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Selective Tumor Cell Apoptosis and Tumor Regressionin CDH17-Positive Colorectal Cancer Models usingBI 905711, a Novel Liver-Sparing TRAILR2 Agonist A C

Juan Manuel García-Martínez1, Shirley Wang1, Cordula Weishaeupl1, Andreas Wernitznig1, Paolo Chetta1,Catarina Pinto2, Jason Ho3, Darrin Dutcher3, Philip N. Gorman3, Rachel Kroe-Barrett3, Joerg Rinnenthal1,Craig Giragossian3, Maria Antonietta Impagnatiello1, I~nigo Tirapu2, Frank Hilberg1, Norbert Kraut1,Mark Pearson1, and Klaus Peter Kuenkele1

ABSTRACT◥

Activation of TRAILR2 has emerged as an important therapeuticconcept in cancer treatment. TRAILR2 agonistic molecules haveonly had limited clinical success, to date, due either to lack of efficacyor hepatotoxicity. BI 905711 is a novel tetravalent bispecific anti-body targeting both TRAILR2 and CDH17 and represents a novelliver-sparing TRAILR2 agonist specifically designed to overcomethe disadvantages of previous strategies. Here, we show thatBI 905711 effectively triggered apoptosis in a broad panel ofCDH17-positive colorectal cancer tumor cells in vitro. Efficientinduction of apoptosis was dependent on the presence of CDH17, asexemplified by the greater than 1,000-fold drop in potency inCDH17-negative cells. BI 905711 demonstrated single-agent tumorregressions in CDH17-positive colorectal cancer xenografts, aneffect that was further enhanced upon combination with irinotecan.

Antitumor efficacy correlated with induction of caspase activation,as measured in both the tumor and plasma. Effective tumor growthinhibition was further demonstrated across a series of differentcolorectal cancer PDXmodels. BI 905711 induced apoptosis in botha cis (same cell) as well as trans (adjacent cell) fashion, translatinginto significant antitumor activity even in xenograft models withheterogeneous CDH17 expression. In summary, we demonstratethat BI 905711 has potent and selective antitumor activity inCDH17-positive colorectal cancer models both in vitro andin vivo. The high prevalence of over 95% CDH17-positive tumorsin patients with colorectal cancer, the molecule preclinical efficacytogether with its potential for a favorable safety profile, support theongoing BI 905711 phase I trial in colorectal cancer and additionalCDH17-positive cancer types (NCT04137289).

IntroductionTumor necrosis factor–related apoptosis-inducing ligand

(TRAIL) is a homotrimeric protein that binds to and activatesspecific cell surface-anchored death receptors, TRAIL-receptor 1(TRAILR1) and TRAIL-receptor 2 (TRAILR2). Ligand bindingleads to receptor-mediated activation of the initiator caspase-8,which subsequently cleaves and activates the effector caspases-3 and-7 to execute apoptotic cell death (1). TRAIL induces apoptosis in abroad range of cancer cell lines and xenograft models, withoutexhibiting significant systemic toxicity in mice or nonhumanprimates (2, 3), driving interest in exploring the potential of theTRAIL pathway as an anticancer therapy.

Several pro-apoptotic agonists against TRAILR2, including mono-clonal antibodies, have been reported. Despite promising preclinicaldata, none of the TRAILR2 agonistic antibodies demonstrated signif-

icant clinical efficacy (4–10). Initial TRAILR2 antibodies required asecondary crosslinking mechanism to enhance their inherently weakagonistic properties (11, 12). Novel strategies were used to induce amore potent and direct activation of TRAILR2. One, a novel TRAILR2agonist tetramer nanobody, TAS266, was able to bind TRAILR2 acrossseveral trimeric receptors leading to TRAILR2 hyper-clustering. Thiscompound demonstrated excellent preclinical in vitro and in vivoactivity (13); however, a clinical trial with TAS266 was terminatedearly due to drug-induced liver toxicity (14), suggesting that the liver isinherently sensitive to potent TRAILR2 activation in humans. Thesedata prompted the development of compounds capable of potentlyinducing TRAILR2 oligomerization and apoptosis in tumor cells whilesparing liver cells.

CDH17 is a cell surface molecule member of the Cadherin super-family of adhesion molecules. In contrast with classical cadherins, itscytoplasmic domain is very short (20 amino acids) and apparentlylacks binding sites necessary for interaction with the cytoskeleton.Under physiological conditions, CDH17 is proposed to regulate thedirection and efficiency of epithelial water transport via trans-interaction with cadherins of neighboring cells (15). CDH17 is over-expressed in the plasma membrane of colorectal and gastrointestinaladenocarcinomas (16) and has been reported as both a tumor bio-marker and oncogene (17). Notably, CDH17 is not expressed inmouseor human liver (18).

In this report, we characterize BI 905711, an optimized CDH17:TRAILR2 bispecific molecule that binds to CDH17 and uses it as ananchor to trigger potent and tissue selective TRAILR2 activation.Accordingly, we demonstrate that BI 905711 is highly efficacious inseveral colorectal xenograft models and its efficacy is strictly depen-dent on CDH17 expression.

1Boehringer Ingelheim Cancer Research Therapeutic Area, Vienna, Austria.2Boehringer Ingelheim Cancer Immunology and Immune Modulation, Vienna,Austria. 3Boehringer Ingelheim Biotherapeutics Discovery Research, Ridgefield,Connecticut.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Juan Manuel García-Martínez, Boehringer IngelheimRCV GmbH and Co. KG, Vienna A-1121, Austria. Phone: 43-180-105-2070; E-mail:juan_manuel.garcia-martinez@boehringer-ingelheim.com

Mol Cancer Ther 2021;20:96–108

doi: 10.1158/1535-7163.MCT-20-0253

�2020 American Association for Cancer Research.

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Materials and MethodsCell lines and cell culture

Cells were grown in cell culture flasks until 70%–80% confluenceusing ATCC-recommended media. Cells were cultured at 37�C and5% CO2, except when Leibovitz’s L-15 medium was used. GP2dCDH17 knockout (KO) cells were generated using the CRISPR/Cas9system. Briefly, three different Cas9 genome editing constructs encod-ing gRNAs for CDH17 were obtained from GenScript. CDH17CRISPR gRNA constructs were transfected to GP2d cells using theX-tremeGENEHPDNA transfection reagent kit (Roche, 0636236001)following the manufacturer’s instructions. Three days post-transfec-tion, cells were cloned. OVCAR-3–mKate2 cells were generatedby transduction with Lentiviral-based labeling reagents (IncucyteNucLight Red, 4625) and cultured in RPMI medium supplementedwith 10% FBS plus 1 mg/mL Puromycin (Sigma, 8833).

Test compounds and molecules sequenceIrinotecan was purchased from Accord Healthcare GmbH

(20 mg/mL, PZN 12422462). TAS 266’ tetrameric nanobody expres-sion vector was produced as described in WO 2011/098520, SEQ IDNo: 032. The sequence source for Lexatumumab’ was the WorldHealth Organization, as listed in the Recommended internationalnonproprietary names, list 57 (19). BI 905711 sequence is describedin WO 2018/115231, SEQ ID No: 212 and SEQ ID No: 218. Thebispecific negative control was generated as a BI 905711-based bis-pecific molecule where the CDH17 building block was substituted byan irrelevant sequence. All molecules were produced at BoehringerIngelheim.

BI 905711 expression and analytical dataBI 905711 was expressed transiently in the CHO-E/pTT5 system.

Transient expression titer ranged from 30 to 60 mg/L in 10 days. Finalsample was 98.9%monomer� 1.0% by analytical SEC. The endotoxinlevel in the purified sample was determined by LAL cartridges (CharlesRiver Laboratories); a standard release threshold of <1 EU/mg ofprotein was applied. Molecular weight of intact BI 905711 wasobserved to be 201 KDa, analysis using LC-TOF-MS.

FACS analysesCells were seeded at 2–3� 106 cells in T80 (ThermoFisher, 178905)

and cultured overnight at 37�C and 5% CO2. The next day, cellswere detached with Versene solution (Gibco, 15040033), centri-fuged at 300 � g for 5 minutes and resuspended in FACS buffercontaining phosphate-buffered saline (PBS; Gibco, 20012–019), 1%FBS, and 0.01% sodium azide (Sigma, 71289). Approximately 2 �105 cells in 50 mL were aliquoted per well into round-bottom 96-wellplates (Costar, 3799), followed by adding anti-CDH17 or anti-TRAILR2 (produced at Boehringer Ingelheim according to WO2010/123874, SEQ ID No: 038 and SEQ ID No: 049 and WO 2002/094880, SEQ ID No: 029 and SEQ ID No: 031), or human IgG(Sigma, I5154) at final concentration of 1 mg/mL. Staining wasperformed on ice for 30 minutes. Cells were washed once with coldFACS buffer, and further incubated with 100 mL per well ofallophycocyanin (APC)-conjugated anti-human IgG Fc Antibody(1:40 pre-diluted in FACS buffer; Biolegend, 409306) on ice foranother 30 minutes. Finally, cells were washed twice with FACSbuffer and analyzed with a BD FACSCanto II system (BD Bios-ciences). A minimum of 10,000 events per well were collected usingFACSDiva software and analyzed using FlowJo version 10.1. Wellscontaining buffer only were included as unstained controls.

CellTiter-Glo luminescent cell viability assayThe cells were plated at 1,000–7,000 cells in 50 mL per well in white

opaque 96-well microtiter plates (PerkinElmer, 6005680) and incu-bated overnight at 37�C and 5% CO2 (except for cells grown inLeibovitz’s Medium). On the following day, serial dilutions of thecorresponding agonists were added and cells were then transferredback to incubator and incubated for an additional 24 or 72 hours asindicated. Cell viability was measured using the CellTiter-Glo lumi-nescent cell viability kit (Promega, G7571) according to the manu-facturer’s instructions. Luminescent signal was recorded with EnSightmultimode plate reader (PerkinElmer). Average background valueswere subtracted from each measurement.

In vitro Caspase-Glo 8 and Caspase-Glo 3/7AssayCOLO 205 cells were plated at 5,000 in 50 mL per well in white

opaque 96-well microtiter plates (PerkinElmer, 6005680) and incu-bated overnight at 37�C and 5% CO2. On the following day, agonistswere added and cells were incubated for an additional 2, 4, 6, or16 hours as indicated. Caspase 8 or caspase 3/7 was determined usingthe Caspase-Glo 8 kit (Promega, G8200) or Caspase-Glo 3/7(Promega, G8090), respectively. Luminescent signal was recordedwith EnSight multimode plate reader (PerkinElmer). Average back-ground values were subtracted from each measurement.

Co-culture in vitro viability assayDLD-1 and OVCAR-3-mKate2 cells were pre-mixed in a 3:1 ratio

(6,000:2,000 cells), 1:1 ratio (4,000:4,000 cells) or 1:3 ratio (2,000:6,000cells) in suspension and plated in 100 mL per well in clear-bottom96-wellmicrotiter plates. In additional plates, OVCAR-3–mKate2 cellsalone were seeded at 4,000 cells in 100 mL per well and used as control.After overnight incubation at 37�C, agonists at a 3-fold of the finalconcentration were added in 50 mL into each well. The plates were thenplaced in the IncuCyte S3 Live-Cell Analysis System (Sartorius) forimage acquisition. Images in experimental wells were captured every2 hours in both phase contrast and the red fluorescent channels. By theuse of the integrated software, the red fluorescent nuclei counts weremeasured to represent the viable OVCAR-3–mKate2 cell number ineach well.

Mouse xenograftsGP2d, COLO 205, and DLD-1 tumors were established by subcu-

taneous injection of tumor cells in 6-week-old athymic femaleBomTac:NMRI-Foxn1nu purchased from Taconic, Denmark. Stan-dardized irradiated diet (PROVIMI KLIBA) and autoclaved tap waterwere provided ad libitum. Subcutaneous microchips implanted underisoflurane anesthesia were used to identify each mouse. Mice wererandomly distributed between the treatment and the vehicle controlgroup when tumors were well established (150–200 mm3) and treat-ment was initiated. A vehicle control group (citrate buffer) wasincluded and applied at the same volume/route. Seven to 10 animalswere included per treatment group. Tumor volume was determinedthree times a week using a caliper and body weight was measured dailyas an indicator of tolerability. Animals were sacrificed at the end of thestudy or when the tumors reached a size of 1,500 mm3. Animals withnecrotic tumors or a bodyweight loss<18%were euthanized for ethicalreasons. All xenograft experiments have been approved by the relevantInstitutional Review Board.

Patient derived colorectal cancer xenograftThe human patient derived colorectal cancer xenografts were

established and characterized at EPO (Experimental Pharmacology

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and Oncology, Berlin-Buch GmbH, Germany; ref. 20). Main clinicalcharacteristic of the tumors and a description of the efficacy exper-imental procedures are described in the Supplementary Material andMethods.

RNA data and human samplesmRNA-expression (TPM, RNAseq) in non-diseased samples and

various cancer entities was obtained from the Genotype-TissueExpression (GTEx) project and the Cancer Genome Atlas database,respectively.

FFPE-blocks of liver samples from different donors with transitionto colorectal cancer metastases were acquired from Indivumed GmbH(Falkenried 88, D-20251 Hamburg, Germany). Commercially avail-able FFPE human normal tissue microarrays and FFPE-blocks ofnormal human organs (Supplementary Materials AndMethods) fromvarious biobanks, meeting all ethical standards and their use foroncological research has been authorized by the donors via informedwritten consent (templates approved by the relevant InstitutionalReview Boards), were used.

IHC for cleaved caspase 3, cleaved caspase 8, CDH17 andTRAILR2

Two-mm thick serial sections of each FFPE-block were prepared ona microtome, put on glass slides and dewaxed. Unmasking solution(Vector Laboratories #H3300) was applied at 121�C/1 bar, followed byblocking steps using 3% H2O2 and subsequently normal goat serum(Vector Laboratories #S-1000) in PBS/2% BSA. The serial sectionswere incubated with the primary antibody [anti-cleaved caspase-3(Asp175) Cell Signaling Technology #9661; anti-cleaved Caspase-8(Asp391; 18C8) Cell Signaling Technology #9496; anti-CDH17 Clone141713 from R&D Systems #MAB1032 and anti-TRAILR2, CellSignaling Technology, #8074] for 1 hour at room temperature, fol-lowed by detection reagent (EnVisionþ, HRP, Rabbit, DAKO #4002)and incubation in DAB-solution. Counterstain was performed usinghematoxylin. Washing steps in PBS were performed where appropri-ate. Corresponding isotype controls were performed in parallel.

Plasma activities of cleaved caspase 3/7EDTA-plasma samples were taken during final sacrifice and imme-

diately frozen in liquid nitrogen. They were stored at �20C untilevaluation with the Caspase-Glo 3/7 assay from Promega # G8091.According to the manufacturer’s instructions.

ResultsCDH17 as a liver-sparing anchor target for TRAILR2 activation incolorectal cancer

We aimed to identify cell surface proteins absent in non-neoplasticliver samples that were expressed at significant levels in one or moremajor cancer types.

We analyzed 188 non-diseased liver tissue samples using mRNAexpression as a classifier. We selected CDH17, as none of the samplesanalyzed had a value higher than 0.24 TPM, with 27% of the samples(51 of 188) displaying less than 0.01 TPM (Fig. 1A).We then examinedthe distribution of CDH17 in normal human organs by IHC using apanel of FFPE-blocks. CDH17 expression was observed in the epi-thelium of the small intestine, colon, and pancreatic ducts. In agree-ment with the RNAanalysis, noCDH17 expressionwas detected in theliver (Fig. 1B).

High expression levels of CDH17 and TRAILR2 were observed incolon (455 samples) and rectum (167 samples) adenocarcinomas (97%

of the samples have > 10TPM for both targets). Additional malignan-cies of the gastrointestinal tract, stomach adenocarcinoma (373 sam-ples), pancreatic adenocarcinoma (178 samples), and esophagealcarcinoma (162 samples) also included relevant populations ofCDH17-high/TRAILR2-high cases (66, 42 and 38% of the samples>10TPM for both targets respectively; Fig. 1A). A significantly lowernumber of related normal tissues (colon, stomach, pancreas andesophagus) displayed high levels of CDH17 and TRAILR2 RNAexpression (Supplementary Fig. S1). IHC analysis of a series ofcolorectal cancer hepatic metastases (n ¼ 10) confirmed widespreadexpression of CDH17. Heterogeneous to diffuse TRAILR2 expressionwas detected in 6 of 10 cases (Fig. 1C; Supplementary Table S1). Noexpression of TRAILR2 by IHC was observed in any normal humantissue analyzed.

Generation of BI 905711, a novel bispecific anti-CDH17 andanti-TRAILR2 antibody

BI 905711 is a novel bispecific molecule with bivalent CDH17 andTRAILR2 binding. The CDH17-building block was derived from amouse hybridoma humanized lead and comprises the conventionalmonoclonal part of the bispecific molecule. The TRAILR2 buildingblock is attached as a scFv to the C-terminus of the molecule. The twobuilding blocks are coupled via a six amino acid Gly-Ser linker. TheL234A/L235A mutation, ablating binding to FcgR and comple-ment (21), was incorporated to avoid CDH17-independent cross-linking (Fig. 2A).

When evaluated using surface plasmon resonance, BI 905711 bindsto human TRAILR2 and human CDH17 with KD values of 176 and33.9 nmol/L, respectively, resulting in an almost 50-fold difference inaffinity. Kinetic and steady-state measurements are summarized inSupplementary Table S2.

BI 905711 does not bind to human TRAILR1, human CDH16, orunrelated charged proteins. Human and cynomolgus TRAILR2 andCDH17 extracellular domain proteins display between 90% and 95%sequence identity. BI 905711 is cross-reactive to the correspondingTRAILR2 and CDH17 cynomolgus orthologues, with similar KD

values (Supplementary Table S2). Mouse and rat show weak sequenceidentities in their extracellular domain compared with the humanproteins [10% and 80% (mouse), 29% and 80% (rat) for TRAILR2 andCDH17, respectively].

BI 905711 induces potent tumor cell apoptosis inCDH17-positive colorectal cancer cell lines in vitro

The COLO 205 cell line was selected as a representative CDH17/TRAILR2 double-positive colorectal cancer cell line. Membraneexpression of both proteins was confirmed by FACS (Fig. 2B). Twoclinical TRAILR2 agonists, the monoclonal antibody lexatumumab(refs. 4, 5; Lexatumumab’), and the more potent tetrameric nanobodyagonist, TAS266 (ref. 14; TAS266’) were included in the assay ascontrols. BI 905711 was 1,000-fold more potent (IC50) than Lexatu-mumab’ (0.0012 vs. 1 nmol/L), being comparable in potency (IC50)and inducing equivalent decreased cell viability (%) when comparedwith TAS266’ (0.0012 nmol/L/91% vs. 0.0024 nmol/L/94%; Fig. 2C).The CDH17 building block of BI 905711, engineered as a conventionalmonoclonal antibody, did not affect the viability of COLO 205 cells(Fig. 2C).

Intracellular caspase activity was assessed to confirm activation ofthe extrinsic apoptotic pathway. Caspase-8 and caspase-3/7 activitywas significantly increased upon treatment with 0.01 nmol/L of BI905711, the concentration inducing maximal effect on cell viability, atthe earliest time points analyzed (2 and 6 hour, respectively; Fig. 2D).

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In a co-culture system of immune activation, in which dendritic cellactivation is measured upon tumor cell death, no effects were observedwith BI 905711, in contrast with culture in the presence of LPS(Supplementary Fig. S2).

Correlation between BI 905711 sensitivity and protein expression ofCDH17 and TRAILR2 was evaluated across a panel of 24 CDH17/TRAILR2 double-positive colorectal cancer cell lines. Minimal per-centage of control (minPOC), corresponding to the minimal number

A

CBSmall intestine (CDH17 IHC) Colon (CDH17 IHC)

Pancreatic ducts (CDH17 IHC) Liver (CDH17 IHC)

CRC metastasis (CDH17 IHC)

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Figure 1.

CDH17 is expressed inmetastases of colorectal cancerwith TRAILR2whereas absent in non-neoplastic human liver tissue.A,mRNA-expression (TPM, RNAseq) levelsfor TRAILR2 (y-axis) and CDH17 (x-axis) in non-diseased liver samples and various cancer entities. The number of samples included in the analysis for each group isindicated in parenthesis. Representative images of CDH17 IHC staining in different non-neoplastic human tissues (B) and CDH17 and TRAILR2 IHC staining in hepaticmetastases of colorectal cancer (C).

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Figure 2.

In vitro characterization of BI 905711. A, Schematic representation of BI 905711. B, Representative FACS histograms showing the relative fluorescence intensity ofCOLO 205 cells stainedwith anti-CDH17 or anti-TRAILR2 antibody (black line) comparedwith the corresponding isotype control (gray histogram). C,COLO 205 cellswere treated for 24 hours with different concentrations of BI 905711, the CDH17 building block of BI 905711 as a conventional monoclonal antibody (CDH17 mAb),Lexatumumab’ and TAS266’ and the effect on cell viability wasmeasured. Curves are representative of three independent experiments. Data are expressed as meanrelative values compared with untreated control plus standard deviation. D, Activation of caspase-8 (left) and caspase-3 (right) after the treatment with BI 905711(0.01 nmol/L) 2/4 and 6/16 hours, respectively. For each time-point, an unpaired t test was performed between the treated values and the untreated controls. In this,��� , P < 0.001. E, XY plot showing the correlation between the in vitro activity of BI 905711 (y-axis) and TAS266’ (x-axis) in a panel of 24 CDH17/TRAILR2 double-positive colorectal cancer cell lines. minPOC corresponds to the minimal number of viable cells detected at any given concentration compared with non-treatedcontrols. The R2 and P values for the corresponding Pearson correlation are shown. Lexatumumab’ and TAS266’ were synthesized from published sequences.

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of viable cells detected at any given compound concentration com-pared with non-treated controls, was used. BI 905711 minPOCinversely correlated with both TRAILR2 and CDH17 expression(Supplementary Fig. S3 and Supplementary Table S3). 54% of celllines were defined as sensitive to BI 905711 (minPOC <35%), 25% ofcells displayed intermediate sensitivity (minPOC between 35% and60%), and 21% of cells were insensitive (minPOC >60%; Fig. 2E,y-axis). The minPOC of BI 905711 and the reference moleculeTAS266’ significantly correlated (Fig. 2E).

BI 905711 is highly selective toward CDH17-positive colorectalcancer cells

CDH17-negative GP2d clones (CDH17-KO GP2d) were generatedto assess the selectivity requirement for BI 905711. Loss of CDH17expression, identified by sequencing, was confirmed by western blotand assessed by FACS analysis (Fig. 3A and C). In agreement with thedifferences in affinity toward CDH17 and TRAILR2, BI 905711 FACSbinding was strongly reduced in CDH17-KO GP2d, signal detected athigher concentrations corresponding to TRAILR2 binding (Fig. 3Eand F). Deletion of CDH17 led to an approximately 1,000-folddecrease in cell killing by BI 905711 (IC50 GP2D and CDH17-KOGP2D cells 0.0002 versus 0.250 nmol/L, respectively; Fig. 3B and D).No differences in TRAILR2 membrane expression or sensitivity toTAS266 was observed (IC50 GP2D and CDH17-KO GP2D cells0.0027 vs. 0.0022 nmol/L, respectively; Fig. 3A–D).

Hep G2 cells, derived from a hepatocellular carcinoma, were usedas a surrogate for TRAIL-sensitive hepatocytes. This cell line wasselected as TRAILR2 expression was not observed in culturedsystems derived from primary human hepatocytes, similar observa-tions were described in ref. 22. TRAILR2 membrane expression andthe lack of CDH17 were confirmed by FACS (Fig. 3G). In contrastwith the reference TAS266’, no effect was observed upon treatmentof HepG2 with any concentrations of BI 905711 concentrationtested (up to 100 nmol/L; Fig. 3H).

In vivo efficacy of BI 905711 in CDH17-positive colorectal cancerxenograft mouse models

Both CDH17 and TRAILR2 are homogeneously expressed inGP2d xenografts (Fig. 4A). In this model, a single dose of BI 905711led to sustained tumor regressions up to 30 days following treat-ment. Subsequent administration at day 36, when only 2/8 tumorswere still in regression, resulted in tumor growth delay that lasteduntil the end of the experiment on day 52 (Fig. 4B). Increasedcleaved caspase-3 and cleaved caspase-8 (IHC) 24 hours after initialadministration indicated induction of tumor cell apoptosis(Fig. 4C). Cleaved caspases 3/7 activity in the plasma correlatedwell with intra-tumoral apoptosis. A statistically significant increaseof cleaved caspases 3/7 plasma activity was also detected 24 hoursafter first treatment (Fig. 4D). Increased caspase activation wasagain detected following the second administration of BI 905711(Supplementary Fig. S4).

DLD-1 cells are less sensitive to TRAILR2 agonism in vitro(Fig. 2E), but display similar homogeneous CDH17 and TRAILR2expression (Fig. 4E). Following initial regression, tumor regrowth wasobserved 10 days after administration of BI 905711. Subsequentadministration of BI 905711 (day 14), caused additional tumor growthdelay, TGI of 67% by day 22 when the control group had to beterminated (Fig. 4F). Combination of BI 905711 and the standard-of-care, irinotecan resulted in a statistically significant tumor growthcontrol, as compared with vehicle and either compound as mono-therapy (Fig. 4F).

BI 905711 activates TRAILR2onbothCDH17-positive tumor cellsand adjacent tumor cells

A bispecific modality, such as BI 905711, can potentially induceapoptosis in both a cis (same cell) as well as trans (adjacent cell)fashion. Evidence for cis-activation was initially supported by the>99% decrease in cell viability observed in cell lines such as COLO 205(Fig. 2C) and GP2d (Fig. 3B) in vitro. Confidence in this hypothesiswas strengthened by tracking individual GP2d cells after BI 905711administration. Analysis of sequential images following treatmentwith BI 905711 administration demonstrated induction of apoptosisin individual cells (Fig. 5A).

The ability of BI 905711 to activate TRAILR2 in adjacent tumor cells(trans-activation) was assessed in Red Fluorescent Protein (RFP)-expressing, CDH17-negative, human ovarian carcinoma cell lineOVCAR-3 (OVCAR-39–mKate2). Similar to parental cells,OVCAR-3–mKate2 cells, were sensitive to TAS266’ but unresponsiveto BI 905711 (Fig. 5B). An additional negative control, a bispecificmolecule where the CDH17-binding domain of BI 905711 was substi-tuted by an irrelevant sequence, had no effects on OVCAR-3–mKate2cell number (Fig. 5B).

Consistent with the hypothesis that BI 905711 is able to mediateTRAILR2 trans-activation, a reduction in the number of detectedOVCAR-3–mKate2 red cells was observed when co-cultured withCDH17-positive DLD-1 cells, in the presence of antibody. Thisreduction was detected in all DLD-1:OVCAR-3–mKate2 seedingratios tested and was comparable with the reduction observed afterTAS266’ administration. Importantly, treatment with the negativecontrol bispecific molecule had no detectable effect (Fig. 5B). Similarresults were observed when images captured over time followingadministration of the different compounds were analyzed (Fig. 5C).

The implication that BI 905711-induced trans-activation canenhance antitumor activity was evaluated in xenograft mouse modelswith heterogeneous CDH17 expression. COLO 205 cells were provenCDH17-positive when cultured in vitro, and represented one of themost sensitive cell lines to BI 905711 administration amongst the 24colorectal cancer cell lines tested (Fig. 2B and E). However, whengrown as a xenograft, heterogeneous CDH17 expression was observed(5%–10% of viable tumor; Fig. 5D). Despite this, BI 905711 admin-istration induced initial tumor regressions and strong growth inhibi-tion by the end of the experiment (day 14; Fig. 5E). Accordingly,increased cleaved caspase-8 and cleaved caspase-3 was observed24 hours after BI 905711 administration. Using a double staining forcleaved caspase-3 and CDH17, we showed that the cleaved caspase 3–positive area in the treated tumors extended beyond the boundary ofthe CDH17-positive area (Fig. 5F). To demonstrate that BI 905711effect was dependent on CDH17 targeting, mice were also treated withthe bispecific molecule where the CDH17 binding domain was substi-tuted by an irrelevant sequence. This negative control failed todemonstrate in vivo efficacy in COLO 205 xenografts, even at doselevels providing over 35-fold plasma concentration when comparedwith BI 905711 efficacious dose (Supplementary Fig. S5).

Effective tumor growth inhibition after single-agent BI 905711administration in colorectal cancer PDX models

The in vivo efficacy of BI 905711 in a more disease-relevantcontext was assessed across a panel of three colorectal cancer PDXmodels. Expression of TRAILR2 and CDH17 was analyzed by IHCafter tumor establishment. In alignment with our prevalence data inhuman samples (Fig. 1), all three models showed a strong andhomogeneous CDH17 expression, with more variation in TRAILR2intensity and expression pattern (Fig. 6A, C, and E). Single-agent

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BI 905711 activity is dependent on CDH17 expression. Representative FACS histograms showing the relative fluorescence intensity of wild-type GP2d (A), CDH17-KOGP2d (C), and Hep G2 (G) cells stained with anti-CDH17 or anti-TRAILR2 antibody (black line) compared with the corresponding isotype control (gray histogram).Wild-type GP2d (B), CDH17-KOGP2d (D), andHepG2 (H) cellswere treated for 72 hourswith different concentrations of BI 905711 and TAS266’ and the effect on cellviability was measured. Data are expressed as mean relative values compared with untreated control plus standard deviation. TAS266’ was synthesized frompublished sequences. E and F, Binding of BI 905711 to wild-type GP2d (E) and CDH17-KO GP2d (F) cells by FACS. Data are expressed as APC median values for thedifferent concentrations of BI 905711 (black circles) and human IgG control (white circles). Curves are representative from three independent experiments.

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administration of BI 905711 on a q14d schedule, resulted insignificant antitumor efficacy in all three models (Fig. 6B, D, andF), with >50% of the tumors remaining in regression on terminationof the experiment (days 18 and 28, respectively) in two of themodels analyzed (Fig. 6B and F).

DiscussionHere, we report the preclinical antitumor efficacy of BI 905711, a

novel bispecificmolecule with bivalent CDH17 andTRAILR2 binding.BI 905711 binds to CDH17 and uses it as an anchor to trigger potent

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BI 905711 induced intratumoral cas-pase activation resulting in antitumorefficacy in CDH17-positive colorectalcancer xenograft mouse models. A,CDH17 and TRAILR2 IHC inGP2d xeno-graft tumors. B, BI 905711 administra-tion (1.67 mg/kg) in GP2d xenografttumors at days 1 and 36. C, IHC forcleaved caspase-8 and cleaved cas-pase-3 24 hours after the first treat-ment and (D) plasma activities forcleaved caspases 3/7 24 hours afterfirst treatment in GP2d xenografttumors. An unpaired t test was per-formed. In this, � , P < 0.05. E, CDH17and TRAILR2 IHC in DLD-1 xenografttumors. F, Antitumor activity ofBI 905711 (1.5 mg/kg, q14d) aloneand in combination with irinotecan(15 mg/kg, q7d) in DLD-1 xenografttumors. All conditions were run withinthe same experiment and individualtreatment groups are representedindividually versus the vehicle group.Dotted lines indicate the day of treat-ment. BI 905711 administration led toan initial tumor regression. Tumor vol-ume values at day 22 are representedas the median plus interquartile range.One-sided non-parametric Mann–Whitney–Wilcoxon U tests wereapplied to compare each treatmentgroup with the control. The P valueswere adjusted for multiple compari-sons according to Bonferroni–Holmand the level of significance was fixedata¼ 5%. In this, �� ,P<0.01 comparedwith the vehicle group. ##, P <0.01; #, P< 0.05 compared with BI 905711 plusthe irinotecan group.

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Cis-and trans-activation of TRAILR2 induced by BI 905711. A, Sequential images of individual GP2d taken every 2 hour after the administration of BI 905711(0.1 nmol/L). Arrows included highlighting single cells. B, OVCAR-3–mKate2 cells were cultured alone or co-cultured with DLD-1 cells at 1:3, 1:1, and 3:1 ratios. Bargraphs represent OVCAR-3–mKate2 cell line viability 48 hours after the administration of a bispecific negative control, BI 905711 and TAS266’ (0.02 nmol/L for allmolecules). Data are expressed as the number of red fluorescent nuclei mean relative values compared with untreated control plus standard deviation. For eachcondition, a one-way ANOVA plus Tukey test for multiple comparison was performed. In this, ��� , P < 0.001; �� , P < 0.01; � , P < 0.05; and ns, not significant. C,Representative images of OVCAR-3–mKate2 cells cultured alone (bottom) or in co-culturewithDLD-1 cells (1:1) at time0 and 12, and 24 hours after treatmentwith thedifferentmolecules. TAS266’was synthesized from published sequences.D,CDH17 and TRAILR2 IHC in COLO 205 xenograft tumors. E, Single dose administration ofBI 905711 (5 mg/kg) in COLO 205 xenograft tumors. Dotted lines indicate the day of treatment. F, Cleaved caspase-3 (brown) and CDH17 (red) IHC in vehicle and BI905711 (1.67 mg/kg)–treated COLO 205 xenograft tumors 24 hours after the treatment. Double staining (left), and the digitally separated CDH17 (middle) andcaspase-3 (right) images are provided.

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BI 905711 antitumor activity in colorectalcancer PDX models. Tumor modelsderived from three different patientswithcolorectal cancer: Co10749 (A and B),Co10809 (C and D), and Co10376 (E andF). Main clinical characteristic of thepatients and tumors of origin are provid-ed in Supplementary Material and Meth-ods. A, C, and E, CDH17 and TRAILR2 IHCin representative tumor samples. B, D,and F, Antitumor activity of BI 905711(1.2 mg/kg, q14d) in all three tumors.Dotted lines indicates day of treatment.Multiple t tests were applied to comparecontrol and treated groups along thedifferent days of treatment. The P valueswere adjusted for multiple comparisonsaccording to the Bonferroni–Dunn meth-od and the level of significance was fixedat a ¼ 5%. In this, ��� , P < 0.001; �� , P <0.01 compared with the vehicle group.

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TRAILR2 activation, resulting in apoptosis and tumor regression in aCDH17-expression dependent manner.

In agreement with published data, CDH17 expression was observedin 100% of the colorectal cancer samples analyzed. In addition,published studies described a high level of concordance in CDH17expression between primary colorectal adenocarcinomas and theircorresponding lung and lymph node metastases (23), suggesting thatCDH17 expression is preserved during the metastatic process.TRAILR2 expression in several solid cancers, including colorectaltumors, has also been described previously (24–26). In agreementwith these published studies, we were able to confirm expression ofTRAILR2 in colorectal cancer hepatic metastases. Altogether, thesedata point to colorectal cancer as a relevant cancer type to evaluate theantitumor activity of BI 905711. Evidence of co-expression of bothtargets in a large subset of gastric and pancreatic cancers suggest that BI905711 may have additional activity in these indications (16, 27–31).Preclinical studies to confirm this hypothesis are currently ongoing.

Using CDH17 as an anchor, BI 905711 is approximately 1,000-foldmore potent than conventional TRAILR2 targeting antibodies, exam-ples of which displayed weak and inconsistent signs of efficacy inhuman clinical trials (4, 5). The activity of these anti-TRAILR2antibodies can be enhanced by secondary crosslinking (32) and theirantitumor activity relies on interaction with Fcg receptors (11). Thesuperior TRAILR2 agonistic activity of BI 905711, independent of anysecondary mechanism, can overcome this deficiency increasing itsprobability to translate into a robust therapeutic activity in patients.

BI 905711 displayed comparable potency with TRAILR2 second-generation agonists, developed to work independently of a secondarycross-linkingmechanism, across a panel of CDH17-positive colorectalcancer cells. This potent in vitro activity translated into single-agentantitumor efficacy in different CDH17-positive colorectal cancerxenograft models, including patient-derived xenografts. Importantly,BI 905711 activity is strictly dependent on CDH17 expression, asdeletion of CDH17 resulted in a approximately 1,000-fold loss ofpotency. In addition, BI 905711 had no significant effect on theviability of Hep G2 cells, a liver derived CDH17-negative cell lineused as surrogate for TRAIL-sensitive hepatocytes. Collectively, andconsidering the lack of detectable CDH17 protein in non-neoplasticliver tissue, our data suggest that BI 905711 has the potential to triggerpotent tumor cell apoptosis while improving the therapeutic window,by sparing hepatotoxicity associatedwith the activation of TRAILR2 inthe liver.

Currently, there are three biologics (ABBV-621, GEN1029, andINBRX-109) designed to induce superior clustering of TRAILR2 inphase-I clinical trials (NCT03082209, NCT03576131, andNCT03715933). As none of them includes a domain or motif thatspecifically enables sparing the targeting of normal liver cells, it isunclear how well these new molecules will overcome the reportedclinical hepatotoxicity associated with TRAILR2 agonism in patients.

Targeted strategies coupling TRAILR2 activation to distinct tumorrestricted antigens have been previously proposed (33–35). This hasresulted in initiation of clinical trials with RG-7386 (33), a bispecificmolecule that co-targets fibroblast activation protein alpha (FAP) toincrease tumor specificity of TRAILR2 activation. The phase-I trial ofRG-7386 has been completed and initial data presented showed asingle partial response among 32 heavily pretreated patients withNSCLC (36). FAP is not expressed directly in the cancer cells inepithelial tumors, such as colorectal cancer, but rather inmesenchymaltumors and on activated fibroblasts, which are prevalent in the tumorstroma (37, 38). Thus, with the exception of mesenchymal tumors,killing can be only achieved at sites of contact between tumor and

stromal cells, where RG-7386 bound to FAP on stromal cells canactivate TRAILR2 on adjacent tumor cells (trans-activation). Tumorcells that are not in direct contact with activated fibroblasts will not beaffected by this treatment. In contrast, BI 905711 is directed byCDH17, an anchor target expressed on tumor cells, increasing thetotal tumor area that can be targeted (cis- and trans-activation).Altogether, these data suggest that the mode of action of BI 905711is a promising approach to target colorectal cancer and overcome thelimitations of current TRAILR2 agonists.

High expression of both targets appears beneficial for BI 905711activity and the use of CDH17 and/or TRAILR2 expression as putativebiomarkers will be further explored in the ongoing clinical trial. Weanticipate that not only the level of CDH17 expression on individualtumor cells, but also, the proportion and distribution of CDH17-positive cells across the entire tumor mass will determine BI 905711activity. However, we believe that BI 905711 can still offer benefit topatients with rather low fractions of CDH17-positive cells in theirtumors, as demonstrated by our data in COLO 205 xenograft.Although high levels of TRAILR2 are expected to be beneficial, thelevel of TRAILR2 expression alone has been shown to incompletelycorrelate with TRAIL sensitivity (39–43). Investigation of other genes/proteins within the extrinsic apoptotic pathway that may modulateTRAIL sensitivity are under active investigation.

Colorectal cancers, with the exception ofmetastatic DNAmismatchrepair/microsatellite instability-high colorectal cancers, have beenlargely refractory to cancer immunotherapies (44). For this reason,we also explored whether BI 905711 as a monotherapy can evoke animmune response. Apoptotic cell death induced by BI 905711 did notactivate dendritic cells by itself. However, it cannot be excluded that BI905711 might be combined with immunotherapy, where the antigenneeded for presentation is provided by cells dying upon BI 905711treatment. Nevertheless, for a productive T-cell activation, the adju-vant signal would need to be provided by the combination partner.TRAIL agonists are also reported to eliminate myeloid-derived sup-pressor cells (45, 46). The selective elimination of those immunosup-pressive cells, potentially via BI 905711-mediated TRAILR2 trans-activation, could remodel the tumor microenvironment and therebyenhance the activity of an immunomodulatory combination partner.

Expression of CDH17 is not tumor-specific and we confirmedCDH17 expression in a subset of normal tissues (small intestine,colon and pancreatic ducts). CDH17-positive non-neoplastic tissuesmay be spared from BI 905711-mediated apoptosis due to their lowersensitivity to TRAILR2 activation, which also contributes to a poten-tially wider therapeutic window. In vitro studies demonstrated that,unlike colorectal cancer cells, freshly isolated normal crypt cells areinsensitive to TRAIL stimulation (47). This hypothesis is furthersupported by the results of preclinical safety studies in cynomolgusmonkeys, where administration of BI 905711waswell tolerated and nogastrointestinal toxicity was detected by clinical observations orhistopathological evaluation (Table S4).

TRAILR2 transactivation may raise concerns regarding unwantedtoxicity in the liver, especially in TRAILR2-positive normal cellssurrounding colon cancer metastases. In the clinical setting, a rim ofdesmoplastic stroma or a layer of reticulin fibers may prevent directcontact between tumor cells and hepatocytes in the large majority ofcases (48). Similar growth patterns were observed in the colorectalcancer liver metastases samples analyzed in this study (SupplementaryTable S1). BI 905711 is designed to spare normal liver tissue; however,some liver damage cannot be absolutely excluded and the ongoingphase I clinical trial in patients with advanced/metastatic colorectalcancer will allow us to confirm the liver-sparing concept.

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In summary, we demonstrate here that BI 905711 potently triggersthe extrinsic apoptosis pathway, specifically in CDH17-positive tumorcells, which translates into strong antitumor activities in differentcolorectal cancer models in vivo, including patient-derived xenografttumors. BI 905711 is a novel bispecificmolecule that shows superiorityto existing TRAILR2 agonists, representing a targeted strategy for thetreatment of colorectal cancer and additional CDH17-positive cancertypes. Together with its potential for a favorable safety profile, thesedata support the ongoing phase I trial of BI 905711 in these patientpopulations (NCT04137289).

Authors’ DisclosuresJ.M. García-Martínez reports grants from Austrian Research Promotion Agency

(grant reference numbers: 839361 and 844335) during the conduct of the study, as wellas a patent for (binding molecules for the treatment of cancer; US20180179287A1)pending to Boehringer Ingelheim, and reports employment with BoehringerIngelheim. S. Wang reports grants from Austrian Research Promotion Agency(grant reference numbers: 839361 and 844335) during the conduct of the study,as well as reports employment with Boehringer Ingelheim. C. Weishaeupl reportsgrants from Austrian Research Promotion Agency (grant reference numbers: 839361and 844335) during the conduct of the study, as well as reports employment withBoehringer Ingelheim. A. Wernitznig reports grants from Austrian ResearchPromotion Agency grants (grant reference numbers: 839361 and 844335) duringthe conduct of the study, as well as a patent for (bindingmolecules for the treatment ofcancer; US20180179287A1) pending to Boehringer Ingelheim, and reportsemployment with Boehringer Ingelheim. C. Pinto reports grants from AustrianResearch Promotion Agency (grant reference numbers: 839361 and 844335)during the conduct of the study, as well as reports employment with BoehringerIngelheim. D. Dutcher reports employment with Boehringer Ingelheim Pharma.M.A. Impagnatiello reports grants from Austrian Research Promotion Agency(839361 and 844335) during the conduct of the study, as well as reportsemployment with Boehringer Ingelheim. I. Tirapu reports grants from AustrianResearch Promotion Agency (grant reference numbers: 839361 and 844335) duringthe conduct of the study, as well as reports employment with Boehringer Ingelheim.F. Hilberg reports personal fees and other from Boehringer Ingelheim RCV(employee) during the conduct of the study, as well as personal fees fromBoehringer Ingelheim RCV (employee) outside the submitted work. N. Krautreports grants from Austrian Research Promotion Agency (grant referencenumbers: 839361 and 844335) during the conduct of the study, as well as reportsemployment with Boehringer Ingelheim. M. Pearson reports grants from Austrian

Research Promotion Agency (grant reference numbers: 839361 and 844335) duringthe conduct of the study, as well as reports employment with Boehringer Ingelheim.K.P. Kuenkele reports a patent for (binding molecules for the treatment of cancer;US20180179287A1) pending. No disclosures were reported by the other authors.

Authors’ ContributionsJ.M. García-Martínez: Conceptualization, formal analysis, supervision,

validation, investigation, visualization, methodology, writing-original draft,writing-review and editing. S. Wang: Formal analysis, validation, investigation,visualization, methodology, writing-review and editing. C. Weishaeupl: Formalanalysis, validation, investigation, methodology. A. Wernitznig: Data curation,software, formal analysis, validation, visualization. P. Chetta: Formal analysis,supervision, validation, investigation, visualization, methodology, writing-reviewand editing. C. Pinto: Formal analysis, validation, investigation, visualization,writing-review and editing. J. Ho: Formal analysis, supervision, validation,investigation, writing-review and editing. D. Dutcher: Formal analysis,validation, investigation. P.N. Gorman: Formal analysis, validation, investigation.R. Kroe-Barrett: Supervision, validation, writing-review and editing. J. Rinnenthal:Formal analysis, supervision, validation, investigation. C. Giragossian: Formalanalysis, supervision, validation, investigation, writing-review and editing.M.A. Impagnatiello: Formal analysis, supervision, validation, investigation.I. Tirapu: Formal analysis, supervision, validation, investigation, visualization,writing-review and editing. F. Hilberg: Formal analysis, supervision, validation,investigation. N. Kraut: Supervision, writing-review and editing. M. Pearson:Supervision, writing-review and editing. K.P. Kuenkele: Conceptualization,supervision, writing-review and editing.

AcknowledgmentsWe thank Dr. Verena Supper for kindly providing the OVCAR-3-mKate2 cells,

and colleagues at Boehringer Ingelheim RCV and Boehringer Ingelheim Biother-apeutics Discovery for scientific and technical support. We also thank MahmoudOuld Kaci and Holly Dursema for insightful discussions. This work has beensupported by the Austrian Research Promotion Agency (grant reference numbers:839361 and 844335).

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 31, 2020; revised August 12, 2020; accepted September 30, 2020;published first October 9, 2020.

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