Translational Cancer Mechanisms and Therapy

Tumor-Localized Costimulatory T-CellEngagement by the 4-1BB/HER2 BispecificAntibody-Anticalin Fusion PRS-343Marlon J. Hinner1, Rachida Siham Bel Aiba1, Thomas J. Jaquin1, Sven Berger1,Manuela Carola D€urr1, Corinna Schlosser1, Andrea Allersdorfer1, Alexander Wiedenmann1,Gabriele Matschiner1, Julia Sch€uler2, Ulrich Moebius1, Christine Rothe1, Louis Matis1, andShane Anthony Olwill1

Abstract

Purpose: 4-1BB (CD137) is a key costimulatory immuno-receptor and promising therapeutic target in cancer. To over-come limitations of current 4-1BB–targeting antibodies, wehave developed PRS-343, a 4-1BB/HER2 bispecific molecule.PRS-343 is designed to facilitate T-cell costimulation by tumor-localized, HER2-dependent 4-1BB clustering and activation.

Experimental Design: PRS-343 was generated by the genet-ic fusion of 4-1BB–specific Anticalin proteins to a variant oftrastuzumab with an engineered IgG4 isotype. Its activity wascharacterized using a panel of in vitro assays and humanizedmouse models. The safety was assessed using ex vivo humancell assays and a toxicity study in cynomolgus monkeys.

Results: PRS-343 targets 4-1BB andHER2 with high affinityand binds both targets simultaneously. 4-1BB–expressingT cells are efficiently costimulated when incubated withPRS-343 in the presence of cancer cells expressing HER2, as

evidenced by increased production of proinflammatory cyto-kines (IL2, GM-CSF, TNFa, and IFNg). In a humanizedmousemodel engrafted with HER2-positive SK-OV-3 tumor cells andhuman peripheral bloodmononuclear cells, PRS-343 leads totumor growth inhibition and a dose-dependent increaseof tumor-infiltrating lymphocytes. In IND-enabling studies,PRS-343 was found to be well tolerated, with no overt toxicityand no relevant drug-related toxicologic findings.

Conclusions: PRS-343 facilitates tumor-localized targetingof T cells by bispecific engagement of HER2 and 4-1BB. Thisapproach has the potential to provide a more localized acti-vation of the immune systemwith higher efficacy and reducedperipheral toxicity compared with current monospecificapproaches. The reported data led to initiation of a phase Iclinical trial with this first-in-class molecule.

See related commentary by Su et al., p. 5732

Introduction4-1BB, also known as CD137, is a costimulatory immune

receptor, a member of the TNF receptor (TNFR) superfamily, andis predominantly expressed on activated CD4þ and CD8þ T cells,activated B cells, and natural killer (NK) cells (3). 4-1BB plays animportant role in the regulation of immune responses andtogether with the fact that it is expressed on tumor-infiltratinglymphocytes (TIL) makes it a promising target for cancer immu-notherapy. The 4-1BB ligand (4-1BBL) is constitutively expressedon several types of antigen-presenting cells (APC; ref. 4). Uponpathway activation, 4-1BB facilitates enhanced proliferation,cytokine production, and cytolytic activity of T and NK cells (5).Recent studies have pointed to the pathway's impact on mito-

chondrial capacity and biogenesis of T cells to explain why 4-1BBagonism could help overcome the immunosuppressive landscapeof the tumor microenvironment (6). While most research hasfocused on 4-1BB in the context of T effector cells, it should benoted that 4-1BB is also expressed on T-regulatory cells where itsrole remains contentious and requires further elucidation (7).4-1BB has also been implicated in promoting a central memoryT-cell response, which may support therapeutic persistence oftumor-specific T cells and resistance to exhaustion in patientstreated with a 4-1BB agonist (8, 9).

The potential therapeutic benefit of 4-1BB costimulation hasbeen demonstrated in multiple preclinical models. The forcedexpression of 4-1BBL on a tumor, for example, leads to tumorrejection (10). Likewise, the forced expression of an anti–4-1BBsingle-chain antibody fragment (scFv) on a tumor leads to aCD4þ

T-cell and NK-cell–dependent elimination of the tumor (11–13).A systemically administered anti–4-1BB antibody has also beendemonstrated to lead to retardation of tumor growth in mousemodels (14).

Human ex vivo data support the potential of 4-1BB as a costi-mulatory receptor in cancer therapy. It has been reported that for Tcells isolated fromhuman tumors, 4-1BB is amarker for those thatare tumor-specific (15). In line with this observation, anti–4-1BBantibodies can be utilized to improve adoptive T-cell therapy(ACT) by augmenting the expansion and activity of CD8þ mel-anoma TILs (16). Further clinical evidence for the importance of

1Research and Development, Pieris Pharmaceuticals GmbH, Freising, Germany.2In Vivo Operations, Oncotest GmbH, Freiburg, Germany.

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

Corresponding Authors: Shane Anthony Olwill, Pieris Pharmaceuticals,Lise-Meitner-Strasse 30, Freising 85354, Germany. Phone: 816-1141-1423;Fax: 49 (0) 8161 14 11 444; E-mail: olwill@pieris.com; and Marlon J. Hinner,marlon.jakob@hinner.de

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4-1BB signaling for a sustained and successful anticancer T-cellresponse is provided in ACT with chimeric antigen receptors(CAR), where inclusion of 4-1BB–signaling elements in the cyto-plasmic domain of CARs has been found to improve durability ofclinical responses (17, 18).

Receptors from the TNFR family in general require higher-orderclustering for efficient downstream signaling activation in theTNFR-expressing cell (19), which usually is not afforded by thesoluble ligand alone, but requires engagement of another cellexpressing the respective ligand on its surface. For example,despite being a homotrimer, soluble 4-1BBL is not capable ofefficiently activating 4-1BB downstream signaling (20). Antibo-dies targeting 4-1BB are either inherently agonistic or often requirea secondarymeans of clustering beyond bivalent binding to allowfor agonistic activity. It is hypothesized that in vivo TNFR agonismby such antibodies is dependent on simultaneous binding to Fcgreceptor–positive cells (21, 22).

The intricate mode of activation of 4-1BB may underlie themodest clinical success obtained with monospecific anti–4-1BBantibodies to date. BMS-663513 (urelumab; ref. 23) has beenhampered by dose-limiting hepatotoxicity, likely due to its ten-dency to systemically activate 4-1BB (1). Initial clinical trials withPF-05082566 (utomilumab; refs. 24, 25) have not shown thesame safety issues as urelumab, yet preclinical characterizationsuggests it is a less potent 4-1BB agonist (2).

To overcome the challenges of targeting the 4-1BB pathwaysystemically, we have generated PRS-343, a bispecific moleculedesigned to activate the pathway in a tumor-localized and-dependent manner. PRS-343 binds 4-1BB and the tumor-associated antigen HER2, and was generated by the recombinantfusion of a 4-1BB–specific Anticalin protein to a HER2-specificantibody. Anticalin proteins are engineered variants of lipocalins,a family of natural extracellular binding proteins. Lipocalins arecharacterized by a highly conserved tertiary structure despite lowamino acid identity. This property coupled to the fact that theypossess free C- and N-termini, which are not required for targetrecognition, allow facile fusion to other proteins, defining a

versatile basis for a multispecific biologics platform (26). Themolecule, PRS-343, is designed to promote 4-1BB clustering bybridging T cells with HER2-positive tumor cells, providing apotent costimulatory signal to tumor antigen–specific T cells,further enhancing T-cell receptor–mediated activity and leadingto tumor destruction. PRS-343–mediated 4-1BB activation is,therefore, biased toward locations in the body where T cells andtumor cells are colocalized, such as in primary tumors with TILs orin lymph nodes containing tumor metastases. Here, we describethe generation and preclinical characterization of PRS-343 withregard to target engagement, biological activity, and safety.

Materials and MethodsEngineering and production of Anticalin proteins andbispecific fusion proteins

Recombinant 4-1BB (R&D Systems) was used as the targetfor phage display selection and ELISA screening of cognateAnticalin protein candidates. A random library based on humanneutrophil-gelatinase–associated lipocalin (NGAL) with highcombinatorial complexity was prepared by concerted mutagen-esis ofmultiple amino acid positions (27). 4-1BB target specificitywas confirmed by ELISA screening and selected Anticalin proteinswere optimized via partial mutagenesis of coding regions fol-lowed by biophysical and functional characterization, whichresulted in selection of a lead candidate J10.

Bispecific antibody-Anticalin fusion proteinswere generated bythe recombinant fusion of the 4-1BB–specific Anticalin protein(J10) to either the heavy or light chain N- or C-terminus of amodified variant of trastuzumab. Specifically, the isotype oftrastuzumab was changed to an IgG4 and additional mutationswere introduced to reduce interactions with activating and inhib-itory Fcg receptors. Mutation S228P, which has been described tosuppress half-antibody exchange (28), was introduced togetherwith mutations F234A and L235A, which have been reportedto reduce binding to FcgRI (29, 30). Bispecific proteins wereobtained by recombinant expression in mammalian cells(HEK293 or CHO) using standard techniques.

Reagents and cell linesRecombinant 4-1BB and HER2 protein (human and cynomol-

gus monkey) were obtained from R&D Systems and Sino Biolog-ical. The cell linesHT-29, SKBR-3, SKOV-3, BxPC-3,MCF-7,MDA-MB-231, MKN-7, NCI-N87, ZR-75-1, A375, A431, A549, JIMT-1,MDA-MB-453, MKN45, SUM-225, and LoVo were obtained fromATCC. Primary cells (human cardiacmyocytes, human epidermalkeratinocytes, human pulmonary fibroblasts, human cardiacfibroblasts, human dermal microvascular endothelial cells,human umbilical vein endothelial cells, human aortic smoothmuscle cells, and human bronchial smooth muscle cells) wereobtained from Promocell. The cells were expanded following cellbank instructions. The HER2 levels were confirmed by QifiKit(Dako) according to the provided protocol andwere standardizedto HER2 levels expressed by SKBR3. The list of the cells, corre-sponding cell bank catalogue number, and their respectiveHER2 expression levels standardized to SKBR3 HER2 levels aresummarized in Supplementary Table S1. CHO cells expressinghuman 4-1BB (h 4-1BB) were established by stable transfectionof h 4-1BB using the Flp-In System (Invitrogen). An NF-kB-Luc2/4-1BB Jurkat cell line was obtained from Promega. Humanperipheral blood mononuclear cells (PBMC) from healthy

Translational Relevance

Immunotherapy with checkpoint inhibitors, such asanti–PD-1 mAbs, has had a major impact on cancer therapy.Although such therapies afford durable responses or evencures, additional therapeutic strategies are still required forthe majority of patients who do not respond or relapse. Theactivation of costimulatory pathways such as 4-1BB has longbeen acknowledged to hold great promise, but clinical effortsthus far have failed to demonstrate broad efficacy and havebeen associated with severe toxicity (1, 2). Herein, we describethe HER2/4-1BB bispecific molecule PRS-343, which aims toovercome these limitations by activating 4-1BB in a tumor-localized manner, sparing the periphery of unwanted toxicity.The data presented herein confirm the desired mode of actionof PRS-343, providing a novel approach to target 4-1BB thatmay prove both safer and more efficacious compared withmonospecific targeting. PRS-343 has the potential to offer analternative therapeutic strategy to patients in multiple HER2-positive indications including bladder, breast, and gastriccancer.

Costimulatory T-Cell Engagement by the 4-1BB

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volunteer donors were isolated from buffy coats by centrifuga-tion through a polysucrose density gradient, and T cells wereisolated from the resulting PBMCs using a pan T-cell isolationKit (Miltenyi Biotec) according to the manufacturer's protocols.The 4-1BB agonist antibody 20H4.9 (corresponding tourelumab)was obtained by recombinant expression in CHO cells usingstandard techniques.

Target-binding studiesBinding of PRS-343 to 4-1BB or HER2 proteins (human or

cynomolgus monkey) was analyzed by ELISA or surface plasmonresonance (SPR).

In the target-binding ELISA, the extracellular domain of thetarget proteins, 4-1BB (human or cynomolgus), HER2 (human)or control protein, was coated onto microtiter plates. EitherPRS-343, the 4-1BB–specific reference Anticalin protein, or thereference anti-HER2 antibody was added and binding wasdetected with peroxidase-conjugated anti-human IgG or anti-NGAL antibody.

For the dual-binding ELISA, humanHER2 (hHER2)was coatedand PRS-343-binding was detected using biotinylated h 4-1BBdetected by peroxidase-labeled ExtrAvidin.

In the SPR affinity assay, biotinylated h 4-1BB or h HER2 wascaptured on a sensor chip CAP using the Biotin CAPture Kit (GE).Determination of PRS-343–binding kinetics and affinity wasperformed by applying four dilutions of PRS-343 to the chipsurface with a flow rate of 30 mL/minute; the sample contact timewas 180 seconds and dissociation time was 1,800 seconds. Datawere fit with a 1:1 binding model.

Binding capability to primary cells, tumor cells, and target-positive engineered cell lines was assessed by flow cytometry.Cells were incubated with PRS-343, reference antibody, or h IgG4asnegative control, andbinding of the testmoleculeswas detectedusing Alexa Fluor 488–labeled goat anti-human IgG antibody.

An IHC assay was performed to assess cross-reactivity ofPRS-343 on human tissue. Briefly, cryo-sections histologicallyprepared at a nominal 5 mm from a panel of 40 frozen humantissues together with positive and negative control material werestained with biotinylated PRS-343 or control. A detailed micro-scopic examination was performed to assess each tissue for anysign of positive staining.

T-cell activationThe impact of HER2 receptor density on PRS-343–mediated

T-cell activation was assessed in coculture experiments using apanel of cell lines expressing different levels of HER2. Cancer celllines representing a range of clinically relevant levels of HER2receptor were tested for their ability to mediate clustering of PRS-343 and subsequent activation of T cells. To evaluate a potentialtherapeuticwindow, cell lines derived fromhealthy tissues knownto express background levels of HER2 were also included. Thelevel of T-cell activation was measured by quantification ofhuman IL2, using an electrochemiluminescence (ECL) immuno-assay (Mesoscale Discovery). Briefly, cancer cells or cells derivedfrom healthy tissue pretreated with 10 mg/mL of mitomycin C(Sigma Aldrich) were seeded in culture plates precoated with anti-CD3 and incubated overnight at 37�C in a humidified 5% CO2

atmosphere. T-cell suspension (5 � 104 cells) together with testarticle was added. The IL2 concentration in the supernatant wasassessed by an ECL assay (using IL2 DuoSet kit; R&D Systems)following 3-day incubation.

Specific activation of the 4-1BB pathway by PRS-343 wasassessed using a luciferase reporter cell assay (Promega), wherea 4-1BB–overexpressing reporter cell line (NF-kB-Luc2/4-1BBJurkat cells) was cocultured with HER2-positive tumor cell linesand where 4-1BB pathway activation was measured byluminescence.

Cytokine release assayThe potential of PRS-343 to induce cytokine release syndrome

was evaluated in vitro using a cytokine release assay (31), wheresoluble or coated (wet or dry-coated) PRS-343was incubatedwithhuman PBMC for 72 hours followed by the quantification of apanel of proinflammatory cytokines (MSD). The anti-CD3 mAbOKT3 (Muromonab-CD3) was used as positive control and ahuman monoclonal IgG4 isotype antibody was used as negativecontrol.

Cynomolgus toxicity studyA 4-week–repeated dose toxicity study (vehicle, 10 mg/kg or

120 mg/kg PRS-343 on days 1, 8, 15, 22, and 29) followed by a4-week recovery period was performed in cynomolgus monkeys.Serum levels of PRS-343 were assessed by an ECL assay. Assess-ment of safety and toxicity was based on standard parameters.

Mouse and cynomolgus monkey pharmacokineticsSingle-dose pharmacokinetics studies were performed in mice

and cynomolgus monkeys. Male CD-1 mice were administeredwith an intravenous injection of PRS-343 (10 mg/kg). Cynomol-gus monkeys (Macaca fascicularis) received a 60-minute infusionof PRS-343 (3 mg/kg) For each study, plasma was collectedpredose and at multiple time points post administration.

PRS-343 plasma concentrations were determined via an ECLassay (MSD) using a dual-binding ELISA. The pharmacokineticparameters were derived by noncompartmental analysis usingPhoenix WinNonlin.

In vivo tumor efficacy modelPRS-343 in vivo activity was evaluated in a SK-OV-3 ovarian

cancer model in human PBMC–reconstituted NOG female mice(NOD.cdPrkdcscidIL2rgtm1.Sug/JicTac, supplied by Taconic), ages5–7weeks. Tumor growthwasmonitored twiceweekly and tumorvolume was calculated as follows: (length � width2)/2. At studyend, plasma was collected for all animals and lymphocytephenotyping was carried out by flow cytometry assessing humancell markers such as CD45 (Invitrogen), CD4, and CD8 (BDBiosciences). To assess TILs, tumors were also collected, fixed,and paraffin-embedded followed by human CD45, CD3, CD4,and CD8 staining. Stained tumor sections were digitally scanned,and the resulting digitalized data were evaluated to determine thepercentage of target-positive (tumor-infiltrating) cells.

Graft versus host disease modelThe potential for PRS-343 to induce systemic graft versus

host disease (GVHD) was assessed. Five- to 7-week-old femaleNOG mice (NOD.cdPrkdcscidIL2rgtm1.Sug/JicTac, supplied byTaconic) were injected with fresh human PBMC and treated withPRS-343 or controls. As a read-out for signs of GVHD, the animalswere routinely checked for changes in body weight or generalhealth. Mice who were euthanized (based on predefined criteria)or spontaneously expiredmice were recorded for survival analysisby each treatment. Data were plotted using a Kaplan–Meier curve.

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Flow cytometryFlow cytometry analyses were carried out on an iQue Screener

(Intellicyt Corporation) equipped with ForeCyt software or withan Attune Focusing Cytometer [blue (488 nm)/violet (405 nm)laser configuration].

Ethical considerationsAll animal experiments and protocols were approved by the

institutional animal welfare body and the relevant local author-ities andwere conducted according to all applicable international,national, and local laws and guidelines. All animal experimentswere approved by the regional Ethics Committees (Germany orthe United Kingdom).

Statistical analysisAll statistics were calculated using GraphPad Prism Version 5

for Windows. Statistical significance was determined using one-way or two-way ANOVA to compare differences among multiplegroups. P values less than 0.05 were considered statisticallysignificant.

ResultsGeneration of PRS-343 by protein engineering and design

A 4-1BB–binding Anticalin protein was obtained by phagedisplay selection and optimized by protein engineering asdescribed inMaterials andMethods. The 4-1BB–specific Anticalinprotein (J10) has an affinity of 2 nmol/L as determined by SPR,binds to human 4-1BB transfected CHO cells, and does notcompete with the binding of 4-1BB ligand to its receptor (datanot shown). AnELISA screenof relatedTNFR superfamily proteins(4-1BBOx40, GITR, TNFRI, TNFRII, CD30, RANK) confirmed J10Anticalin protein specifically binds to 4-1BB (Supplementary Fig.S1)

As the HER2-binding, tumor-targeting building block of a4-1BB/HER2 bispecific, we utilized a modified variant of trastu-zumab. The formatting flexibility offered by the Anticalin tech-nology facilitated the generation of various Antibody-anticalinfusion formats as depicted in Supplementary Fig. S 1. We hypoth-esized that the distance between T-cell and tumor cell–bindingsites may impact the ability of the 4-1BB/HER2 bispecific toappropriately cluster the 4-1BB receptor and thus activate thepathway. Therefore, the 4-1BB–engaging Anticalin protein wasgenetically fused either to the N- or C-terminus of the anti-HER2antibody heavy or light chain resulting in the generation of fourdifferent geometries of the fusion protein covering a range ofbinding distances. The antibody Fc region was engineered (seeMaterials and Methods for details) to exclude the risk of NK-driven antibody-directed cellular cytotoxicity (ADCC) against4-1BB–positive cells or undesired, non–tumor-target activationof 4-1BB–positive lymphocytes via FcgR-derived cross-linking.The interaction with the neonatal Fc receptor (FcRn) was retainedto support a prolonged terminal plasma half-life.

While all formats behaved similarly from the perspective ofbiophysical and target-binding properties, we observed a markeddifference in their ability to elicit T-cell activation (SupplementaryFig. S2). On the basis of these data, a single format was selectedand is described herein. The lead bispecific fusion molecule,PRS-343, was constructed via the genetic fusion of the 4-1BB–specific Anticalin protein to the C-terminus of the heavy chain of

the trastuzumab IgG4 variant, connected by a flexible, nonim-munogenic (G4S)3 linker sequence, as depicted in Fig. 1.

Human target bindingThe interaction of PRS-343 with its human targets was inves-

tigated by ELISA, SPR, and FACS. As assessed by SPR, PRS-343binds to recombinant human HER2 with high affinity (Kd ¼0.3 nmol/L), similar to the parental antibody trastuzumab. Thebinding affinity of PRS-343 to human monomeric 4-1BB wasdetermined to be 5 nmol/L (Fig. 1).

In a FACS assay, PRS-343 was found to bind HER2-expressingMCF-7 cells with an EC50 of 7.4 nmol/L. Binding to CHO cellstransfected with human 4-1BB was determined with an EC50 of6.2 nmol/L (Fig. 1).

Using a dual-binding ELISA, PRS-343 was found to be capableof binding both targets simultaneously, which is a prerequisite forits envisioned mode of action.

PRS-343 displays reduced cross-reactivity to cyno 4-1BB incomparison with human 4-1BB as shown by ELISA (Fig. 1K). AnSPR experiment also demonstrated that binding of PRS-343 tocyno 4-1BB strongly depends on target densities (data notshown).

Further SPR experiments demonstrated that PRS-343 does notbind to Fcg receptors but binding to the neonatal Fc receptor(FcRn) is retained. In accordance with its lack of interaction withFcg receptors, PRS-343 does not elicit any ADCC activity whencoincubated with human PBMCs and HER2-positive breast car-cinoma cells (data not shown).

A tissue cross-reactivity study using a panel of 40 human tissues(n¼ 3donors) each demonstrated that PRS-343binds as expectedfor a molecule targeting both HER2 and 4-1BB in a bispecificmanner. Epithelial cells of various tissues showed strong positiv-ity for binding PRS-343, which is in accordance with publisheddata on HER2 expression in human tissues (32). The observedlymphocyte staining is in agreement with the expected binding ofPRS-343 to 4-1BB on this cell type because 4-1BB has beenreported to be (inducibly) expressed on lymphocytes (33).

In vitro activity of PRS-343The ability of PRS-343 to costimulate T cells in a HER2-

dependentmanner was investigated in vitro using a reporter assay.The reporter cell assay was performed to investigate whether

PRS-343 is capable of inducingHER2-dependent 4-1BB clusteringon T cells. In this assay, NF-kB-Luc2/4-1BB Jurkat cells werecocultured with a HER2-positive tumor cell line. Successful clus-tering of 4-1BB on the Jurkat cell surface is expected to lead toTRAF-2 (and TRAF-1)-mediated activation of NF-kB (reviewed inref. 34), which in the presence of a luciferase substrate can bedetected by increased luminescence.

Indeed, coincubation of up to 10 nmol/L PRS-343 with theJurkat reporter cell line and the HER2-positive NCI-N87 cell lineled to a 20-fold increase of NF-kB luciferase reporter activity withan EC50 of 50 pmol/L (Fig. 2A). Furthermore, in a wash-outexperiment where PRS-343 was added to various cancer cell linesovernight prior to a media exchange, HER2-specific 4-1BB acti-vation was maintained (Supplementary Fig. S4). When using thelow HER2-expressing MKN-45 or Hep-G2 cell lines, the NF-Bactivity was similar to background and control values. A bell-shaped response was observed when concentrations of up to1 mmol/L PRS-343 were applied in this type of assay (data notshown).

Costimulatory T-Cell Engagement by the 4-1BB

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These results demonstrate that PRS-343 facilitates 4-1BB path-way activation only in the presence of HER2-overexpressing cells.

HER2-dependent costimulatory T-cell activationTo investigate whether this mechanism can be applied to

effectively costimulate T cells, a coculture assay was developedusing human T cells and HER2-expressing cell lines. Here, the Tcells receive a primary T-cell receptor stimulus via anti-CD3antibody activation, and PRS-343 induced costimulation can bequantified by measuring supernatant levels of proinflammatorycytokines. Using a multiplex assay, we first investigated whichcytokineswereproducedbyT cells costimulatedbyPRS-343 in thepresence of HER2-expressing cells (Supplementary Fig. S5). The

strongest fold increase over background was observed for IL2.Statistically significant increase in GM-CSF, IFNg , and TNFawas observed. IL4 and IL6 supernatant levels were increasedalbeit absolute cytokine levels were low. There was no PRS-343–dependent response observed for IL1b, IL8, IL10, and IL12.Given the robust nature of the IL2 response, it was selected as amarker of activation for future assays. In the presence of HER2-positive cell lines, a dose-dependent induction of IL2 wasobserved with PRS-343 (Fig. 2B and C). When the experimentwas performed with cell lines expressing basal levels of HER2, noPRS-343–dependent IL2 induction was observed. To furtherconfirm that PRS-343 activity is dependent on HER2 binding,additional assays were performed in the presence of an excess of

Figure 1.

PRS-343 design and target and cell binding. PRS-343 structure (A) and design (B). HER2-binding studies were performed by ELISA (C) or by FACS (D) usingMCF-7 cells. Similarly, 4-1BB–binding studies were performed by ELISA (F) or by FACS (G) using 4-1BB–overexpressing CHO cells. Representative sensograms ofSPR experiments are shown using immobilized hHER2 (E) or immobilized h4-1BB (H) and allowed determination of PRS-343 on-, off-rate, and KD (I). Dualbinding: PRS-343 is capable of binding both targets at the same time as determined in a simultaneous binding ELISA (J). Cross-reactivity: PRS-343 displaysreduced cross-reactivity to cynomolgus monkey 4-1BB (K).

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trastuzumab (200 nmol/L). Under these conditions, the excesstrastuzumab competed for binding to HER2 and was found toabrogate PRS-343–induced T-cell costimulation, further support-

ing its envisaged mode of action (Fig. 2C), whereas the control4-1BB agonistic antibody 20H4.9 consistently induced IL2 secre-tion irrespective of the coincubated cell lines (Fig. 2C).

Figure 2.

PRS-343 cell–based activity. Concentration dependence of PRS-343–mediated T-cell activation. Dose-dependent T-cell costimulation by PRS-343 againstNCI-N87 (HER2 high), MKN45 (HER2 low), and HepG2 (HER2 null) cell lines, or without tumor cells, using a 4-1BB overexpressing Jurkat NF-kB reporter cell line(A) or purified human T cells (B). IL2 secretion induced by human T cells costimulated by PRS-343, isotype control, trastuzumab, PRS-343 in excess oftrastuzumab, and anti–4-1BB antibody in the presence of cells lines with expressing various HER2 levels (NCI-N87; SK-BR-3 and SUM-225-CW (HER2 high);JIMT-1 (HER2 medium) and MCF-7, MKN45 and MDA-MB-231 (HER2 low; C). All data depicted here are representative illustrations of experiments carried outwith minimum two different donors. Statistical analysis: �, P < 0.05; �� , P < 0.01; and ��� , P < 0.001, using one-way ANOVAwith Dunnet multiple comparison test.

Costimulatory T-Cell Engagement by the 4-1BB

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Impact of HER2 expression levelTo examine the HER2 expression level threshold required for

successful PRS-343–induced T-cell costimulation, the assay wasperformed with a panel of cell lines of variable HER2-positivity(Table S1). TheHER2 cell surface expression level was determinedby quantitative FACS and is reported as the specific anti–HER2-antibody–binding capacity. For the purpose of comparison,HER2 expression was normalized relative to a reference cell line,SK-BR-3. The cancer cell lines selected represent a wide range ofHER2 expression levels ranging from approximately 1% to 200%of the reference value. In contrast, cell lines from healthy tissuesexpress HER2 at a much lower and more confined level, withrelative HER2 expression around 1 % of HER2 expression on SK-BR-3 (Table S1). These cancer- and healthy tissue–derived celllines were then applied in the previously described costimulatoryT-cell activation assay to identify a potential HER2 thresholdrequired for T-cell costimulation.

A selection of experimental results based on IL2 as the readoutand utilizing tumor cell lines and primary cell types is shownin Fig. 3. These data confirm that IL2 secretion strongly correlateswith HER2 expression. Assessing the maximum level of IL2secretion compared with control, we show that cancer cells witha high level of HER2 induced significantly increased IL2 secretion.For cell lines described as having a more intermediate level ofHER2 (e.g., MCF-7 or MKN-45), we could also observe signifi-cantly increased IL2 secretion in a proportion of the donors tested.Both the cancer cell lines expressing basal levels of HER2 (such asBx-PC-3 or MDA-MB-231) and the tissue-derived primary cellsdid not induce a significant amount of IL2 secretion, supportingthe hypothesis that PRS-343 requires HER2 expression at supra-physiologic levels on target cells to costimulate T cells.

Cytokine release assayTo investigate the risk of systemic cytokine release in patients

induced by PRS-343, an in vitro cytokine release assay (31) wascarried out with PBMCs from 12 healthy control donors. Incontrast to the anti-CD3–positive control antibody OKT3,PRS-343 did not induce a significant increase of cytokines overbackground whenwet coated onto plates or when incubated withPBMCs in solution. When PRS-343 was air-dried, a modest butstatistically significant increase of the cytokines IFNg , GM-CSF,IL4, and IL8 was observed only at the highest concentration(i.e.,100 mg/mL). Relative to the maximum increase induced byOKT-3 (10 mg/mL, air dried), the levels induced by PRS-343 were0.2% for GM-CSF, 0.1% for IFNg , 6% for IL4, and 13% for IL8(Supplementary Fig. S3).

Pharmacokinetics in mice and cynomolgus monkeysPharmacokinetics was evaluated following a single intravenous

administration of PRS-343 by bolus injection of male CD-1 miceat 10 mg/kg, or upon a 60-minute intravenous infusion to malecynomolgus monkeys at 3 mg/kg. The plasma concentrations inboth species were found to decline in a biphasic manner (Fig. 4).The terminal elimination half-life was >14 days in mouse. Incynomolgus monkeys, a terminal elimination half-life of approx-imately 4 days was observed. In conclusion, the determined PKparameters for PRS-343 demonstrate that it behaves similar to aconventional mAb in preclinical models.

Humanized mouse tumor modelThe in vivo activity of PRS-343 was evaluated in a humanized

mouse model using immunocompromised mice subcutaneouslyengrafted with the SK-OV-3 cell line as a HER2-positive tumor

Figure 3.

Coculture system data showing correlation between HER2 expression level and T-cell activation (IL2 secretion) via PRS-343. A, Collated data from cocultureexperiments show that significant T-cell activation (as measured by increased IL2) is observed in the presence of HER2-positive cancer cell lines and not primarytissue cell lines. Effect size (x-axis) and significance (y-axis) of PRS-343 on IL2 levels as compared with isotype control. Dotted line shows the significance cutoffcorresponding to P¼ 0.05. B, Data shows that increasing levels of HER2 correlate with enhanced IL2 secretion (R2¼ 0.46; P¼ 0.002). Shaded area indicatesconfidence interval for a linear fit.

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xenograft. This cell line was chosen based on HER2 positivity,trastuzumab sensitivity, and the ability to grow homogenously asa subcutaneous xenograft in the presence of human PBMC with-out being directly eliminated. Mice with established tumors(>100 mm3) were grouped and treated with test articles on aweekly dosing schedule. Median tumor volumes over time foreach of the treatment groups are shown in Fig. 5A. PRS-343displayed dose-dependent antitumor efficacy with doses rangingfrom 4 mg to 100 mg (approximately 0.2mg/kg to 5mg/kg), whilethe 200-mg dose (approximately 10 mg/kg) did not furtherenhance tumor regression. Equimolar dosing of the trastuzu-mab-IgG4 control antibody displayed comparable antitumorefficacy to that of PRS-343. The model has been previouslydescribed as being responsive to anti-HER2 therapy and helpsconfirm that HER2-mediated antitumor activity is preserved inPRS-343. Interestingly, the 4-1BB agonist (20H4.9) antibody wasunable to block tumor growth in this model.

Flow cytometry analysis of the human immune cell populationin the peripheral blood of the mice revealed very little differencesbetween the PRS-343–treated groups and control groups. How-ever, in mice treated with the 4-1BB agonist antibody (20H4.9), aperipheral expansion of human CD8þ cells was observed(Fig. 5B), demonstrating a systemic impact of the agent.

To assess the 4-1BB–mediated effect of PRS-343, excisedtumors from treated mice were analyzed for immune infiltration.PRS-343 administration led to a significant increase in humanCD45þ lymphocytes in tumor tissue (Fig. 5C) compared with thecontrol groups. While assessing the immune cell subtypes infil-tratedwithin the tumors, we could observe that PRS-343–inducedinfiltrate consisted predominantly of CD8þ T cells (Fig. 5C).Importantly, there was no significant increase in immune cellinfiltrates in the excised tumors from mice treated with thetrastuzumab-IgG4 when compared with controls.

In immune-compromisedmice engrafted with human PBMCs,GVHD is expected to occur due to the human PBMCs destroyingmouse tissue and organs resulting in progressive weight loss andmortality (35). In this humanized xenograft study, we observedthat the 4-1BB agonist antibody accelerated GVHD-induced mor-tality, while the administration of PRS-343 or the isotype controlantibody did not influence weight loss or the median survival ofthe mice. To confirm this observation, we carried out a dedicatedexperiment using larger group sizes (n ¼ 15) of non–tumor-bearing mice. This study confirmed that weight loss and survivalfollowing PRS-343 treatmentwere comparablewith that observedin the IgG4 isotype control–treated animals, while treatment with

the systemically active 4-1BB agonist led to both significantlygreater weight loss and shorter survival compared with control(Fig. 5D).

Taken together, these data show that PRS-343 provides dualactivity by increasing the number of TILs coupled with directtumor growth inhibition by bispecific targeting of 4-1BB andHER2. Notably, the tumor growth inhibition provided by target-ing HER2 did not require any ADCC, as both PRS-343 and thetrastuzumab-IgG4 control lack the ability to interact withFc-gamma receptors on NK cells that ADCC would require.

Safety of PRS-343 in cynomolgus monkeysThe safety of PRS-343 was investigated in a 4-week GLP-

compliant toxicity study in cynomolgus monkeys. It is importantto take into account the reduced cross-reactivity of PRS-343 tocynomolgus 4-1BB, which impacts the ability of this study topredict 4-1BB–related toxicity. PRS-343 was administered weeklyas an intravenous infusion of 120-minute duration at doses of 10and 120 mg/kg over 4 weeks, followed by a recovery phase in thecontrol and high-dose group to evaluate any potential delayedonset or reversibility of toxicity.

Overall, PRS-343 was well tolerated at both doses tested, withno significant findings. No unscheduled deaths occurred. Therewere no changes in standard parameters such as clinical observa-tions, body weight, ophthalmology, rectal temperatures, clinicalchemistry, hematology, coagulation tests, urinalysis parameters,or serum cytokines, as well as ECG. Furthermore, no toxicolog-ically relevant organweight or organ weight ratio changes, and nomacroscopic or microscopic findings were observed, indicatingthat treatment with PRS-343 over four weeks did not lead to anysystemic toxicity. In addition, no evidence of delayed onsettoxicity was noted at the end of the 4-week recovery phase.Toxicokinetic analysis demonstrated dose-proportional systemicexposure at both dose levels upon first and last dose. Two ofsixteen animals developed anti-drug antibodies (ADAs) thatpersisted throughout the study while three others showed atransient ADA response. No gender-related toxicity differenceswere noted in the study.

Discussion4-1BB (CD137) is a key costimulatory immunoreceptor and a

member of the TNFR superfamily. The preclinical and clinicaldemonstration of the potential therapeutic benefit of 4-1BB costi-mulation has spurred the development of therapeutic antibodiestargeting 4-1BB, utomilumab (24, 25), and urelumab (23).Utomilumab is a fully humanized IgG2 mAb that binds 4-1BB ina manner that blocks the binding of endogenous 4-1BBL to 4-1BBand that appears well tolerated as a monotherapy (36) and incombination with rituximab; however, modest activity has beenobserved (37). Urelumab is an IgG4 mAb that, in contrast to uto-milumab, binds 4-1BB in amanner that does not interfere with the4-1BB/4-1BBL interaction. While an initial trial reported modestclinical activity (38), a follow-up study was stopped because ofhepatotoxicity (1). These data indicate that systemic activation of4-1BB with a potent agonist may lead to prohibitive toxicity, sup-porting the rationale for tumor-localized targeting of the pathway.

Although multiple lines of evidence suggest that 4-1BB is ahighly promising therapeutic target in cancer, current systemicantibody-based approaches are not designed to achieve a tumor-target–driven activation and are likely to display toxicity due to

Figure 4.

Pharmacokinetic profile of PRS-343 in murine (A) and primate (B) speciesfollowing single-dose intravenous administration.

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Figure 5.

PRS-343–mediated antitumor activity in human PBMCs reconstituted xenograft SK-OV-3 tumor–bearing mice. A, Tumor volume was measured at indicatedtimes through day 20 and plotted as group mean� SEM. B, Lymphocyte phenotyping of the peripheral blood for human cell markers CD45 and CD8 wasperformed by flow cytometry. All data were plotted as group mean� SEM. C, TILs were assessed by IHC staining of excised tumors using the humanmarkersCD45, CD3, CD4, and CD8. D, For each cell type, fold-increase infiltration over isotype control was plotted as column graph plot� SEM. For each condition,between 3 and 5 tumors were analyzed. E, Kaplan–Meier curves plotting the survival of human PBMC reconstituted non–tumor-bearing mice following weeklydosing of PRS-343 or controls.

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peripheral T-cell and NK-cell activation. We hypothesized thatbiotherapeutics addressing this pathway should efficiently acti-vate the immune costimulatory target, but its activation should berestricted to the tumor microenvironment (TME) to avoid sys-temic effects and unwanted toxicity.

PRS-343 is an Anticalin-antibody fusion protein with dualspecificity for both 4-1BB and the tumor antigen HER2. PRS-343was designed to promote 4-1BB clustering by bridging 4-1BB–positive T cells with HER2-positive tumor cells, thereby providingapotent costimulatory signal to tumor antigen–specific T cells.Onthe basis of its differentiated mechanism of action, PRS-343 hasthe potential to expand therapeutic options to HER2-positivetumors including those who may not be responsive to conven-tional antibody or small-molecule–based HER2 inhibitors.

Anticalin proteins are 18 kDaprotein therapeutics derived fromhuman lipocalins. We utilized phage display to generate anAnticalin protein binding to 4-1BB with high affinity and spec-ificity. PRS-343 was generated by genetic fusion of the 4-1BB–specific Anticalin protein to a variant of the HER2-targeting mAbtrastuzumab with an engineered IgG4 backbone. We demonstrat-ed the benefits of our bispecific platform's flexible formatting,allowing for functional testing of multiple bispecific geometriesand the demonstration that the 4-1BB Anticalin protein fused tothe heavy-chain C-terminus of the antibody was functionally themost active in cell-based assays.

Binding studies using SPR, ELISA, and FACS showed thatPRS-343 displays similar potency against each target comparedwith parental building blocks. Simultaneous binding of bothtargets was also demonstrated. PRS-343 displays cross-reactivityto cynomolgus HER2 similar to trastuzumab but with reducedcross-reactivity to cynomolgus 4-1BB. PRS-343 induces 4-1BBclustering and downstream signaling in a Jurkat NF-kB reportercell line in the presence of HER2-positive cells with a potency ofapproximately 50 pmol/L (EC50) as well as IL2 production in acostimulatory T-cell activation assay in the presence of HER2-positiveNCI-N87 cells, with a potency of 35 pmol/L.Weobservedabell-shaped response both in the JurkatNF-kB reporter assay andthe primary T-cell activation assay, which is in accord withexpectations as a response requires the formation of a ternarycomplex of the tumor cell target HER2, the drug PRS-343 and theT-cell receptor 4-1BB, that can be disrupted when components areindividually saturated with drug. The effect can be rationalized bya mathematical model recently described for ternary complexformation in biological systems and depends on the concentra-tions of all binding partners and their affinities to each other (39).

The pharmacology of PRS-343 was investigated by furtherex vivo T-cell costimulation assays based on mixed culture ofhuman T cells and cell lines. The cell panel was selected to covera broad range of HER2-expressing cells derived from cancer tissueas well as from different healthy tissue origins. Of note, three ofthe cell lines capable of costimulating T cells in aHER2-dependentmanner have been described as being resistant [(40–42)] inpreclinical models to trastuzumab (SUM-225) or even bothtrastuzumab and lapatinib (JIMT-1, MDA-MB-453), demonstrat-ing the potential of PRS-343 to provide a therapeutic option forpatients with innate or acquired resistance to HER2-targetedtherapy. The risk of PRS-343–mediated systemic 4-1BB activationand concomitant toxicity was investigated in a cytokine releaseassay, indicating that 4-1BB clustering leads to negligible cytokinerelease by T cells in the absence of a primary T-cell receptorstimulus. Together with the costimulation experiments, these

results indicate that PRS-343 is able to activate T cells only whenthese are engaging a target cell that expressesHER2 at a level that isusually only encountered in malignant, tumorous tissue, andwhen the T cells are activated at the same time via the T-cellreceptor, for example, by recognizing a tumor antigen.

In vivo proof-of-concept data utilizing a humanized SK-OV-3mouse model support the desired mode of action of PRS-343in vivo. PRS-343 exhibits both direct cytotoxicity via monospecifictargeting of tumor-expressed HER2 and activates 4-1BB via bis-pecific targeting of tumoral HER2 and 4-1BB on human lympho-cytes. These data also highlight the differentiating features over amonospecific 4-1BB–targeting constitutive agonist. PRS-343leads to an increase in the frequency of human lymphocytes inthe tumor, but does not affect the human lymphocyte frequencyin the peripheral blood, which correlates with an unchanged timecourse of GVHD-induced morbidity and mortality comparedwith controls. In contrast, the monospecific 4-1BB–targetingagonist antibody 20H4.9 leads to an expansion of human lym-phocytes in the peripheral blood and a concomitant accelerationof GVHD-induced morbidity and mortality, despite lacking anyactivity in the TME, as evidencedbyno significant increase inT-cellinfiltration in the TME when compared with control. The data onPRS-343 reported here support its further evaluation either as asingle-agent or combination therapy. Indeed, a preclinical ratio-nale for combining 4-1BB agonismwith checkpoint blockade hasbeen demonstrated (43, 44). PRS-343 is the first bispecific 4-1BBagonist to enter the clinic, and a phase I dose escalation study inpatients with HER2-positive advanced or metastatic solid tumorsis ongoing (NCT03330561). A clinical trial evaluating PRS-343 incombination with atezolizumab (anti–PD-L1) has also com-menced (NCT03650348).

Disclosure of Potential Conflicts of InterestM. Hinner, R.S. Bel Aiba, T.J. Jaquin, S. Berger, M.C. D€urr, C. Schlosser,

A. Allersdorfer, G. Matschiner, U. Moebius, C. Rothe, L. Matis, and S.A. Olwillhold ownership interest (including patents) in Pieris Pharmaceuticals. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M.J. Hinner, R.S. Bel Aiba, S. Berger, U. Moebius,C. Rothe, L. Matis, S.A. OlwillDevelopment of methodology: M.J. Hinner, R.S. Bel Aiba, T.J. Jaquin,C. Schlosser, A. Allersdorfer, A. Wiedenmann, J. Sch€uler, U. Moebius,C. Rothe, S.A. OlwillAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.J. Hinner, R.S. Bel Aiba, T.J. Jaquin, S. Berger,C. Schlosser, A. Allersdorfer, A. Wiedenmann, J. Sch€ulerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.J. Hinner, R.S. Bel Aiba, T.J. Jaquin, S. Berger,M.C. D€urr, C. Schlosser, A. Allersdorfer, A. Wiedenmann, J. Sch€uler, C. Rothe,S.A. OlwillWriting, review, and/or revision of themanuscript:M.J. Hinner, R.S. Bel Aiba,T.J. Jaquin, S. Berger, M.C. D€urr, C. Schlosser, A. Allersdorfer, G. Matschiner,J. Sch€uler, L. Matis, S.A. OlwillAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Schlosser, A. Allersdorfer, A. Wiedenmann,J. Sch€uler, L. MatisStudy supervision: M.J. Hinner, U. Moebius, C. Rothe, L. Matis, S.A. Olwill

AcknowledgmentsWe would like to thank the extended Pieris team for their support

in generating and reviewing data related to the PRS-343 project. We wouldlike to specifically thank Tanya Aneichyk for her assistance in data

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interpretation and presentation. The research funding was provided byPieris Pharmaceuticals.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedNovember 8, 2018; revisedMarch 14, 2019; acceptedMay 22, 2019;published first May 28, 2019.

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2019;25:5878-5889. Published OnlineFirst May 28, 2019.Clin Cancer Res   Marlon J. Hinner, Rachida Siham Bel Aiba, Thomas J. Jaquin, et al.   4-1BB/HER2 Bispecific Antibody-Anticalin Fusion PRS-343Tumor-Localized Costimulatory T-Cell Engagement by the

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Published OnlineFirst May 28, 2019; DOI: 10.1158/1078-0432.CCR-18-3654