apoptotic tumor cells to the infiltrating T-cells was antibodies. Treatment with DLL3/CD3 ITE induced infiltration of visualized by costaining of tumor tissues for cleaved caspase 3 and CD3 þ þ CD3 T-cells into the SHP-77 xenograft tissue. Infiltrating CD3 (Fig. 4G). T-cells upregulated PD-1 and LAG-3, and SHP-77 tumor cells upregulated PD-L1 (Fig. 5). Dexamethasone only minimally affects DLL3/CD3 ITE In a following study (Study III), we examined the ability of the The impact of dexamethasone on the activity of the DLL3/CD3 ITE DLL3/CD3 ITE to modulate the inflammatory environment in the was assessed by adding increasing concentrations of dexamethasone tumor tissue. SHP-77 tumor tissues from animals were collected 8 days ranging from 1 to 1,000 ng/mL to the multiparametric cytotoxicity after administration of one dose of 0.25 mg/kg ITE i.v., and sections of assay simultaneously with 30 mg/mL DLL3/CD3 ITE. Treatment formalin-fixed and paraffin-embedded tissues were immunohisto- with dexamethasone led to a dose-dependent reduction of DLL3/ chemically stained with anti-CD3, anti-CD4, anti-CD8, anti–PD-1, CD3–induced T-cell redirected lysis of DLL3-positive SHP-77 cells anti–PD-L1, and anti-cC3 (cleaved caspase 3) antibodies. A statisti- with one out of three PBMC donors (Supplementary Fig. S4A). The þ þ þ cally significant increase in CD4 and CD8 T-cells (Fig. 6A–C)in DLL3/CD3 ITE–induced upregulation of CD25 on CD4 T-cells DLL3/CD3 ITE–treated animals compared with the vehicle-treated was also reduced with one out of three donors, whereas dexameth- þ animals was observed. In this study, we did not see an increase of PD-1 asone did not have an effect on CD25 expression on CD8 T-cells expression (Fig. 6D) and only a nonstatistically significant trend of (Supplementary Fig. S4B and S4C). The DLL3/CD3-induced

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A Novel DLL3-Targeted IgG-Like T-Cell Engager

Figure 4. DLL3/CD3 ITE monotherapy induces dose-dependent efﬁcacy in an SHP-77 xenograft in a human T-cell reconstituted mouse model. A, Dose dependency of DLL3/ CD3 ITE–induced antitumor activity in an SHP-77 xenograft model in NOG mice reconstituted with human CD3þ T-cells (Study I). B, Antitumor activity of DLL3/CD3 ITE in vivo is retained after stop of treatment (Study II). C, Pharmacokinetic proﬁle of DLL3/CD3 ITE in C57BL/6 mice.

secretion of IFNg (Supplementary Fig. S4D) and MCP-1 (Supple- DLL3-expressing HEK293 cells in presence of cynomolgus monkey mentary Fig. S4E), by PBMCs in presence of DLL3-positive SHP-77 PBMCs is shown in Supplementary Fig. S5. Pharmacokinetic studies cells, was reduced in the presence of dexamethasone with all three showed that the half-life of the DLL3/CD3 ITE is 10 days. tested donors.

DLL3/CD3 pharmacokinetics in nonhuman primates Discussion To support pharmacokinetic and safety assessment in nonhuman Despite the high unmet medical need for new treatments for SCLC primates, the DLL3/CD3 ITE was designed to be cross-reactive to and the highly attractive tumor-speciﬁc expression of DLL3 for a cynomolgus monkey DLL3 and CD3. The ability of the DLL3/CD3 ITE targeted therapy, the development of such a therapy has yet to be to induce T-cell redirected lysis of recombinant cynomolgus monkey realized. The ﬁrst approach for targeting DLL3 with an antibody-based

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Figure 5. Representative example of IHC analysis of xenograft tumors at the end of Study II for CD3, PD-1, PD-L1, and LAG-3 (brown staining).

approach was Rova-T (rovalpituzumab tesirine), a humanized anti- SCLC patients with DLL3-positive tumors (22). The toxicities DLL3 monoclonal antibody conjugated to a DNA-damaging pyrro- observed with Rova-T, such as thrombocytopenia, nausea, fatigue, lobenzodiazepine dimer toxin (6, 7). In a phase I trial including vomiting, edema, and effusions (7), have also been observed with patients with small cell lung or large cell neuroendocrine tumors that vadastuximab talirine, an anti-CD33 antibody–drug conjugate (23), have previously relapsed after platinum-based chemotherapy, 18% of and are considered drug- but not DLL3-related. patients had a confirmed objective response rate and a manageable T-cell engagers have a completely different mechanism of action to safety profile (7). However, in a phase II study, Rova-T showed only antibody–drug conjugates and have shown high potency and efficacy modest activity [median overall survival (OS) of 5.7 months in patients with a promising safety profile in hematologic malignancies (14, 15). with high DLL3 expression vs. 5.6 months in all patients] with However, the development of bispecific T-cell engagers for solid associated toxicities of grades 3 to 5 in third-line and beyond (3Lþ) tumors presents new challenges. Firstly, given the potency of the

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A Novel DLL3-Targeted IgG-Like T-Cell Engager

Figure 6. DLL3/CD3 ITE induces infiltration of T-cells. DLL3/CD3 ITE monotherapy induces infiltration of T-cells and upregulation of PD-(L)1 into tumor tissue resulting in apoptosis of tumor cells in an SHP-77 xenograft. IHC analysis of xenograft tumors on day 8 (Study III) for (A) CD3þ,(B) CD4þ,(C) CD8þ T-cells, (D) PD-1–positive T-cells, (E) PD-L1–positive, and (F) cleaved caspase 3 tumor area. G, Representative example of IHC costaining of a xenograft tumor from one mouse for CD3 (brown) and cleaved caspase 3 (dark red). mode of action, a tumor selectively expressed cell surface antigen is antigen–related cell adhesion molecule 5), respectively, are widely required to ensure optimal efficacy and safety. So far, bispecific T-cell expressed. Secondly, the BiTE molecules have a very short half-life and engagers for solid tumors such as solitomab and MEDI-565 for the have to be administered by continuous intravenous infusion, for treatment of gastrointestinal cancers did not result in a sufficient example. As such, opportunities to extend the half-life of T-cell therapeutic window in clinical trials (24, 25), as the antigens EpCAM engagers while maintaining the potent pharmacological effect have (epithelial cell adhesion molecule) and CEA (carcinoembryonic been sought to improve the dosing convenience for patients.

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In this article, we describe a novel approach for targeting the tumor- DLL3/CD3 ITE treatment, but the treatment could result in reduced selective DLL3 antigen and treating DLL3-expressing SCLC using T-cell cytotoxic potential of DLL3/CD3 ITE. redirection. We have designed a highly DLL3-selective novel IgG-like According to the DLL3-expression profile, the DLL3/CD3 ITE has T-cell engaging bispecific antibody with an optimal pharmacologic the potential to be developed in additional indications with high unmet profile, and antibody-like in vivo stability and half-life. In contrast to medical need, such as glioma (33), medullary thyroid cancer (34), AMG 757 (26), a half-life extended BiTE consisting of a bispecificscFv gastrointestinal neuroendocrine malignancies (35), dispersed neuro- fused to an Fc, the DLL3/CD3 ITE has an IgG-like architecture which endocrine tumors of the pancreas (34), small cell bladder cancer (36), may pose a lower immunogenicity risk in humans. DLL3/CD3 ITE Merkel-cell carcinoma (37), and neuroendocrine prostate cancer (38). induces potent T-cell redirected lysis of DLL3-positive SCLC cell lines in Taken together, the DLL3/CD3 ITE provides a novel drug candidate a dose, E:T ratio, and time-dependent manner, with EC50 values in the for the treatment of SCLC as monotherapy with upside potential in þ þ low ng/mL range by redirecting CD4 and CD8 T-cells toward DLL3- other neuroendocrine tumor types. postitive SCLC cells without lysing DLL3-negative target cells. As a result of the formation of the cytolytic synapse, T-cells secrete proinflamma- Disclosure of Potential Conflicts of Interest tory cytokines and proliferate. In vivo, DLL3/CD3 ITE monotherapy S. Hipp reports grants from Basisprogramm grants of the Austrian Research leads to infiltration of T-cells into tumor tissue and converts a nonin- Promotion Agency (FFG; 860968, 869530, and 875923) and personal fees from flamed (cold) into an inflamed (hot) tumor environment, leading to Boehringer Ingelheim (employment) during the conduct of the study; in addition, she has filed a patent application for DLL3-CD3 bispecific antibodies. The filed patent tumor cell apoptosis and resulting in strong tumor regressions with þ application is owned by Boehringer Ingelheim and relates to generation and use of complete tumor eradication in several mice in a human CD3 T-cell DLL3-CD3 bispecific antibodies. V. Voynov reports personal fees from Boehringer humanized SHP-77 xenograft model. Ingelheim (employment) during the conduct of the study; in addition, he has filed a In the phase II TRINITY study with Rova-T, cutoffs for analysis of patent application for DLL3-CD3 bispecific antibodies. The filed patent application is DLL3 expression in SCLC tissues of patients were defined as DLL3- owned by Boehringer Ingelheim and relates to generation and use of DLL3-CD3 fi positive when tissues contained ≥25% of DLL3-positive tumor cells, bispeci c antibodies. B. Drobits-Handl reports grants from Basisprogramm grants of ≥ the Austrian Research Promotion Agency (FFG; 860968, 869530, and 875923) and and as DLL3-high when tissues contained 75% of DLL3-positive personal fees from Boehringer Ingelheim (employment) during the conduct of the tumor cells (22). In our study, we could detect DLL3 expression in cell study. C. Giragossian reports personal fees from Boehringer Ingelheim (employment) pellets of SCLC cell lines expressing more than 500 DLL3 molecules per during the conduct of the study. F. Trapani reports grants from Austrian research cell using the same qualified IHC assay (Ventana, SP347; ref. 9) Promotion Agency (FFG; 860968, 869530, and 875923) and personal fees from previously validated for the determination of DLL3 expression levels Boehringer-Ingelheim (employment) during the conduct of the study. J.M. Scheer has fi fi fi in the Rova-T phase II study (22). In in vitro cytotoxicity assays, cell led a patent application for DLL3-CD3 bispeci c antibodies. The led patent application is owned by Boehringer Ingelheim and relates to generation and use of lines with immunohistochemically detectable DLL3 levels correlate DLL3-CD3 bispecific antibodies. P.J. Adam reports grants from Basisprogramm with higher DLL3/CD3 ITE potency, suggesting that the detection of grants of the Austrian Research Promotion Agency (FFG; 860968, 869530, and DLL3 expression using the SP347 IHC assay may be a promising 875923) and personal fees from Boehringer Ingelheim (employment) during the biomarker for patient selection that should be evaluated in clinical conduct of the study; in addition, he has filed a patent application for DLL3-CD3 trials. bispecific antibodies. The filed patent application is owned by Boehringer Ingelheim fi The FDA recently approved the PD-1 inhibitor pembrolizumab and relates to generation and use of DLL3-CD3 bispeci c antibodies. No potential conflicts of interest were disclosed by the other authors. for the treatment of patients with SCLC who experienced disease progression after two or more prior lines of therapy based on two Authors’ Contributions clinical studies showing a 19% overall objective response rate (27). S. Hipp: Conceptualization, supervision, methodology, writing-original draft, In our preclinical studies, we observed an upregulation of PD-(L)1 writing-review and editing, devised and designed the main conceptual idea of the and LAG-3 expression in vivo as a result of the inflamed environ- study and wrote the manuscript. V. Voynov: Supervision, methodology, designed the ment induced by treatment with the DLL3/CD3 ITE, which is in line molecule and established the production process. B. Drobits-Handl: Supervision, with observations from preclinical studies with other T-cell enga- methodology, writing-review and editing, designed and supervised in vivo studies, gers, e.g., CEA BiTE, CEA TCB, AMG330, and Her2- and analyzed, assembled, and interpreted data. C. Giragossian: Supervision, methodology, designed and supervised PK studies, and analyzed, assembled, and TDB (16, 28–30). Furthermore, in a clinical trial, evidence of interpreted data. F. Trapani: Supervision, methodology, designed and supervised enhanced activity of RO6958688, a CEA-targeted T-cell engager, IHC studies, and analyzed, assembled, and interpreted data. A.E. Nixon: Supervision, in combination with atezolizumab with a manageable safety profile methodology, supported the molecule design principles. J.M. Scheer: Supervision, has been reported (18). In future studies of the DLL3/CD3 ITE, it methodology. P.J. Adam: Supervision, writing-review and editing, supported the will be important to look at the impact on other immune checkpoint design and conceptual idea of the study and contributed to the writing and critical molecules. evaluation of the manuscript. As activation of T-cells and cytokine secretion is a characteristic of Acknowledgments the mode of action of T-cell engagers, which in some cases can also We thank Christoph Albrecht, Ilse Apfler, Kathrin Bauer, Oliver Bergner, Erica cause clinical side effects such as fever and hypotension, it might be Bolella, Richard Liedauer, Irene Schweiger, Abdallah Souabni, and Christian desirable to reduce cytokine secretion as much as possible without Walterskirchen for their experimental support and help for this study, and all negatively influencing the cytotoxic potential of T-cells. Glucocorti- colleagues from Biotherapeutics Discovery for all aspects of molecule discovery. coids have been described to inhibit inflammatory responses such as This study was supported by the Basisprogramm grants of the Austrian Research cytokines (31), and a preclinical study using the glucocorticoid dexa- Promotion Agency (FFG; 860968, 869530, and 875923). methasone in combination with blinatumomab reported that dexamethasone can qualify as a potential comedication for TcE thera- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance pies (32). The data obtained in this study reveal that dexamethasone with 18 U.S.C. Section 1734 solely to indicate this fact. can reduce DLL3/CD3 ITE–induced secretion of IFNg and MCP-1, and therefore support the use of dexamethasone to treat potential Received March 9, 2020; revised May 12, 2020; accepted June 15, 2020; infusion reactions or cytokine release syndromes associated with published first June 18, 2020.

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A Bispecific DLL3/CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer

Susanne Hipp, Vladimir Voynov, Barbara Drobits-Handl, et al.

Clin Cancer Res Published OnlineFirst June 18, 2020.

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