ﬁc dll3/cd3 igg-like t-cell engaging antibody induces ......2020/08/18 · in this article, we...

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

A Bispecific DLL3/CD3 IgG-Like T-Cell EngagingAntibody Induces Antitumor Responses in Small CellLung 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 andaggressive subtype of lung carcinoma characterized by highlychemotherapy-resistant recurrence in the majority of patients.To effectively treat SCLC, we have developed a unique and novelIgG-like T-cell engaging bispecific antibody (ITE) that potentlyredirects T-cells to specifically lyse SCLC cells expressing Delta-likeligand 3 (DLL3), an antigen that is frequently expressed on the cellsurface of SCLC cells, with no to very little detectable expression innormal tissues.

Experimental Design: The antitumor activity and mode ofaction of DLL3/CD3 ITE was evaluated in vitro using SCLC celllines and primary human effector cells and in vivo in an SCLCxenograft model reconstituted with human CD3þ T-cells.

Results: Selective binding of DLL3/CD3 ITE to DLL3-positivetumor cells and T-cells induces formation of an immunologicalsynapse resulting in tumor cell lysis and activation of T-cells. Ina humanT-cell engrafted xenograftmodel, theDLL3/CD3 ITE leadsto an increase in infiltration of T-cells into the tumor tissue resultingin apoptosis of the tumor cells and tumor regression. Consistentwith the mode of action, the DLL3/CD3 ITE treatment led toupregulation of PD-1, PD-L1, and LAG-3.

Conclusions: This study highlights the ability of the DLL3/CD3 ITEto induce strictly DLL3-dependent T-cell redirected lysis of tumor cellsand recruitment of T-cells into noninflamed tumor tissues leading totumorregression inapreclinical invivomodel.Thesedatasupportclinicaltesting of the DLL3/CD3 ITE in patients with SCLC.

IntroductionLung cancer remains the number one cause of cancer-related deaths

in both men and women in the United States with over 220,000 newcases diagnosed annually (1). Out of these, small cell lung cancer(SCLC), a poorly differentiated neuroendocrine tumor, accounts forroughly 10% to 15%of cases (2). SCLC is themost lethal and aggressivesubtype of lung carcinoma with 5-year survival rates below 7%characterized by highly chemotherapy-resistant recurrence within12 months in the majority of patients (2).

Delta-like ligand 3 (DLL3) is amember of the Notch receptor ligandfamily that plays a critical role for Notch signaling during embryonaldevelopment and is functionally distinct from the related Notch familymembers DLL1 and DLL4, as demonstrated in animal studies inxenopus laevis and mus musculus. In mice, DLL3 is expressed bypostmitotic cells during somitogenesis (3). However, DLL3 is notpresented on the surface, but instead interacts with Notch1 in the late

endocytic compartment preventing Notch1 from reaching the cellsurface (4). This finding has been confirmed in in vitro studies bydemonstrating that DLL3 does not activate Notch signaling throughbinding to Notch expressed on the cell surface, but rather acts as aNotch antagonist by suppressingNotch signaling in a cell-autonomousmanner (5). DLL3 expression has been described in SCLC tissuesamples (6). In normal tissues, DLL3 is expressed during embryonaldevelopment with highest expression in fetal brain, but is absent inadult normal tissues (6). Although in developmental processes DLL3 isexpressed intracellularly, in SCLC tumors with DLL3 overexpression,DLL3 escapes to the cell surface which makes it targetable withantibody-based therapies (6). IHC studies indicate a high prevalenceof DLL3 expression in SCLC tissues from Caucasian and Japanesepatients, with 76% to 88% of tumors comprising at least 1% of DLL3-positive tumor cells and 32% to 67% of patients with tumors com-prising 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 toinduce the formation of a cytolytic synapse in an MHC-independentmanner by binding concomitantly to a respective antigen on the cellsurface of tumor cells and to CD3 on T-cells, and direct their cytolyticactivity selectively to the tumor cells. After formation of thecytolytic synapse, the T-cells produce perforin and granzyme B,leading to apoptosis of the tumor cells. Activation of T-cells leadsto transient release of cytokines, which engages other immune cellsand broadens the immune response against the tumor tissueleading to conversion of a noninflamed (cold) to an inflamed(hot) tumor environment, infiltration and proliferation of T-cells,and serial killing of tumor cells (10–13). After recent clinicalsuccesses of bispecific T-cell engagers with a short half-life (BiTE)for the treatment of hematologic malignancies (14, 15), the nextgeneration of T-cell engagers incorporating half-life extension forincreased dosing convenience for patients for the treatment of solidtumors is emerging (13, 16–18).

1Boehringer Ingelheim Pharmaceuticals, Inc., Cancer Immunology & ImmuneModulation, Ridgefield, Connecticut. 2Boehringer Ingelheim Pharmaceuticals,Inc., Biotherapeutics Discovery, Ridgefield, Connecticut. 3Boehringer IngelheimRCV, GmbH & Co KG., Cancer Pharmacology and Disease Positioning, Vienna,Austria. 4Boehringer Ingelheim RCV, GmbH & Co KG., Oncology TranslationalScience, Vienna, Austria. 5Boehringer Ingelheim RCV, GmbH & Co KG., CancerImmunology & Immune Modulation, Vienna, Austria.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Susanne Hipp, Boehringer Ingelheim Pharmaceuticals,Inc., 900 Ridgebury Rd./P.O. Box 368, Ridgefield, CT 06877-0368. Phone: 203-798-4567; E-mail: [email protected]

Clin Cancer Res 2020;XX:XX–XX

doi: 10.1158/1078-0432.CCR-20-0926

�2020 American Association for Cancer Research.

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In this article, 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 strictlyDLL3-dependent lysis of tumor cells and T-cell infiltration into tumortissue leading to complete tumor regression and an inflamed tumorenvironment in vivo.

Materials and MethodsEngineering, expression, and purification of DLL3/CD3 ITE

Single B-cell technology from mice immunized with the recombi-nant extracellular domain of human DLL3 generated the anti-DLL3antibody. Themurine anti-CD3 antibody described previously was thesource of the anti-CD3 arm used in the test article (19). Humanizationand further sequence optimization of both variable regions wasperformed using a Fab expression vector system according to methodspreviously described (20). In brief, closely matching human germlinesidentified in silico and libraries of Fab variants based on thesegermlines were evaluated for binding to the target, and percent humanand Epivax (in silico predictive tool for potential immunogenicity)scores. Subsequently, the optimized variable regions with drug-likeproperties (high percentage of human sequence, minimal chemicalliabilities, and reduced immunogenicity potential) were formatted inan ITE-bispecific expression construct using pTT5 expression vector(National ResearchCouncil, Canada) with commonmolecular biologytechniques. The ITE is a two-chain heterodimeric bispecific anti-body with Fab domains that contain polypeptide linkers to assurecorrect light-heavy chain pairing and an Fc domain engineered forheterodimerization and efficient purification (21). The DLL3/CD3ITE produced in CHO-E (National Research Council, Canada)cells by transient transfection with DLL3:CD3 DNA plasmid massratio of 1:3 enabled efficient purification of heterodimeric speciesas follows. After 10-day culturing, the supernatant was harvestedand purified by Protein A affinity chromatography using MabSelectcolumn (GE Healthcare). The bispecific antibody was furtherpurified to homogeneity by cation exchange chromatography usinga Poros 50 HS column (Applied Biosystems). The purified proteinwas concentrated and stored in 50 mmol/L sodium acetate and100 mmol/L NaCl, pH 5.0 buffer. Analytical characterizationby intact mass spectrometry, SDS-PAGE, and size-exclusion chroma-tography showed that the purified antibody was the expectedheterodimeric mass and contained minimal high-molecular-weightaggregate (less than 1%) and no contaminating homodimericspecies.

Cell lines and cell cultureAll cell lines were obtained from the ATCC and cultured as

described in the supplier's description. Recombinant human DLL1,DLL4, and cynomolgus monkey DLL3-expressing HEK293 cell lineswere generated by transfection of HEK293 cells using Lipofectamine(Thermo Fisher Scientific) with pcDNA3.1(þ) (Thermo Fisher Scien-tific) containing the coding sequences including an N-terminal FLAGtag between the signal sequence and the extracellular domain. All celllines were tested to exclude mycoplasma contamination and main-tained in culture for a maximum of 20 passages.

mRNA sequencing of human SCLC tissues and SCLC cell linesRNA was isolated using the TRI Reagent (Sigma) and RNeasy Mini

Kit (Qiagen). Five hundred nanograms RNA was subjected to librarypreparation using the TruSeq RNA Library Prep Kit v2 with poly-Aselection (Illumina). The Library was then multiplexed and sequencedusing a HiSeq device (Illumina) using paired end sequencing. Allhuman SCLC tissues were obtained after receiving written-informedconsent from the patients.

Isolation of peripheral blood mononuclear cells and T-cellsFresh buffy coats from healthy volunteers were obtained from the

Austrian Red Cross after receiving written-informed consent in accor-dance with theDeclaration of Helsinki andwith approval of the federalethical committee in Austria. Peripheral blood mononuclear cells(PBMC) were purified from buffy coats by density gradient centrifu-gation. T-cell subsets were purified from PBMCs using the respectiveT-cell subset isolation kits (Miltenyi Biotech) according to themanufacturer's instructions. Catalog numbers of T-cell subset kits arelisted in Supplementary Table S1.

Cell binding analysisCells were incubated in the presence of DLL3/CD3 ITE on ice for 25

minutes at 4�C. Subsequently, cells were washed 2 times in FACSbuffer and stained with phycoerythrin-conjugated goat anti-humanIgG secondary antibody (Sigma-Aldrich) diluted 1:200 in FACS bufferfor 25minutes at 4�C. Samples weremeasured by flow cytometry on anFACS Canto II instrument (Becton Dickinson) and analyzed withFlowJo V10 software (Becton Dickinson).

Quantification of DLL3 cell surface molecules on SCLC celllines

Quantification of DLL3 cell surface molecules expressed on SCLCcell lines was performed by flow cytometry using the QIFIKIT(Agilent) according to the manufacturer's instructions.

Cytotoxicity, T-cell activation, T-cell proliferation, and cytokinesecretion assays

Cytotoxicity, activation, degranulation, and proliferation ofT-cells, and cytokine secretion were analyzed in multiparametriccytotoxicity assays.

Redirected T-cell cytotoxicity was assessed by quantification oflactate dehydrogenase (LDH) concentrations in the cell culture super-natant using the Cytotoxicity Detection KitPLUS (Sigma-Aldrich)according to themanufacturer's instructions. Monitoring of specificityof lysis in the presence of DLL3-positive and DLL3-negative cells wasassessed by monitoring of depletion of cells labeled with carboxy-fluorescein diacetate succinimidyl ester (CFDA-SE) or CellTraceViolet (CTV; both Thermo Fisher Scientific).

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

Translational Relevance

Small cell lung cancer (SCLC) is the most lethal and aggressivesubtype of lung carcinoma characterized by a high relapse rate tochemotherapy in a majority of patients. Delta-like ligand 3 (DLL3)represents a promising antigen for targeted therapy of SCLC. Thisarticle provides mechanistic insights into the mode of action of theDLL3/CD3 ITE, a novel IgG-like bispecific T-cell engaging anti-body currently in preclinical development. The DLL3/CD3 ITEinduces highly selective lysis of tumor cells and subsequent acti-vation and proliferation of T cells. In a preclinical in vivomodel, theDLL3/CD3 ITE induces infiltration ofCD4þ andCD8þT-cells intononinflamed tumors leading to a more inflamed tumor environ-ment and resulting in complete tumor regression.

Hipp et al.

Clin Cancer Res; 2020 CLINICAL CANCER RESEARCHOF2




additionally stained with anti-CD3 (BioLegend). Cytokine con-centrations were analyzed from supernatants of cytotoxicity assaysusing the V-PLEX Assays (Mesoscale Discovery) following themanufacturer's instructions.

Mouse xenograft studiesAll animal studies were approved by the internal ethics committee

and the responsible local governmental committee. Antitumor activitywas evaluated in 8-week-old female NOD.CgPrkdcscidIl2rgtm1Sug/JicTac mice (Taconic). Note that 2.5 � 106 SHP-77 cells weresuspended in 1 � PBS/5% FCS and injected subcutaneously into theright dorsal flank of animals that had been sublethally irradiated with 2Gy. Thirteen days later, when tumors reached a size between 71 and118 mm3, mice were randomized in groups, and 2 � 107 in vitro–expanded human T-cells (T-cell isolation, activation/expansion kitsfrom Miltenyi Biotec) were injected into the peritoneal cavity. Treat-ment with DLL3/CD3 ITE was initiated 3 days after T-cell injection(Supplementary Fig. S1). Tumor size was measured by an electroniccaliper and calculated according to the formula “tumor volume ¼length � diameter2 � p/6.” Regressions were defined as a relativetumor volume < 1 when normalized to the tumor volume at the start oftreatment. One-sided nonparametric Mann–Whitney–WilcoxonU tests were applied to compare the treatment group with the vehiclegroup. The level of significance was fixed at a ¼ 5%.

IHC analysisMouse tumor tissues were harvested when animals were termi-

nated, fixed overnight in 4% neutral-buffered formaldehyde, andembedded in paraffin. Note that 2-mm-thick serial sections wereprepared on a microtome, put on glass slides, and subsequentlydewaxed. SHP-77 tumor tissues were stained with anti-humanCD3 (Roche Diagnostics), anti-human CD4 (Roche Diagnostics),anti-human CD8 (Roche Diagnostics), anti-human PD-1 (CellSignaling Technology), anti-human LAG-3 (Novus Biologicals),anti-human PD-L1 (Abcam), anti-human cleaved Caspase 3 (CST),or anti-human DLL3 (Ventana SP347) antibodies. Two-sided non-parametric Mann–Whitney–Wilcoxon U tests were applied tocompare the treatment group with the vehicle group.

Pharmacokinetic studiesSerum concentrations of DLL3/CD3 ITE were determined in an

ELISA-based assay using anti-human IgG capture (Novus) and detec-tion (MyBiosource) reagents. Data were treated as a naïve pool andfitted to a linear two-compartment model using a log-additive errormodel in Phoenix 64 WinNonlin 7.0.

ResultsMolecule discovery and mode of action of the DLL3/CD3 ITE

The DLL3/CD3 ITE is based on a heterodimeric IgG scaffold thatincorporates flexible peptide linkers between light and heavy chainsand is designed to bind concurrently to DLL3 on tumor cells and CD3on T-cells (Fig. 1A).

Binding specificity of DLL3/CD3 ITE for human DLL3 and CD3was verified by using DLL3-positive SCLC cell lines with endogenousDLL3 expression, DLL3-negative cell lines, purified T-cells, andrecombinant cell lines expressing human DLL1 or DLL4, the closesthomologs of DLL3. Cell-bound DLL3/CD3 ITE was detected by flowcytometry. DLL3/CD3 ITE binds to DLL3-positive SCLC cell linesSHP-77 and NCI-H82, and CD3-positive purified human T-cells, butnot to DLL3-negative cell lines RKO-E6, HL-60, and recombinant

HEK293 cell lines expressing human DLL1 or DLL4 (SupplementaryFig. S2). DLL3/CD3 ITE–induced T-cell redirected lysis is strictlydependent on DLL3 expression and presence of T-cells (Fig. 1B).Donor dependency was observed in a cytotoxicity assay with PBMCsfrom 23 different human donors and the SCLC cell line SHP-77 with ageometricmeanEC50 of 5.5 ng/mLwith a range from0.7 to 20.8 ng/mL(Fig. 1C).

The potency of the DLL3/CD3 ITE–induced lysis of SHP-77 cellsdepends on the E:T ratio as indicated by the range of EC50 valuesranging from 1 ng/mL at an E:T of 30:1 to 129 ng/mL at an E:T of 2:1withmaximal lysis activity observed at an E:T ratio of ≥ 10:1 (Fig. 1D).The selectivity of the DLL3/CD3 ITE–induced lysis of DLL3-positivecells was further evaluated by coculture of PBMCs, CTV-labeledDLL3-positive SHP-77 cells, and CFDA-SE–labeled DLL3-negativeHL-60 cells at a ratio of 20:1:1, and increasing concentrations ofDLL3/CD3 ITE for 72 hours. The number of viable SHP-77 andHL-60 cells was determined by flow cytometry. The DLL3/CD3 ITEdid not induce lysis of DLL3-negative HL-60 cells, when coculturedwith DLL3-positive SHP-77 cells. The slight reduction of viable targetcell numbers at high concentrations of DLL3/CD3 ITE is considerednegligible compared with the strong reduction of SHP-77 cells(Fig. 1E). The DLL3/CD3 ITE lyses cells through induction ofapoptosis of SHP-77 cells, as demonstrated by the increase of AnnexinV binding and PI incorporation in CTV-labeled SHP-77 cells aftercocultivationwith PBMCs and increasing concentration ofDLL3/CD3ITE for 24 hours (Fig. 1F). Further analysis of themode of action of theDLL3/CD3 ITE confirmed DLL3-dependent secretion of MCP-1(Supplementary Fig. S3A) and IFNg (Supplementary Fig. S3B), andproliferation of T-cells (Fig. 1G).

In further cytotoxicity experiments, we confirmed that CD4þ aswellas CD8þ T-cell subsets contributes to the DLL3/CD3 ITE–inducedT-cell redirected lysis of SHP-77 cells in a time-dependent manner.T-cell redirected lysis by PBMCs (Fig. 2A), pan T-cells (Fig. 2B), andCD8þ T-cells (Fig. 2D) became already apparent after 48 hours whenCD4þ T-cells (Fig. 2C) still showed only minor lysis. After 72 hours,both CD4þ and CD8þT-cells contributed comparably with the lysis ofSHP-77 cells. In coculture experiments with purified subsets of T-cells,we demonstrated that CD4þ central memory (CD4þ/CD45RA�/CD197þ), CD4þ effector memory (CD4þ/CD45RA�/CD197�),CD8þ memory (CD8þ/CD45RA�), CD8þCD45RAþ (CD8þ/CD45RAþ/CD197þ), as well as naïve T-cells (CD4þ/CD45RAþ andCD8þCD45RAþ) are involved in the DLL3/CD3 ITE–induced T-cellredirected lysis of SHP-77 cells (Fig. 2E–I).

Correlation of DLL3 expression with DLL3/CD3 ITE–inducedT-cell redirected lysis

DLL3 mRNA expression was analyzed by RNA sequencing inprimary tumor tissues from 20 patients with SCLC and eight SCLCcell lines. The mRNA expression ranged from 30 to 370 TPM(transcripts per million) in tumor tissues and 20 to 230 TPM in thecell lines (Fig. 3A). Cell pellets of three representative SCLC cell lineswith low (NCI-H2286),medium (NCI-H82), andhigh (SHP-77)DLL3expression levels were generated and analyzed by an IHC assay(SP347) qualified for assessing DLL3 protein expression in earlyclinical trials with Rova-T (9). DLL3 protein expression was detectablefor the SHP-77 and NCI-H82 cell pellets, but not the NCI-H2286 cellpellet (Fig. 3B). To analyze DLL3/CD3 ITE–induced T-cell redirectedlysis of the eight SCLC cell lines, each cell line was cocultured withPBMCs and increasing concentrations of DLL3/CD3 ITE. The DLL3/CD3 ITE–induced T-cell redirected lysis of all tested SCLC cell lines ina dose-dependent manner, but the level of DLL3/CD3 ITE–induced

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

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Selectivity of DLL3/CD3 ITE.A, Schematic graphic of DLL3/CD3 ITE.B–E,Cytotoxicitywas determinedbyLDH release relative to a control containing 3%TritonX-100after 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 barsrepresent 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 incoculturewithDLL3-negativeHL-60 cells. HumanPBMCswere cocultivatedwith SHP-77 and/orHL-60 cells, and increasing concentrations ofDLL3/CD3 ITE at an E:Tratio 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-77cells and increasing concentrations ofDLL3/CD3 ITE at anE:T of 10:1 for 24 hours. Cellswere stainedwith anti-CD3,AnnexinV, andPI and analyzed byflowcytometry.Each datapoint represents a single measurement. G, Proliferation of T-cells. Representative example.

Hipp et al.





T-cell redirected lysis depended on the tumor cell line and the DLL3expression levels, indicated by the range of EC50 concentrations from5.9 to 24 ng/mL (Fig. 3C; Supplementary Table S3). Immunohisto-chemically detectable DLL3 levels correlated with higher DLL3/CD3ITE potency in the SHP-77 and NCI-H82 cell lines. However, lysisactivity could also be detected using the NCI-H2286 cell line for whichno DLL3 protein could be detected immunohistochemically, indicat-ing the potential ofDLL3/CD3 ITE to induce lysis of cells with very lowDLL3 expression levels.

DLL3/CD3 ITE–induced antitumor activity and modulation ofT-cell infiltration in vivo

In vivo efficacy studies were conducted in a subcutaneous humanSHP-77 xenograft model in CD3þ T-cell humanized mice (Fig. 4Aand 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(Fig. 4C).

In a first efficacy study (Study I), the DLL3/CD3 ITE was admin-istered at doses of 0.025 and 0.25 mg/kg i.v., and tumor growth wasmonitored until day 21. Although treatment with 0.025mg/kg inducedtumor regression in 3 out of 5 animals at day 21 (P < 0.05), a strongerantitumor response was observed with a dose of 0.25mg/kg and tumorregression in all animals (5/5) already at day 9. Notably, four out of fivetumors were still in regression at the end of the study (P < 0.05;Fig. 4A). In a second efficacy study (Study II) with a different humanT-cell donor, DLL3/CD3 ITE monotherapy induced a statisticallysignificant tumor growth inhibition (P< 0.05 at day 15) comparedwiththe vehicle control group. Sustained tumor regressions could beobserved for most of the tumors from day 15 (7/9 animals) until day36 (8/9 animals). Although treatment was stopped after 4 cycles (day22), tumor regression was maintained. Two out of the eight animalswith tumor regression still showed complete tumor clearance (tumornot palpable anymore) at the end of the study (day 36), 14 days after thelast dose (Fig. 4B).

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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 redirectedlysis 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 (Eand F) CD4þ, (E) CD4þ centralmemory, (F) CD4þ effectormemory, (G andH) CD8þ, (G) CD8þmemory, (H) CD8þCD45þ effector, or (I) naïve T-cells. Each datapointrepresents the mean of duplicate measurements, and error bars represent the SD.






Tumor tissues from mice treated with vehicle or 0.25 mg/kgDLL3/CD3 ITE in Study II were collected at the day of termination,and tissue sections were stained with anti-CD3, PD-1, and PD-L1antibodies. Treatment with DLL3/CD3 ITE induced infiltration ofCD3þ T-cells into the SHP-77 xenograft tissue. Infiltrating CD3þ

T-cells upregulated PD-1 and LAG-3, and SHP-77 tumor cellsupregulated PD-L1 (Fig. 5).

In a following study (Study III), we examined the ability of theDLL3/CD3 ITE to modulate the inflammatory environment in thetumor tissue. SHP-77 tumor tissues from animals were collected 8 daysafter administration of one dose of 0.25 mg/kg ITE i.v., and sections offormalin-fixed and paraffin-embedded tissues were immunohisto-chemically stained with anti-CD3, anti-CD4, anti-CD8, anti–PD-1,anti–PD-L1, and anti-cC3 (cleaved caspase 3) antibodies. A statisti-cally significant increase in CD4þ and CD8þ T-cells (Fig. 6A–C) inDLL3/CD3 ITE–treated animals compared with the vehicle-treatedanimals was observed. In this study, we did not see an increase of PD-1expression (Fig. 6D) and only a nonstatistically significant trend of

increased PD-L1 (Fig. 6E) expression. In the tumor cells, a statisticallysignificant increase of cleaved caspase 3 was detected (Fig. 4F). Theproximity of the apoptotic tumor cells to the infiltrating T-cells wasvisualized by costaining of tumor tissues for cleaved caspase 3 andCD3(Fig. 4G).

Dexamethasone only minimally affects DLL3/CD3 ITEThe impact of dexamethasone on the activity of the DLL3/CD3 ITE

was assessed by adding increasing concentrations of dexamethasoneranging from 1 to 1,000 ng/mL to the multiparametric cytotoxicityassay simultaneously with 30 mg/mL DLL3/CD3 ITE. Treatmentwith dexamethasone led to a dose-dependent reduction of DLL3/CD3–induced T-cell redirected lysis of DLL3-positive SHP-77 cellswith one out of three PBMC donors (Supplementary Fig. S4A). TheDLL3/CD3 ITE–induced upregulation of CD25 on CD4þ T-cellswas also reduced with one out of three donors, whereas dexameth-asone did not have an effect on CD25 expression on CD8þ T-cells(Supplementary Fig. S4B and S4C). The DLL3/CD3-induced

Figure 3.

Correlation of DLL3 expression and DLL3/CD3 ITE–induced T-cell redirected lysis.A,DLL3mRNAexpression in 20SCLC tissuesand eight SCLC cell lines. B, IHC detectionof DLL3 using cell pellets of three represen-tative SCLC cell lines. C, Representativeexample of dose-dependent DLL3/CD3ITE–induced lysis of eight SCLC cell lines(E:T 10:1). Cytotoxicity was determined byLDH release relative to a control containing3% Triton X-100 after coculture of PBMCsand eight SCLC cell lines at an E:T ratio of 10:1for 72 hours. Each datapoint represents themean of duplicate measurements, and errorbars represent the SDofmaximal lysis versusDLL3 molecules on the cell surface.

Hipp et al.





secretion of IFNg (Supplementary Fig. S4D) and MCP-1 (Supple-mentary Fig. S4E), by PBMCs in presence of DLL3-positive SHP-77cells, was reduced in the presence of dexamethasone with all threetested donors.

DLL3/CD3 pharmacokinetics in nonhuman primatesTo support pharmacokinetic and safety assessment in nonhuman

primates, the DLL3/CD3 ITE was designed to be cross-reactive tocynomolgusmonkeyDLL3 andCD3. The ability of theDLL3/CD3 ITEto induce T-cell redirected lysis of recombinant cynomolgus monkey

DLL3-expressing HEK293 cells in presence of cynomolgus monkeyPBMCs is shown in Supplementary Fig. S5. Pharmacokinetic studiesshowed that the half-life of the DLL3/CD3 ITE is 10 days.

DiscussionDespite the high unmet medical need for new treatments for SCLC

and the highly attractive tumor-specific expression of DLL3 for atargeted therapy, the development of such a therapy has yet to berealized. The first approach for targetingDLL3with an antibody-based

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 NOGmice reconstituted with human CD3þ T-cells (Study I). B,Antitumor activity of DLL3/CD3ITE in vivo is retained after stop of treatment (Study II). C, Pharmacokinetic profile of DLL3/CD3 ITE in C57BL/6 mice.






approach was Rova-T (rovalpituzumab tesirine), a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrro-lobenzodiazepine dimer toxin (6, 7). In a phase I trial includingpatients with small cell lung or large cell neuroendocrine tumors thathave previously relapsed after platinum-based chemotherapy, 18% ofpatients had a confirmed objective response rate and a manageablesafety profile (7). However, in a phase II study, Rova-T showed onlymodest activity [median overall survival (OS) of 5.7months in patientswith high DLL3 expression vs. 5.6 months in all patients] withassociated toxicities of grades 3 to 5 in third-line and beyond (3Lþ)

SCLC patients with DLL3-positive tumors (22). The toxicitiesobserved with Rova-T, such as thrombocytopenia, nausea, fatigue,vomiting, edema, and effusions (7), have also been observed withvadastuximab 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 toantibody–drug conjugates and have shown high potency and efficacywith a promising safety profile in hematologic malignancies (14, 15).However, the development of bispecific T-cell engagers for solidtumors presents new challenges. Firstly, given the potency of the

Figure 5.

Representative example of IHC analysis of xenografttumors at the end of Study II for CD3, PD-1, PD-L1, andLAG-3 (brown staining).

Hipp et al.





mode of action, a tumor selectively expressed cell surface antigen isrequired to ensure optimal efficacy and safety. So far, bispecific T-cellengagers for solid tumors such as solitomab and MEDI-565 for thetreatment of gastrointestinal cancers did not result in a sufficienttherapeutic window in clinical trials (24, 25), as the antigens EpCAM(epithelial cell adhesion molecule) and CEA (carcinoembryonic

antigen–related cell adhesion molecule 5), respectively, are widelyexpressed. Secondly, the BiTEmolecules have a very short half-life andhave to be administered by continuous intravenous infusion, forexample. As such, opportunities to extend the half-life of T-cellengagers while maintaining the potent pharmacological effect havebeen sought to improve the dosing convenience for patients.

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 inapoptosis 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–positiveT-cells, (E) PD-L1–positive, and (F) cleaved caspase 3 tumor area.G,Representative example of IHC costaining of a xenograft tumor fromonemouse for CD3 (brown)and cleaved caspase 3 (dark red).






In this article, we describe a novel approach for targeting the tumor-selective DLL3 antigen and treatingDLL3-expressing SCLC using T-cellredirection. We have designed a highly DLL3-selective novel IgG-likeT-cell engaging bispecific antibody with an optimal pharmacologicprofile, and antibody-like in vivo stability and half-life. In contrast toAMG 757 (26), a half-life extended BiTE consisting of a bispecific scFvfused to an Fc, the DLL3/CD3 ITE has an IgG-like architecture whichmay pose a lower immunogenicity risk in humans. DLL3/CD3 ITEinduces potent T-cell redirected lysis ofDLL3-positive SCLC cell lines ina dose, E:T ratio, and time-dependent manner, with EC50 values in thelow ng/mL range by redirecting CD4þ and CD8þ T-cells toward DLL3-postitive SCLCcellswithout lysingDLL3-negative target cells.As a resultof the formation of the cytolytic synapse, T-cells secrete proinflamma-tory cytokines and proliferate. In vivo, DLL3/CD3 ITE monotherapyleads to infiltration of T-cells into tumor tissue and converts a nonin-flamed (cold) into an inflamed (hot) tumor environment, leading totumor cell apoptosis and resulting in strong tumor regressions withcomplete tumor eradication in several mice in a human CD3þ T-cellhumanized SHP-77 xenograft model.

In the phase II TRINITY study with Rova-T, cutoffs for analysis ofDLL3 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-positivetumor cells (22). In our study, we could detect DLL3 expression in cellpellets of SCLC cell lines expressingmore than 500DLL3molecules percell using the same qualified IHC assay (Ventana, SP347; ref. 9)previously validated for the determination of DLL3 expression levelsin the Rova-T phase II study (22). In in vitro cytotoxicity assays, celllines with immunohistochemically detectable DLL3 levels correlatewith higher DLL3/CD3 ITE potency, suggesting that the detection ofDLL3 expression using the SP347 IHC assay may be a promisingbiomarker for patient selection that should be evaluated in clinicaltrials.

The FDA recently approved the PD-1 inhibitor pembrolizumabfor the treatment of patients with SCLC who experienced diseaseprogression after two or more prior lines of therapy based on twoclinical studies showing a 19% overall objective response rate (27).In our preclinical studies, we observed an upregulation of PD-(L)1and LAG-3 expression in vivo as a result of the inflamed environ-ment induced by treatment with the DLL3/CD3 ITE, which is in linewith observations from preclinical studies with other T-cell enga-gers, e.g., CEA BiTE, CEA TCB, AMG330, and Her2-TDB (16, 28–30). Furthermore, in a clinical trial, evidence ofenhanced activity of RO6958688, a CEA-targeted T-cell engager,in combination with atezolizumab with a manageable safety profilehas been reported (18). In future studies of the DLL3/CD3 ITE, itwill be important to look at the impact on other immune checkpointmolecules.

As activation of T-cells and cytokine secretion is a characteristic ofthe mode of action of T-cell engagers, which in some cases can alsocause clinical side effects such as fever and hypotension, it might bedesirable to reduce cytokine secretion as much as possible withoutnegatively influencing the cytotoxic potential of T-cells. Glucocorti-coids have been described to inhibit inflammatory responses such ascytokines (31), and a preclinical study using the glucocorticoid dexa-methasone in combination with blinatumomab reported that dexa-methasone can qualify as a potential comedication for TcE thera-pies (32). The data obtained in this study reveal that dexamethasonecan reduce DLL3/CD3 ITE–induced secretion of IFNg and MCP-1,and therefore support the use of dexamethasone to treat potentialinfusion reactions or cytokine release syndromes associated with

DLL3/CD3 ITE treatment, but the treatment could result in reducedcytotoxic potential of DLL3/CD3 ITE.

According to the DLL3-expression profile, the DLL3/CD3 ITE hasthe potential to be developed in additional indications with high unmetmedical need, such as glioma (33), medullary thyroid cancer (34),gastrointestinal neuroendocrine malignancies (35), dispersed neuro-endocrine tumors of the pancreas (34), small cell bladder cancer (36),Merkel-cell carcinoma (37), and neuroendocrine prostate cancer (38).

Taken together, theDLL3/CD3 ITE provides a novel drug candidatefor the treatment of SCLC as monotherapy with upside potential inother neuroendocrine tumor types.

Disclosure of Potential Conflicts of InterestS. Hipp reports grants from Basisprogramm grants of the Austrian Research

Promotion Agency (FFG; 860968, 869530, and 875923) and personal fees fromBoehringer Ingelheim (employment) during the conduct of the study; in addition, shehas filed a patent application for DLL3-CD3 bispecific antibodies. The filed patentapplication is owned by Boehringer Ingelheim and relates to generation and use ofDLL3-CD3 bispecific antibodies. V. Voynov reports personal fees from BoehringerIngelheim (employment) during the conduct of the study; in addition, he has filed apatent application for DLL3-CD3 bispecific antibodies. The filed patent application isowned by Boehringer Ingelheim and relates to generation and use of DLL3-CD3bispecific antibodies. B. Drobits-Handl reports grants from Basisprogramm grants ofthe Austrian Research Promotion Agency (FFG; 860968, 869530, and 875923) andpersonal fees from Boehringer Ingelheim (employment) during the conduct of thestudy. C. Giragossian reports personal fees fromBoehringer Ingelheim (employment)during the conduct of the study. F. Trapani reports grants from Austrian researchPromotion Agency (FFG; 860968, 869530, and 875923) and personal fees fromBoehringer-Ingelheim (employment) during the conduct of the study. J.M. Scheer hasfiled a patent application for DLL3-CD3 bispecific antibodies. The filed patentapplication is owned by Boehringer Ingelheim and relates to generation and use ofDLL3-CD3 bispecific antibodies. P.J. Adam reports grants from Basisprogrammgrants of the Austrian Research Promotion Agency (FFG; 860968, 869530, and875923) and personal fees from Boehringer Ingelheim (employment) during theconduct of the study; in addition, he has filed a patent application for DLL3-CD3bispecific antibodies. The filed patent application is owned by Boehringer Ingelheimand relates to generation and use of DLL3-CD3 bispecific antibodies. No potentialconflicts of interest were disclosed by the other authors.

Authors’ ContributionsS. Hipp: Conceptualization, supervision, methodology, writing-original draft,

writing-review and editing, devised and designed the main conceptual idea of thestudy and wrote the manuscript.V. Voynov: Supervision, methodology, designed themolecule and established the production process. B. Drobits-Handl: Supervision,methodology, writing-review and editing, designed and supervised in vivo studies,and analyzed, assembled, and interpreted data. C. Giragossian: Supervision,methodology, designed and supervised PK studies, and analyzed, assembled, andinterpreted data. F. Trapani: Supervision, methodology, designed and supervisedIHC studies, and analyzed, assembled, and interpreted data.A.E.Nixon: Supervision,methodology, supported the molecule design principles. J.M. Scheer: Supervision,methodology. P.J. Adam: Supervision, writing-review and editing, supported thedesign and conceptual idea of the study and contributed to the writing and criticalevaluation of the manuscript.

AcknowledgmentsWe thank Christoph Albrecht, Ilse Apfler, Kathrin Bauer, Oliver Bergner, Erica

Bolella, Richard Liedauer, Irene Schweiger, Abdallah Souabni, and ChristianWalterskirchen for their experimental support and help for this study, and allcolleagues from Biotherapeutics Discovery for all aspects of molecule discovery.

This study was supported by the Basisprogramm grants of the Austrian ResearchPromotion Agency (FFG; 860968, 869530, and 875923).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 9, 2020; revised May 12, 2020; accepted June 15, 2020;published first June 18, 2020.

Hipp et al.





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Published OnlineFirst June 18, 2020.Clin Cancer Res Susanne Hipp, Vladimir Voynov, Barbara Drobits-Handl, et al. Induces Antitumor Responses in Small Cell Lung CancerA Bispecific DLL3/CD3 IgG-Like T-Cell Engaging Antibody

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