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.

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.

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.

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).

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.

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 human and Epivax (in silico predictive tool for potential immunogenicity) scores. Subsequently, the optimized variable regions with drug-like properties (high percentage of human sequence, minimal chemical liabilities, and reduced immunogenicity potential) were formatted in an ITE bispecific expression construct using pTT5 expression vector (National Research Council, Canada) with common molecular biology techniques. The ITE is a two-chain heterodimeric bispecific antibody with Fab domains that contain polypeptide linkers to assure correct light-heavy chain pairing and an Fc domain engineered for heterodimerization and efficient purification (21). The DLL3/CD3 ITE produced in

CHO-E (National Research Council, Canada) cells by transient transfection with DLL3:CD3

DNA plasmid mass ratio of 1:3 enabled efficient purification of heterodimeric species as follows.

After 10-day culturing, the supernatant was harvested and purified by Protein A affinity chromatography using MabSelect™ column (GE Healthcare, Princeton, NJ). The bispecific antibody was further purified to homogeneity by cation exchange chromatography using a Poros

50 HS column (Applied Biosystems, Foster City, CA). The purified protein was concentrated and stored in 50 mM Sodium Acetate and 100 mM NaCl, pH 5.0 buffer. Analytical characterization by intact mass spectrometry, SDS-PAGE and size-exclusion chromatography showed that the

purified antibody was the expected heterodimeric mass and contained minimal high molecular weight aggregate (less than 1%) and no contaminating homodimeric species.

Cell lines and cell culture

All cell lines were obtained from ATCC (Manassas, VA, USA) and cultured as described in the supplier’s description. Recombinant human DLL1, DLL4, and cynomolgus monkey DLL3 expressing HEK293 cell lines, were generated by transfection of HEK293 cells using

LipofectamineTM (Thermo Fisher Scientific, Linz Austria) with pcDNATM3.1(+) (Thermo Fisher

Scientific, Linz, Austria) containing the coding sequences including an N-terminal FLAG tag between the signal sequence and the extracellular domain. All cell lines were tested to exclude mycoplasma contamination and maintained in culture for a maximum of 20 passages.

mRNA Sequencing of Human SCLC tissues and SCLC cell lines

RNA was isolated using the TRI Reagent (Sigma) and RNeasy Mini Kit (Qiagen, Hamburg,

Germany). Five-hundred nanograms RNA was subjected to library preparation using the TruSeq

RNA Library Prep Kit v2 with poly-A selection (Illumina, San Diego, CA). The Library was then multiplexed and sequenced using a HiSeq device (Illumina) using paired end sequencing. All human SCLC tissues were obtained after receiving written informed consent from the patients.

Isolation of PBMCs and T-cells

Fresh buffy coats from healthy volunteers were obtained from the 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. PBMCs were purified from buffy coats by density gradient centrifugation. T-cell subsets were purified from PBMCs using the respective T-

cell subset isolation kits (Miltenyi Biotech, Bergisch Gladbach, Germany) according for manufacturer’s instructions. Catalog numbers of T-cell subset kits are listed in Table S1.

Cell binding analysis

Cells were incubated in presence of DLL3/CD3 ITE on ice for 25 min at 4 °C. Subsequently cells were washed two times in FACS buffer and stained with PE-conjugated goat-anti human IgG secondary antibody (Sigma-Aldrich, Taufkirchen, Germany) diluted 1:200 in FACS buffer for 25 min at 4 °C. Samples were measured by flow cytometry on a FACS Canto II instrument (Becton

Dickinson, San Jose, CA, USA) and analyzed with FlowJo V10 software (Becton Dickinson, San

Jose, CA, USA).

Quantification of DLL3 cell surface molecules on SCLC cell lines

Quantification of DLL3 cell surface molecules expressed on SCLC cell lines was performed by flow cytometry using the QIFIKIT® (Agilent, Santa Clara, CA, USA) according of manufacturer’s instructions.

Cytotoxicity, T-cell activation, T-cell proliferation, Cytokine secretion assays

Cytotoxicity, activation, degranulation, and proliferation of T-cells, and cytokine secretion was analyzed in multiparametric cytotoxicity assays.

Redirected T-cell cytotoxicity was assessed by quantification of LDH concentrations in the cell culture supernatant using the Cytotoxicity Detection KitPLUS (Sigma-Aldrich, Taufkirchen,

Germany) according to manufacturer’s instructions. Monitoring of specificity of lysis in presence of DLL3-positive and DLL3-negative cells was assessed by monitoring of depletion of cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) or CellTraceTM Violet

(CTV) (both Thermo Fisher Scientific, Linz, Austria).

For analysis of T-cell proliferation, the PBMCs and T-cells were labeled with CFDA-SE (Becton

Dickinson, San Jose, CA, USA) and T-cells were additionally stained with anti-CD3 (BioLegend,

San Diego, CA, USA). Cytokine concentrations were analyzed from supernatants of cytotoxicity assays using the V-PLEX Assays (Mesoscale Discovery, Rockville, MA, USA) following the manufacturer’s instructions.

Mouse xenograft studies

All animal studies were approved by the internal ethics committee and the responsible local governmental committee. Antitumor activity was evaluated in eight week-old female

NOD.CgPrkdcscidIl2rgtm1Sug/JicTac mice (Taconic, Denmark). 2.5x106 SHP-77 cells were suspended in 1 x PBS/ 5% FCS and injected subcutaneously into the right dorsal flank of animals that had been sub-lethally irradiated with 2 Gy. Thirteen days later, when tumors reached a size between 71-118 mm³, mice were randomized in groups and 2 × 107 in vitro expanded human T-cells (T-cell isolation, activation/expansion kits from Miltenyi Biotec) were injected into the peritoneal cavity. Treatment with DLL3/CD3 ITE was initiated three days after

T-cell injection (Figure S1). Tumor size was measured by an electronic caliper and calculated according to the formula “tumor volume = length*diameter2*π/6”. Regressions were defined as a relative tumor volume < 1 when normalized to the tumor volume at the start of treatment. One- sided non-parametric Mann-Whitney-Wilcoxon U-tests were applied to compare the treatment group with the vehicle group. The level of significance was fixed at α = 5%.

Immunohistochemical analysis

Mouse tumor tissues were harvested when animals were terminated, fixed overnight in 4% neutral-buffered formaldehyde, and embedded in paraffin. 2 μm thick serial sections were prepared on a microtome, put on glass slides and subsequently dewaxed. SHP-77 tumor tissues were stained with anti-human CD3 (Roche Diagnostics, Tucson, AZ, USA), anti-human

CD4 (Roche Diagnostics, Tucson, AZ, USA), anti-human CD8 (Roche Diagnostics, Tucson, AZ,

USA), anti-human PD-1 (Cell signaling Technology, Danvers, MA, USA), anti-human LAG-3

(Novus Biologicals, Littleton, CO, USA), anti-human PD-L1 (Abcam, Cambridge, MA, USA), anti-human cleaved Caspase 3 (CST, Danvers, MA, USA), or anti-human DLL3 (Ventana

SP347, Tucson, AZ, USA) antibodies. Two-sided non-parametric Mann-Whitney-Wilcoxon U- tests were applied to compare the treatment group with the vehicle group.

Pharmacokinetic studies

Serum concentrations of DLL3/CD3 ITE were determined in an ELISA-based assay using anti- human IgG capture (Novus, Centennial, CO, USA) and detection (MyBiosource, San Diego, CA,

USA) reagents. Data were treated as a naïve pool and fitted to a linear two-compartment model using a log-additive error model in Phoenix 64 WinNonlin 7.0.

Results Molecule discovery and mode of action of the DLL3/CD3 ITE The DLL3/CD3 ITE is based on a heterodimeric IgG scaffold that incorporates flexible peptide linkers between light and heavy chains and is designed to bind concurrently to DLL3 on tumor cells and CD3 on T-cells (Figure 1A).

Binding specificity of DLL3/CD3 ITE for human DLL3 and CD3 was verified by using DLL3- positive SCLC cell lines with endogenous DLL3-expression, DLL3-negative cell lines, purified T- cells, and recombinant cell lines expressing human DLL1 or DLL4, the closest homologues of

DLL3. Cell bound DLL3/CD3 ITE was detected by flow cytometry. DLL3/CD3 ITE binds to DLL3- positive SCLC cell lines SHP-77 and NCI-H82, and CD3-positive purified human T-cells, but not to DLL3-negative cell lines RKO-E6, HL-60, and recombinant HEK293 cell lines expressing human DLL1 or DLL4 (Figure S2). DLL3/CD3 ITE-induced T-cell redirected lysis is strictly dependent on DLL3-expression and presence of T-cells (Figure 1B). Donor-dependency was observed in a cytotoxicity assay with PBMCs from 23 different human donors and the SCLC cell line SHP-77 with a geometric mean EC50 of 5.5 ng/ml with a range from 0.7 to 20.8 ng/ml

(Figure 1C).

The potency of the DLL3/CD3 ITE-induced lysis of SHP-77 cells depends on the E:T ratio as indicated by the range of EC50 values ranging from 1 ng/ml at an E:T of 30:1 to 129 ng/ml at an

E:T of 2:1 with maximal lysis activity observed at an E:T ratio of ≥ 10:1 (Figure 1D). The selectivity of the DLL3/CD3 ITE-induced lysis of DLL3-positive cells was further evaluated by co-culture of PBMCs, CTV-labeled DLL3-positive SHP-77 cells and CFDA-SE-labeled DLL3- negative HL-60 cells at a ratio of 20:1:1, and increasing concentrations of DLL3/CD3 ITE for 72 hours. The number of viable SHP-77 and HL-60 cells was determined by flow cytometry. The

DLL3/CD3 ITE did not induce lysis of DLL3-negative HL-60 cells, when co-cultured with DLL3- positive SHP-77 cells. The slight reduction of viable target cell numbers at high concentrations of DLL3/CD3 ITE is considered negligible compared to the strong reduction of SHP-77 cells

(Figure 1E). The DLL3/CD3 ITE lyses cells through induction of apoptosis of SHP-77 cells, as demonstrated by the increase of Annexin V binding and PI incorporation in CTV-labeled SHP-77 cells after co-cultivation with PBMCs and increasing concentration of DLL3/CD3 ITE for 24 hours (Figure 1F). Further analysis of the mode of action of the DLL3/CD3 ITE confirmed DLL3- dependent secretion of MCP-1 (Figure S3A) and IFN-γ (Figure S3B), and proliferation of T-cells

(Figure 1G).

In further cytotoxicity experiments we confirmed that CD4+ as well as CD8+ T-cell subsets contribute to the DLL3/CD3 ITE-induced T-cell redirected lysis of SHP-77 cells in a time- dependent manner. T-cell redirected lysis by PBMCs (Figure 2A), pan T-cells (Figure 2B), and

CD8+ T-cells (Figure 2D) became already apparent after 48 hours when CD4+ T-cells (Figure 2C) still showed only minor lysis. After 72 hours, both, CD4+ and CD8+ T-cells, contributed comparably to the lysis of SHP-77 cells. In co-culture experiments with purified subsets of T- cells we demonstrated that CD4+ Central Memory (CD4+/CD45RA-/CD197+), CD4+ Effector

Memory (CD4+/CD45RA-/CD197-), CD8+ Memory (CD8+/CD45RA-), CD8+CD45RA+

(CD8+/CD45RA+/CD197+) as well as naϊve T-cells (CD4+/CD45RA+ and CD8+CD45RA+) are involved in the DLL3/CD3 ITE-induced T-cell redirected lysis of SHP-77 cells (Figures 2E-I).

Correlation of DLL3 expression with DLL3/CD3 ITE-induced T-cell redirected lysis

DLL3 mRNA expression was analyzed by RNA sequencing in primary tumor tissues from 20

SCLC patients and eight SCLC cell lines. The mRNA expression ranged from 30 to 370 TPM

(transcripts per million) in tumor tissues and 20 to 230 TPM in the cell lines (Figure 3A). Cell pellets of three representative SCLC cell lines with low (NCI-H2286), medium (NCI-H82) and high (SHP-77) DLL3 expression levels were generated and analyzed by an immunohistochemical assay (SP347) qualified for assessing DLL3 protein expression in early clinical trials with Rova-T (9). DLL3 protein expression was detectable for the SHP-77 and NCI-

H82 cell pellets, but not the NCI-H2286 cell pellet (Figure 3B). To analyze DLL3/CD3 ITE- induced T-cell redirected lysis of the eight SCLC cell lines, each cell line was co-cultured with

PBMCs and increasing concentrations of DLL3/CD3 ITE. The DLL3/CD3 ITE induced T-cell redirected lysis of all tested SCLC cell lines in a dose-dependent manner but the level of

DLL3/CD3 ITE-induced T-cell redirected lysis depended on the tumor cell line and the DLL3 expression levels, indicated by the range of EC50 concentrations from 5.9 ng/ml to 24 ng/ml

(Figure 3C, Table S3). Immunohistochemically detectable DLL3 levels correlated with higher

DLL3/CD3 ITE potency in the SHP-77 and NCI-H82 cell lines. However, lysis activity could also be detected using the NCI-H2286 cell line for which no DLL3 protein could be detected immunohistochemically, indicating the potential of DLL3/CD3 ITE to induce lysis of cells with very low DLL3 expression levels.

DLL3/CD3 ITE-induced anti-tumor activity and modulation of T-cell infiltration in vivo

In vivo efficacy studies were conducted in a subcutaneous human SHP-77 xenograft model in

CD3+ T-cell humanized mice (Figures 4A,B) where the DLL3/CD3 ITE was administered in a weekly (q7d) regimen supported by its long half-life of 20 days in C57/BL6 mice (Figure 4C).

In a first efficacy study (Study I), the DLL3/CD3 ITE was administered at doses of 0.025 and

0.25 mg/kg, i.v., and tumor growth was monitored until day 21. While treatment with 0.025 mg/kg induced tumor regression in three out of five animals at day 21 (p<0.05), a stronger anti- tumor response was observed with a dose of 0.25 mg/kg and tumor regression in all animals

(5/5) already at day 9. Notably, four out of five tumors were still in regression at the end of the study (p<0.05) (Figure 4A). In a second efficacy study (Study II) with a different human T-cell donor, DLL3/CD3 ITE monotherapy induced a statistically significant tumor growth inhibition (p

<0.05 at day 15) compared to the vehicle control group. Sustained tumor regressions could be observed for most of the tumors from day 15 (7/9 animals) until day 36 (8/9 animals). Although treatment was stopped after 4 cycles (day 22), tumor regression was maintained. Two out of the eight animals with tumor regression still showed complete tumor clearance (tumor not palpable anymore) at the end of the study (day 36), 14 days after the last dose (Figure 4B).

Tumor tissues from mice treated with vehicle or 0.25 mg/kg DLL3/CD3 ITE in Study II were collected at the day of termination and tissue sections were stained with anti-CD3, PD-1 and

PD-L1 antibodies. Treatment with DLL3/CD3 ITE induced infiltration of CD3+ T-cells into the

SHP-77 xenograft tissue. Infiltrating CD3+ T-cells upregulated PD-1 and LAG-3, and SHP-77 tumor cells upregulated PD-L1 (Figure 5).

In a following study (Study III), we examined the ability of the DLL3/CD3 ITE to modulate the inflammatory environment in the tumor tissue. SHP-77 tumor tissues from animals were collected eight days after administration of one dose of 0.25 mg/kg ITE i.v., and sections of formalin fixed and paraffin embedded tissues were immunohistochemically stained with anti-

CD3, anti-CD4, anti-CD8, anti-PD-1, anti-PD-L1 and anti-cC3 (cleaved caspase 3) antibodies. A statistically significant increase in CD4+ and CD8+ T-cells (Figure 6A-C) in DLL3/CD3 ITE treated animals compared with the vehicle treated animals was observed. In this study, we did not see an increase of PD-1 expression (Figure 6D) and only a non-statistically significant trend of increased PD-L1 (Figure 6E) expression. In the tumor cells a statistically significant increase

of cleaved caspase 3 was detected (Figure 4F). The proximity of the apoptotic tumor cells to the infiltrating T-cells was visualized by co-staining of tumor tissues for cleaved caspase 3 and CD3

(Figure 4G).

Dexamethasone only minimally impacts DLL3/CD3 ITE

The impact of dexamethasone on the activity of the DLL3/CD3 ITE was assessed by adding increasing concentrations of dexamethasone ranging from 1 to 1,000 ng/ml to the multiparametric cytotoxicity assay simultaneously with 30 μg/ml DLL3/CD3 ITE. Treatment with dexamethasone led to a dose dependent reduction of DLL3-CD3-induced T-cell redirected lysis of DLL3-positive SHP-77 cells with one out of three PBMC donors (Figure S4A). The DLL3/CD3

ITE-induced upregulation of CD25 on CD4+ T-cells was also reduced with one out of three donors, whereas dexamethasone did not have an effect on CD25 expression on CD8+ T-cells

(Figure S4B,C). The DLL3/CD3-induced secretion of IFN-γ (Figure S4D) and MCP-1 (Figure

S4E), by PBMCs in presence of DLL3-positive SHP-77 cells was reduced in presence of dexamethasone with all three tested donors.

DLL3/CD3 pharmacokinetics in non-human primates

To support pharmacokinetic and safety assessment in non-human primates, the DLL3/CD3 ITE was designed to be cross-reactive to cynomolgus monkey DLL3 and CD3. The ability of the

DLL3/CD3 ITE to induce T-cell redirected lysis of recombinant cynomolgus monkey DLL3- expressing HEK293 cells in presence of cynomolgus monkey PBMCs is shown in Figure S5.

Pharmacokinetic studies showed that the half-life of the DLL3/CD3 ITE is 10 days.

Discussion Despite the high unmet medical need for new treatments for SCLC and the highly attractive tumor specific expression of DLL3 for a targeted therapy, the development of such a therapy has yet to be realized. The first approach for targeting DLL3 with an antibody-based approach was Rova-T (rovalpituzumab tesirine), a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine dimer toxin (6,7). In a Phase I trial including patients with small cell lung or large-cell neuroendocrine tumors that have previously relapsed after platinum-based chemotherapy, 18% of patients had a confirmed objective response rate and a manageable safety profile (7). However, in a Phase II study, Rova-T showed only modest activity (median OS of 5.7 months in patients with high DLL3-expression versus 5.6 months in all patients) with associated toxicities of Grade 3-5 in third-line and beyond (3L+) SCLC patients with DLL3-positive tumors (22). The toxicities observed with Rova-T, such as thrombocytopenia, nausea, fatigue, vomiting, edema and effusions (7), have also been observed with vadastuximab talirine, an anti-CD33 antibody drug conjugate (23), and are considered drug- but not DLL3-related.

T-cell engagers have a completely different mechanism of action to antibody drug-conjugates and have shown high potency and efficacy with a promising safety profile in hematologic malignancies (14,15). However, the development of bispecific T-cell engagers for solid tumors presents new challenges. Firstly, given the potency of the mode of action, a tumor selectively expressed cell surface antigen is required to ensure optimal efficacy and safety. So far, bispecific T-cell engagers for solid tumors such as solitomab and MEDI-565 for the treatment of gastrointestinal cancers did not result in a sufficient therapeutic window in clinical trials (24,25), as the antigens EpCAM and CEA, respectively, are widely expressed. Secondly, the BiTE® molecules have a very short half-life and have to be administered by continuous intravenous infusion, for example. As such, opportunities to extend the half-life of T-cell engagers whilst

maintaining the potent pharmacological effect have been sought to improve the dosing convenience for patients.

In this manuscript, we describe a novel approach for targeting the tumor selective DLL3 antigen and treating DLL3-expressing SCLC using T-cell redirection. We have designed a highly DLL3- selective novel IgG-like T-cell engaging bi-specific antibody with an optimal pharmacological profile, and antibody-like in vivo stability and half-life. In contrast to AMG 757 (26), a half-life extended (HLE) BiTE consisting of a bispecific scFv fused to an Fc, the DLL3/CD3 ITE has an

IgG-like architecture which may pose a lower immunogenicity risk in humans. DLL3/CD3 ITE induces potent T-cell redirected lysis of DLL3-positive SCLC cell lines in a dose, E:T ratio, and

+ + time-dependent manner, with EC50 values in the low ng/ml range by redirecting CD4 and CD8

T-cells towards DLL3-postitive SCLC cells without lysing DLL3-negative target cells. As a result of the formation of the cytolytic synapse, T-cells secrete pro-inflammatory cytokines and proliferate. In vivo, DLL3/CD3 ITE monotherapy leads to infiltration of T-cells into tumor tissue and converts a non-inflamed (cold) into an inflamed (hot) tumor environment, leading to tumor cell apoptosis and resulting in strong tumor regressions with complete tumor eradication in several mice in a human CD3+ T-cell humanized SHP-77 xenograft model.

In the Phase II TRINITY study with Rova-T, cut-offs for analysis of DLL3 expression in SCLC tissues of patients were defined as DLL3-positive when tissues contained ≥ 25% of DLL3- positive tumor cells, and as DLL3-high when tissues contained ≥ 75% of DLL3-positive tumor cells (22). In our study, we could detect DLL3 expression in cell pellets of SCLC cell lines expressing more than 500 DLL3 molecules per cell using the same qualified immunohistochemistry assay (Ventana, SP347) (9) previously validated for the determination of

DLL3 expression levels in the Rova-T Phase II study (22). In in vitro cytotoxicity assays, cell lines with immunohistochemically detectable DLL3 levels correlate with higher DLL3/CD3 ITE

potency, suggesting that the detection of DLL3 expression using the SP347 IHC assay may be a promising biomarker for patient selection that should be evaluated in clinical trials.

The FDA recently approved the PD-1 inhibitor Pembrolizumab for the treatment of SCLC patients who experienced disease progression after two or more prior lines of therapy based on two clinical studies showing a 19% overall objective response rate (27). In our pre-clinical studies, we observed an upregulation of PD-(L)1 and LAG-3 expression in vivo as a result of the inflamed environment induced by treatment with the DLL3-CD3 ITE, which is in line with observations from preclinical studies with other T-cell engagers, e.g. CEA BiTE, CEA TCB,

AMG330, Her2-TDB (16,28-30). Furthermore, in a clinical trial, evidence of enhanced activity of

RO6958688, a CEA targeted T-cell engager, in combination with atezolizumab with a manageable safety profile has been reported (18). In future studies of the DLL3/CD3 ITE it will be important to look at the impact on other immune checkpoint molecules.

As activation of T-cells and cytokine secretion is a characteristic of the mode of action of T-cell engagers, which in some cases can also cause clinical side effects such as fever and hypotension, it might be desirable to reduce cytokine secretion as much as possible without negatively influencing the cytotoxic potential of T-cells. Glucocorticoids have been described to inhibit inflammatory responses such as cytokines (31), and a preclinical study using the glucocorticoid dexamethasone in combination with blinatumomab reported that dexamethasone can qualify as a potential co-medication for TcE therapies (32). The data obtained in this study reveal that dexamethasone can reduce DLL3/CD3 ITE-induced secretion of IFN-γ and MCP-1, and therefore support the use of dexamethasone to treat potential infusion reactions or cytokine release syndromes associated with DLL3/CD3 ITE treatment, but the treatment could result in reduced cytotoxic potential of DLL3/CD3 ITE.

According to the DLL3-expression profile, the DLL3/CD3 ITE has the potential to be developed in additional indications with high unmet medical need, such as glioma (33), medullary thyroid

cancer (34), gastrointestinal neuroendocrine malignancies (35), dispersed neuroendocrine tumors of the pancreas (34), small cell bladder cancer (36), Merkel-cell carcinoma (37), and neuroendocrine prostate cancer (38).

Taken together the DLL3/CD3 ITE provides a novel drug candidate for the treatment of SCLC as monotherapy with upside potential in other neuroendocrine tumor types.

Abbreviations cC3: Cleaved Caspase 3; CFDA-SE: Carboxyfluorescein diacetate succinimidyl ester; CTV:

CellTraceTMViolet; DLL3: Delta-like-ligand 3; FCS: fetal calf serum; Gy: Gray; ITE: IgG-like T-cell engager; i.v.: intravenous; q7d: every 7 day dosing; LDH: Lactate dehydrogenase; MoA: Mode of Action; PBMCs: Peripheral blood mononuclear cells; PI: Propidium iodide; SD: standard deviation; SCLC: Small cell lung cancer

Acknowledgments

We thank Christoph Albrecht, Ilse Apfler, Kathrin Bauer, Oliver Bergner, Erica Bolella, Richard Liedauer, Irene Schweiger, Abdallah Souabni, and Christian Walterskirchen for their experimental support and help for this study, and all colleagues from Biotherapeutics Discovery for all aspects of molecule discovery. This study was supported by the Basisprogramm grants of the Austrian Research Promotion Agency (FFG; 860968, 869530 and 875923).

References

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69(1):7-34 doi 10.3322/caac.21551. 2. Bunn PA, Jr., Minna JD, Augustyn A, Gazdar AF, Ouadah Y, Krasnow MA, et al. Small Cell Lung Cancer: Can Recent Advances in Biology and Molecular Biology Be Translated into Improved Outcomes? J Thorac Oncol 2016;11(4):453-74 doi 10.1016/j.jtho.2016.01.012. 3. Dunwoodie SL, Henrique D, Harrison SM, Beddington RS. Mouse Dll3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo. Development 1997;124(16):3065-76. 4. Chapman G, Sparrow DB, Kremmer E, Dunwoodie SL. Notch inhibition by the ligand DELTA-LIKE 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis. Hum Mol Genet 2011;20(5):905-16 doi 10.1093/hmg/ddq529. 5. Ladi E, Nichols JT, Ge W, Miyamoto A, Yao C, Yang LT, et al. The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands. J Cell Biol 2005;170(6):983- 92 doi 10.1083/jcb.200503113. 6. Saunders LR, Bankovich AJ, Anderson WC, Aujay MA, Bheddah S, Black K, et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med 2015;7(302):302ra136 doi 10.1126/scitranslmed.aac9459. 7. Rudin CM, Pietanza MC, Bauer TM, Ready N, Morgensztern D, Glisson BS, et al. Rovalpituzumab tesirine, a DLL3-targeted antibody-drug conjugate, in recurrent small-cell lung cancer: a first-in-human, first-in-class, open-label, phase 1 study. Lancet Oncol 2017;18(1):42-51 doi 10.1016/s1470-2045(16)30565-4. 8. Tanaka K, Isse K, Fujihira T, Takenoyama M, Saunders L, Bheddah S, et al. Prevalence of Delta-like protein 3 expression in patients with small cell lung cancer. Lung Cancer 2018;115:116-20 doi 10.1016/j.lungcan.2017.11.018. 9. Huang RSP, Holmes BF, Powell C, Marati RV, Tyree D, Admire B, et al. Delta- like Protein 3 Prevalence in Small Cell Lung Cancer and DLL3 (SP347) Assay Characteristics. Arch Pathol Lab Med 2019;143(11):1373-7 doi 10.5858/arpa.2018-0497-OA. 10. Gruen M, Bommert K, Bargou RC. T-cell-mediated lysis of B cells induced by a CD19xCD3 bispecific single-chain antibody is perforin dependent and death receptor independent. Cancer Immunol Immunother 2004;53(7):625-32 doi 10.1007/s00262-003-0496-2. 11. Hoffmann P, Hofmeister R, Brischwein K, Brandl C, Crommer S, Bargou R, et al. Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3- bispecific single-chain antibody construct. Int J Cancer 2005;115(1):98-104 doi 10.1002/ijc.20908. 12. Hipp S, Tai YT, Blanset D, Deegen P, Wahl J, Thomas O, et al. A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo. Leukemia 2017;31(10):2278 doi 10.1038/leu.2017.219.

13. Mathur D, Root AR, Bugaj-Gaweda B, Bisulco S, Tan X, Fang W, et al. A Novel GUCY2C-CD3 T cell Engaging Bispecific construct (PF-07062119) for the Treatment of Gastrointestinal Cancers. Clin Cancer Res 2020 doi 10.1158/1078- 0432.Ccr-19-3275. 14. Sanford M. Blinatumomab: first global approval. Drugs 2015;75(3):321-7 doi 10.1007/s40265-015-0356-3. 15. Topp MS, Duell J, Zugmaier G, Attal M, Moreau P, Langer C, et al. Anti-B-Cell Maturation Antigen BiTE Molecule AMG 420 Induces Responses in Multiple Myeloma. J Clin Oncol 2020:Jco1902657 doi 10.1200/jco.19.02657. 16. Bacac M, Fauti T, Sam J, Colombetti S, Weinzierl T, Ouaret D, et al. A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors. Clin Cancer Res 2016;22(13):3286-97 doi 10.1158/1078-0432.Ccr-15-1696. 17. Hernandez-Hoyos G, Sewell T, Bader R, Bannink J, Chenault RA, Daugherty M, et al. MOR209/ES414, a Novel Bispecific Antibody Targeting PSMA for the Treatment of Metastatic Castration-Resistant Prostate Cancer. Mol Cancer Ther 2016;15(9):2155-65 doi 10.1158/1535-7163.Mct-15-0242. 18. Tabernero J, Melero I, Ros W, Argiles G, Marabelle A, Rodriguez-Ruiz M, et al. Phase Ia and Ib studies of the novel carcinoembryonic antigen (CEA) T-cell bispecific (CEA CD3 TCB) antibody as a single agent and in combination with atezolizumab: Preliminary efficacy and safety in patients with metastatic colorectal cancer (mCRC). Journal of Clinical Oncology 2017;35((May 20, 2017)):1 doi 10.1200/JCO.2017.35.15_suppl.3002. 19. Pessano S, Oettgen H, Bhan AK, Terhorst C. The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits. Embo j 1985;4(2):337-44. 20. Singh S, Kroe-Barrett RR, Canada KA, Zhu X, Sepulveda E, Wu H, et al. Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody. MAbs 2015;7(4):778-91 doi 10.1080/19420862.2015.1032491. 21. Venkataramani S, Low S, Weigle B, Dutcher D, Jerath K, Menzenski M, et al. Design and characterization of Zweimab and Doppelmab, high affinity dual antagonistic anti-TSLP/IL13 bispecific antibodies. Biochem Biophys Res Commun 2018;504(1):19-24 doi 10.1016/j.bbrc.2018.08.064. 22. Morgensztern D, Besse B, Greillier L, Santana-Davila R, Ready N, Hann CL, et al. Efficacy and Safety of Rovalpituzumab Tesirine in Third-Line and Beyond Patients with DLL3-Expressing, Relapsed/Refractory Small-Cell Lung Cancer: Results From the Phase II TRINITY Study. Clin Cancer Res 2019;25(23):6958- 66 doi 10.1158/1078-0432.Ccr-19-1133. 23. Fathi AT, Erba HP, Lancet JE, Stein EM, Ravandi F, Faderl S, et al. A phase 1 trial of vadastuximab talirine combined with hypomethylating agents in patients with CD33-positive AML. Blood 2018;132(11):1125-33 doi 10.1182/blood-2018- 03-841171. 24. Pishvaian M, Morse MA, McDevitt J, Norton JD, Ren S, Robbie GJ, et al. Phase 1 Dose Escalation Study of MEDI-565, a Bispecific T-Cell Engager that Targets Human Carcinoembryonic Antigen, in Patients With Advanced Gastrointestinal

Adenocarcinomas. Clin Colorectal Cancer 2016;15(4):345-51 doi 10.1016/j.clcc.2016.07.009. 25. Kebenko M, Goebeler ME, Wolf M, Hasenburg A, Seggewiss-Bernhardt R, Ritter B, et al. A multicenter phase 1 study of solitomab (MT110, AMG 110), a bispecific EpCAM/CD3 T-cell engager (BiTE(R)) antibody construct, in patients with refractory solid tumors. Oncoimmunology 2018;7(8):e1450710 doi 10.1080/2162402x.2018.1450710. 26. Giffin JM, Lobenhofer EK, Cooke K, Raum T, Stevens J, Beltran PJ, et al. BiTE® antibody constructs for the treatment of SCLC [abstract]. . Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3632 2017 doi 10.1158/1538-7445.AM2017-3632. 27. Chung HC, Piha-Paul SA, Lopez-Martin J, Schellens JHM, Kao S, Miller WH, Jr., et al. Pembrolizumab After Two or More Lines of Previous Therapy in Patients With Recurrent or Metastatic SCLC: Results From the KEYNOTE-028 and KEYNOTE-158 Studies. J Thorac Oncol 2019 doi 10.1016/j.jtho.2019.12.109. 28. Osada T, Patel SP, Hammond SA, Osada K, Morse MA, Lyerly HK. CEA/CD3- bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1. Cancer Immunol Immunother 2015;64(6):677-88 doi 10.1007/s00262-015-1671-y. 29. Krupka C, Kufer P, Kischel R, Zugmaier G, Lichtenegger FS, Kohnke T, et al. Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism. Leukemia 2016;30(2):484-91 doi 10.1038/leu.2015.214. 30. Junttila TT, Li J, Johnston J, Hristopoulos M, Clark R, Ellerman D, et al. Antitumor efficacy of a bispecific antibody that targets HER2 and activates T cells. Cancer Res 2014;74(19):5561-71 doi 10.1158/0008-5472.Can-13-3622-t. 31. Franchimont D, Kino T, Galon J, Meduri GU, Chrousos G. Glucocorticoids and inflammation revisited: the state of the art. NIH clinical staff conference. Neuroimmunomodulation 2002;10(5):247-60 doi 10.1159/000069969. 32. Brandl C, Haas C, d'Argouges S, Fisch T, Kufer P, Brischwein K, et al. The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct. Cancer Immunol Immunother 2007;56(10):1551-63 doi 10.1007/s00262-007-0298-z. 33. Spino M, Kurz SC, Chiriboga L, Serrano J, Zeck B, Sen N, et al. Cell Surface Notch Ligand DLL3 is a Therapeutic Target in Isocitrate Dehydrogenase-mutant Glioma. Clin Cancer Res 2019;25(4):1261-71 doi 10.1158/1078-0432.Ccr-18- 2312. 34. Saunders LR WS, Bheddah S, Isse K, Fong S, Pysz MA, et al. Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC). Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC Philadelphia (PA): AACR 2017;Cancer Res 2017;77(13 Suppl):Abstract nr 3093. doi 10.1158/1538-7445.

35. Matsuo K, Taniguchi K, Hamamoto H, Ito Y, Futaki S, Inomata Y, et al. Delta-like 3 localizes to neuroendocrine cells and plays a pivotal role in gastrointestinal neuroendocrine malignancy. Cancer Sci 2019;110(10):3122-31 doi 10.1111/cas.14157. 36. Koshkin VS, Garcia JA, Reynolds J, Elson P, Magi-Galluzzi C, McKenney JK, et al. Transcriptomic and Protein Analysis of Small-cell Bladder Cancer (SCBC) Identifies Prognostic Biomarkers and DLL3 as a Relevant Therapeutic Target. Clin Cancer Res 2019;25(1):210-21 doi 10.1158/1078-0432.Ccr-18-1278. 37. Xie H, Kaye FJ, Isse K, Sun Y, Ramoth J, French DM, et al. Delta-like Protein 3 Expression and Targeting in Merkel Cell Carcinoma. Oncologist 2020 doi 10.1634/theoncologist.2019-0877. 38. Puca L, Gavyert K, Sailer V, Conteduca V, Dardenne E, Sigouros M, et al. Delta- like protein 3 expression and therapeutic targeting in neuroendocrine prostate cancer. Sci Transl Med 2019;11(484) doi 10.1126/scitranslmed.aav0891.

Legends

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 co-culture of PBMCs and SCLC cell lines at the indicated E:T ratios for 72 hours. Each datapoint represents the mean of duplicate measurements, 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 co-culture with DLL3-negative HL-60 cells. Human PBMCs were co-cultivated 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 co-culture was monitored by flow cytometry. Each datapoint represents a single measurement. (F) DLL3/CD3 ITE-induced apoptosis of SCLC cells. Human PBMCs were co-cultivated 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 analysed by flow cytometry. Each datapoint represents a single measurement. (G) Proliferation of T-cells. Representative example.

Figure 2 CD4+ and CD8+ T-cells contribute to DLL3/CD3 ITE-mediated T-cell redirected lysis of SHP-77 cells Time-Dependency of DLL3/ITE -induced T-cell Redirected Lysis of SHP-77 cells by (A) PBMCs, (B) pan T-cells, (C) CD4+ or (D) CD8+ T-cells. DLL3/CD3 ITE-induced T-cell redirected lysis of SHP-77 cells by purified human (E,F) CD4+, (E) CD4+ central memory, (F) CD4+ effector memory, (G,H) CD8+, (G) CD8+ memory, (H) CD8+CD45+ effector, or (I) naϊve T-cells. Each datapoint represents the mean of duplicate measurements, error bars represent the SD.

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) Immunohistochemical 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 co-culture 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, error bars represent the SD of maximal lysis versus DLL3 molecules on the cell surface.

Figure 4 DLL3/CD3 ITE monotherapy induces dose-dependent efficacy in a SHP-77 xenograft in a human T-cell reconstituted mouse model (A) Dose-dependency of DLL3/CD3 ITE-induced anti-tumor activity in a SHP-77 xenograft model in NOG mice reconstituted with human CD3+ T-cells (Study I). (B) Anti- tumor 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.

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

Figure 6 DLL3/CD3 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 a SHP-77 xenograft Immunohistochemical analysis of xenograft tumors on day 8 (Study III) for (A) CD3+, (B) CD4+, (C) CD8+ T-cells, and (D) PD-1-positive T-cells, (E) PD-L1-positive, and (F) cleaved Caspase 3 tumor area. (G) Representative example of of immunohistochemical co-staining of a xenograft tumor from one mouse for CD3 (brown) and cleaved Caspase-3 (dark red).

A Bispecific DLL3/CD3 IgG-like T-cell Antibody induces anti-tumor responses in Small Cell Lung Cancer

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

Clin Cancer Res Published OnlineFirst June 18, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-20-0926

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/06/18/1078-0432.CCR-20-0926.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2020/06/18/1078-0432.CCR-20-0926. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.