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

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A Bispecific DLL3/CD3 Igg-Like T-Cell Antibody Induces Anti-Tumor Responses in Small Cell Lung Cancer Author Manuscript Published OnlineFirst on June 18, 2020; DOI: 10.1158/1078-0432.CCR-20-0926 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title Page Title: A Bispecific DLL3/CD3 IgG-like T-cell Antibody induces anti-tumor responses in Small Cell Lung Cancer Running title: A Novel DLL3-targeted IgG-like T-cell Engager Authors and affiliations: Susanne Hipp1, Vladimir Voynov2, Barbara Drobits-Handl3, Craig Giragossian2, Francesca Trapani4, Andrew E. Nixon2, Justin M. Scheer2, and Paul J. Adam5 1 Boehringer Ingelheim Pharmaceuticals, Inc., Cancer Immunology & Immune Modulation, Ridgefield, CT, USA 2 Boehringer Ingelheim Pharmaceuticals, Inc., Biotherapeutics Discovery, Ridgefield, CT, USA 3 Boehringer Ingelheim RCV, GmbH & Co KG., Cancer Pharmacology and Disease Positioning, Vienna, Austria 4 Boehringer Ingelheim RCV, GmbH & Co KG., Oncology Translational Science, Vienna, Austria 5 Boehringer Ingelheim RCV, GmbH & Co KG., Cancer Immunology & Immune Modulation, Vienna, Austria Corresponding author: Susanne Hipp 900 Ridgebury Rd./P.O. Box 368 Ridgefield, CT06877-0368, USA Phone: 203-798-4567 Email: [email protected] Conflict of interest disclosure: All authors are employees of Boehringer Ingelheim affiliates. Boehringer Ingelheim has filed patent applications on aspects of this work. 1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 18, 2020; DOI: 10.1158/1078-0432.CCR-20-0926 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational relevance Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by a high relapse rate to chemotherapy in a majority of patients. Delta-like ligand 3 (DLL3) represents a promising antigen for targeted therapy of SCLC. This article provides mechanistic insights into the mode of action of the DLL3/CD3 ITE, a novel IgG-like bispecific T- cell engaging (ITE) antibody currently in preclinical development. The DLL3/CD3 ITE induces highly selective lysis of tumor cells and subsequent activation and proliferation of T-cells. In a pre-clinical in vivo model, the DLL3/CD3 ITE induces infiltration of CD4+ and CD8+ T-cells into non-inflamed tumors leading to a more inflamed tumor environment and resulting in complete tumor regression. 2 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 18, 2020; DOI: 10.1158/1078-0432.CCR-20-0926 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by highly chemotherapy resistant recurrence in the majority of patients. To effectively treat SCLC we have developed a unique and novel IgG-like T-cell engaging bispecific antibody (ITE) that potently re-directs T-cells to specifically lyse SCLC cells expressing Delta-like ligand 3 (DLL3), an antigen that is frequently expressed on the cell surface of SCLC cells, with no to very little detectable expression in normal tissues. Experimental Design: The anti-tumor activity and mode of action of DLL3/CD3 ITE was evaluated in vitro using SCLC cell lines and primary human effector cells and in vivo in a SCLC xenograft model reconstituted with human CD3+ T-cells. Results: Selective binding of DLL3/CD3 ITE to DLL3-positive tumor cells and T-cells induces formation of an immunological synapse resulting in tumor cell lysis and activation of T-cells. In a human T-cell engrafted xenograft model, the DLL3/CD3 ITE leads to an increase in infiltration of T-cells into the tumor tissue resulting in apoptosis of the tumor cells and tumor regression. Consistent with the mode of action, the DLL3/CD3 ITE treatment led to upregulation of PD-1, PD-L1, and LAG-3. Conclusion: This study highlights the ability of the DLL3/CD3 ITE to induce strictly DLL3- dependent T-cell re-directed lysis of tumor cells and recruitment of T-cells into non-inflamed tumor tissues leading to tumor regression in a pre-clinical in vivo model. These data support clinical testing of the DLL3/CD3 ITE in SCLC patients. 3 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 18, 2020; DOI: 10.1158/1078-0432.CCR-20-0926 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Lung cancer remains the number one cause of cancer-related deaths in both men and women in the United States with over 220,000 new cases diagnosed annually (1). Out of these, small cell lung cancer (SCLC), a poorly differentiated neuroendocrine tumor, accounts for roughly 10- 15% of cases (2). SCLC is the most lethal and aggressive subtype of lung carcinoma with 5- year survival rates below 7% characterized by highly chemotherapy resistant recurrence within 12 months in the majority of patients (2). DLL3 (Delta-like ligand 3) is a member of the Notch receptor ligand family that plays a critical role for Notch signaling during embryonal development and is functionally distinct from the related Notch family members DLL1 and DLL4, as demonstrated in animal studies in xenopus laevis and mus musculus. In mice, DLL3 is expressed by postmitotic cells during somitogenesis (3). However, DLL3 is not presented on the surface, but instead interacts with Notch1 in the late endocytic compartment preventing Notch1 from reaching the cell surface (4). This finding has been confirmed in in vitro studies by demonstrating that DLL3 does not activate Notch signaling through binding to Notch expressed on the cell surface, but rather acts as a Notch antagonist by suppressing Notch signaling in a cell-autonomous manner (5). DLL3 expression has been described in SCLC tissue samples (6). In normal tissues, DLL3 is expressed during embryonal development with highest expression in fetal brain, but is absent in adult normal tissues (6). Whilst in developmental processes DLL3 is expressed intracellularly, in SCLC tumors with DLL3 overexpression, DLL3 escapes to the cell surface which makes it targetable with antibody- based therapies (6). Immunohistochemistry studies indicate a high prevalence of DLL3 expression in SCLC tissues from Caucasian and Japanese patients, with 76-88 % of tumors comprising at least 1% of DLL3-positive tumor cells and 32-67% of patients with tumors comprising more than 50% positive tumor cells (7-9). 4 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 18, 2020; DOI: 10.1158/1078-0432.CCR-20-0926 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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 a MHC-independent manner by binding concomitantly to a respective antigen on the cell surface of tumor cells and to CD3 on T-cells, and direct their cytolytic activity selectively to the tumor cells. After formation of the cytolytic synapse, the T-cells produce perforin and granzyme B, leading to apoptosis of the tumor cells. Activation of T-cells leads to transient release of cytokines, which engages other immune cells and broadens the immune response against the tumor tissue leading to conversion of a non-inflamed (cold) to an inflamed (hot) tumor environment, infiltration and proliferation of T-cells, and serial killing of tumor cells (10-13). After recent clinical successes of bispecific T-cell engagers with a short half-life (BiTEs®) for the treatment of hematological malignancies (14,15), the next generation of T-cell engagers incorporating half-life extension for increased dosing convenience for patients for the treatment of solid tumors is emerging (13,16-18). In this manuscript, we describe the preclinical profile of a novel half-life extended DLL3/CD3 IgG-like T-cell engager (DLL3/CD3 ITE). DLL3/CD3 ITE monotherapy treatment induces potent and strictly DLL3-dependent lysis of tumor cells and T-cell infiltration into tumor tissue leading to complete tumor regression and an inflamed tumor environment in vivo. 5 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 18, 2020; DOI: 10.1158/1078-0432.CCR-20-0926 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Material and Methods Engineering, expression and purification of DLL3/CD3 ITE Single B-cell technology from mice immunized with the recombinant extracellular domain of human DLL3 generated the anti-DLL3 antibody. The murine anti-CD3 antibody described previously was the source of the anti-CD3 arm used in the test article (19). Humanization and further sequence optimization of both variable regions was performed using a Fab expression vector system according to methods previously described (20). In brief, closely matching human germlines identified in silico and libraries of Fab variants based on these germlines were evaluated for binding to the target, and percent
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