Tumor-Localized Costimulatory T Cell Engagement by the 4-1BB/HER2 Bispecific Antibody-Anticalin Fusion PRS-343 Authors: Marlon J

Author Manuscript Published OnlineFirst on May 28, 2019; DOI: 10.1158/1078-0432.CCR-18-3654 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Tumor-localized costimulatory T cell engagement by the 4-1BB/HER2 bispecific antibody-Anticalin fusion PRS-343 Authors: Marlon J. Hinner1*, Rachida Siham Bel Aiba1, Thomas Jaquin1, Sven Berger1, Manuela Dürr1, Corinna Schlosser1, Andrea Allersdorfer1, Alexander Wiedenmann1, Gabriele Matschiner1, Julia Schüler2, Ulrich Moebius1, Christine Rothe1, Louis Matis1, Shane.A. Olwill1*

Affiliations: 1 Research and Development, Pieris Pharmaceuticals GmbH, Freising, Germany. 2 In Vivo Operations, Oncotest GmbH, Freiburg, Germany. *Corresponding authors: Shane A. Olwill, Pieris Pharmaceuticals GmbH, Lise-Meitner-Strasse 30, 85354 Freising, Germany. Phone: +49-8161-1411400; email [email protected]. Marlon J. Hinner; email [email protected].

Abstract:

Purpose: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have 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 genetic fusion of 4-1BB-specific Anticalin® proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. Results: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS- 343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL-2, GM-CSF, TNF- and IFN-In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human PBMCs, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicological findings. Conclusion: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared to current monospecific approaches. The reported data led to initiation of a Phase 1 clinical trial with this first-in-class molecule.

Running Title: Costimulatory T-cell engagement by the 4-1BB/HER2 bispecific PRS-343 molecule

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2019; DOI: 10.1158/1078-0432.CCR-18-3654 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Translational Relevance Immunotherapy with checkpoint inhibitors, such as anti-PD-1 monoclonal antibodies, has had a major impact on cancer therapy. While such therapies afford durable responses or even cures, additional therapeutic strategies are still required for the majority of patients who do not respond or relapse. The activation of costimulatory pathways such as 4-1BB has long been acknowledged to hold great promise, but clinical efforts thus far have failed to demonstrate broad efficacy and have been associated with severe toxicity (1, 2). Herein we describe the HER2/4-1BB bispecific molecule PRS-343, which aims to overcome 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 action of PRS-343, providing a novel approach to target 4-1BB that may prove both safer and more efficacious compared to monospecific targeting. PRS-343 has the potential to offer an alternative therapeutic strategy to patients in multiple HER2-positive indications including bladder, breast, and gastric cancer.

Introduction 4-1BB, also known as CD137, is a costimulatory immune receptor, a member of the tumor necrosis factor receptor (TNFR) super-family, and is predominantly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells (3). 4-1BB plays an important role in the regulation of immune responses and together with the fact that it is expressed on tumor infiltrating lymphocytes makes it a promising target for cancer immunotherapy. The 4-1BB ligand (4-1BBL) is constitutively expressed on several types of antigen-presenting cells (APC) (4). Upon pathway 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 mitochondrial capacity and biogenesis of T cells to explain why 4-1BB agonism could help overcome the immunosuppressive landscape of the tumor microenvironment (6). While most research has focused on 4-1BB in the context of T effector cells it should be noted that 4-1BB is also expressed on T regulatory cells where its role remains contentious and requires further elucidation (7) . 4-1BB has also been implicated in promoting a central memory T cell response which may support therapeutic persistence of tumor specific T cells and resistance to exhaustion in patients treated with a 4-1BB agonist (8, 9). The potential therapeutic benefit of 4-1BB costimulation has been demonstrated in multiple preclinical models. The forced expression of 4-1BBL on a tumor, for example, leads to tumor rejection (10). Likewise, the forced expression of an anti-4-1BB single chain antibody fragment (scFv) on a tumor leads to a CD4+ T-cell and NK-cell dependent elimination of the tumor (11- 13). A systemically administered anti-4-1BB antibody has also been demonstrated to lead to retardation of tumor growth in mouse models (14). Human ex vivo data support the potential of 4-1BB as a costimulatory receptor in cancer therapy. It has been reported that for T cells isolated from human tumors, 4-1BB is a marker for those that are tumor-specific (15). In line with this observation, anti-4-1BB antibodies can be utilized to improve adoptive T-cell therapy (ACT) by augmenting the expansion and activity of CD8+ melanoma tumor-infiltrating lymphocytes (16). Further clinical evidence for the importance of 4- 1BB signaling for a sustained and successful anti-cancer T cell response is provided in ACT with

chimeric antigen receptors (CARs), where inclusion of 4-1BB-signaling elements in the cytoplasmic domain of CARs has been found to improve durability of clinical responses (17, 18). Receptors from the TNF receptor family in general require higher-order clustering for efficient downstream signaling activation in the TNF receptor expressing cell (19), which usually is not afforded by the soluble ligand alone, but requires engagement of another cell expressing the respective ligand on its surface. For example, despite being a homotrimer, soluble 4-1BBL is not capable of efficiently activating 4-1BB downstream signaling (20). Antibodies targeting 4-1BB are either inherently agonistic or often require a secondary means of clustering beyond bivalent binding to allow for agonistic activity. It is hypothesized that in vivo TNF receptor agonism by such antibodies is dependent on simultaneous binding to Fc receptor-positive cells (21, 22). The intricate mode of activation of 4-1BB may underlie the modest clinical success obtained with monospecific anti-4-1BB antibodies to date. BMS-663513 (urelumab) (23) has been hampered by dose-limiting hepatotoxicity, likely due to its tendency to systemically activate 4- 1BB (1). Initial clinical trials with PF-05082566 (utomilumab) (24, 25) have not shown the same safety issues as urelumab profile, yet preclinical characterization suggests it is a less potent 4- 1BB agonist (2). To overcome the challenges of targeting the 4-1BB pathway systemically, we have generated PRS-343, a bispecific molecule designed 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 recombinant fusion of a 4-1BB-specific Anticalin protein to a HER2-specific antibody. Anticalin proteins are engineered variants of lipocalins, a family of natural extracellular binding proteins. Lipocalins are characterized by a highly conserved tertiary structure despite low amino acid identity. This property coupled to the fact that they possess free C- and N-termini, which are not required for target recognition, allow facile fusion to other proteins, defining a versatile basis for a multispecific biologics platform (26). The molecule, PRS-343, is designed to promote 4-1BB clustering by bridging T cells with HER2-positive tumor cells, providing a potent costimulatory signal to tumor antigen-specific T cells, further enhancing T cell receptor (TCR)-mediated activity and leading to tumor destruction. PRS-343- mediated 4-1BB activation is, therefore, biased towards locations in the body where T cells and tumor cells are co-localized, such as in primary tumors with tumor-infiltrating lymphocytes (TIL) or in lymph-nodes containing tumor metastases. Here, we describe the generation and preclinical characterization of PRS-343 with regard to target engagement, biological activity and safety.

Materials and Methods

Engineering and production of Anticalin proteins and bispecific fusion proteins Recombinant 4-1BB (R&D Systems) was used as the target for phage display selection and ELISA screening of cognate Anticalin protein candidates. A random library based on human neutrophil-gelatinase associated lipocalin (NGAL) with high combinatorial complexity was prepared by concerted mutagenesis of multiple amino acid positions (27). 4-1BB target specificity was confirmed by ELISA screening and selected Anticalin proteins were optimized

via partial mutagenesis of coding regions followed by biophysical and functional characterization, which resulted in selection of a lead candidate J10. Bispecific antibody-Anticalin fusion proteins were generated by the recombinant fusion of the 4- 1BB-specific Anticalin protein (J10) to either the heavy or light chain N- or C-terminus of a modified variant of trastuzumab. Specifically, the isotype of trastuzumab was changed to an IgG4 and additional mutations were introduced to reduce interactions with activating and inhibitory Fc-receptors. These were the mutation S228P, which has been described to suppress half-antibody exchange (28), and the mutations F234A and L235A which have been reported to reduce binding to FcRI, were introduced (29, 30). Bispecific proteins were obtained by recombinant expression in mammalian cells (HEK293 or CHO) using standard techniques. Reagents and cell lines Recombinant 4-1BB and HER2 protein (human and cynomolgus monkey) were obtained from R&D Systems and Sino Biological. The cell lines HT-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 from ATCC. Primary cells (human cardiac myocytes, human epidermal keratinocytes, human pulmonary fibroblasts, human cardiac fibroblasts, human dermal microvascular endothelial cells, human umbilical vein endothelial cells, human aortic smooth muscle cells and human bronchial smooth muscle cells) were obtained from Promocell. The cells were expanded following cell bank instructions. The HER2 levels were confirmed by QifiKit (Dako) according to the provided protocol and were standardized to HER2 levels expressed by SKBR3. The list of the cells, corresponding cell bank catalogue number and their respective HER2 expression levels standardized to SKBR3 HER2 levels are summarized in Supplementary Table S1. CHO cells expressing human 4-1BB (h 4- 1BB) were established by stable transfection of h 4-1BB using the Flp-In system (Invitrogen). An NF-ĸB-Luc2/4-1BB Jurkat cell line was obtained from Promega. Human peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors were isolated from buffy coats by centrifugation through a polysucrose density gradient, and T cells were isolated from the resulting PBMCs using a pan T cell isolation Kit (Miltenyi Biotec) according to the manufacturer´s protocols. The 4-1BB agonist antibody 20H4.9 (corresponding to urelumab) was obtained by recombinant expression in CHO cells using standard techniques. Target-binding studies

Binding of PRS-343 to 4-1BB or HER2 proteins (human or cynomolgus monkey) was analyzed by enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR). In the target-binding ELISA, the extracellular domain of the target proteins, 4-1BB (human or cynomolgus), HER2 (human) or control protein, was coated onto microtiter plates. Either PRS- 343, the 4-1BB specific reference Anticalin protein or the reference anti-HER2 antibody was added and binding was detected with peroxidase conjugated anti-human IgG or anti-NGAL antibody. For the dual-binding ELISA, human HER2 (h HER2) was coated and PRS-343 binding was detected using biotinylated h 4-1BB detected by peroxidase-labeled ExtrAvidin. In the SPR affinity assay, biotinylated h 4-1BB or h HER2 was captured on a sensor chip CAP using the Biotin CAPture Kit (GE). Determination of PRS-343 binding kinetics and affinity was performed by applying four dilutions of PRS-343 to the chip surface with a flow rate of

30 µL/min; the sample contact time was 180 s and dissociation time was 1800 s. Data were 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 IgG4 as negative control, and binding of the test molecules was detected using Alexa Fluor 488 labeled goat anti-human IgG antibody. An immunohistochemical assay was performed to assess cross reactivity of PRS-343 on human tissue. Briefly, cryo-sections histologically prepared at a nominal 5 μm from a panel of 40 frozen human tissues together with positive and negative control material were stained with biotinylated PRS-343 or control. A detailed microscopic examination was performed to assess each tissue for any sign of positive staining.

T-cell activation The impact of HER2 receptor density on PRS-343-mediated T-cell activation was assessed in co- culture experiments using a panel of cell lines expressing different levels of HER2. Cancer cell lines representing a range of clinically relevant levels of HER2 receptor were tested for their ability to mediate clustering of PRS-343 and subsequent activation of T-cells. To evaluate a potential therapeutic window cell lines derived from healthy tissues known to express background levels of HER2 were also included. The level of T-cell activation was measured by quantification of human IL-2, using an electrochemiluminescence (ECL) immunoassay (Mesoscale Discovery). Briefly, cancer cells or cells derived from healthy tissue pretreated with 10 µg/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 x 104 cells) together with test article were added. The IL-2 concentration in the supernatant was assessed by an ECL assay (using IL-2 DuoSet kit; R&D Systems) following 3 days incubation. Specific activation of the 4-1BB pathway by PRS-343 was assessed using a luciferase reporter cell assay (Promega), where a 4-1BB-overexpressing reporter cell line (NF-ĸB-Luc2/4-1BB Jurkat cells) was co-cultured with HER2-positive tumor cell lines and where 4-1BB pathway activation was measured by luminescence. Cytokine release assay The potential of PRS-343 to induce cytokine release syndrome was evaluated in vitro using a cytokine release assay (31), where soluble or coated (wet or dry coated) PRS-343 was incubated with human PBMC for 72 hours followed by the quantification of a panel of pro-inflammatory cytokines (MSD). The anti-CD3 monoclonal antibody OKT3 (Muromonab-CD3) was used as positive control and a human monoclonal IgG4 isotype antibody was used as negative control. Cynomolgus Toxicity Study A 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 a 4-week recovery period was performed in cynomolgus monkeys. Serum levels of PRS-343 were assessed by an ECL assay. Assessment of safety and toxicity was based on standard parameters.

Mouse and cynomolgus monkey pharmacokinetics Single dose pharmacokinetics studies were performed in mice and cynomolgus monkeys. Male CD-1 mice were administered with an intravenous injection of PRS-343 (10 mg/kg). Cynomolgus monkeys (Macaca fascicularis) received a 60-min infusion of PRS-343 (3 mg/kg) For each study plasma was collected predose and at multiple timepoints post administration. PRS-343 plasma concentrations were determined via an ECL assay (MSD) using a dual-binding ELISA. The pharmacokinetic parameters were derived by non-compartmental analysis using Phoenix WinNonlin. In vivo tumor efficacy model PRS-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, Denmark), aged 5-7 weeks. Tumor growth was monitored twice weekly and tumor volume was calculated as follows: (length x width2)/2. At study end, plasma was collected for all animals and lymphocyte phenotyping was carried out by flow cytometry assessing human cell markers such as CD45 (Invitrogen), CD4 and CD8 (BD Biosciences). To assess tumor infiltrating lymphocytes, 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 the percentage of target positive (tumor- infiltrating) cells.

Graft Versus Host Disease (GVHD) model The potential for PRS-343 to induce systemic GVHD was assessed. 5-7-week-old female NOG mice (NOD.cdPrkdcscidIL2rgtm1.Sug/JicTac, supplied by Taconic, Denmark) were injected with fresh human PBMC and treated with PRS-343 or controls. As a read-out for signs of GVHD, the animals were routinely checked for changes in body weight or general health. Mice who were euthanized (based on predefined criteria) or spontaneously expired mice were recorded for survival analysis by each treatment. Data were plotted using a Kaplan Meier curve.

Flow cytometry Flow cytometry analyses were carried out on an iQue Screener (Intellicyt Corporation) equipped with ForeCyt software or with an Attune Focusing Cytometer (blue (488 nm)/violet (405 nm) laser configuration).

Ethical considerations

All animal experiments and protocols were approved by the institutional animal welfare body and the relevant local authorities and were conducted according to all applicable international, national and local laws and guidelines. All animal experiments were approved by the regional Ethics Committees (Germany or the UK).

Statistical analysis

All 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 multiple groups. P values less than 0.05 were considered statistically significant.

Results Generation of PRS-343 by Protein Engineering and Design A 4-1BB binding Anticalin protein was obtained by phage display selection and optimized by protein engineering as described in Materials and Methods. The 4-1BB specific Anticalin protein (J10) has an affinity of 2 nM as determined by SPR, binds to human 4-1BB transfected CHO cells and does not compete with the binding of 4-1BB ligand to its receptor (data not shown). An ELISA screen of related TNFR superfamily proteins (4-1BB Ox40, GITR, TNFRI, TNFRII, CD30, RANK) confirmed J10 Anticalin protein specifically binds to 4-1BB (Figure S1) As the HER2-binding, tumor-targeting building block of a 4-1BB/HER2 bispecific, we utilized a modified variant of trastuzumab. The formatting flexibility offered by the Anticalin technology facilitated the generation of various Antibody-anticalin fusion formats as depicted in Figure S 1. We hypothesized that the distance between T cell and tumor cell binding sites may impact the ability of the 4-1BB/HER2 bispecific to appropriately cluster the 4-1BB receptor and thus activate the pathway. Therefore, the 4-1BB-engaging Anticalin protein was genetically fused either to the N- or C-terminus of the anti-HER2 antibody heavy or light chain resulting in the generation of four different geometries of the fusion protein covering a range of binding distances. The antibody Fc region was engineered (see Materials and Methods for details) to exclude the risk of NK-driven ADCC against 4-1BB-positive cells or undesired, non-tumor- target activation of 4-1BB-positive lymphocytes via FcR derived crosslinking. The interaction with the neonatal Fc receptor (FcRn) was retained to support a prolonged terminal plasma half- life. While all formats behaved similarly from the perspective of biophysical and target binding properties, we observed a marked difference in their ability to elicit T cell activation (Figure S2). Based on these data a single format was selected and 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, non-immunogenic (G4S)3 linker sequence, as depicted in Figure 1.

Human target binding The interaction of PRS-343 with its human targets was investigated by ELISA, surface plasmon resonance (SPR) and FACS. As assessed by SPR, PRS-343 binds to recombinant human HER2 with high affinity (KD = 0.3 nM), similar to the parental antibody trastuzumab. The binding affinity of PRS-343 to human monomeric 4-1BB was determined to be 5 nM (Figure 1). In a FACS assay, PRS-343 was found to bind HER2-expressing MCF-7 cells with an EC50 of 7.4 nM. Binding to CHO cells transfected with human 4-1BB was determined with an EC50 of 6.2 nM (Figure 1).

Using a dual binding ELISA, PRS-343 was found to be capable of binding both targets simultaneously, which is a prerequisite for its envisioned mode of action. PRS-343 displays reduced cross-reactivity to cyno 4-1BB in comparison to human 4-1BB as shown by ELISA (Figure 1K). An SPR experiment also demonstrated that binding of PRS-343 to cyno 4-1BB strongly depends on target densities (data not shown). Further SPR experiments demonstrated that PRS-343 does not bind to Fc receptors but binding to the neonatal Fc receptor (FcRn) is retained. In accordance with its lack of interaction with Fc receptors, PRS-343 does not elicit any ADCC activity when co-incubated with human PBMCs and HER2-positive breast carcinoma cells (data not shown). A tissue cross-reactivity study using a panel of 40 human tissues (n=3 donors) each demonstrated that PRS-343 binds as expected for a molecule targeting both HER2 and 4-1BB in a bispecific manner. Epithelial cells of various tissues showed strong positivity for binding PRS-343 which is in accordance with published data on HER2 expression in human tissues (32). The observed lymphocyte staining is in agreement with the expected binding of PRS-343 to 4-1BB on this cell type since 4-1BB has been reported to be (inducibly) expressed on lymphocytes (33). In vitro activity of PRS-343 The ability of PRS-343 to costimulate T cells in a HER2-dependent manner was investigated in vitro using a reporter assay. The reporter cell assay was performed to investigate whether PRS-343 is capable of inducing HER2-dependent 4-1BB clustering on T cells. In this assay, NF-ĸB-Luc2/4-1BB Jurkat cells were co-cultured with a HER2-positive tumor cell line. Successful clustering of 4-1BB on the Jurkat cell surface is expected to lead to TRAF-2 (and TRAF-1) mediated activation of Nf-B (reviewed in reference (34)), which in the presence of a luciferase substrate can be detected by increased luminescence. Indeed, co-incubation of up to 10 nM PRS-343 with the Jurkat reporter cell line and the HER2- positive NCI-N87 cell line led to a 20-fold increase of NF-κB luciferase reporter activity with an EC50 of 50 pM (Figure 2A). Furthermore, in a wash-out experiment where PRS-343 was added to various cancer cell lines overnight prior to a media exchange, HER2 specific 4-1BB activation was maintained (Figure S4). When using the low HER2-expressing MKN-45 or Hep-G2 cell lines, the NF-κB activity was similar to background and control values. A bell-shaped response was observed when concentrations of up to 1µM PRS-343 were applied in this type of assay (data not shown). These results demonstrate that PRS-343 facilitates 4-1BB pathway activation only in the presence of HER2-overexpressing cells. HER2-dependent costimulatory T cell activation To investigate whether this mechanism can be applied to effectively costimulate T cells, a co- culture assay was developed using human T cells and HER2-expressing cell lines. Here, the T cells receive a primary T cell receptor stimulus via anti-CD3 antibody activation, and PRS-343 induced costimulation can be quantified by measuring supernatant levels of pro-inflammatory cytokines. Using a multiplex assay, we first investigated which cytokines were produced by T cells co-stimulated by PRS-343 in the presence of HER2-expressing cells (Figure S5). The

strongest fold increase over background was observed for IL-2. Statistically significant increases in GM-CSF, IFN and TNFwereobserved. IL-4 and IL-6 supernatant levels were increased albeit absolute cytokine levels were low. There was no PRS-343-dependent response observed for IL-1, IL-8, IL-10 and IL-12. Given the robust nature of the IL-2 response it was selected for as a marker of activation for future assays. In the presence of HER2 positive cell lines, a dose- dependent induction of IL-2 was observed with PRS-343 (Figure 2B and C). When the experiment was performed with cell lines expressing basal levels of HER2, no PRS-343- dependent IL-2 induction was observed. To further confirm that PRS-343 activity is dependent on HER2 binding, additional assays were performed in the presence of an excess of trastuzumab (200 nM). Under these conditions, the excess trastuzumab competed for binding to HER2 and was found to abrogate PRS-343 induced T cell costimulation, further supporting its envisaged mode of action (Figure 2C), whereas the control 4-1BB agonistic antibody 20H4.9 consistently induced IL2 secretion irrespective of the co-incubated cell lines (Figure 2C).

Impact of HER2 expression level To examine the HER2 expression level threshold required for successful PRS-343-induced T cell costimulation, the assay was performed with a panel of cell lines of variable HER2-positivity (Table S1). The HER2 cell surface expression level was determined by quantitative FACS and is reported as the specific anti-HER2-antibody binding capacity (sABC). 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 of HER2 expression levels ranging from approximately 1% to 200% of the reference value. In contrast, cell lines from healthy tissues express HER2 at a much lower and more confined level, with relative HER2 expression around 1 % of HER2 expression on SK-BR-3 (Table S1). These cancer- and healthy tissue-derived cell lines were then applied in the previously described costimulatory T cell activation assay to identify a potential HER2 threshold required for T cell costimulation. A selection of experimental results based on IL2 as the readout and utilizing tumor cell lines and primary cell types is shown in Figure 3. These data confirm that IL2 secretion strongly correlates with HER2 expression. Assessing the maximum level of IL2 secretion compared to control, we show that cancer cells with a high level of HER2 induced significantly increased IL2 secretion. For cell lines described as having a more intermediate level of HER2 (e.g. MCF-7 or MKN-45), we could also observe significantly increased IL2 secretion in a proportion of the donors tested. Both the cancer cell lines expressing basal levels of HER2 (such as Bx-PC-3 or MDA-MB-231) and the tissue-derived primary cells did not induce a significant amount of IL2 secretion, supporting the hypothesis that PRS-343 requires HER2 expression at supraphysiologic levels on target cells to costimulate T cells. Cytokine release assay To investigate the risk of systemic cytokine release in patients induced by PRS-343, an in vitro cytokine release assay (31) was carried out with peripheral blood mononuclear cells (PBMCs) from 12 healthy control donors. In contrast to the anti-CD3 positive control antibody OKT3, PRS-343 did not induce a significant increase of cytokines over background when wet coated onto plates or when incubated with PBMCs in solution. When PRS-343 was air-dried, a modest but statistically significant increase of the cytokines IFN-y, GM-CSF, IL-4 and IL-8 was

observed only at the highest concentration (i.e.100 µg/mL). Relative to the maximum increase induced by OKT-3 (10 µg/mL, air dried), the levels induced by PRS-343 were 0.2% for GM- CSF, 0.1% for IFN-ɣ, 6% for IL-4 and 13% for IL-8 (Figure S3).

Pharmacokinetics in mice and cynomolgus monkeys Pharmacokinetics was evaluated following a single i.v. administration of PRS-343 by bolus injection of male CD-1 mice at 10 mg/kg, or upon a 60-minute i.v. infusion to male cynomolgus monkeys at 3 mg/kg. The plasma concentrations in both species were found to decline in a biphasic manner (Figure 4). The terminal elimination half-life was >14 days in mouse. In cynomolgus monkeys, a terminal elimination half-life of approximately 4 days was observed. In conclusion, the determined PK parameters for PRS-343 demonstrate that it behaves similar to a conventional monoclonal antibody in preclinical models.

Humanized Mouse Tumor Model The in vivo activity of PRS-343 was evaluated in a humanized mouse model using immunocompromised mice subcutaneously engrafted with the SK-OV-3 cell line as a HER2- positive tumor xenograft. This cell line was chosen based on HER2 positivity, trastuzumab sensitivity, and the ability to grow homogenously as a subcutaneous xenograft in the presence of human PBMC without being directly eliminated. Mice with established tumors (> 100mm3) were grouped and treated with test articles on a weekly dosing schedule. Median tumor volumes over time for each of the treatment groups are shown in Figure 5A. PRS-343 displayed dose- dependent anti-tumor efficacy with doses ranging from 4µg to 100 µg (approximately 0.2 mg/kg to 5 mg/kg), while the 200 µg dose (approximately 10 mg/kg) did not further enhance tumor regression. Equimolar dosing of the trastuzumab-IgG4 control antibody displayed comparable anti-tumor efficacy to that of PRS-343. The model has been previously described as being responsive to anti-HER2 therapy and helps confirm that HER2-mediated anti-tumor activity is preserved in PRS-343. Interestingly, the 4-1BB agonist (20H4.9) antibody was unable to block tumor growth in this model. Flow cytometry analysis of the human immune cell population in the peripheral blood of the mice revealed very little differences between the PRS-343-treated groups and control groups. However, in mice treated with the 4-1BB agonist antibody (20H4.9), a peripheral expansion of human CD8-positive cells was observed (Figure 5B), demonstrating a systemic impact of the agent. To assess the 4-1BB-mediated effect of PRS-343, excised tumors from treated mice were analyzed for immune infiltration. PRS-343 administration led to a significant increase in human CD45-positive lymphocytes in tumor tissue (Figure 5C) compared to the control groups. While assessing the immune cell subtypes infiltrated within the tumors, we could observe that PRS- 343-induced infiltrate consisted predominantly of CD8 positive T cells (Figure 5C). Importantly, there was no significant increase in immune cell infiltrates in the excised tumors from mice treated with the trastuzumab-IgG4 when compared to controls. In immune-compromised mice engrafted with human PBMCs, graft-versus-host disease (GVHD) is expected to occur due to the human PBMCs destroying mouse tissue and organs resulting in

progressive weight loss and mortality (35). In this humanized xenograft study, we observed that the 4-1BB agonist antibody accelerated GVHD-induced mortality, while the administration of PRS-343 or the isotype control antibody did not influence weight loss or the median survival of the mice. To confirm this observation, we carried out a dedicated experiment using larger group sizes (n=15) of non-tumor bearing mice. This study confirmed that weight loss and survival following PRS-343 treatment were comparable to that observed in the IgG4 isotype control treated animals, while treatment with the systemically active 4-1BB agonist led to both significantly greater weight loss and shorter survival compared to control (Figure 5D). Taken together, these data show that PRS-343 provides dual activity by increasing the number of tumor-infiltrating lymphocytes coupled with direct tumor growth inhibition by bispecific targeting of 4-1BB and HER2. Notably, the tumor growth inhibition provided by targeting HER2 did not require any antibody directed cellular cytotoxicity (ADCC), as both PRS-343 and the trastuzumab-IgG4 control lack the ability to interact with Fc-gamma receptors on NK cells that ADCC would require.

Safety of PRS-343 in cynomolgus monkeys The safety of PRS-343 was investigated in a 4-week GLP-compliant toxicity study in cynomolgus monkeys. It is important to take into account the reduced cross-reactivity of PRS- 343 to cynomolgus 4-1BB which impacts the ability of this study to predict 4-1BB-related toxicity. PRS-343 was administered weekly as an intravenous infusion of 120 min duration at doses of 10 and 120 mg/kg over 4 weeks, followed by a recovery phase in the control and high dose group to evaluate any potential delayed onset or reversibility of toxicity. Overall, PRS-343 was well tolerated at both doses tested, with no significant findings. No unscheduled deaths occurred. There were no changes in standard parameters such as clinical observations, body weight, ophthalmology, rectal temperatures, clinical chemistry, hematology, coagulation tests, urinalysis parameters or serum cytokines as well as ECG. Furthermore, no toxicologically relevant organ weight or organ weight ratio changes, and no macroscopic or microscopic findings were observed, indicating that treatment with PRS-343 over four weeks did not lead to any systemic toxicity. In addition, no evidence of delayed onset toxicity was noted at the end of the four-week recovery phase. Toxicokinetic analysis demonstrated dose-proportional systemic exposure at both dose levels upon first and last dose. Two animals out of sixteen developed ADA’s that persisted throughout the study while three others showed a transient ADA response. No gender-related toxicity differences were noted in the study.

Discussion 4-1BB (CD137) is a key costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. The preclinical and clinical demonstration of the potential therapeutic benefit of 4-1BB costimulation has spurred the development of therapeutic antibodies targeting 4-1BB, utomilumab (24, 25) and urelumab (23). Utomilumab is a fully humanized IgG2 monoclonal antibody that binds 4-1BB in a manner that blocks the binding of endogenous 4- 1BBL to 4-1BB and that appears well tolerated as a monotherapy (36) and in combination with rituximab; however, modest activity has been observed (37). Urelumab is an IgG4 monoclonal antibody that, in contrast to utomilumab, binds 4-1BB in a manner that does not interfere with

the 4-1BB / 4-1BBL interaction. While an initial trial reported modest clinical activity (38), a follow-up study was stopped due to hepatotoxicity (1). These data indicate that systemic activation of 4-1BB with a potent agonist may lead to prohibitive toxicity, supporting the rationale for tumor-localized targeting of the pathway. While multiple lines of evidence suggest that 4-1BB is a highly promising therapeutic target in cancer, current systemic antibody-based approaches are not designed to achieve a tumor-target driven activation and are likely to display toxicity due to peripheral T cell and NK cell activation. We hypothesized that biotherapeutics addressing this pathway should efficiently activate the immune costimulatory target, but its activation should be restricted to the tumor microenvironment (TME) to avoid systemic effects and unwanted toxicity. PRS-343 is an Anticalin-antibody fusion protein with dual specificity for both 4-1BB and the tumor antigen HER2. PRS-343 was designed to promote 4-1BB clustering by bridging 4-1BB- positive T-cells with HER2-positive tumor cells, thereby providing a potent costimulatory signal to tumor antigen-specific T-cells. Based on its differentiated mechanism of action PRS-343 has the potential to expand therapeutic options to HER2-positive tumors including those who may not be responsive to conventional antibody or small molecule based HER2 inhibitors. Anticalin® proteins are 18 kDa protein therapeutics derived from human lipocalins. We utilized phage display to generate an Anticalin protein binding to 4-1BB with high affinity and specificity. PRS-343 was generated by genetic fusion of the 4-1BB-specific Anticalin protein to a variant of the HER2-targeting monoclonal antibody trastuzumab with an engineered IgG4 backbone. We demonstrated the benefits of our bispecific platform’s flexible formatting, allowing for functional testing of multiple bispecific geometries and the demonstration that the 4-1BB Anticalin protein fused to the heavy-chain C-terminus of the antibody was functionally the most active in cell-based assays. Binding studies using SPR, ELISA and FACS showed that PRS-343 displays similar potency against each target compared to parental building blocks. Simultaneous binding of both targets was also demonstrated. PRS-343 displays cross-reactivity to cynomolgus HER2 similar to trastuzumab but with reduced cross-reactivity to cynomolgus 4-1BB. PRS-343 induces 4-1BB clustering and downstream signaling in a Jurkat Nf-kB reporter cell line in the presence of HER2-positive cells with a potency of approximately 50 pM (EC50) as well as IL-2 production in a costimulatory T cell activation assay in the presence of HER2-positive NCI-N87 cells, with a potency of 35pM. We observed a bell-shaped response both in the Jurkat Nf-kB reporter assay and the primary T-cell activation assay which is in accord with expectations as a response requires the formation of a ternary complex of the tumor cell target HER2, the drug PRS-343 and the T-cell receptor 4-1BB, that can be disrupted when components are individually saturated with drug. The effect can be rationalized by a mathematical model recently described for ternary complex formation in biological systems and depends on the concentrations of all binding partners and their affinities to each other (39), The pharmacology of PRS-343 was investigated by further ex vivo T-cell costimulation assays based on mixed culture of human T-cells and cell lines. The cell panel was selected to cover a broad range of HER2 expressing cells derived from cancer tissue as well as from different healthy tissue origins. Of note, three of the cell lines capable of costimulating T-cells in a HER2-dependent manner have been described as being resistant [(40-42)] in preclinical models to trastuzumab (SUM-225) or even both trastuzumab and lapatinib (JIMT-1, MDA-MB-453),

demonstrating the potential of PRS-343 to provide a therapeutic option for patients with innate or acquired resistance to HER2-targeted therapy. The risk of PRS-343-mediated systemic 4-1BB activation and concomitant toxicity was investigated in a cytokine release assay, indicating that 4-1BB clustering leads to negligible cytokine release by T-cells in the absence of a primary T- cell receptor stimulus. Together with the costimulation experiments, these results indicate that PRS-343 is able to activate T cells only when these are engaging a target cell that expresses HER2 at a level that is usually only encountered in malignant, tumorous tissue, and when the T- cells are activated at the same time via the T cell receptor, e.g. by recognizing a tumor antigen. In vivo proof of concept data utilizing a humanized SK-OV-3 mouse model support the desired mode of action of PRS-343 in vivo. PRS-343 exhibits both direct cytotoxicity via monospecific targeting of tumor expressed HER2 and activates 4-1BB via bispecific targeting of tumoral HER2 and 4-1BB on human lymphocytes. These data also highlight the differentiating features over a monospecific 4-1BB-targeting constitutive agonist. PRS-343 leads to an increase in the frequency of human lymphocytes in the tumor, but does not affect the human lymphocyte frequency in the peripheral blood, which correlates with an unchanged time course of GVHD- induced morbidity and mortality compared to controls. In contrast, the monospecific 4-1BB- targeting agonist antibody 20H4.9 leads to an expansion of human lymphocytes in the peripheral blood and a concomitant acceleration of GVHD-induced morbidity and mortality, despite lacking any activity in the TME, as evidenced by no significant increase in T cell infiltration in the TME when compared to control. The data on PRS-343 reported here support its further evaluation either as a single agent or combination therapy. Indeed, a preclinical rationale for combining 4- 1BB agonism with checkpoint blockade has been demonstrated (43, 44). PRS-343 is the first bispecific 4-1BB agonist to enter the clinic, and a Phase 1 dose escalation study in patients with HER2-positive advanced or metastatic solid tumors is ongoing (NCT03330561). A clinical trial evaluating PRS-343 in combination with atezolizumab (anti-PD-L1) has also commenced (NCT03650348).

Disclosure of Potential Conflicts of Interest All authors except JS are or were employees of Pieris Pharmaceuticals, Inc. or its subsidiaries and received compensation from and hold ownership interest in Pieris Pharmaceuticals, Inc. or its subsidiaries. JS is an employee of Oncotest GmbH, a subsidiary of Charles River, Inc., which received funding to perform the mouse studies described in this publication.

Authors' Contributions Conception and design: Development of methodology: Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Writing, review, and/or revision of the manuscript:

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Study supervision:

Acknowledgments We would like to thank the extended Pieris team for their support in generating and reviewing data related to the PRS-343 project. We would like to specifically thank Tanya Aneichyk for her assistance in data interpretation and presentation.

Grant Support The research funding was provided by Pieris Pharmaceuticals. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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) using MCF-7 cells. Similarly, 4-1BB binding studies were performed by ELISA (F) or by FACS (G) using 4-1BB overexpressing CHO cells. Representative sensograms of SPR experiments are shown using immobilized hHER2 (E) or immobilized h4-1BB (H) and allowed determination of PRS-343 on-, off-rate and KD (I). Dual binding: PRS-343 is capable of binding both targets at the same time as determined in a simultaneous binding ELISA (J). Cross-reactivity: PRS-343 displays reduced cross-reactivity to cynomolgus monkey 4-1BB (K).

Figure 2: PRS-343 cell-based activity. Concentration dependence of PRS-343 mediated T-cell activation. Dose-dependent T-cell costimulation by PRS-343 against NCI-N87 (HER2 high), MKN45 (HER2 low) and HepG2 (HER2 null) cell lines, or without tumor cells, using a 4-1BB over-expressing 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 of trastuzumab 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 illustration of experiments carried out with minimum two different donors. Statistical analysis: * p<0.05; ** p<0.01 and *** p<0.001, using 1way ANNOVA with Dunnet’s Multiple comparison test.

Figure 3: Co-culture system data showing correlation between HER2 expression level and T-cell activation (IL2 secretion) via PRS-343. (A) Collated data from co-culture experiments show that significant T-cell activation (as measured by increased IL2) is observed in the presence of HER2 positive cancer cell lines and not primary tissue cell lines. Effect size (x-axis) and significance (y-axis) of PRS-343 on IL2 levels as compared to isotype control. Dotted line shows the significance cut-off corresponding to p-value = 0.05. (B) Data shows that increasing levels of HER2 correlate with enhanced IL2 secretion (R2=0.46; p = 0.002). Shaded area indicates confidence interval for a linear fit.

Figure 4: Pharmacokinetic profile of PRS-343 in murine (A) and primate (B) species following single dose i.v. administration.

Figure 5: PRS-343 mediated antitumor activity in human PBMCs reconstituted xenograft SK-OV-3 tumor- bearing mice. (A) Tumor volume was measured at indicated times through Day 20 and plotted as group mean ± SEM. (B) Lymphocyte phenotyping of the peripheral blood for human cell markers CD45 and CD8 was performed by flow cytometry. All data were plotted as group mean ± SEM. (C) Tumor infiltrating lymphocytes were assessed by immunohistochemistry staining of excised tumors using the human markers (CD45, CD3, CD4 and CD8). (D) For each cell type, fold increase infiltration over isotype control were plotted as column graph plot ± SEM. For each condition, between 3 and 5 tumors were analyzed. (E) Kaplan Meir curves plotting the survival of human PBMC reconstituted non-tumor bearing mice following weekly dosing of PRS-343 or controls.

References

1. Ascierto PA, Simeone E, Sznol M, Fu YX, Melero I. Clinical experiences with anti-CD137 and anti-PD1 therapeutic antibodies. Seminars in oncology 2010;37: 508-16. 2. Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood;131: 49-57. 3. Li SY, Liu Y. Immunotherapy of melanoma with the immune costimulatory monoclonal antibodies targeting CD137. Clinical pharmacology : advances and applications 2013;5: 47-53. 4. Wang C, Lin GH, McPherson AJ, Watts TH. Immune regulation by 4-1BB and 4-1BBL: complexities and challenges. Immunological reviews 2009;229: 192-215. 5. Melero I, Johnston JV, Shufford WW, Mittler RS, Chen L. NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cellular immunology 1998;190: 167-72. 6. Teijeira A, Labiano S, Garasa S, et al. Mitochondrial Morphological and Functional Reprogramming Following CD137 (4-1BB) Costimulation. Cancer immunology research 2018;6: 798-811. 7. Bartkowiak T, Curran MA. 4-1BB Agonists: Multi-Potent Potentiators of Tumor Immunity. Frontiers in oncology 2015;5: 117. 8. Bertram EM, Lau P, Watts TH. Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4- 1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection. Journal of immunology 2002;168: 3777-85. 9. Kawalekar OU, O'Connor RS, Fraietta JA, et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. Immunity 2016;44: 380-90. 10. Melero I, Bach N, Hellstrom KE, Aruffo A, Mittler RS, Chen L. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. European journal of immunology 1998;28: 1116-21. 11. Ye Z, Hellstrom I, Hayden-Ledbetter M, Dahlin A, Ledbetter JA, Hellstrom KE. Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB. Nature medicine 2002;8: 343-8. 12. Zhang H, Knutson KL, Hellstrom KE, Disis ML, Hellstrom I. Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv-expressing whole-cell tumor vaccine compared with CD137- specific monoclonal antibody infusion. Molecular cancer therapeutics 2006;5: 149-55. 13. Yang Y, Yang S, Ye Z, et al. Tumor cells expressing anti-CD137 scFv induce a tumor-destructive environment. Cancer research 2007;67: 2339-44. 14. Martinet O, Divino CM, Zang Y, et al. T cell activation with systemic agonistic antibody versus local 4- 1BB ligand gene delivery combined with interleukin-12 eradicate liver metastases of breast cancer. Gene therapy 2002;9: 786-92. 15. Ye Q, Song DG, Poussin M, et al. CD137 accurately identifies and enriches for naturally occurring tumor- reactive T cells in tumor. Clinical cancer research : an official journal of the American Association for Cancer Research 2014;20: 44-55. 16. Chacon JA, Wu RC, Sukhumalchandra P, et al. Co-stimulation through 4-1BB/CD137 improves the expansion and function of CD8(+) melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy. PloS one 2013;8: e60031. 17. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. The New England journal of medicine 2014;371: 1507-17. 18. Kalos M, Levine BL, Porter DL, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Science translational medicine 2011;3: 95ra73. 19. Vanamee ES, Faustman DL. Structural principles of tumor necrosis factor superfamily signaling. Science signaling 2018;11. 20. Wyzgol A, Muller N, Fick A, et al. Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid- induced TNF receptor ligand. Journal of immunology 2009;183: 1851-61. 21. Bulliard Y, Jolicoeur R, Windman M, et al. Activating Fc gamma receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies. The Journal of experimental medicine 2013;210: 1685- 93.

22. Bulliard Y, Jolicoeur R, Zhang J, Dranoff G, Wilson NS, Brogdon JL. OX40 engagement depletes intratumoral Tregs via activating FcgammaRs, leading to antitumor efficacy. Immunology and cell biology 2014;92: 475-80. 23. Jure-Kunkel M, Hefta LJ, Santoro M, Ganguly S, Halk EL; Fully human antibodies against human 4-1BB. United States of America patent US7288638 B2. 2007. 24. Fisher TS, Kamperschroer C, Oliphant T, et al. Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer immunology, immunotherapy : CII 2012;61: 1721-33. 25. Ahrens B, Baxi SM, Fisher TS, et al.; 4-1BB binding molecules patent US8821867 B2. 2014. 26. Rothe C, Skerra A. Anticalin((R)) Proteins as Therapeutic Agents in Human Diseases. BioDrugs 2018;32: 233-43. 27. Skerra A. Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. The FEBS journal 2008;275: 2677-83. 28. Lewis KB, Meengs B, Bondensgaard K, et al. Comparison of the ability of wild type and stabilized human IgG(4) to undergo Fab arm exchange with endogenous IgG(4)in vitro and in vivo. Molecular immunology 2009;46: 3488-94. 29. Vafa O, Gilliland GL, Brezski RJ, et al. An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations. Methods 2014;65: 114-26. 30. Glaesner W, Vick AM, Millican R, et al. Engineering and characterization of the long-acting glucagon-like peptide-1 analogue LY2189265, an Fc fusion protein. Diabetes/metabolism research and reviews 2010;26: 287-96. 31. Stebbings R, Findlay L, Edwards C, et al. "Cytokine storm" in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics. Journal of immunology 2007;179: 3325-31. 32. Press MF, Cordon-Cardo C, Slamon DJ. Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 1990;5: 953-62. 33. Kwon B. CD137-CD137 Ligand Interactions in Inflammation. Immune network 2009;9: 84-9. 34. Sanchez-Paulete AR, Labiano S, Rodriguez-Ruiz ME, et al. Deciphering CD137 (4-1BB) signaling in T- cell costimulation for translation into successful cancer immunotherapy. European journal of immunology 2016;46: 513-22. 35. Sanmamed MF, Rodriguez I, Schalper KA, et al. Nivolumab and Urelumab Enhance Antitumor Activity of Human T Lymphocytes Engrafted in Rag2-/-IL2Rgammanull Immunodeficient Mice. Cancer research 2015;75: 3466-78. 36. Segal NH, Gopal AK, Bhatia S, et al. A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer. J Clin Oncol 2014;32: suppl; abstr 3007. 37. Gopal AK, Bartlett NL, Levy R, et al. A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL. J Clin Oncol 2015;33: suppl; abstr 3004. 38. Sznol M, Hodi FS, Margolin K, et al. Phase I study of BMS-663513, a fully human anti-CD137 agonist monoclonal antibody, in patients (pts) with advanced cancer (CA) [abstract]. J Clin Oncol 2008;26. 39. Douglass EF, Jr., Miller CJ, Sparer G, Shapiro H, Spiegel DA. A comprehensive mathematical model for three-body binding equilibria. Journal of the American Chemical Society 2013;135: 6092-9. 40. O'Brien NA, Browne BC, Chow L, et al. Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab but not lapatinib. Molecular cancer therapeutics 2010;9: 1489-502. 41. Ginestier C, Adelaide J, Goncalves A, et al. ERBB2 phosphorylation and trastuzumab sensitivity of breast cancer cell lines. Oncogene 2007;26: 7163-9. 42. Tanner M, Kapanen AI, Junttila T, et al. Characterization of a novel cell line established from a patient with Herceptin-resistant breast cancer. Molecular cancer therapeutics 2004;3: 1585-92. 43. Perez-Ruiz E, Etxeberria I, Rodriguez-Ruiz ME, Melero I. Anti-CD137 and PD-1/PD-L1 Antibodies En Route toward Clinical Synergy. Clinical cancer research : an official journal of the American Association for Cancer Research 2017;23: 5326-8. 44. Wei H, Zhao L, Li W, et al. Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin. PloS one 2013;8: e84927.

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Tumor-localized costimulatory T cell engagement by the 4-1BB/HER2 bispecific antibody-Anticalin fusion PRS-343

Marlon J Hinner, Rachida Siham Bel Aiba, Thomas J Jaquin, et al.

Clin Cancer Res Published OnlineFirst May 28, 2019.

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